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TABLE OF CONTENTS
WELCOME...................................................................................................................................2
CAMPUS MAP.............................................................................................................................3
AGENDA AT A GLANCE..............................................................................................................4
TUC MAPS..................................................................................................................................5
POSTER SESSION ASSIGNMENTS.........................................................................................6-7
CONFERENCE DAILY SCHEDULE.........................................................................................8-12
LIST OF ABSTRACTS..........................................................................................................13-37
NOTES................................................................................................................................38-39
ABOUT International Workshop on Opportunistic Protists - 14 : August 9-12, 2017After the human immunodeficiency virus (HIV)/AIDS was recognized in the 1980s as a pandemic impacting the United States, Western Europe, and later Sub-Saharan Africa, it was discovered that in many cases, several microbes, rather than HIV itself, were the direct cause of mortality. In fact, an initial diagnostic criteria for AIDS was the presence of Pneumocystis Pneumonia (PCP) or other Opportunistic Infections (OI). Given the importance of these microbes in AIDS, immunodeficiency-associated diseases, and in newer susceptible populations, such as those receiving new biological therapies that manipulate the immune system (alpha-TNF inhibitors, e.g.), OIs caused by several eukaryotic protists have received the attention of scientists and clinicians worldwide. Collectively and singularly, these organisms present a significant clinical management challenge because of their intractability to standard experimental approaches. They represent diverse taxa such as the micro-Fungi (Pneumocystis, Microsporidia, Candida, Histoplasma), Apicomplexans (Cryptosporidium, Cyclospora, Toxoplasma), and other unicellular eukaryotes (free-living amebae, Blastocystis). All of these OIs present similar challenges to investigators. The primary difficulty facing researchers in these areas is the lack of in vitro axenic mass culture methods that would allow rapid organism proliferation and indefinite sub-cultivation. Various immune-deficient animal models and co-cultivation methods with animal cell lines have been developed, but these alternatives have not eliminated the challenges of low organism yields, serial sub-cultivation, and facile genetic manipulations. Hence, despite “brute force,” expensive, labor-intensive, and creative research approaches, there remain many unanswered questions and an ongoing need for research on these pathogenic organisms. Hence, our gathering at the University of Cincinnati to explore research.
The objectives of IWOP-14 are, therefore, to:(1) Provide a venue for presentation and exchange of the latest research advances in the study of these OIs;(2) Craft an environment of support and collegiality for trainees and minorities in these areas of research;(3) Disseminate advances and challenges for increased input from the greater scientific community;(4) Recruit investigators to these poorly developed areas from those that are more advanced.
TABLE OF CONTENTS
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WELCOME
Welcome to the 14th International Workshop on Opportunistic Protists! It is striking to realize that this Workshop is one of a series that began in 1988 in Bristol, England and has spanned almost 3 decades of scientific research focused on unusual and oftentimes fastidious protists. Remarkable progress in these fields has been facilitated by these meetings and we expect the same from the 14th. We would like to welcome all of our international attendees as we know such travel can be difficult and we appreciate your extra efforts to get here. Thanks to our national attendees as well, sometimes getting from point A to point B is not easy even with the US. We especially want to thank our students and trainees for participating. You are the future of research and we hope you take the opportunity to interact with your peers as well as experienced investigators during the Workshop. The program policy of the 14th IWOP is the same as it was at IWOP 1, an open meeting where anyone can attend and present their findings on the opportunistic protist(s) they are studying. We anticipate presentations on the leading edge of research in all areas, as such work has been presented at previous Workshops. The University of Cincinnati is a great venue. Forbes Magazine says UC is among the world's most beautiful campuses. We are a premier academic institution and a public research powerhouse where more than 44,000 undergraduate and graduate students are empowered daily to challenge and change the world through some serious real-world learning opportunities. As the largest employer in the region, UC’s economic impact is $4 billion. Founded in 1819, our urban university has inspired countless transformative ideas. Over the years, UC researchers have defeated polio, invented Benadryl and even dreamed up the very idea of cooperative education. And it is that sort of innovation that drives hundreds of programs here, dozens of which are ranked in the Top 50 by U.S. News and World Report. Cincinnati, Ohio is a great venue for our Workshop but also hosts many great restaurants and attractions. Information has been included in your complimentary backpacks, but more points of interest can be found in the on-line Visitor’s Guide: http://cincinnatiusa.com/Cincinnati-visitors-guide/. Cincinnati has a great history of beer brewing due to its strong German heritage. We encourage you to explore our newly revitalized area close to downtown, called “Over the Rhine” or OTR, a homage to that heritage. There are many walking tours included in the Visitor’s guide that will take you on historic journeys. Be sure to try some of the signature dishes of the area, such as Skyline chili and Graeter’s ice cream. We have planned signature lunches and dinners that include some of the regional fare. Remember, you are a stone’s throw away from Kentucky which provides more great State Parks and attractions. We would like to recognize and thank our sponsors: the National Institute for Allergy and Infectious Diseases which funded the Conference grant and will support travel awards for trainees; the Cincinnati Center for Clinical and Translational Science and Training which also funded travel awards; and Cidara Therapeutics, which supported the purchase of the Workshop backpacks. Florine Postell, from the UC Conference and Event Services, has been the primary moving force in getting this Workshop organized and we are grateful for her expert help. She will be around the Workshop and, if you have a question, feel free to ask her or your organizers. So, enjoy, learn, and experience the best in research! Melanie T. Cushion and Alexey Porollo Co-Chairs and site organizers for IWOP 14 Louis M. Weiss, M.D., Co-Chair Organizing Committee: Enrique J. Calderon (Spain), Jacob Lorenzo-Morales (Canary Islands, Spain), Olga Matos (Portugal), Anthony P. Sinai (USA), Lihua Xiao (USA)
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UC CAMPUS MAP
Key: Hotels - proximity to TUC Shuttle - stop/drop off location Arrows - walking paths to TUC TUC Cinema - star
Access to Campus Instructions: Walking from either hotel to TUC ~ 10-15 minutes MainStreet/Eden Bearcat Shuttle Bus picks up at Kingsgate Hotel ~ 15 minutes CCM/Eden Bearcat Shuttle Bus picks up by Eden Garage ~ 15 minutes Shuttle Moble App provides real-time shuttle location en route: https://uc.doublemap.com/map/
Parking
Hotels
Main/Eden Shuttle Stop
CCM/Eden Shuttle Stop
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WEDNESDAY, AUGUST 9
6:30 pm – 9:00 pm Registration and Welcome Reception Kingsgate Marriott Hotel Caminetto Restaurant Event Room, Main Level
THURSDAY, AUGUST 10
8:00 am – 3:00 pm Registration Tangeman University CenterCinema Lobby, 2nd Level
7:30 am – 9:00 am Continental Breakfast Cinema 9:00 am Opening Remarks Cinema 9:15 am – 10:30 am Platform Session 1 Cinema10:30 am – 10:45 am Break and Refreshments Cinema10:45 am – 12:15 pm Platform Session 2 Cinema12:15 pm – 2:00 pm Open Time and Lunch Buffet Food Court 2:00 pm – 3:30 pm Round Table Discussion Cinema 3:30 pm – 5:30 pm Poster Session A and Break and Refreshments Atrium, 3rd Floor Dinner at your leisure 6:30 pm Brew Tour Bus Pick Up Kingsgate Marriott Hotel
Street Level
FRIDAY, AUGUST 11
8:00 am – 12:00 pm Registration Tangeman University CenterCinema Lobby, 2nd Level
7:30 am – 9:00 am Continental Breakfast Cinema 9:00 am – 10:15 am Platform Session 3 Cinema10:15 am – 10:45 am Break and Refreshments Cinema10:45 am – 12:30 pm Platform Session 4 Cinema12:30 pm – 2:00 pm Lunch Buffet Food Court 2:00 pm – 3:00 pm Platform Session 5 Cinema 3:00 pm – 5:00 pm Poster Session B and Break and Refreshments Atrium, 3rd Floor 5:00 pm – 6:30 pm Open Time 6:30 pm – 10:00 pm Cocktails (cash bar) and Dinner 400 ABC 4th Level
SATURDAY, AUGUST 12
8:30 am – 11:30 am Continental Breakfast Cinema 8:30 am Open Topic Round Table Cinema12:00 pm End of Conference TUC
AGENDA AT A GLANCE
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TUC FLOOR MAPS
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POSTER SESSIONS
Location Abstract # Title Location Abstract # Title
1 1709 Binding of Pneumocystis carinii to the Epithelial Cell Receptor GRP78 20 1693
Comparison of Pneumocystis nucleic acid and antibody profiles and their associations with other respiratory pathogens in two Austrian pig herds
2 1712 Development of repetitive DNA‐based genotyping tools for the “brain‐eating” ameba Naegleria fowleri 21 1714 Epidemiological investigation of coccidiosis in yaks on
the Qinghai Tibet Plateau of China
3 1713Differential Response of the NLRP3 Inflammasome to Fungal and Bacterial Antigens: Crosstalk between Innate and Adaptive Immune Response
22 1715Changing epidemiology of primary amebic Meningoencephalitis in the United States: What have we learned in recent years?
4 1718A previously undescribed Pneumocystis jirovecii Cytochrome b Mutation Associated with Atovaquone Exposure and putative Resistance
23 1722Application of synthetic recombinant multi‐epitope antigens of Pneumocystis jirovecii in the immunodiagnosis of Pneumocystosis
5 1719 Pneumocystis primary infection: P. jirovecii detection and genomic diversity 24 1723
Evaluation of protein disulfide isomerase (PDI) functions in Toxoplasma‐host invasion mechanism using anti‐human PDI monoclonal antibodies (MAbs)
6 1724 Cloning and expression of MIC3 protein from Toxoplasma gondii 25 1725 In vitro assay of plant extracts from Guiné‐Bissau against
Toxoplasma gondii.
7 1726 Gold Bionanoconjugate‐based Point‐of‐Care Platform for Serological Diagnosis of Pneumocystis Pneumonia 26 1734
Development of a quantitative pharmacokinetic‐pharmacodynamics model of Pneumocystis treatment in mice
8 1737Risk factors and epidemiological features of Pneumocystis Pneumonia in patients without HIV infection in Spain
27 1738 Cryptosporidium oocysts do not require activation in stomach for successful intestinal infection
9 1739 Cryptosporidium spp. in wild rodents of genus Rattus 28 1743
Study of the sensitivity to caspofungin of the Pneumocystis jiroveci i 1,3‐ß glucan synthase catalytic subunit
10 1740 Native and introduced squirrels in Italy host different Cryptosporidium spp. 29 1747
Immunocompetent mice treated with a Bruton’s Tyrosine Kinase Inhibitor (Ibrutinib) are more susceptible to Pneumocystis infection
11 1741 Diversity and biology of Cryptosporidium in ducks and geese in the Czech Republic 30 1753 Duration of storage in liquid nitrogen and effect on the
viability of Pneumocystis isolates
12 1742Revision of effect of adaptive immune response to control microsporidiosis induced by Encephalitozoon cuniculi
31 1754 The effects of gender and strain on Pneumocystis murina Pneumonia in mice
13 1744 Disseminated Encephalitozoon cuniculi infection in patients with hip implant loosening 32 1755
Expression of genes by Pneumocystis murina after treatment with the echinocandin, anidulafungin reveal up‐regulation of genes involved in meiosis and stress and down‐regulation of genes associated with homeostasis
14 1745Prevalence and genotypic characterization of Pneumocystis jirovecii among patients with various respiratory diseases in Poland
33 1708 Investigation of Coccidium species and diagnosis of dairy calves Coccidiosis at Sichuan Province
15 1746 Evolution and diversity of the large surface protein family in the compacted genomes of Pneumocystis 34 1760 The trophic forms of Pneumocystis suppress
macrophage cytokine production
16 1752Prevalence of microsporidia, Cryptosporidium spp. and Giardia intestinalis in children with gastrointestinal diseases and different immunological status
35 1763 Enhancing viability of Pneumocystis in suspension culture by the addition of nutritional supplements
17 1757 Toxoplasma depends on lysosomal consumption of autophagosomes for persistent infection 36 Late Breakers
18 1764 Cryptosporidium infecting North American and European cricetids 37 Late Breakers
19 1765Diagnosis and subtype analysis of Blastocystis isolates from symptomatic and asymptomatic patients in Lower Silesia, Poland
POSTER SESSION A (Thursday) POSTER SESSION B (Friday)
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POSTER LOCATIONS
IWOP14 SESSIONS
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WEDNESDAY
6:30 pm – 9:00 pm ................................................................................................................ Registration and Welcome Reception Kingsgate Marriott Hotel
Caminetto Restaurant Event Room, Main Level
THURSDAY
8:00 am – 3:00 pm ..................................................................................................................................................... ....Registration Tangeman University Center
Cinema Lobby, 2nd Level
7:30 am – 9:00 am .........................................................................................................................................Continental Breakfast
9:00 am ...................................................................................................................................................................Opening Remarks
9:15 am – 10:30 am .............................................................................................................................................. Platform Session 1Session Co-chairs: Louis Weiss and Alexey Porollo
SESSION 1: PROTIST ADAPTATIONS TO THE HOST ENVIRONMENT
9:15 - 9:30
Abstract 1731: Genomic divergence of Pneumocystis populations during adaptation to mammalsPresenter: Cisse, Ousmane; National Institutes of Health
9:30 - 9:45
Abstract 1759: The art of adaptation of Pneumocystis: Insights from studies of the Pneumocystis genomePresenter: Ma, Liang; National Institutes of Health
9:45 - 10:00
Abstract 1751: Mechanisms of surface antigenic variation in the human pathogenic fungus Pneumocystis jiroveciiPresenter: Hauser, Philippe; Lausanne University Hospital
10:00 - 10:15
Abstract 1750: Further evidence of primary homothallism in Pneumocystis speciesPresenter: Hauser, Philippe; Lausanne University Hospital
10:15 - 10:30
Abstract 1767: Functional genomics of the mouse model of Pneumocystis pneumoniaPresenter: Porollo, Alexey; Cincinnati Children’s Hospital Medical Center
10:30 am –10:45 am....................................................................................................................................Break and RefreshmentsTUC Food Court
IWOP14 SESSIONS
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10:45 am – 12:15 pm ........................................................................................................................................... Platform Session 2Session Co-chairs: Anthony Sinai and Philippe Hauser
SESSION 2: HOST: PARASITE INTERACTIONS
10:45 - 11:00
Abstract 1710: Pneumocystis-host defense interactions: A tale of two CLRsPresenter: Limper, Andrew; Mayo Clinic
11:00 - 11:15
Open due to cancellation
11:15 - 11:30
Abstract 1756: Membrane active determinants of Toxoplasma egressPresenter: Carruthers, Vern; University of Michigan
11:30 - 11:45
Abstract 1721: Microsporidian polar tube protein 4 (PTP4)Presenter: Weiss, Louis; Albert Einstein College of Medicine
11:45 - 12:00
Abstract 1720: Pneumocystis jirovecii colonization in preterm infants and their mothers: Prevalence and clinical implications
Presenter: Calderon, Enrique J; Instituto de Biomedicina de Sevilla, Hospital Universitario Virgen del Rocío/ CSIC/Universidad de Sevilla, and CIBER de Epidemiología y Salud Pública
12:00 - 12:15
Abstract 1736: Pneumocystis jirovecii colonization in pregnancyPresenter: Calderon, Enrique J; Instituto de Biomedicina de Sevilla, Hospital Universitario Virgen del Rocío/ CSIC/Universidad de Sevilla, and CIBER de Epidemiología y Salud Pública
12:15 pm – 2:00 pm.........................................................................................................................................Open Time and LunchTUC Food Court
2:00 pm – 3:30 pm...........................................................................................................................................................Round TableSession Co-chairs: Joseph Kovacs and Andrew Limper
Topic: “What is the role of Pneumocystis in immune suppression associated lung disease in children with systemic arthritis?”
Presenter: Vivian Saper, MD; Stanford School of Medicine; Pediatric Rheumatology, Allergy, Asthma, Immunology; Clinical Associate Professor, AdjunctProgram in Immunology, Researcher
3:30 pm – 5:30 pm .......................................................................................................................Poster Session and Refreshments Atrium, 3rd Level
Dinner at your leisure (list of choices at the registration desk)or
Brewery Tour by Bus (6:30 pm meet the bus at Kingsgate Hotel street level for departure)pre-registration required
IWOP14 SESSIONS
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FRIDAY
8:00 am – 12:00 pm ................................................................................................................................................... ....RegistrationTangeman University Center
Cinema Lobby, 2nd Level
7:30 am – 9:00 am..........................................................................................................................................Continental Breakfast
9:00 am – 10:30 am ...............................................................................................................................................Platform Session 3Session Co-chairs: Vern Caruthers and Lihua Xiao
SESSION 3: METABOLIC AND MOLECULAR MECHANISMS
9:00 - 9:15
Abstract 1732: The glucan phosphatase, TgLaforin, regulates amylopectin metabolism in both T. gondii tachyzoites and bradyzoites
Presenter: Sinai, Anthony; University of Kentucky College of Medicine 9:15 - 9:30
Abstract 1733: Ablation of an OTU-family deubiquitinase exposes the underlying regulation governing the plasticity of cell cycle progression in Toxoplasma gondii
Presenter: Dhara, Animesh; University of Kentucky College of Medicine
9:30 - 9:45
Abstract 1735: Characterization of p57, a stage-specific antigen of Pneumocystis murinaPresenter: Kovacs, Joseph; National Institute of Health
9:45 - 10:00
Abstract 1717: Involvement of the parasitophorous vacuole membrane (PVM) in the Cryptosporidium metabolisms?
Presenter: Zhu, Guan; Texas A&M University
10:00 - 10:15
Abstract 1730: Cryopreservation of Cryptosporidium oocysts using the ultra-rapid vitrification techniquePresenter: Jaskiewicz, Justyna; Cummings School of Veterinary Medicine at Tufts University
10:15 am – 10:45 am ..................................................................................................................................Break and RefreshmentsTUC Food Court
IWOP14 SESSIONS
11
10:45 am – 12:30 pm ............................................................................................................................................Platform Session 4Session Co-chairs: Enrique J. Calderon and Olga Matos
SESSION 4: NEW THERAPEUTIC APPROACHES AND MODELS OF INFECTIONS
10:45 - 11:00
Abstract 1705: Mechanisms of action of vitamin D as supplemental therapy for Pneumocystis pneumoniaPresenter: Lee, Chao-Hung; Indiana University School of Medicine
11:00 - 11:15
Abstract 1706: Pseudoloma neurophilia (Microsporidia) in laboratory zebrafishPresenter: Kent, Michael; Oregon State University
11:15 - 11:30
Abstract 1761: A vaccine strategy to prevent Pneumocystis coinfection in a non-human primate model of HIVPresenter: Rabacal, Whitney; University of Georgia
11:30 - 11:45
Abstract 1766: Ibrutinib reduces the inflammatory response of alveolar macrophages to P. murinaPresenter: Linke, Michael; University of Cincinnati/Cincinnati Veterans Affairs Medical Center
11:45 - 12:00
Abstract 1748: Profiling gene expression of Pneumocystis murina after treatment with anidulafunginPresenter: Cushion, Melanie T.; University of Cincinnati/Cincinnati Veterans Affairs Medical Center
12:00 - 12:15
Abstract 1749: Prevention of Pneumocystis pneumonia (PCP) by the novel long-acting Echinocandin, CD101: Implications for life cycle
Presenter: Cushion, Melanie T.; University of Cincinnati/Cincinnati Veterans Affairs Medical Center
12:15 - 12:30
Abstract 1716: Zebrafish as an in vivo model for toxoplasmosisPresenter: Sanders, Justin; Oregon State University
12:30 pm – 2:00 pm ...................................................................................................................................................................Lunch TUC Food Court
IWOP14 SESSIONS
12
2:00 pm – 3:00 pm ................................................................................................................................................Platform Session 5 Session Co-chairs: Guan Zhu and Michael Kent
SESSION 5: EPIDEMIOLOGY OF ZOONOTIC INFECTIONS
2:00 - 2:15
Abstract 1702: Genetic and biological similarity of Cyclospora cayetanensis to cecum-infecting Eimeria spp. as revealed by comparative genomic analysis
Presenter: Feng, Yaoyu; South China Agricultural University 2:15 - 2:30
Abstract 1707: Comparative genomics of an emerging Cryptosporidium hominis outbreak subtype in the United States
Presenter: Xiao, Lihua; Centers for Disease Control and Prevention
2:30 - 2:45
Abstract 1758: Glycan triggers of trophozoite development in Cryptosporidium Presenter: McEvoy, John; North Dakota State University
2:45 - 3:00
Abstract 1762: Molecular characterization of Enterocytozoon bieneusi in wild carnivores in SpainPresenter: Santin, Monica; United States Department of Agriculture
3:00 pm – 5:00 pm ...................................................................................................................................................Poster Session BAtrium, 3rd Level
5:00 pm – 6:30 pm .....................................................................................................................................................................Open
6:30 pm – 10:00 pm.........................................................................................................................................Reception and Dinner Room 400 ABC, 4th Level
SATURDAY
8:30 am – 12:00 pm. ................................................................................................................................... ....Continental Breakfastand Open Topic Round Table
Cinema Lobby, 2nd Level
12:00 pm..........................................................................................................................................................Conference concludes
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# 1693
Title: Comparison of Pneumocystis nucleic acid and antibody profiles and their associations with other respiratory pathogens in two Austrian pig herds
Pneumocystiscarinii f. sp. suis (PCS) nucleic acid and antibody profiles in two Austrian-farrow-to-finish farms were investigated. Their associations with porcine circovirus type 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza virus (SIV), Haemophilusparasuis (HPS), and Mycoplasmahyopneumoniae (MH) were evaluated. Respiratory specimen (n=169) and serum samples (n=126) of pigs from different age classes (suckling piglets 1+3 weeks old, weaning piglets 2+3 months old, fattening pigs 4 months old, and sows) with respiratory disorders were collected. The pathogens were analyzed by PCR, antibody titres against Pneumocystis were determined by XpressBio Pneumocystiscarinii swine ELISA. In farm A, PCS infection occurred early in life. The suckling piglets were already affected in the 1st week of life and the pigs remained positive until the 3rd month of life. In farm B, pigs were infected later between 3 and 4 months of age. The maximum PCS nucleic acid load in farm A was 109 copies/ml BALF, whereas in farm B the PCS burden was significantly lower with 107 copies/ml BALF. Anti-PCS antibodies were present in sows, as maternal antibodies in suckling piglets and as immunological reaction after infection. In both farms, PCS infection was accompanied by several co-infections. In farm A, there were concurrent infections with PRRSV, a virulent HPS strain, and MH. In farm B, PCS was accompanied by infections with SIV, MH, and non-virulent HPS. The results clearly show that the PCS profiles can vary between farms. PCS can contribute to respiratory disorders of pigs of different age classes as soon as the proliferation of the fungus is triggered by co-infections causing immunosuppression. Nevertheless, younger pigs seem to be more susceptible and show higher PCS burdens. As a consequence, PCS should probably be considered as contributing co-factor in the interaction of respiratory pathogens.
Author: Weissenbacher-Lang, Christiane, (University of Veterinary Medicine Vienna, Institute of Pathology and Forensic Veterinary Medicine)Co-Authors: Nedorost, Nora (University of Veterinary Medicine Vienna, Vienna, WN, Austria); Knecht, Christian (University of Veterinary Medicine, Vienna, WN, Austria); Hennig-Pauka, Isabel (University of Veterinary Medicine Vienna, Vienna, WN, Austria); Huber, Mathias; Voglmayr, Thomas (Traunkreis Vet Clinic, Ried im Traunkreis, OO, Austria); Weissenböck, Herbert (University of Veterinary Medicine Vienna, Vienna, WN, Austria)Presentation Type: Poster
# 1702
Title: Genetic and biological similarity of Cyclospora cayetanensis to cecum-infecting Eimeria spp. as revealed by comparative genomic analysis
Cyclospora cayetanensis is an emerging foodborne parasite that has received high attentions because of the massive foodborne outbreaks they caused in recent years. The investigation of foodborne outbreaks of cyclosporiasis has been hampered by a lack of genetic data and poor understanding of pathogen biology. We sequenced the whole genome of C. cayetanensis from a patient in China using Illumina 100-bp paired-end technology. The apicoplast and mitochondrial genomes of C. cayetanensis are highly similar to those of cecum-infecting avian Eimeria spp. in both gene organization and sequences. A comparative analysis of the nuclear genomes has confirmed the similarities in genome organization, metabolic capabilities and potential invasion mechanism between C. cayetanensis and Eimeria tenella. Propanoyl-CoA degradation, GPI anchor biosynthesis, and N-glycosylation are some apparent metabolic differences between C. cayetanensis and E. tenella. The similar repertoire of host cell invasion-related proteins possessed by all coccidia suggests that C. cayetanensis has an invasion process similar to the one in T. gondii and E. tenella. However, the significant reduction in the number of identifiable rhoptry protein kinases, phosphatases and serine protease inhibitors indicates that monoxenous coccidia, especially C. cayetanensis, have limited capabilities or use a different system to regulate host cell nuclear activities. C. cayetanensis does not possess any cluster of genes encoding the TA4-type SAG surface antigens seen in E. tenella, and may use a different family of surface antigens in initial host cell interactions. Based on the whole genome sequence data, a multilocus sequence typing tool was developed, with 2 to 10 geographically segregated sequence types at each of 5 selected loci. This new tool could be useful in case linkage and infection/contamination source tracking.
Author: Feng, Yaoyu (South China Agricultural University)Presentation Type: Platform
# 1705
Title: Mechanisms of action of vitamin D as supplemental therapy for Pneumocystis pneumoniaThe combination of trimethoprim and sulfamethoxazole (TMP-SMX) is the most effective regimen for therapy of Pneumocystis pneumonia (PcP). As many patients with PcP are allergic or do not respond to it, efforts have been devoted to develop alternative therapies for PcP. We have found that the combination of all-trans retinoic acid (ATRA) and primaquine (PMQ) is effective for PcP therapy. Since ATRA also has adverse effects, we tested the combination of vitamin D3 (VitD3; 300 IU/kg/day) and PMQ (5 mg/kg/day) and found it to be as effective as TMP-SMX for therapy of PcP. In this study, we investigated the mechanisms by which vitamin D enhances the efficacy of PMQ. Results showed that vitamin D supplementation increased the number of CD11c+ cells; suppressed the production of pro-inflammatory cytokines (TNF-a, IFNg, and IL-6) and iNOS; and enhanced the expression of genes related to anti-oxidation (glutamate-cysteine ligase modifier subunit and glutathione reductase), anti-microbial peptides (cathelicidin), and autophage (ATG5 and Beclin-1). These results suggest that the main action of vitamin D is enhancing the ability of the host to defend Pneumocystis infection.
Author: Lee, Chao-Hung (Indiana University School of Medicine)Co-Authors: Lei, Guang-Shen (Indiana University School of Medicine, Indianapolis, IN, United States); Zhang, Chen (Indiana University School of Medicine, Indianapolis, IN, United States)Presentation Type: Platform
LIST OF ABSTRACTS
14
# 1706
Title: Pseudoloma neurophilia (Microsporidia) in laboratory zebrafishAs the zebrafish has grown in popularity as a research model for numerous fields of study, so too has awareness of the impacts of underlying chronic infections on research endpoints using this fish. Zebrafish is commonly infected by the microsporidium, Pseudoloma neurophilia. We previously showed that the parasite is vertically transmitted through infected eggs. Hence, it is not surprising that the parasite is present in about half of the zebrafish research facilities based on submissions to the Zebrafish International Resource Center diagnostic service over the last 17 years. The use of zebrafish in behavioral studies has exploded over the past two decades. They have become models for human anxiety, memory, and learning, as well as for specific neurobehavioral diseases such as schizophrenia, autism, and Parkinson’s disease. As the species name implies, the parasite targets the central nervous system, where it infects primarily the hindbrain and spinal white matter, the griseum centrale, and the reticular formation. These are structures associated with the startle response, anxiety, and aversion learning, all of which are important factors in behavioral studies. We conducted three separate behavior studies (shoaling inter-fish distance, habituation to startling stimuli, and capture avoidance). Infected fish showed inhibited habituation to startling stimuli (tapping), enhanced netting evasion, and tighter shoaling patterns. These results, taken together, indicate that P. neurophilia influences zebrafish behavior generally and may specifically potentiate anxiety and hypervigilance. Due to the nature of most behavioral studies utilizing zebrafish, P. neurophilia is a source of non-protocol induced variation. On a positive note, the zebrafish/P. neurophilia system offers a robust model for studying effects of parasitism on behavior as the infection is easy to control and initiate in the laboratory, and the genomes of both the host and parasite are known.
Author: Kent, Michael (Oregon State University)Co-Authors: Sanders, Justin; Spagnoli, Sean (Oregon State University, Corvallis, OR, United States)Presentation Type: Platform
# 1707
Title: Comparative genomics of an emerging Cryptosporidium hominis outbreak subtype in the United StatesCryptosporidium is the leading cause of waterborne disease outbreaks in industrialized nations. In the United States, an estimated 748,000 cryptosporidiosis cases occur annually, and since 2004, the annual incidence of nationally notified cryptosporidiosis has risen threefold in the country. Accompanying the recent increase was the switching of the dominant Cryptosporidium hominis outbreak subtype from IbA10G2 to IaA28R4. Since 2009, a new C. hominis subtype, IfA12G1R5, appeared as a cause of cryptosporidiosis outbreaks in the United Sates and has now become of dominant subtype in the country. To understand to evolution of virulent C. hominis subtypes, we sequenced the genome of 83 C. hominis isolates, including 46 isolates of the IfA12G1R5 subtype. At the whole genome level, there are three major variants (IfA12G1R5a, IfA12G1R5b, and IfA12G1R5c) of IfA12G1R5, which have ~1,000-2,000 SNPs compared with IaA28R4, with most sequence differences in mucin genes (cgd6_1080 and cgd2_430-cgd2_450). In addition, most IfA12G1R5 isolates have a gene content identical to those of IaA28R4, with the absence of cgd2_4380 and presence of cgd6_5470. In contrast, there are >4,000 SNPs between IfA12G1R5 and IbA10G2, which has cgd2_4380 but no cgd6_5470. Among the three major IfA12G1R5 variants, IfA12G1R5b appears to be the initial recombinant of the Ia subtype family (IaA13R3, IaA15R3, or IaA28R4 subtype), IfA12G1R5a is the dominant one currently in circulation, and IfA12G1R5c is probably the newest progeny. The latter has acquired cgd2_4380 (possibly from IbA10G2) and new sequence type at the 5’ end of chromosome 6. Thus, the new subtype IfA12G1R5 appears to be mixed progenies of several crosses of dominant subtypes previously circulating in the United States, and genetic recombination appears to be driven force for the emergence of virulent C. hominis subtypes.
Author: Xiao, Lihua (Waterborne Disease Prevention Branch; Division of Foodborne, Waterborne, and Environmental Diseases; Centers for Disease Control and Prevention)Co-Authors: Wang, Yuanfei (Waterborne Disease Prevention Branch; Division of Foodborne, Waterborne, and Environmental Diseases; Centers for Disease Control and Prevention, Atlanta, GA, United States and School of Resources and Environmental Engineering, East China University of Science and Technology, Shanghai, China); Roellig, Dawn M. (Waterborne Disease Prevention Branch; Division of Foodborne, Waterborne, and Environmental Diseases; Centers for Disease Control and Prevention, Atlanta, GA, United States); Feng, Yaoyu (School of Resources and Environmental Engineering, East China University of Science and Technology, Shanghai, China)Presentation Type: Platform
# 1708
Title: Investigation of Coccidium species and diagnosis of dairy calves Coccidiosis at Sichuan ProvinceFor known of Eimeria species of dairy calves, the 240 fecal samples from Holstein and Chinese Simmental calves on 12 dairy cattle farms were collected at Sichuan Province, China. Egg floating Method was used to collect coccidium oocystes. Then, the identification of oocystes was performed according to morphology using microexamination. On the other hand, the oocystes in 2.5%-potassium dichromate solution were hatched at 37? and in incubator for 3-8 days. Their features, including oocyste shape, oocyste wall, micropore through oocyste wall, oocyste residual bodyes, sporangiums shape, sporozoites, sporangium residula bodes, etc., were observed under microscope. The results showed that there were 6 species, Eimeria abramovi, E. alabamensis, E. bukidnonensis, E. Cylindrical,E. Ellipsoidallis and E. Zürnii.On the 5 dairy cattle farms, the 41 dairy calves which were diagnosed as no other disease except for possible coccidiosis were divided into three groups. According to their fecal characteristics, group A of 21 dairy calves didn’t showed diarrhea, etc., group B of 12 dairy calves showed diarrhea and group C of 11 dairy calve had mucosanguineous feces. McMaster mother was used to count the oocystes in the feces from the dairy calves respectively. Then, their OPG levels were obtained after of processed by statistical method. The results were that the OPD levels of Group A, Group B and Group C were 1352 ± 1458.266, 8788 ± 2863.085 and 21159±23204.400 respectively. On the basis of the OPG levels and the fecal characteristics, the decision reference standard of dairy calves coccidiosis was made, which was low-grade infection which was OPG=1000, medi-grade infection which was 1000?OPG=5000 and high-grade infection which was OPG?5000. We suggest that the dairy calves with OPG? 5000 will need treatment at Sichuan Province.
Author: Dangjin, Liao (Sichuan Animal Science Academy)Co-Authors: Ye, Yonggang (Sichuan Animal Science Academy, Chengdu, China)Presentation Type: Poster
LIST OF ABSTRACTS
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# 1709
Title: Binding of Pneumocystis carinii to the epithelial cell receptor GRP78Pneumocystis pneumonia (PCP) remains an important life-threatening infection in patients with AIDS and malignancy caused by Pneumocystis jirovecii (Pj). Although a number of reports suggest the importance of lung macrophages and their surface receptors in organism/cell wall ligand binding leading to downstream immune signaling and subsequent inflammatory responses, little is known about the initial binding/colonization of the airway epithelium. Our prior studies have documented cell-signaling events that occur following binding of the organisms to lung epithelial cells. However, the various binding receptors that mediate Pneumocystis attachment to lung surfaces have not yet been fully defined. Accordingly, we sought to determine the receptors present in host lung epithelial cells that might be important for Pneumocystis spp. binding. Using an affinity chromatography approach, we have identified glucose-regulated protein 78 (GRP78) as a potential novel host receptor that may have relevance in initial lung colonization by the fungus. Pneumocystis carinii (Pc) organisms bound not only GRP78 in the rat lung epithelial RLE-6TN cell line, but also in primary rat airway epithelial cells (AEC) as well. Furthermore, Pc bound CHO1 cells overexpressing surface GRP78 more than the parent CHO1 line alone, suggesting the importance of the Pc/GR78 protein interaction in mediating organism attachment. Future studies will examine if specific GRP78 antibodies can block the Pc-host cell interactions and also determining the potential ligand(s) on the organism surface that interacts with GRP78. These results provide initial insights into the interactions of Pneumocystis spp. with host lung epithelium.
Author: Kottom, T.J. (Thoracic Diseases Research Unit; Mayo Clinic)Co-Authors: Hebrink, D.M.; Limper, Andrew (Thoracic Diseases Research Unit; Mayo Clinic, Rochester, MN, United States) Presentation Type: Poster
# 1710
Title: Pneumocystis-Host Defense Interactions: A Tale of Two CLRsPneumocystis jirovecii continues to cause life-threatening pneumonia in patients with HIV and other immunocompromised conditions. Host defenses against Pneumocystis involve clearance of the organism, but also initiate activation of deleterious host inflammation leading to respiratory impairment. Our group and others have conducted studies to define host receptor mechanisms involved in mediating host defense against Pneumocystis. The CLR Dectin-1 binds to surface beta-glucans present on cyst forms of Pneumocystis, mediating clearance of the organism, but also strongly activating lung inflammation through translocation of NF- kB with expression of inflammatory cytokines including TNF-alpha and MIP-2. The beta-glucans surface components are present nearly exclusively on cyst forms, but are lacking from the trophic forms. Accordingly, we further sought to identify receptors that interact with MSG/gpA present on the surface of the abundant trophic forms as well as cyst forms. In this light, we identified the novel CLR Mincle as an important receptor that binds MSG/gpA and demonstrated that Mincle mediates host cell inflammatory responses through Syk activation leading to an NF- kB translocation and activation of inflammatory cytokine generation. We recently studied CD4 depleted Mincle knockout mice with Pneumocystis and demonstrated that these animals exhibited significantly impaired clearance of the organism compared to wild type controls despite up regulation of Dectin-1 and other CLR receptors. In addition, host inflammatory responses were modulated with upregulation of the anti-inflammatory molecule IL-1ra during Pneumocystis pneumonia in the absence of Mincle. Our studies indicate strong and complementary interactions of Dectin-1 and Mincle in mediating clearance of the organism, as well as in maintaining the balance of information during infection.
Author: Limper, Andrew ( Mayo Clinic) Co-Authors: Kottom, T.J.; Hebrink, D.M.; Nandakumar, Viji (Thoracic Diseases Research Unit; Mayo Clinic, Rochester, MN, United States)Presentation Type: Platform
# 1712
Title: Development of repetitive DNA-based genotyping tools for the “brain-eating” ameba Naegleria fowleriThe “brain-eating” free-living ameba, Naegleria fowleri, causes a rare brain infection, primary amebic meningoencephalitis (PAM), which is almost always fatal. The ameba is found globally in warm fresh waters, hot springs, and waterparks. Recently, N. fowleri has been found to colonize piped public drinking water systems in the United States, which was linked to the death of a young child. It is unclear why PAM cases are rare while people are likely to be routinely exposed to N. fowleri. Variability in the ameba’s virulence may explain this. However, a genotyping system capable of categorizing N. fowleri strains based on their virulence does not exist. We performed Illumina Hiseq during 2015-2016 to accomplish whole genome sequencing (WGS) of 38 N. fowleri strains representing all three USA genotypes isolated from clinical and environmental samples. Bioinformatics analysis of WGS data identified repetitive DNA sequences, single nucleotide polymorphisms, and other unique DNA sequences that can be used to develop powerful genotyping tools. In this study, we have been testing about 100 loci containing repetitive DNA sequences and verifying their usefulness to develop a reliable genotyping tool for the N. fowleri. One of our goals here is to identify markers (loci) that will categorize N. fowleri strains into 5-10 different genotypes. Some of the loci tested so far showed promise, and were able to detect additional variation within a particular existing USA genotype. An improved genotyping tool will help (a) survey circulating genotypes of N. fowleri in the natural environment, (b) link a PAM case to its environmental source, and (c) understand whether certain strains of N. fowleri are more virulent (disease-causing) than others.
Author: Ali, Ibne (Centers for Disease Control and Prevention)Co-Authors: Kelley, Alyssa; Roy, Shantanu (Centers for Disease Control and Prevention, Atlanta, GA, United States)Presentation Type: Poster
LIST OF ABSTRACTS
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# 1713
Title: Differential response of the NLRP3 inflammasome to fungal and bacterial antigens: Crosstalk between innate and adaptive immune response
The NLRP3 inflammasome is activated in response to different bacterial, viral and fungal pathogens and serves as modulator of different pattern recognition receptors signaling pathways. One of the main functions of the NLRP3 is to participate in IL-1ß maturation important in the host defense against Pneumocystis and other fungal infections. However, dysregulation of the NLRP3 and IL-1ß secretion are also implicated in the pathophysiology of many auto-inflammatory disorders. Oftentimes inflammatory flares are preceded by infectious illnesses questioning the role of infection in autoimmune exacerbations. However, the exact role that infection or even colonization plays as triggers of inflammation is still not fully understood. Herein, we have investigated the role of the NLRP3 in human circulating B-lymphocytes and identified that the NLRP3 is essential for two independent processes, pro-inflammatory cytokine and antibody regulation. Our results showed that ß-glucan stimulated B-lymphocytes secreted IL-1ß which was partially mediated by Dectin-1 activation via SYK and the transcription factors NF-kB and AP-1. IL-1ß was regulated by the NLRP3 inflammasome, which was dependent on ATP, potassium efflux and Caspase-1 (CASP1). Interestingly, B-lymphocytes activated by unmethylated CpG motifs, found in bacterial DNA, failed to induce IL-1ß even in the presence of ATP. However, B-lymphocyte stimulation by CpG resulted in NLRP3 and CASP1 activation and the production and secretion of IgM antibodies. Furthermore, CpG-stimulated IgM secretion unlike b-glucan-mediated IL-1ß production was mediated by the mammalian target of rapamycin. Inhibition of the NLRP3 and the mTOR pathway in CpG activated B-lymphocytes resulted in impaired IgM secretion suggesting their participation in antibody regulation. In conclusion, this study describes a differential response of the NLRP3 to fungal and bacterial antigens and identifies the NLRP3 inflammasome of human circulating B-lymphocytes as modulator of the innate and adaptive immune systems.
Author: Ali, Mohamed (Mayo Clinic)Co-Authors: Dasari, Harika; Limper, Andrew; Carmona, Eva (Mayo Clinic, Rochester, MN, United States)Presentation Type: Poster
# 1714
Title: Epidemiological investigation of coccidiosis in yaks on the Qinghai Tibet Plateau of ChinaAn observational study was conducted to determine the prevalence of coccidial infection in yaks on the Qinghai-Tibet Plateau of China. A total of 865 fecal samples from 9 counties from September to October 2015 was examined, and oocysts were identified to the species level on the basis of morphological features. The results showed: the positive rate of coccidiosis in 9 counties was 100%, and the total infection rate was 31.1% (269/865). According to different age statistics, the infection rate of calves within 6 month old years was 49.15% (146/295). The infection rate of yak at 1.5 years old was 29.07% (84/289), and the infection rate of adult yaks was 13.8% (39/281). The fecal oocyst count per gram (OPG) was 20~5160, and the mean OPG was 262. 14 different species of Eimeria were identified, the species detected and their prevalence values included the following: Eimeria zuernii (50.93%) , E.pellita(36.43%) , E.canadensis(29.94%) , E.bovis(25.28%) , E.cyli-ndrica(19.7%) , E.subspherica(16.73%) , E.ellipsoidali(17.47%), E.Brasiliensis(13.38%) E.wyomingensis(10.41%) , E.Alabamensis(8.92%) , E.illinoisensis(7.06%) , E.auburnensis(5.58%), E.Bombayansis(4.83%) , E. bukidnonensis(3.72%). The majority of yaks showed infection, E. Züernii , E. Pellita and E. canadensis were the most prevalent species in yaks in Qinghai province, and the infection was more serious in less than 6 month old yaks. This may be the key to yak coccidiosis prevention and control.
Author: Cai, Jinzhong (Qinghai Academy of Animal and Veterinary Science)Co-Authors: Li, Chunhua (Qinghai Academy of Animal and Veterinary Science, Xining, China); Huang, Bing (Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Science, Shanghai, China); Lei, Mengtong (Qinghai Academy of Animal and Veterinary Science, Xining, China); Dong, Hui (Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Science, Shanghai, China)Presentation Type: Poster
# 1715
Title: Changing epidemiology of primary amebic meningoencephalitis in the United States: What have we learned in recent years?
Primary amebic meningoencephalitis (PAM) is a fulminant central nervous system infection caused by the thermophilic free-living ameba, Naegleria fowleri, which thrives in warm freshwater. Historically, case reports tended to come from southern-tier states in persons exposed to recreational freshwater. CDC surveillance has documented substantial changes in the epidemiology of PAM in the United States over the past five years. We analyzed data from CDC’s free-living ameba surveillance system for laboratory-confirmed PAM cases from 1962–2016, and describe, in detail, cases from 2010–2016 to examine the recent epidemiology of PAM in the United States. There have been 143 cases of PAM reported in the United States from 1962 through 2016. During 2010–2016, 29 cases of PAM were reported to CDC (4 in 2010, 5 each in 2011 and 2012, 4 in 2013, 1 in 2014, 5 in 2015, and 5 in 2016). Cases were reported from 15 states and territories. Sixty-three percent of cases were female and the median age was 11 years (range: 4–56 years). Twenty-four (83%) cases had exposure to recreational freshwater from a lake, reservoir, river, stream, or ditch during their incubation period. Five (17%) cases were exposed to piped water via nasal irrigation using a neti pot (2 cases), nasal irrigation for ritual ablution (1), play on a backyard waterslide (1), and swimming in a poorly maintained pool using warm water piped overland (1). The epidemiology of PAM in the United States is evolving. While CDC continues to see cases with recreational freshwater exposures, we have now documented cases associated with the use of piped water, bringing to light the threat posed by Naegleria colonizing building plumbing and water distribution systems. Standardized surveillance and reporting of amebic encephalitis, including PAM, is crucial to understanding the changing epidemiology.
Author: Cope, Jennifer (Centers for Disease Control)Presentation Type: Poster
LIST OF ABSTRACTS
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# 1716
Title: Zebrafish as an in vivo model for toxoplasmosisIn spite of the cosmopolitan nature and intensive study of Toxoplasma gondii, no drugs exist to treat the chronic stage of the parasite. The development of a zebrafish model for T. gondii infection model provides the advantage of small size to conserve reagents for testing new antimicrobial agents with the potential of identifying new compounds for treatment potentially leading to a definitive cure of the infection. In 2015, we adapted zebrafish to 37°C and injected them intraperitoneally with two strains of T. gondii and observed them for 7 days post injection. Moribund fish were examined by histology for the presence of T. gondii development. Intracellular parasites were observed in fish beginning at 5 days post injection. The pattern of infection observed was similar to that found in mammalian infection, with parasites developing in several tissues including the brain. This was the first observation of T. gondii infection in a fish. We have subsequently worked to improve the survival of adult zebrafish at 37°C and have adapted this work to transparent casper zebrafish to allow for visualization of fluorescent parasites in whole animals. Using this system, we have conducted experiments demonstrating proof of principle for using the model in drug discovery, demonstrating that sulfadiazine is effective in treating acute infection in zebrafish. Fluorescently labeled tachyzoites (RH:YFP) were injected into casper zebrafish. Two groups were treated with either 400 mg/ml sulfadiazine or lowered temperature to 35 C at 2 days post-exposure (dpe). Six of 9 untreated fish died or became moribund by 6 dpe, and exhibited severe infections, determined by whole fish examination. In contrast, fish treated with either sulfadazine or moved to 35 C showed minimal mortality, with infection observed in only one fish from each group.
Author: Sanders, Justin; (Oregon State University)Co-Authors: Kent, Michael (Oregon State University, Corvalis, OR, United States) Presentation Type: Platform
# 1717
Title: Involvement of the parasitophorous vacuole membrane (PVM) in the Cryptosporidium metabolisms?The host-cell derived parasitophorous vacuole membrane (PVM) forms a physical barrier between the intracellularly developing Cryptosporidium parasite and the environment such as gastrointestinal lumen. The unique PVM is apparently a vital structure for the parasite, but the biology of the PVM is still poorly understood. Around two decades ago, we have observed that the PVM contains many undefined proteins derived from C. parvum. We later localized several parasite proteins to the PVM, including a long-chain fatty acyl-CoA binding protein (ACBP), one of the two oxysterol-binding protein (OSBP)-related proteins (ORP1), and a long chain fatty acid elongase (LCE). More recently, we further observed that one of the three fatty acyl-CoA synthetases (ACS2) and a lactate dehydrogenase (LDH) from C. parvum were also localized to the PVM. These observations strongly imply that the host cell-derived PVM is actively involved in the parasite lipid metabolism and lactate fermentation, for which further investigations might help to provide new insight into the biological role of PVM.
Author: Zhu, Guan (Texas A&M University)Presentation Type: Platform
# 1718
Title: A previously undescribed Pneumocystis jirovecii cytochrome b mutation associated with atovaquone expo-sure and putative resistance
Several mutations at the cytochrome b gene of Pneumocystis jirovecii (P.jirovecii) associated with atovaquone exposure have already been reported. Atovaquone is frequently used as a second line regimen for Pneumocystis pneumonia (PCP) prophylaxis at the heart transplantation unit of Bichat University Hospital (Paris, France). In this context, our objective was to genotype at the cytochrome b gene P.jirovecii isolates obtained from heart transplant recipients monitored at this hospital. Twenty-one P. jirovecii DNA isolates obtained from 10 heart transplant recipients and 11 unlinked control patients who were contemporaneously diagnosed with Pneumocystis infections were examined. The cytochrome b gene was amplified and sequenced from both strands. Sequences were aligned and compared with the reference sequence. A medical chart survey was also performed to determine whether the patients had past history of atovaquone prophylaxis or treatment. Cytochrome bc1 complex modelling was performed to characterize the molecular basis underlying putative resistance of P. jirovecii organims to atovaquone.P.jirovecii genotyping was successful in 9 out of the 10 heart transplant recipients and in the 11 unlinked patients. Seven out of the 10 heart transplant recipients and none of the 11 unlinked patients had past history of atovaquone exposure respectively (7/10 vs. 0/11, p<0.01). Type CYB2 harboring a previously undescribed mutation on position 350 (C350T) was identified in the 9 heart transplant recipients whereas it was not identified in the second patient group (9/9 vs. 0/11, p <0.01). This transition C350T represents a non-synonymous mutation with an amino acid change Ala 144 Val located at the Qo site which is the binding site of atovaquone. The modeling analysis supports the hypothesis that this mutation confers atovaquone resistance through decreased cavity volume for atovaquone binding.This newly described non-synonymous mutation C350T at the cytochrome b gene of P.jirovecii may be associated with atovaquone resistance.
Author: Le Gal, Solène (Brest University Hospital)Co-Authors: Argy, Nicolas (Hôpital Bichat - Claude Bernard, Paris, France); Song, Zehua (Université Paris-Sud, Gif sur Yvette, France); Kao, Wei-Chun; Hunte, Carola (University of Freiburg, Freiburg, Germany); Vindrios, William; Yazdanpanah, Yazdan; Lucet, Jean-Christophe; Houzé, Sandrine (Hôpital Bichat-Claude Bernard, Paris, France); Clain, Jérôme (Université Paris Descartes, Paris, France); Nevez, Gilles (Brest University Hospital, Brest, France)Presentation Type: Poster
LIST OF ABSTRACTS
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# 1719
Title: Pneumocystis primary infection: P. jirovecii detection and genomic diversityPneumocystis jirovecii (P. jirovecii) primary infection occurs with a high incidence during the first two years of life in humans. Nonetheless, data on genomic characteristics of P. jirovecii in this patient population are still scarce, while molecular typing of P. jirovecii has mostly been performed in immunosuppressed patients with PCP. The present report describes P. jirovecii detection in 196 symptomatic immunocompetent infants followed up in the Brest University Hospital, Brest, France. MLST genotyping of P. jirovecii was performed in the positive infants with primary infection and in a population of immunosuppressed adults developing PCP, these two patient populations being managed in the same hospital. P. jirovecii specimens were examined at the mitochondrial large subunit rRNA (mtLSUrRNA), cytochrome (CYB), and superoxide dismutase (SOD) genes. These loci were amplified and sequenced from both strands. Sequences were aligned and compared with the reference sequences. P. jirovecii was detected in 36 infants (18.4%) with a peak of detection at the age of 30-90 days. The genotyping gave positive results in 26 infants and in 20 adults with PCP. Twenty different MLST genotypes were identified, 9 in the infants developing P. jirovecii primary infection and 11 in the immunosuppressed adults with PCP. The MLST method allowed discriminating the patients in 2 groups according to their presentation of P. jirovecii infection and their age. Indeed, none of the MLST genotypes identified in the first patient group were found in the second patient group. These results provide additional data on primary infection and suggest that i) infants develop primary infection in the first months of life as previously observed in Chile, France, and Denmark, ii) infants and adults harbor different P. jirovecii MLST genotypes in Brest iii) cycles of P. jirovecii acquisition and transmission in the two patient populations may occur independently.
Author: Le Gal, Solène (Brest University Hospital)Co-Authors: Guillaud-Saumur, Thibaud (Université de Bretagne Occidentale, Brest, France); Cros, Pierrick (Brest University Hospital, Brest, France); Virmaux, Michèle (Université de Bretagne Occidentale, Brest, France); Vallet, Sophie (Brest University Hospital, Brest, France); Pougnet, Laurence (Université de Bretagne Occidentale, Brest, France); de Parscau du Plessix, Loïc (Brest University Hospital, Brest, France); Vargas, Sergio (Universi-dad de Chile, Santiago, Chile); Nevez, Gilles (Brest University Hospital, Brest, France)Presentation Type: Poster
# 1720
Title: Pneumocystis jirovecii colonization in preterm infants and their mothers: prevalence and clinical implicationsPneumocystis jirovecii is an atypical opportunistic fungus with lung tropism and strong host species specificity that cause interstitial plasma cell pneumonia, the first human disease associated with Pneumocystis infection, in premature and malnourished babies. Even though, serologic studies have shown that children are exposed to Pneumocystis early in life the epidemiology of human Pneumocystis infection and the host–microorganism relationship in infancy remain poorly understood. The aim of the present study was to investigate the prevalence of P. jirovecii colonization in preterm neonates and its possible association with medical complications. A prospective observational study of preterm newborns (birth weight <1500 g and/or gestational age <32 weeks) and their mothers was carried out. Identification of P. jirovecii colonization was performed by mean of molecular techniques in nasal aspirated samples of newborn and oropharyngeal washings of their mothers. A total of 128 preterm neonates and their mothers were included in the study. Pneumocystis DNA was identified in 25% (95% CI: 17.8%-33.7%) of newborns studied and in 32.9% (95% CI: 23.1-43.9%) of their mothers. A significant increase of respiratory distress syndrome in newborn colonized group, even after adjusting for confounding factors (odds ratio 2.7 [95% CI: 1.0-7.5]; p=0.04), was observed. There were an increased in retinopathy of prematurity and bronchopulmonary dysplasia at a postmenstrual age of 36 weeks among preterm infants colonized by P. jirovecii but without statistical significance. No differences were observed in other medical conditions between the two groups. P. jirovecii colonization is frequent in both preterm newborns and their mothers and could be a risk factor to develop respiratory distress syndrome among preterm infants.
Author: Calderon, Enrique J (Instituto de Biomedicina de Sevilla, Hospital Universitario Virgen del Rocío/ CSIC/Universidad de Sevilla, and CIBER de Epidemiología y Salud Pública)Co-Authors: Friaza, Vicente; Rojas, Pilar; Moreno, Fidel; Garcia, Elisa; de la Horra, Carmen; Pereira, Estefania (Instituto de Biomedicina de Sevilla, Hospital Universitario Virgen del Rocío/ CSIC/Universidad de Sevilla, and CIBER de Epidemiología y Salud Pública, Seville, Spain); Vargas, Sergio (Universidad de Chile, Santiago, Chile); Medrano, Francisco Javier; Pavon, Antonio (Instituto de Biomedicina de Sevilla, Hospital Universitario Virgen del Rocío/ CSIC/Universidad de Sevilla, and CIBER de Epidemiología y Salud Pública, Seville, Spain)Presentation Type: Platform
LIST OF ABSTRACTS
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# 1721
Title: Microsporidian polar tube protein 4 (PTP4)Microsporidia have been identified as pathogens that have important effects on our health, food security and economy. A key to the success of these obligate intracellular pathogens is their unique invasion organelle, the polar tube, which delivers the nucleus containing sporoplasm into host cells during invasion. Due to the size of the polar tube, the rapidity of polar tube discharge and sporoplasm passage, and the absence of genetic techniques for the manipulation of microsporidia, study of this organelle has been difficult and there is relatively little known regarding polar tube formation and the function of the proteins making up this structure. Herein, we have characterized polar tuibe protein 4 (PTP4) from the microsporidium Encephalitozoon hellem and found that a monoclonal antibody to PTP4 labels the tip of the polar tube suggesting that PTP4 might be involved in a direct interaction with host cell proteins during invasion. Further analyses employing indirect immunofluorescence (IFA), enzyme-linked immunosorbent (ELISA) and fluorescence-activated cell sorting (FACS) assays confirmed that PTP4 binds to mammalian cells. The addition of either recombinant PTP4 protein or anti-PTP4 antibody reduced microsporidian infection of its host cells in vitro. Proteomic analysis of PTP4 bound to host cell membranes purified by immunoprecipitation identified transferrin receptor 1 (TfR1) as a potential host cell interacting partner for PTP4. Additional experiments revealed that knocking out TfR1, adding TfR1 recombinant protein into cell culture, or adding anti-TfR1 antibody into cell culture significantly reduced microsporidian infection rates. These results indicate that PTP4 is an important protein competent of the polar tube involved in the mechanism of host cell infection utilized by these pathogens.
Author: Weiss, Louis (Albert Einstein College of Medicine)Co-Authors: Han, Bing (State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing, China; Department of Pathol-ogy, Albert Einstein College of Medicine, Bronx, NY, United States; Key Laboratory for Sericulture Functional Genomics and Biotechnology of Agricultural Ministry, Southwest University, Chongqing, China); Polonais, Valérie (Université Clermont Auvergne, Laboratoire “Microorganismes : Génome et Environnement, BP 10448, F-63000 Clermont-Ferrand, France; CNRS, UMR 6023, LMGE, F-63171 Aubière, France); Sugi, Tatsuki (De-partment of Pathology, Albert Einstein College of Medicine, Bronx, NY, United States); Yakubu, Rama (Department of Pathology, Albert Einstein College of Medicine, Bronx, NY, United States); Takvorian, Peter M. (Department of Biological Sciences, Rutgers University, Newark, NJ, United States); Cali, Ann (Department of Biological Sciences, Rutgers University, Newark, NJ, United States); Maier, Keith (Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY, United States); Long, Mengxian (State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing, China; Key Laboratory for Sericulture Functional Genomics and Biotechnology of Agricultural Ministry, Southwest Uni-versity, Chongqing, China; Levy, Matthew (Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY, United States); Tanowitz, Herbert B. (Department of Pathology, Albert Einstein College of Medicine, Bronx, NY, United States; Department of Medicine, Albert Einstein Col-lege of Medicine, Bronx, NY, United States); Pan, Guoqing (State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing, China; Key Laboratory for Sericulture Functional Genomics and Biotechnology of Agricultural Ministry, Southwest University, Chongqing, China); Delbac, Frédéric (Université Clermont Auvergne, Laboratoire “Microorganismes : Génome et Environnement, BP 10448, F-63000 Clermont-Ferrand, France.; CNRS, UMR 6023, LMGE, F-63171 Aubière, France); Zhou, Zeyang (State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing, China; College of Life Sciences, Chongqing Normal University, Chongqing, China; Key Laboratory for Sericulture Func-tional Genomics and Biotechnology of Agricultural Ministry, Southwest University, Chongqing, China); Weiss Louis M. (Department of Pathology, Albert Einstein College of Medicine, Bronx, NY, United States; Department of Medicine, Albert Einstein College of Medicine, Bronx, NY, United States)Presentation Type: Platform
# 1722
Title: Application of synthetic recombinant multi-epitope antigens of Pneumocystis jirovecii in the immunodiagno-sis of Pneumocystosis
Pneumocystis jirovecii (Pj) pneumonia (PcP) is an important morbidity/mortality factor in immunocompromised patients, whose diagnosis requires respiratory specimens obtained by invasive and costly techniques. Thus, the development of a serological approach would be a significant clinical advance. Specific surface proteins such as the Major Surface Glycoprotein (MSG) and kexin-like serine protease (KEX1), are highly-specific of Pj and have reactive antigenic properties. Hence, newly recombinant synthetic (multi-epitopes) antigens (RSA), involving antigenic regions of these proteins, were designed, synthetized, purified and tested as tools to identify suitable serological markers of Pj infection. A synthetic antigen with three reactive MSG epitopes1 and one with three KEX1 reactive epitopes were designed based on the study of the immunogenicity of the carboxyl-terminal domain of MSG and the entire sequence of KEX1. These RSAs were cloned and expressed in E. coli XJb (DE3) bacteria and the purification process occurred by affinity chromatography with immobilized nickel ions. The purified RSAs were applied as antigenic tools in indirect ELISA assays that were optimized for detection of circulating antibodies anti-Pj in serum specimens of patients (N=122) previously classified as PcP cases (n=56), Pj colonized carriers (n=30) and Pj negative cases (n=36). Assays results showed that the IgM anti-Pj levels were statistically increased in patients with PcP, compared with patients without PcP, with both RSAs (p<0.001 with KEX1 RSA and p=0.003 with MSG RSA). Results also showed that the MSG RSA and the KEX1 RSA allow differentiation between PcP cases and Pj colonized carriers (p=0.014 and p=0.04, respectively) and that the KEX1 RSA also allows differentiation between PcP cases and Pj negative cases (p<0.001). Thus, the two RSAs showed applicability as biomarkers of Pj infection and may be used in a serological platform for PcP diagnosis. Acknowledgements: Partially supported by FCT (SFRH/BD/108433/2015) and FCT- UID/Multi/04413/2013.
Author: Tomás, Ana Luisa (Global Health and Tropical Medicine, GHTM, Instituto de Higiene e Medicina Tropical, IHMT, Universidade Nova de Lisboa)Co-Authors: Cardoso, Fernando (Global Health and Tropical Medicine, GHTM, Instituto de Higiene e Medicina Tropical, IHMT, Universidade Nova de Lisboa, Lisboa, Portugal); De Sousa, Bruno (CINEICC, Faculdade de Psicologia e de Ciências da Educação, Universidade de Coimbra, Coimbra, Portugal); Franco, Ricardo (REQUIMTE/UCIBIO, Faculdade de Ciências e Tecnologia, FCT, Universidade NOVA de Lisboa, Almada, Portugal); Matos, Olga (Global Health and Tropical Medicine, GHTM, Instituto de Higiene e Medicina Tropical, IHMT, Universidade Nova de Lisboa, Por, Lisboa, Portugal)Presentation Type: Poster
LIST OF ABSTRACTS
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# 1723
Title: Evaluation of protein disulfide isomerase (PDI) functions in Toxoplasma-host invasion mechanism using anti-human PDI monoclonal antibodies (MAbs)
Toxoplasmosis is an infectious disease with worldwide distribution caused by Toxoplasma gondii. This opportunistic pathogen remains a serious threat to human health, reflecting the importance to continue and stimulate clinical and basic research on this challenging microorganism. Several molecules have been described as good drug targets’ candidates to help in designing innovative therapeutic strategies against T. gondii infection. T. gondii protein disulfide isomerase (PDI) members are among these promising drug molecular targets, as recent data indicates an important role of parasite’s PDI on early steps of parasite invasion mechanism. The main goal of this study is to address the functional roles of PDI on Toxoplasma gondii-host interplay, in the context of acute infection, and evaluating their usefulness as drug-targets using anti-human PDI commercial monoclonal antibodies (MAbs), on in vitro culture systems. A preliminary analysis of the data based on computational exploration of the T. gondii genome database for human-orthologous PDI family members revealed a degree of homology between the amino acid sequences described for human PDI and Toxoplasma. The results obtained from indirect immunolabeling assays using immunofluorescence techniques with anti-human PDI MAbs (PDI, PDIA6, PDIA3, glucose-regulated proteins/immunoglobulin heavy-chain binding protein [GRP78/BiP], GRP94 and Calnexin [CNX]) suggest both cross-reaction with target cell-lines and Toxoplasma tachyzoites, being suitable for their profile identification. Different patterns of immunolabeling were observed in the distinct types of analyzed samples (Toxoplasma infected and non-infected Human Foreskin Fibroblast - HFF and Human Embryonic Kidney - HEK293 cells, and in single tachyzoites), according to the MAb used. The functional confirmation of the characterized PDI involved in the host-pathogen interaction, were performed by (short hairpin) shRNA silencing of PDIA3 and PDIA6 target-genes to get complementary evidence of these specific proteins in Toxoplasma-host interactions. Down-regulation of the suppressed genes were validated by Western blot.Acknowledgments: Supported by FCT UID/Multi/04413/2013 and VIH/SAU/0019/2011.
Author: Lobo, Maria Luisa (Medical Parasitology Unit, GHMT, IHMT/UNL)Co-Authors: Novo, Carlos (Medical Parasitology Unit, IHMT/UNL, Lisboa, Portugal); Ramalho, José S. (CEDOC, Nova Medical School, Lisboa, Portu-gal); Matos, Olga (Global Health and Tropical Medicine, GHTM, Instituto de Higiene e Medicina Tropical, IHMT, Universidade Nova de Lisboa, Por, Lisboa, Portugal)Presentation Type: Poster
# 1724
Title: Cloning and expression of MIC3 protein from Toxoplasma gondiiToxoplasma gondii is an opportunistic apicomplexan protozoon that can cause devastating disease in immunosuppressed patients and congenital infection. The diagnosis of toxoplasmosis is usually done by observing the parasite in biological samples or by the detection of specific IgM and IgG against T. gondii antigens in the patient’s serum. The improvement of toxoplasmosis diagnostic techniques and the differentiation between the infection stages can be achieved using recombinant antigens. This study aims to use the micronemal protein MIC3 recombinant antigens in the serodiagnosis of toxoplasmosis. Total Toxoplasma RNA was isolated using the Tri-Reagent method and genes encoding MIC3 were amplified. The recombinant protein was cloned into the expression vector pLATE 31, through ligation independent cloning (LIC) technology. The samples were sequenced and showed homology with the sequence of T. gondii MIC3 protein, stored in GenBank’s database. An expression study with IPTG was performed in different E. coli BL21 (DE3) strains: Star, XJB, RIPL and PlysS transformed with pLATE 31. Since this vector enabled the production of the recombinant antigen MIC3 with a polyhistidine tail end, MIC3 was purified by high-affinity chromatography with immobilized nickel ions, testing several tampons with different pH values. The eluted samples were analyzed by ELISA, SDS-PAGE electrophoresis. A dot-blot and a western-blot were also performed to show that the recombinant protein MIC3 was present and results show that E. coli BL21 XJB (DE3) and Sodium Phosphate buffer pH 7.5 used in the purification is the best combination to purify the recombinant protein MIC3. Acknowledgements: Partially supported by FCT- UID/Multi/04413/2013 and VIH/SAU/0019/2011.
Author: Mota, Cátia (Global Health and Tropical Medicine, GHTM, Instituto de Higiene e Medicina Tropical, IHMT Universidade Nova de Lisboa, UNL)Co-Authors: Cardoso, Fernando (Global Health and Tropical Medicine, GHTM, Instituto de Higiene e Medicina Tropical, IHMT, Universidade Nova de Lisboa, Lisboa, Portugal); Matos, Olga (Global Health and Tropical Medicine, GHTM, Instituto de Higiene e Medicina Tropical, IHMT, Universi-dade Nova de Lisboa, Por, Lisboa, Portugal) Presentation Type: Poster
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# 1725
Title: In vitro assay of plant extracts from Guiné-Bissau against Toxoplasma gondii.Toxoplasma gondii infection causes toxoplasmosis, an infectious disease with worldwide prevalence. The limited efficiency of drugs against this infection, their side effects and the potential appearance of resistant strains make the search of novel drugs an essential need. Traditional herbal plants are used by people to cure a large number of parasitic disorders. Plants were collected at the Bolana-Bijagós region of Guinea-Bissau (West Africa) and a methanolic plant extract library was prepared and fractionated using Diaion HP 20 chromatographic resin. The methanolic library and the fractions were tested as potential sources of new compounds with high activity and low toxicity, using the Vero, 3T3 and HeLa cell lines with a MTT cytoxicity assay. T. gondii tachizoites were also tested with these libraries using a XTT cytotoxicity assay. Preliminary results show inhibitory activity of the methanolic extracts from the following plants: Combretum micrathum, Zanthoxylum leprieurii, Faidherbia albida and Hymenocardia acida to T. gondii in an in vitro assay. Assays to identify key compounds from these plants with anti-T. gondii activity are in progress.Acknowledgements: Partially supported by FCT- UID/Multi/04413/2013.
Author: Silva, Liliana (Global Health and Tropical Medicine, GHTM, Instituto de Higiene e Medicina Tropical, IHMT Universidade Nova de Lisboa, UNL)Co-Authors: Valente, Cláudia (Global Health and Tropical Medicine, GHTM, Instituto de Higiene e Medicina Tropical, IHMT Universidade Nova de Lisboa, UNL, Lisboa, Portugal); Cardoso, Fernando (Global Health and Tropical Medicine, GHTM, Instituto de Higiene e Medicina Tropical, IHMT, Universidade Nova de Lisboa, Lisboa, Portugal); Catarino, Luis (cE3c, Faculdade de Ciências, ULisboa, Lisboa, Portugal); Indjai, Bucar (INEP, Bissau, Guinea-Bissau); Matos, Olga (Global Health and Tropical Medicine, GHTM, Instituto de Higiene e Medicina Tropical, IHMT, Universidade Nova de Lisboa, Por, Lisboa, Portugal)Presentation Type: Poster
# 1726
Title: Gold bionanoconjugate-based point-of-care platform for serological diagnosis of Pneumocystis pneumoniaPneumocystis jirovecii (Pj) pneumonia (PcP) is a major HIV-related illness, rising among immunocompromised non-HIV patients. Presently, diagnosis relies on the detection of P. jirovecii in respiratory specimens obtained by invasive techniques. An early diagnostic method using biological specimens obtained non-invasively is highly desirable. Rapid diagnostic tests (RDTs) using gold nanoparticle (AuNP)-based bionanoconjugates allow a fast and inexpensive point-of-care diagnosis of P. jirovecii in serum samples.1 This work aims to develop AuNP-based bionanoconjugates for an immunochromatographic RDT, allowing the detection of anti-P. jirovecii circulating antibodies in patient sera. 2 In this test, the binding moieties to the anti-P. jirovecii antibodies, are bionanoconjugates formed by spherical AuNPs functionalized with 11-mercaptoundecanoic acid (MUA), and a multi-epitope synthetic recombinant antigen. This recombinant antigen was produced from the MSG protein of P. jirovecii, using an expression vector containing the coding sequence for the antigen.3 Techniques such as agarose gel electrophoresis, zeta potential and nanoparticle-tracking analysis were used to characterize the capacity of such bionanoconjugates to capture anti-P. jirovecii antibodies in the serum of Pneumocystis-infected patients.1 These stable bionanoconjugates will be the basis for a RDT platform with potential in point-of-care applications.2* Partially supported by Gilead GÉNESE-PGG/001/2014.1 Cavadas, M. et al. (2016) Part. Part. Syst. Charact., 33: 906–9152 Koczula K. M., Gallota A. (2016) Essays in Biochemistry, 60: 111-1203 Tomás, A.L. et al. (2016) Sci. Rep., 6(36287): 1-8
Author: Pinto, Mafalda (REQUIMTE/UCIBIO, Faculdade de Ciências e Tecnologia, Universidade NOVA de Lisboa)Co-Authors: Tomás, Ana Luisa (Global Health and Tropical Medicine, GHTM, Instituto de Higiene e Medicina Tropical, IHMT, Universidade Nova de Lisboa, Lisboa, Portugal); Peixoto de Almeida, Miguel (LAQV-REQUIMTE, Faculdade de Ciências, Universidade do Porto, Porto, Portugal); Cardoso, Fernando (Global Health and Tropical Medicine, GHTM, Instituto de Higiene e Medicina Tropical, IHMT, Universidade Nova de Lisboa, Lisboa, Portugal); Pereira, Eulália (LAQV-REQUIMTE, Faculdade de Ciências, Universidade do Porto, Porto, Portugal); Matos, Olga (Global Health and Tropical Medicine, GHTM, Instituto de Higiene e Medicina Tropical, IHMT, Universidade Nova de Lisboa, Por, Lisboa, Portugal); Franco, Ricardo (1 REQUIMTE/UCIBIO, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade NOVA de Lisboa, Almada, Portugal)Presentation Type: Poster
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# 1727
Title: Immune suppression associated lung disease in children with systemic onsetjJuvenile idiopathic arthritisSystemic onset juvenile idiopathic arthritis (sJIA) and similar illnesses are typically associated with pleuritis, but not parenchymal lung disease. However, since the introduction of IL-1 and IL-6 inhibitors as treatment, there appears to be an increase in often fatal lung disease. Here, we report on 58 recent cases. Our series suggests that a striking and unusual clinical and pathologic pattern characterizes this disease. Median age of onset in this series is 3 years. No geographic, seasonal or racial clustering is noted. The overall fatality rate to date exceeds 36%. Lung disease onset is frequently characterized by acute pronounced hypoxia, with a relative paucity of respiratory symptoms. Extensive lung disease in some instances was found unexpectedly on autopsy. Acute erythematous digital clubbing (69%), pruritic eczematous dermatitis (39%), peripheral eosinophilia (25%) and lymphocytopenia (96%) precede the clinical diagnosis of lung disease. Medication exposure prior to lung disease includes IL-1 and/or IL-6 inhibitors in 88% of cases. Glucocorticoids and other immune suppressants were variably employed. Pathology, when available, demonstrated primarily a pulmonary alveolar proteinosis/endogenous lipoid pneumonia pattern (over 70%) with or without pulmonary vascular changes, and BAL was neutrophilic. Infectious work up on BAL or lung tissue was either negative or failed to adequately explain the extent of disease. Other than two cases of histoplasmosis, evaluation for pneumocystis (PCP) and other fungi was negative by direct microscopy and by DFA, when performed. Prior to lung disease, only one case received PCP prophylaxis. Subsequent to lung disease, lack of PCP prophylaxis was associated with a 71% fatality rate. PCP testing by rtPCR was positive in four samples without exposure to trimethoprim sulfamethoxazole. Despite risk factors for PCP and the suggestion that PCP may be involved, the role of PCP, and relative roles of other infections, underlying inflammatory illness and immune suppression remain unknown.
Author: Saper, Vivian (Stanford University School of Medicine)Co-Authors: Deutsch, Gail (Seattle Children’s Hospital, Seattle, WA, United States); Sunduram, Vandana (Stanford University Medical Center, Palo Alto, CA, United States); Mellins, Elizabeth (Stanford University School of Medicine, Stanford, CA, United States)Presentation Type: Platform
# 1730
Title: Cryopreservation of Cryptosporidium oocysts using the ultra-rapid vitrification technique.Lack of a method for cryopreservation of the enteric protozoa Cryptosporidium, poses a serious technical limitation in basic and applied research on this parasite. A major obstacle in cryopreservation of the parasite is the impermeable nature of the oocyst wall that prevents exclusion of water and inclusion of cryoprotectants necessary to protect sporozoite viability during freezing. We have successfully characterized permeability of the oocyst wall to both water and variety of intracellular cryoprotectants in relation to oocysts age and to other key variable parameters. In a set of experiments, water was excluded in response to the extracellular hyperosmotic gradient of trehalose solution, while permeability to cryoprotectants was achieved by alteration of the oocyst wall structure by bleaching at different temperatures. Here we report the development of a successful cryopreservation protocols yielding viable and infectious oocysts upon thawing. Pre-bleached C.parvum oocysts were frozen using the ultra-rapid vitrification technique in liquid nitrogen, following 10 minutes dehydration in 1M trehalose solution and 0 or 20 minutes incubation in 30% DMSO. The recovery rate of thawed oocysts was at ~70% and ~50% respectively by means of propidium iodide inclusion and by the excystation assay which yielded morphologically viable intact sporozoites. Frozen oocysts successfully established infection in INF-? knockout mice at 6-7 dpi in comparison to 5dpi for the unfrozen parent oocyst. The ongoing work aims at establishing asimplified vitrification procedure using commercially available devices such as insemination straws and cryovials.Funded by the BMGF.
Author: Jaskiewicz, Justyna (Cummings School of Veterinary Medicine at Tufts University) Co-Authors: Sandlin, Rebecca (Massachusetts General Hospital, Charlestown, MA, United States); Widmer, Giovanni (Tufts Cummings School of Veterinary Medicine at Tufts University, North Grafton, MA, United States); Swei, Anisa (Center for Engineering in Medicine, Massachusetts General Hospital, Boston, MA, United States); Toner, Mehmet (Center for Engineering in Medicine, Massachusetts General Hospital, Boston, MA, United States); Tzipori, Saul (Tufts Cummings School of Veterinary Medicine at Tufts University, North Grafton, MA, United States)Presentation Type: Platform
# 1731
Title: Genomic divergence of Pneumocystis populations during adaptation to mammalsPneumocystis species form a group of uncultivable host-specific fungal parasites of mammals. Host specificity is one of the most unique and critical features of a parasite, as it directly reflects adaptation to available resources as well as development of mechanisms to subvert host defenses. The genetic basis of this process in Pneumocystis is unknown. Comparative analysis of species divergence can provide insights into the unique evolutionary adaptation of each species to their respective mammalian hosts. To investigate the patterns of genomic divergence at a population level, we analyzed whole genome sequencing data from 53 individual hosts for three Pneumocystis species infecting humans, mice and rats. Here we show that the streamlined genomes of these species have a bipartite architecture, with a core mappable portion (defined as genomic regions conserved in all three species, representing ~72% of the genomes) exhibiting an unexpectedly low level of genetic differentiation (FST < 0.4). The subtelomeres and a few intra-chromosomal regions, which correspond primarily to multigenic families including surface glycoproteins (Msg), display a high level of differentiation (FST > 0.8) caused by a combined effect of genetic drift and diversifying selection. We found no evidence of genetic flow among species that could explain the reduced level of genetic differentiation, which suggests ancient reproductive isolation of these species. Our analysis also uncovered small sets of novel lineage-specific differentiated genomic regions that potentially contribute to host specificity. Since subtelomeres primarily encode Msgs and related proteins, and these proteins are likely involved in avoiding host defenses, this suggests that host specificity is driven at least in part by a host-parasite arms race via an accelerated evolution of small fractions of the genomes.
Author: Cisse, Ousmane (National Institutes of Health)Co-Authors: Ma, Liang (National Institutes of Health, Bethesda, MD, United States); Cuomo, Christina (Broad Institute of Harvard and Massachu-setts Institute of Technology, Cambridge, MA, United States); Kovacs, Joseph (National Institutes of Health, Bethesda, MD, United States) Presentation Type: Platform
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# 1732
Title: The glucan phosphatase, TgLaforin, regulates amylopectin metabolism in both T. gondii tachyzoites and bradyzoites
A distinguishing characteristic of cyst forming Apicomplexa is the accumulation of amylopectin (starch) granules (AGs) within bradyzoites. The AG levels within encysted T. gondii bradyzoites is highly variable suggesting their accumulation and depletion is under homeostatic control. In light of our recent findings that encysted bradyzoites retain replicative capacity, we propose that AGs serve as a glucose cache for energy intensive processes. Land plants liberate glucose from insoluble starch by utilizing a cycle of phosphorylation/dephosphorylation of the starch surface. The T. gondii genome encodes all of these activities needed for starch turnover. These include a glucan water dikinase (TgGWD: TgME49_214260), a glucan phosphatase (TgME49_205290), and multiple amylases. Glucan phosphatases possess a carbohydrate binding module (CBM) and a dual specificity phosphatase domain (DSP). Bioinformatic analysis of TgLaforin revealed the presence of an atypical CBM and a canonical DSP. We expressed a codon optimized TgLaforin fusion to an N-terminal SUMO protein to facilitate folding and solubility in E. coli. Enzymatic activity assays using the recombinant TgLaforin with both pNPP and potato amylopectin as substrates confirmed that TgLaforin is an active glucan phosphatase. We have demonstrated that, surprisingly, a CRISPR/Cas9 knockout of TgLaforin in tachyzoites results in massive starch accumulation replication arrest, a phenotype that takes over 10 days to manifest. This suggests that the regulation of amylopectin turnover may play a crucial role in both tachyzoites and bradyzoites. We are currently developing conditional KO/KD lines to assess the importance of TgLaforin in both tachyzoites and bradyzoites in cell culture and in vivo.
Author: Sinai, Anthony (University of Kentucky College of Medicine)Co-Authors: Murphy, Robert; Dhara, Animesh; Watts, Elizabeth; Brizzee, Corey; Stewart, Travis; Gentry, Matthew (University of Kentucky College of Medicine, Lexington, KY, United States)Presentation Type: Platform # 1733
Title: Ablation of an OTU-family deubiquitinase exposes the underlying regulation governing the plasticity of cell cycle progression in Toxoplasma gondii
Toxoplasma encodes the capacity for distinct architectures underlying cell cycle progression in a life cycle stage dependent manner. Replication in intermediates hosts occurs by endodyogeny whereas a hybrid of schizogony and endopolygeny occurs in the gut of the definitive feline host. Here we characterized the consequence of the loss of a cell cycle regulated Ovarian TUmor (OTU-family) deubiquitinase, TgOTUD3A (TGGT1_258780) in T. gondii tachyzoites. Rather than being detrimental, mutant parasites out-competed the wild type. This phenotype was due to roughly one third of TgOTUD3A-KO tachyzoites exhibiting deviations from endodyogeny by employing replication strategies producing 3, 4 or 5 viable progeny within a gravid mother instead of the usual 2. We established the mechanistic basis underlying these altered replication strategies to be a transient dysregulation of centrosome duplication causing a transient loss of the stoichiometry between the inner and outer cores, resulting in a failure to correctly engage the spindle checkpoint at the attainment of 2N ploidy. This resulting dysregulation manifests as deviations in the normal transitions from S-phase to mitosis (S/M) (endopolygeny-like) or M to cytokinesis (M/C) (schizogony-like). Notably, these imbalances are corrected prior to cytokinesis resulting in the generation of normal progeny. Our findings suggest that decisions regarding the utilization of a specific cell cycle architecture are controlled by a ubiquitin-mediated mechanism dependent on the absolute threshold levels of an as yet unknown target(s). The TgOTUD3A-KO provides a broader understanding of schizogony and endopolygeny, two uniquely Apicomplexan cell cycle architectures used by Plasmodium spp. and Sarcocystis spp. respectively.
Author: Dhara, Animesh (University of Kentucky College of Medicine)Co-Authors: de Paula Baptista, Rodrigo; Kissinger, Jessica C. (University of Georgia, Athens, GA, United States); Snow, E. Charles (University of Kentucky, Lexington, KY, United States); Sinai, Anthony (University of Kentucky College of Medicine, Lexington, KY, United States)Presentation Type: Platform
# 1734
Title: Development of a quantitative pharmacokinetic-pharmacodynamics model of Pneumocystis treatment in mice
Residing extracellularly in lung alveoli, the yeast-like fungi Pneumocystis cause lethal infection (Pneumocystis pneumonia, PCP) in hosts with impaired immune systems. The current therapies for PCP, such as trimethoprim-sulfamethoxazole (TMP-SMX) and Atovaquone, suffer from significant treatment failures as well as side effects. Hence, novel therapeutic approaches such as newly developed drugs or new combination of available drugs are needed for this common opportunistic infection. For both purposes, dose optimization would be critical to achieve maximal therapeutic benefits. Quantitative systems pharmacological (QSP) modelling, which utilizes measured pharmacokinetic and pharmacodynamics data to predict the effect of novel treatment regimes, promises to facilitate the process of dose optimization. Indeed, QSP modeling has been explored for dose optimization of drugs against other infectious diseases (e.g. tuberculosis). In this study, we have constructed a QSP module which describes the distribution and decay of four different drugs (Anidulafungin, Caspofungin, Micafungin and TMP-SMX) as well as the proliferation, transformation and death of Pneumocystis in mice. In addition to summarizing these available kinetic profiles into a consistent dynamical framework, the model was also used to predict the PK profiles of unobserved treatment regimes. Furthermore, the model provides novel insight on the accumulation dynamics of Pneumocystis when different treatment protocols are applied. In this way, the model serves as a promising tool for analyzing dynamical data of Pneumocystis infection and facilitating the design of novel therapies.
Author: Liu, Guansheng (University of Cincinnati)Presentation Type: Poster
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# 1735
Title: Characterization of p57, a stage-specific antigen of Pneumocystis murinaOur recent sequencing of 3 Pneumocystis genomes led to the identification of multiple genes predicted to be surface proteins, many of which belong to the Msg superfamily. In P. murina, one such protein is encoded by 3 highly conserved genes present in the subtelomeric region of 3 chromosomes. The encoded proteins, termed p57, are 95-99% conserved, have a predicted MW of ~57 kD, and contain a single Msg domain (N1). To characterize the expression of this protein, one recombinant p57 gene was expressed in E. coli and mammalian cells. By SDS-PAGE analysis, rp57 migrated with an apparent MW of ~98 kD, suggesting it ran aberrantly. Sera from immunocompetent mice that cleared Pneumocystis infection or were immunized with P. murina lysates reacted with rp57 by immunoblot analysis, demonstrating that p57 is antigenic. Sera from mice immunized with rp57 reacted by immunoblot with rp57 as well as a native P. murina antigen of ~120 kD; however, splenocytes from these mice did not proliferate in response to crude P. murina antigens. Immunofluorescent labeling of partially purified intact P. murina identified many small organisms, demonstrating that p57 is a surface protein; cysts did not appear to be labeled. Immunolabeling of fixed lung sections again showed expression by smaller organisms. Cysts did not express p57 on their surface, but it was expressed on intracystic bodies in some cysts. Trophic forms expressed either Msg or p57, but rarely both. Thus, p57 appears to be a stage specific antigen of P. murina that is expressed exclusively on intracystic bodies and small trophic forms that likely were recently released from cysts. Given that intracystic bodies are protected from host immune responses, we hypothesize that they express p57 rather than Msg to conserve resources; following their release, expression shifts to Msg to facilitate evasion of host immune responses
Author: Bishop, Lisa (National Institutes of Health)Co-Authors: Davis, A. Sally; Gamez, Monica; Bradshaw, Kaitlyn (Kansas State University College of Veterinary Medicine, Manhattan, KS, United States); Cisse, Ousmane; Wang, Honghui; Ma, Liang; Kovacs, Joseph (National Institutes of Health, Bethesda, MD, United States)Presentation Type: Platform
# 1736
Title: Pneumocystis jirovecii colonization in pregnancyPneumocystis jirovecii is an atypical opportunistic fungus with strong host species specificity that cause pneumonia in immunosuppressed individual. However, Pneumocystis colonization is not restricted to those who are severely immunocompromised. Molecular techniques have shown that Pneumocystis colonization is common in other segments of the population that are immunocompetent or display a lesser degree of immune compromise. In this sense, the pregnancy could constitute a risk factor for Pneumocystis colonization, due to the mechanisms of immunological tolerance during this period. The aims of this study were to know the prevalence and clinical implications of Pneumocystis colonization in pregnant women. A prospective observational study of pregnant women and their newborn children was conducted. Healthy non-pregnant women in child-bearing age were included as controls. Identification of Pneumocystis colonization was performed by mean of molecular techniques in oropharyngeal washings from women and in nasal aspirated samples in newborn children. A total of 82 women at the time of their deliveries and their newborn children were included in the study. Also, 36 matched non-pregnant women were studied. Pneumocystis DNA was identified in 39% of pregnant women vs 13.9% in non-pregnant women (p = 0.007). The prevalence of Pneumocystis in newborn children was 29.7% showing a close correlation with mothers’ status. The mean duration of gestation in women colonized by Pneumocystis was 31.5 ± 5.5 weeks vs 34.4 ± 5.3 weeks in the case of non-colonized mothers (p = 0.017). A linear regression analysis confirmed the effect of Pneumocystis colonization on duration of gestation (p = 0.01) and showed that was similar to effect of diabetes mellitus. Pregnancy is a risk factor for Pneumocystis colonization and could be a potential cause of prematurity hitherto unknown. However, future studies are needed to further define the role of Pneumocystis colonization during pregnancy.
Author: Calderon, Enrique J. (Instituto de Biomedicina de Sevilla, Hospital Universitario Virgen del Rocío/ CSIC/Universidad de Sevilla, and CIBER de Epidemiología y Salud Pública)Co-Authors: Rojas, Pilar; Moreno, Fidel; Lopez, Rafael; de la Horra, Carmen; Pereira, Estefania; (Instituto de Biomedicina de Sevilla, Hospital Universitario Virgen del Rocío/ CSIC/Universidad de Sevilla, and CIBER de Epidemiología y Salud Pública, Seville, Spain); Lopez, Luis; (Hospital Universitario Virgen del Rocío, Seville, Spain); Praena, Juan Manuel (Universidad de Sevilla, Seville, Spain); Garcia, Elisa (Instituto de Biomedicina de Sevilla, Hospital Universitario Virgen del Rocío/ CSIC/Universidad de Sevilla, and CIBER de Epidemiología y Salud Pública, Seville, Spain); Res-paldiza, Nieves (Instituto de Biomedicina de Sevilla, Hospital Universitario Virgen del Rocío/ CSIC/Universidad de Sevilla, Seville, Spain); Medrano, Francisco Javier; Pavon, Antonio; Friaza, Vicente (Instituto de Biomedicina de Sevilla, Hospital Universitario Virgen del Rocío/ CSIC/Universidad de Sevilla, and CIBER de Epidemiología y Salud Pública, Seville, Spain)Presentation Type: Platform
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# 1737
Title: Risk factors and epidemiological features of Pneumocystis pneumonia in patients without HIV infection in Spain
The incidence of Pneumocystis pneumonia (PcP) seem to be growing among non-HIV patients, whose risk features are worse defined. Our aim was to evaluate the incidence, risk factors and epidemiological features of PcP in non-HIV patients in Spain. MATERIAL AND METHODS Design: Observational cross-sectional study. Population: hospitalized patients in Spain whose main discharge diagnosis was PcP (CIE-9-MC 136.3), and who were non HIV-infected, from 2008 to 2012. During the study period a total of 4554 PcP patients was recorded, 1204 (26, 4%) in patients without HIV infection. The average incidence of PcP was 19,4 cases per million, remaining globally stable during the study period, increasing from 4,4 to 6,3 in non HIV-patients and decreasing among HIV-infected patients from 15,5 to 13,4 per million. Annual changes in the variables analyzed are shown in the table. Risk factors for developing PcP were identified in 85,5% of cases, being the more frequent haematological malignancies (29%), respiratory illnesses (15,9%), and non-haematological neoplasm (14,9%). No seasonal differences in PcP incidence were found. CONCLUSIONS: (1) PcP is still being a frequent disease in Spain, with a steady incidence due to increasing trend in rates for clinical cases in non HIV-patients, and a parallel decrease in HIV-related cases; (2) Mortality among non HIV-patients still remain high, with an increasing of age and PcP-related costs during the study period, despite not increasing in complexity nor stay duration; (3) Main risk factors for PcP in non HIV-patients are haematological malignancies, chronic respiratory illnesses and solid tumors, but there is still a high number of cases with no predisposing factors identified; (4) In our research, no seasonal differences in the incidence of PcP were seen.
Author: Pereira, Estefania (Instituto de Biomedicina de Sevilla, Hospital Universitario Virgen del Rocío/ CSIC/Universidad de Sevilla, and CIBER de Epidemiología y Salud Pública)Co-Authors: Lopez, Rafael; Ruiz, Francisco; Moreno, Fidel; Calero Bernal, Maria Luz; Martínez- Risquez, Maria Teresa; Calderon, Enrique J; Me-drano, Francisco Javier (Instituto de Biomedicina de Sevilla, Hospital Universitario Virgen del Rocío/ CSIC/Universidad de Sevilla, and CIBER de Epidemiología y Salud Pública, Seville, Spain)Presentation Type: Poster
# 1738
Title: Cryptosporidium oocysts do not require activation in stomach for successful intestinal infectionCryptosporidium are protozoan parasites that infect the gastrointestinal epithelium of various vertebrate hosts. The genus has two major phylogenetic groups: a gastric group that infect the epithelium of the stomach and an intestinal group that infect the epithelium of the small and large intestine. Cryptosporidium are transmitted by the faecal-oral route and infect epithelial cells following excystation of the environmental oocyst stage. It has been proposed that excystation of intestinal species is triggered by exposure to the acidic stomach contents, although this has not been verified experimentally. This study aimed to determine whether exposure to stomach contents is necessary for in vivo infection by the intestinal speciesC. parvum and whether passage through the intestine is necessary for the gastric species C. proliferans to cause infection. It was shown that purified and non-purified oocysts of C. parvum were infectious for SCID mice following surgical inoculation directly into different parts of the small intestine, demonstrating that passage through the stomach is not necessary for infection by this intestinal species. Inoculation of the jejunum resulted in a course of infection similar to oral inoculation. Cryptosporidium proliferans was infectious for naïve SCID mice following surgical extraction from the stomach of infected SCID mice, demonstrating that passage through the small intestine is not necessary for infection by this gastric species. However, surgical inoculation of C. proliferans oocysts directly into the intestinum tenue did not cause infection. This study was funded by the Czech Science Foundation (project No. 15-01090S) and a project of the Grant Agency of the University of South Bohemia (project No. 002/2016/Z).
Author: Kvac, Martin (Biology Centre CAS)Presentation Type: Poster
# 1739
Title: Cryptosporidium spp. in wild rodents of genus RattusMany rodents, including genus Rattus, host parasites from the genus Cryptosporidium. Prior to 2007, studies on Cryptosporidium used mostly microscopic detection methods, which could not differentiate species/genotypes. Using molecular detection approaches, C. parvum, C. muris, C. tyzzeri, C. scrofarum, C. meleagridis, C. suis-like, four Rattus host-specific genotypes (rat genotype I – IV), and many other unnamed genotypes have been detected in rats. In the present study, 788 faecal samples were collected from 8 countries (Czech Republic, Slovakia, Kenya, Cameroon, Cambodia, Thailand, Philippines and New Zealand) and examined for presence of Cryptosporidium by microscopy (aniline-carbol-methyl violet staining) and PCR/sequence analysis of the 18S rRNA and actin genes. Oocysts and Cryptosporidium specific DNA was detected in 5 and 107 samples by microscopy and PCR, respectively. Sequence and phylogenetic analyses showed the presence of rat genotype I (n=34), rat genotype II (n=8), rat genotype III (n=16), rat genotype IV (n=30), C. andersoni (n=3), C. muris (n=8), C. proliferans (n=2), C. ryanae (n=1), C. serpentis (n=1), and C. suis-like (n=4). Oocysts of rat genotype I and IV from naturally infected Rattus norvegicus were infectious only for the same host species, and were not infectious for Mus musculus, Meriones unguiculatus. The prepatent period of rat genotype I and rat genotype IV was 4 and 3 days, respectively. Oocysts of both genotypes were shed intermittently and the patent period ranged between 14?26 days. Histology and electron microscopy analyses revealed the presence of developmental stages of rat genotype I or rat genotype IV attached to the microvilli in the jejunum and ileum of infected rats and there was no pathology associated with the infection. Experimentally infected rats showed no clinical signs of cryptosporidiosis. This study was funded from Grant Agency of University of South Bohemia (072/2017/Z) and the Czech Science Foundation (15-01090S).
Author: Kvac, Martin (Biology Centre CAS)Presentation Type: Poster
LIST OF ABSTRACTS
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# 1740
Title: Native and introduced squirrels in Italy host different Cryptosporidium spp. Many Cryptosporidium spp. have a narrow host specificity and geographical distribution. The present study was undertaken to describe Cryptosporidium spp. infection in tree squirrels from 17 locations in Northern Italy. A total of 357 squirrels were examined, including species native to Europe (Eurasian red squirrels (Sciurus vulgaris); n=123), and species introduced from North America (eastern grey squirrels (Sciuruscarolinensis); n=162) and Southeast Asia (Pallas’ squirrels (Callosciuruserythraeus); n=72). Faecal samples were examined for the presence of Cryptosporidium infection by microscopy (flotation method) and PCR/sequence analysis of the Cryptosporidium 18S rRNA, actin, and gp60 genes. Despite the overlapping ranges of native and introduced tree squirrel species in the study area, they host different Cryptosporidium spp.. Eurasian red squirrels were exclusively infected with Cryptosporidium ferret genotype (13/123) belonging to three novel gp60 subtypes, VIIIb–VIIId. Eastern grey squirrels hosted C. ubiquitum subtype XIIb (n=2), Cryptosporidium skunk genotype subtype XVIa (n=3), and chipmunk genotype I subtype XIVa (n=1). Cryptosporidium chipmunk genotype I subtype XIVa was also found in two Pallas’ squirrels. Comparing data from this and previous studies we propose that Cryptosporidium skunk genotype, chipmunk genotype I, and possibly C. ubiquitum subtype XIIb were introduced to Europe with eastern grey squirrels. Cryptosporidium chipmunk genotype I and ferret genotype were associated with high intensity infections, but there was no association between infection with Cryptosporidium spp. and diarrhoea. All Cryptosporidium spp. infecting tree squirrels in Italy are associated with human disease, and because tree squirrels live in close proximity to humans, squirrels may be a significant reservoir of zoonotic infectionsThis study was funded by the Czech Science Foundation (15-01090S), Ministry of Education, Youth and Sports of the Czech Republic (LTAUSA17165), and by Grant Agency of the University of South Bohemia (002/2016/Z). We would like to thank LIFE09 NAT/IT/00095 EC-SQUARE.
Author: Kvac, Martin (Biology Centre CAS)Presentation Type: Poster
# 1741
Title: Diversity and biology of Cryptosporidium in ducks and geese in the Czech RepublicCryptosporidium is one of the most prevalent parasitic infections in domestic and wild birds. The study was conducted to determine the prevalence, biology and genetic diversity of Cryptosporidium in birds of order Anseriformes in the Czech Republic. A total of 337 fecal samples were obtained from the genera Anas (307) and Anser (30) and examined for presence of Cryptosporidium specific DNA by PCR amplification and sequence analysis of SSU rRNA, actin and HSP70 genes. A total 37 positive cases of infection with Cryptosporidium were detected using nested PCR. Phylogenetic analyses based on three loci revealed the presence of five genetically distinct cryptosporidia: C. avium (4), C. baileyi (11), Cryptosporidium avian genotype III (5), duck genotype (8) and a novel genotype operationally named duck genotype II (9). Both duck genotype and duck genotype II were successfully transmitted to ducklings. The prepatent period of both genotypes was 4 DPI using PCR. Oocysts of duck genotype were microscopically detected 4 DPI; in contrast, oocysts of duck genotype II were not detected until 6 DPI. Molecular methods, histology and electron microscopy revealed the presence of infection in small intestine, with predilection to duodenum and ileum. This study was funded from Grant Agency of University of South Bohemia (082/2017/Z) and the Czech Science Foundation (15-01090S).
Author: Kvac, Martin (Biology Centre CAS)Presentation Type: Poster
# 1742
Title: Revision of effect of adaptive immune response to control microsporidiosis induced by Encephalitozoon cuniculi
This study determined the effect of Albendazole on Encephalitozoon cuniculi genotype II course of infection in immunocompetent BALB/c and C57Bl6 mice and immunodefficient SCID, CD4-/-, and CD8-/- mice. PCR and qPCR were used to detect and quantify E. cuniculi infection in tissues throughout the body.Following oral inoculation, an acute infection, characterized by intense dissemination of microsporidia into most organs, developed in both immunocompetent and immunocompromised mice. Immunocompetent and CD4-/- mice subsequently developed a chronic, non-lethal infection, whereas CD8-/- and SCID mice developed a lethal infection. The administration of Abendazole during the acute phase of infection reduced microsporidia to non-detectable levels in immunocompetent and CD4-/- mice, and this effect persisted beyond the treatment course. Albendazole reduced the microsporidia infection in SCID and CD8-/- mice; however, in contrast to observations in immunocompetent and CD4-/- mice, microsporidia infection increased following the cessation of Albendazole treatment. The post-treatment infection in SCID mice was lethal, similar to that in untreated SCID mice. However, post-treatment, CD8-/- mice surprisingly survived untill the end of the experiment, despite a massive microsporidia burden (up to 109 spores per 1 g of tissue), suggesting a mechanism other than cell mediated cytotoxicity is responsible for survival following Albendazole treatment. Understanding how microsporidia survive in hosts despite a competent immune response and how infected hosts tolerate an enormous spore burden in their tissues without any clinical signs could significantly contribute to research related to human health. This study was funded from the Czech Science Foundation (17-12871S).
Author: Sak, Bohumil (Biology Centre CAS, Institute of Parasitology)Presentation Type: Poster
LIST OF ABSTRACTS
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# 1743
Title: Study of the sensitivity to caspofungin of the Pneumocystis jirovecii 1,3-ss glucan synthase catalytic subunitEchinocandins inhibit the catalytic subunit Gsc1 of the enzymatic complex responsible for the synthesis of 1,3-ß glucan, an essential constituent of the fungal cell wall. Studies in rodent models suggested the efficacy of the echinocandin caspofungin to treat Pneumocystis pneumonia. However, its efficacy against Pneumocystis jirovecii infecting humans remains a controversial issue. The aim of this study was to study the sensitivity of the P. jirovecii Gsc1 to caspofungin. In absence of an established in vitro culture method for P. jirovecii, we used functional complementation of the S. cerevisiae GSC1 deletant. In order to study the sensitivity of the P. jirovecii Gsc1, spot test on medium supplemented or not with caspofungin was used. In the fungal pathogen Candida albicans, resistance to echinocandins has been reported to be conferred by point mutations leading to amino acid substitutions. We used site-directed mutagenesis to introduce the corresponding mutations in the P. jirovecii gsc1 gene leading to F714S and S718P substitutions. The S. cerevisiae GSC1 deletant showed an increased sensitivity to caspofungin compared to the wild type. Complementation with the S. cerevisiae GSC1 gene led to a total restoration of the wild type growth. In presence of the P. jirovecii gsc1 gene, a partial restoration of the wild type growth was observed. The latter restoration increased in presence of one substitution (S718P), and increased more in the presence of the two substitutions (F714S + S718P). The decrease of sensitivity conferred by the substitutions shows that the P. jirovecii Gsc1 catalytic subunit is sensitive to caspofungin to some level. We plan to quantify this sensitivity in order to evaluate its possible usefulness in the clinical practice. Caspofungin targets only P. jirovecii asci, not the trophic forms, and thus might be useful only in combination with Bactrim.
Author: Luraschi, Amanda (Hospital and University of Lausanne)Co-Authors: Hauser, Philippe (Lausanne University Hospital, Lausanne, Switzerland) Presentation Type: Poster
# 1744
Title: Disseminated Encephalitozoon cuniculi infection in patients with hip implant looseningPeriprosthetic osteolysis is the most common complication after primary hip arthroplasty, and can lead to implant loosening and arthroplasty revision. With the increasing incidence of total hip replacement, implant destabilization cases present a challenging problem. Among causes of hip joint stability loss, the most common are aseptic mechanical and biological, resulting in a local inflammatory response to particulate wear debris. Moreover, infectious agents such as bacteria and fungi are particularly devastating, leading to osteolysis and implant destabilization. Since microsporidia, widespread opportunistic fungal pathogens, has been shown to disseminate to the most of human tissues, such agent also cannot be excluded. Here we aimed to determine the occurrence of disseminated Encephalitozoon spp. infections among immunocompetent patients following total hip arthroplasty, both primary (THA group) and revision (RHA group), resulting in periprosthetic osteolysis. We examined urine sediments, stool and periprosthetic tissues obtained from a total of 53 individuals, 30 THA and 23 RHA, who had been under the care of the Department of Physiotherapy, Wroclaw Medical University (Wroclaw, Poland). Genus-specific nested PCR followed by genotyping revealed 10 patients who had Encephalitozoon-positive periprosthetic tissues, 9 (39%) after revision and one (3.3%) after primary hip arthroplasty. Among the tissue-positive RHA patients, 7 had a positive urine sample and one had a positive stool sample. Encephalitozoon cuniculi genotype II was identified in 88.8% (16/18) of samples. Two urine samples were positive for a novel Encephalitozoon sp.Our results indicate that E. cuniculi should be considered as a contributing cause of periprosthetic osteolysis and implant destabilization after hip replacement, especially with regard to the increasing evidence of widespread microsporidial infection in immunocompetent people.Funding: National Science Centre, Poland (DEC-2012/05/D/NZ6/00615) and Grant for Young Scientists, Wroclaw Medical University (Pbmn191).
Author: Kicia, Marta (Wroclaw Medical University)Co-Authors: Kopacz, Zaneta; Wesolowska, Maria (Wroclaw Medical University, Wroclaw, Poland); Kvac, Martin (Biology Centre CAS, Ceske Budejo-vice, Czech Republic); Sak, Bohumil (Biology Centre CAS, Institute of Parasitology, Ceske Budejovice, Czech Republic); Sokulska, Magdalena; Cebulski, Kamil; Hendrich, Andrzej; Pozowski, Andrzej (Wroclaw Medical University, Wroclaw, Poland)Presentation Type: Poster
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# 1745
Title: Prevalence and genotypic characterization of Pneumocystis jirovecii among patients with various respiratory diseases in Poland
Pneumocystis jirovecii is a unicellular fungus locating in human lungs. Its infections may induce the development of symptoms of opportunistic disease, Pneumocystis pneumonia (PcP), primarily in immunosuppressed patients. Immunocompetent individuals may become colonized and potentially serve as a reservoir of the pathogen in the population. Moreover, an asymptomatic infection may be associated with the risk and severity of certain respiratory diseases. Therefore, the aim of our study was to investigate the prevalence of P. jirovecii among patients with various respiratory diseases, followed by the analysis of genotypic variants occurring in identified organisms. Bronchial washings were taken from 105 patients who had been under the care of the Department of Pulmonology and Lung Cancer of Wroclaw Medical University. Detection of P. jirovecii presence was performed both by microscopic (indirect immunofluorescence, IF) and molecular (nested PCR) methods. Multilocus sequence typing involving three loci – mtLSU rRNA, CYB,SOD – was used for genotypic characterization.DNA of P. jirovecii was detected in 17 of the 105 (16.2%) subjects studied. Moreover, eight of these 17 patients (47%) were confirmed as Pneumocystis-positive by IF staining as well. The low number of cysts observed along with the lack of symptoms suggested colonization. Comparison of P.jirovecii-positive and -negative patients has shown that the only factor statistically correlated with infection was an immunosuppressive treatment. Multilocus typing revealed eight genotypes, with Pj 1 and Pj 2 as the most common. Moreover, it has been shown that infection with certain genotypic variants might be associated with the type of underlying respiratory disease. In conclusion, our study has confirmed that patients with various respiratory diseases, especially when immunosuppressed, are at risk of Pneumocystis colonization. Furthermore, specific genetic polymorphisms might be associated with certain underlying conditions.Funding: National Science Centre, Poland (DEC-2012/05/D/NZ6/00615) and Grant for Young Scientists, Wroclaw Medical University (Pbmn191).
Author: Sokulska, Magdalena (Wroclaw Medical University)Co-Authors: Kicia, Marta; Wesolowska, Maria; Piesiak, Pawel; Kowal, Aneta (Wroclaw Medical University, Wroclaw, Poland); Lobo, Maria Luisa (Medical Parasitology Unit, GHMT, IHMT/UNL, Lisboa, Portugal); Kopacz, Zaneta; Hendrich, Andrzej (Wroclaw Medical University, Wroclaw, Po-land); Matos, Olga (Global Health and Tropical Medicine, GHTM, Instituto de Higiene e Medicina Tropical, IHMT, Universidade Nova de Lisboa, Por, Lisboa, Portugal)Presentation Type: Poster
# 1746
Title: Evolution and diversity of the large surface protein family in the compacted genomes of PneumocystisPneumocystis, a major opportunistic pathogen in immunodeficient patients, contains abundant surface proteins encoded by a multi-copy gene family, termed the major surface protein (Msg) gene superfamily (including Msg-A, -B, -C, -D and -E families). The msg sequences account for 3-6% of an otherwise highly compacted genome, suggesting an essential role in the organism’s survival. Recent genome sequencing has permitted identification of a nearly complete set of msg genes in P. murina, P. carinii and P. jirovecii, which infects mice, rats and human, respectively. In this report we provide an update on identification and classification of Msg superfamily members, including those of P. wakefieldiae, and describe the Msg domain structure and characteristics of each individual Msg family or subfamily. Based on our analysis, msg genes show not only conservation among msg families or subfamilies across different Pneumocystis species, but also species-specific expansions or contractions. Notably, the classical Msg (included in the Msg-A1 subfamily, whose expression is controlled by the upstream conserved sequence or UCS) is highly conserved among all Pneumocystis species while P. jirovecii shows significant expansions of three families or subfamilies (Msg-A3, Msg-B and Msg-D) but a complete loss of the Msr (Msg-A2) subfamily, and P. murina shows an expansion of the Msg-C family as well as a subset of the Msg-A1 subfamily, in which each member contains a leader sequence very similar to but clearly different from UCS. The versatility of these proteins may mirror their association with a variety of functions, including antigenic variation to allow immune evasion, mediation of life-cycle development, optimization of cell mobility and adhesion ability, adaptation to specific host niches, and modulation of response to environmental stresses. Our analysis provides a rich source of information that lays the foundation for the continued experimental exploration of the function of the Msg superfamily in Pneumocystis biology.
Author: Ma, Liang (National Institutes of Health)Co-Authors: Chen, Zehua (Broad Institute of Harvard and Massachusetts Institute of Technology, Cambridge, MA, United States); Huang, Da Wei (National Cancer Institute, Bethesda, MD, United States); Cisse, Ousmane; Peng, Li; Kutty, Geetha; Bishop, Lisa; Liu, Yueqin (National Institutes of Health, Bethesda, MD, United States); Cushion, Melanie T. (University of Cincinnati, Cincinnati, OH, United States); Cuomo, Christina (Broad Institute of Harvard and Massachusetts Institute of Technology, Cambridge, MA, United States); Kovacs, Joseph (National Institutes of Health, Bethesda, MD, United States)Presentation Type: Poster
LIST OF ABSTRACTS
29
# 1747
Title: Immunocompetent mice treated with a Bruton’s tyrosine kinase inhibitor (Ibrutinib) are more susceptible to Pneumocystis infection
Recently it was shown that treatment of chronic lymphocytic leukemia (CLL) patients with the Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib increased susceptibility to Pneumocystis pneumonia (PCP). All the patients had a CD4+ T-cell count >500/µL suggesting that the susceptibility to PCP is related to the BTK inhibition by ibrutinib. To develop an animal model to study the mechanism of action of the increased susceptibility, we examined the development of PCP in immunocompetent mice treated with ibrutinib. Experimental mice were treated with 12.5 mg/kg ibrutinib for 7 days while control mice were not treated. Both groups of mice were then infected with 2 x 106Pneumocystis murina (P. murina) organisms by intranasal inoculation and treated mice were continued on ibrutinib treatment for the remainder of study. At day 14 post inoculation, the treated mice showed significantly more P. murina in their lungs than the control mice by reverse transcriptase real-time PCR targeting the mtLSU gene; ~9677nuclei per PCR reaction were detected in the treated mice vs. 478 in the control mice. Also, significantly fewer B-cells were isolated from the spleens of the treated mice than control mice by flow cytometry; ~4 x 106 B-cells in the treated mice vs. 7.8 x 106 in the control mice. These results demonstrate that the mouse model may be used to study the mechanism of action of increased susceptibility to PCP caused by ibrutinib and suggest that the effect of ibrutinib on B cell maturation may be related to the increased susceptibility.
Author: Ashbaugh, Alan (University of Cincinnati College of Medicine; Cincinnati Education and Research for Veterans Foundation)Co-Authors: Linke, Michael J. (Veterans Affairs Medical Center, Medical Research Service, Cincinnati, OH, United States); Cushion, Melanie T. (University of Cincinnati College of Medicine, Cincinnati, OH, United States; Veterans Affairs Medical Center, Medical Research Service, Cincinnati, OH, United States)Presentation Type: Poster
# 1748
Title: Profiling gene expression of Pneumocystis murina after treatment with anidulafunginThe echinocandins are a relatively new class of anti-fungal agents that target ß-1,3-D-glucan (BD) biosynthesis. In Pneumocystis species, treatment with these drugs deplete the asci life cycle stage which contain BD, but large numbers of the trophic life cycle stage which do not express BD, remain in the infected lungs. Notably, the morphology of the lingering trophic forms is aberrant and lacking in structure. In the present study, to understand the metabolism of the persisting trophic forms, gene expression profiles of Pneumocystis murina were compared from infected mice untreated and treated with anidulafungin for 2 weeks. Over 60 P. murina genes were found significantly up- or down-regulated. Among these, homologs associated with sexual replication comprise the strongest up-regulated gene signals in the treated mice as well as genes involved with cell remodeling and stress. The up-regulation of the P. murina ß-1,3-D-glucan endohydrolase was notable as it may explain the disappearance of the asci in the lungs of treated mice as it could use the BD in the asci walls as substrate and the lead to its degradation. Down-regulated signals include genes involved in cell replication, genome stability, ribosomal biogenesis, and the Pneumocystis-specific genes encoding the major surface glycoproteins. These data indicate that the anidulafungin treated P. murina are attempting to undergo sexual replication due to a stressed environment, but fail to do so because of a blockade in BD synthesis. Anidulafungin-treated mice are unable to transmit the infection. When anidulafungin was used as prophylactic agent, the infection was prevented. It appears that asci are a necessary and required part of the life cycle stage of Pneumocystis and BD is a requirement for full progression through its life cycle via sexual replication.
Author: Cushion, Melanie T. (Department of Internal Medicine, University of Cincinnati College of Medicine; The Veterans Affairs Medical Center, Medical Research Service)Co-Authors: Ashbaugh Alan; Lynch, Keeley; Sayson, Steven E. (Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, OH, United States; The Veterans Affairs Medical Center, Medical Research Service, Cincinnati, OH, United States); Linke, Michael J. (Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, OH, United States); Tisdale, Nikeya (Department of In-ternal Medicine, University of Cincinnati College of Medicine, Cincinnati, OH, United States; The Veterans Affairs Medical Center, Medical Research Service, Cincinnati, OH, United States); Porollo, Alexey (Division of Biomedical Informatics, Cincinnati Children’s Hospital Medical Center, Cincin-nati, OH, United States) Presentation Type: Platform
LIST OF ABSTRACTS
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# 1749
Title: Prevention of Pneumocystis pneumonia (PCP) by the novel long-acting echinocandin, CD101: implications for life cycle
PCP does not respond to standard antifungal therapy and there are few treatment alternatives. Previously, we reported that echinocandins were not suitable candidates for monotherapy because although asci were depleted, large numbers of trophic forms remained after 3 weeks of treatment, and when released from therapy, asci re-appeared and replication resumed. Asci formation may be essential for the Pneumocystis life cycle. The requirement for asci to establish infection and the efficacy of CD101, a novel long-acting echinocandin, to prevent PCP, were explored in this study.C3H/HeN mice were infected with P. murina by intranasal inoculation. Mice were immunosuppressed by dexamethasone (4 mg/L) in acidified drinking water. Groups included: untreated control, TMP/SMX 50/250 mg/kg/3X/wk; and CD101 at 20-, 2-, and 0.2- mg/kg individually at 1X and 3X/wk. All drugs were administered at the time mice were inoculated. After 6 weeks, mice were sacrificed, lungs homogenized and slides prepared for quantification of trophic forms by rapid Wright-Giemsa stain and for asci by cresyl echt violet staining. Efficacy was based on the reduction of organism burden between treatment and untreated control groups. Counts were log transformed and analyzed by ANOVA and Student-Newman Keuls for multiple comparisons (GraphPadPrismv.6).Prophylaxis with CD101 showed a statistically significant reduction in nuclei levels at all doses except for the 0.2 mg/kg/1x/week group vs the untreated control group. Three of the treatment groups were as efficacious as TMP/SMX with no nuclei observed by microscopic evaluation. All CD101 treatment groups showed a statistically significant reduction in asci levels vs the untreated group. There was no difference in efficacy between 5 of the CD101 treatment groups and TMP/SMX, with no asci observed microscopically. CD101 inhibits the formation of asci which appears to be critical for replication of Pneumocystis and represents a viable candidate for prophylactic therapy of PCP.
Author: Cushion, Melanie T. (Cincinnati VAMC, Medical Research Service; University of Cincinnati College of Medicine)Co-Authors: Ashbaugh Alan; Lynch Keeley; Linke Michael J. (Cincinnati VAMC, Medical Research Service, Cincinnati, OH, United States; University of Cincinnati College of Medicine, Department of Internal Medicine, Cincinnati, OH, United States)Presentation Type: Platform
# 1750
Title: Further evidence of primary homothallism in Pneumocystis speciesComparative genomics suggested that the mode of sexual reproduction of Pneumocystis species is primary homothallism (Almeida et al 2015). However, this is a working hypothesis derived from computational analyses, which, in addition, is based on the genome sequence of single isolates of Pneumocystis jirovecii and Pneumocystis carinii. In the present study, we tested this hypothesis in the wet laboratory by several approaches.Results: The function of the matMc gene of P. jirovecii and P. carinii was assessed by restoration of sporulation of the corresponding null mutant of fission yeast. Homology searches excluded the presence of alpha 1 and amphipathic alpha-helix MAT transcription factors in Pneumocystis species. Long-range PCR confirmed that the three MAT genes of P. jirovecii are present on a single DNA molecule in several isolates, which is compatible only with homothallism. As expected from primary homothallism, RNAseq analysis suggested that all three MAT genes are expressed during P. jirovecii infection. Reverse transcriptase PCR confirmed this finding in several isolates. The results further support that primary homothallism is the mode of reproduction of Pneumocystis species. This mode might be advantageous because it favors sexual reproduction by eliminating the search for a compatible partner, while avoiding deleterious mutations and increasing genetic diversity.
Author: Hauser, Philippe (Lausanne University Hospital)Co-Authors: Richard, Sophie (Lausanne University Hospital, Lausanne, Switzerland); Almeida, Joao (Faculdade de Ciências e Tecnologia, Uni-versidade Nova de Lisboa, Lisbon, Portugal); Cissé, Ousmane (Lausanne University Hospital, Lausanne, Switzerland); Luraschi, Amanda (Hospital and University of Lausanne, Switzerland); Nielsen, Olaf (University of Copenhagen, Denmark); Pagni, Marco (Swiss Institute of Bioinformatics, Lausanne, Switzerland)Presentation Type: Platform
LIST OF ABSTRACTS
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# 1751
Title: Mechanisms of surface antigenic variation in the human pathogenic fungus Pneumocystis jiroveciiMicrobial pathogens commonly escape the human immune system by varying surface proteins. We investigated the mechanisms used for that purpose by Pneumocystis jirovecii. Long-read PacBio sequencing was used to assemble a set of subtelomeres of a single P. jirovecii strain from a bronchoalveolar lavage fluid specimen of a single patient. A total of 113 genes encoding surface proteins were identified on 37 contigs, including 28 pseudogenes. These genes formed a subtelomeric gene superfamily which included five families encoding adhesive GPI-anchored glycoproteins, and one family encoding excreted glycoproteins. Numerical analyses suggested that diversification of the glycoproteins relies on mosaic genes created by ectopic recombination, and occurs only within each family. DNA motifs suggested that all genes are expressed independently, except those of the family encoding the most abundant surface glycoproteins which are subject to mutually exclusive expression. PCR analyses showed that exchange of the expressed gene of the latter family occurs frequently, possibly favoured by the location of the genes proximal to the telomere because this allows concomitant telomere exchange.Our observations suggest that (i) the structure of P. jirovecii cell surface is made of a complex mixture of different glycoproteins, (ii) genetic mosaicism ensures variation of the glycoproteins, and (iii) the strategy of the fungus consists in the continuous production of new subpopulations composed of cells which are antigenically different. This strategy is unique among human pathogens and may be associated to the particular niche within lungs which tolerates the presence of low abundant fungi within the natural microbiota.
Author: Hauser, Philippe (Lausanne University Hospital)Co-Authors: Emanuel, Schmid-Siegert (Swiss Institute of Bioinformatics, Lausanne, Switzerland); Richard, Sophie (Lausanne University Hospital, Lausanne, Switzerland); Luraschi, Amanda (Hospital and University of Lausanne, Switzerland); Mühlethaler, Konrad (Universität Bern, Berne, Swit-zerland); Pagni, Marco (Swiss Institute of Bioinformatics, Lausanne, Switzerland)Presentation Type: Platform
# 1752
Title: Prevalence of microsporidia, Cryptosporidium spp. and Giardia intestinalis in children with gastrointestinal diseases and different immunological status
Children are considered among groups at the highest risk of infections due to their unique immunological status. Moreover, the lack of proper hygiene habits and close contact with animals favor infections, especially transmitted by fecal-oral route. Furthermore, in children with immune disorders the risk of infection is even higher. The aim of our study was to examine prevalence of three widely distributed pathogens: microsporidia, Cryptosporidium spp. and Giardia intestinalis, in stool samples obtained from different children groups both immunocompetent and with immunodeficiency. We examined 221 children, 176 immunocompetent (76 with gastrointestinal diseases, including Inflammatory Bowel Disease (IBD), and 100 healthy preschool children) and 45 with immunosuppression (6 with different primary immunodeficiency (PI) and 39 with the acquired immunodeficiency, resulting from pharmacological treatment after allogeneic hematopoietic cell transplantation with graft versus host diseases (GvHD)).Preliminary research was performed by genus-specific nested PCR followed by genotyping. The pathogens were confirmed in 7.2% (16/221) of all examined patients. The highest prevalence was found for microsporidia (6.8%, 15/221). Cryptosporidium spp. was detected only in one patient (0.45%, 1/221). No G. intestinalis infection was confirmed. Among groups tested, the highest prevalence of infection was shown for GvHD (12.8%, 5/39 infected with Enterocytozoon bieneusi, 7.7%, 3/39 with Encephalitozoon cuniculi) and IBD (9.2%, 7/76, infected with E. bieneusi, 1.3%, 1/76 with Cryptosporidium spp.). No infection were found in PI and healthy preschool groups. Statistical analysis revealed that immunosuppressed children, especially with acquired immunodeficiency, are infected more often than immunocompetent ones (p=0.002). Our findings highlight the necessity of consideration of the above-mentioned pathogens’ infection among children, especially with impaired immunity, which is particularly important due to the lack of effective therapeutic solutions.Funding: Grants for Young Scientists, Wroclaw Medical University (STM.A060.17.038 and Pbmn191).
Author: Kopacz, Zaneta (Wroclaw Medical University)Co-Authors: Wesolowska, Maria; Akutko, Katarzyna; Iwanczak, Barbara; Lewandowicz-Uszynska, Aleksandra; Kalwak, Krzysztof (Wroclaw Medical University, Wroclaw, Poland); Kvac, Martin (Biology Centre CAS, Ceske Budejovice, Czech Republic); Sak, Bohumil (Biology Centre CAS, Institute of Parasitology, Ceske Budejovice, Czech Republic); Sokulska, Magdalena; Hendrich, Andrzej; Kicia, Marta (Wroclaw Medical University, Wroclaw, Poland)Presentation Type: Poster
LIST OF ABSTRACTS
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# 1753
Title: Duration of storage in liquid nitrogen and effect on the viability of Pneumocystis isolatesPneumocystis, a genus of fungal pathogens, is the cause of the lung infections in mammals and a lethal pneumonia in humans termed PCP (Pneumocystis Pneumonia), which can be fatal if left untreated. Research of these fungi has been hindered by a lack of a continuous in vitro culture system, though short term cultures can sustain limited growth and viability. Previously, our laboratory showed that cryopreservation of Pneumocystis isolates had no effect on the ATP content of the populations using a bioluminescent assay used to determine viability. Cryopreservation provided a standard source of organisms that could be evaluated for microbial contaminants and overall ATP content prior to use, which was a vast improvement over the use of freshly isolated Pneumocystis which often contained microbial contaminants that ruined in vitro assays. In the present study, the long term effects of cryopreservation were evaluated by assessing the ATP content of isolates from different years. The ATP content of the original inoculum was compared to that of the cryopreserved isolates from 0 to 17 years. The data showed a correlation between time in cryopreservation and the degree of viability, with decreases in ATP content associated with increased duration of cryopreservation. These data will be useful in the further standardization of in vitro assays.
Author: Smith, Arthur (University of Cincinnati Research)Presentation Type: Poster
# 1754
Title: The effects of gender and strain on Pneumocystis murina pneumonia in miceThe yeast-like fungal pathogen Pneumocystis spp. can cause a lethal pneumonia (PCP) in mammalian hosts with weak immune systems. Though studies have used both male and female rodents to study various aspects of PCP, few if any have directly compared results from both genders. The NIH implemented a monitoring system for sex inclusion in basic and translational research studies in 2014. Per this mandate, we examined the progression of Pneumocystis murina infection in 3 mouse strains in both male and female animals. Results showed higher infection burdens in females compared to their male counterparts in 2 of the 3 mouse strains and differences in burdens among the 3 mouse strains. These outcomes reinforce the need to investigate disease progression and therapeutic efficacies in both sexes and different genetic backgrounds. It is anticipated that gender will likely play an important role in other areas of research, such as host responses to Pneumocystis.
Author: Tisdale, Nikeya (University of Cincinnati College of Medicine; Cincinnati VAMC, Medical Research Service)Co-Authors: Ashbaugh, Alan; Lynch, Keeley, Collins, Margaret S., Cushion, Melanie T.* (University of Cincinnati College of Medicine, Department of Internal Medicine, Division of Infectious Diseases, Cincinnati, OH, United States; Cincinnati VAMC, Medical Research Service, Cincinnati, OH, United States)Presentation Type: Poster
# 1755
Title: Expression of genes by Pneumocystis murina after treatment with the echinocandin, anidulafungin reveal up-regulation of genes involved in meiosis and stress and down-regulation of genes associated with homeostasis
Pneumocystis pneumonia (PCP) is an infection caused by an opportunistic fungus from the Pneumocystis genus in immunocompromised mammalian hosts. Pneumocystis spp. have distinct features which differs from other species of fungi, such as the presence of cholesterol, instead of ergosterol, in the cell membrane. These distinct differences of Pneumocystis render commonly used fungal therapeutics ineffective at treating PCP. Additionally, the inability to culture these fungi outside of the mammalian host results in limited development of therapeutic options and poor understanding of the bi-phasic lifecycle. Pneumocystis spp. reproduce both asexually and sexually, establishing trophic forms and meiotically-formed asci, respectively. Unlike the trophic form, the ascus contains an abundance of a cell wall component, ß-1,3-D-glucan. Data from previous studies showed a depletion of asci in P. murina-infected mice after anidulafungin treatment, which inhibits ß-1,3-D-glucan synthesis. Interestingly, anidulafungin-treated mice were unable to transmit the infection, likely due to the lack of ascus formation, but retained a population of viable tropic forms. To elucidate the effects of anidulafungin and better our understanding of bi-phasic life cycle changes in P. murina, the expression profiles of P. murina-infected mice treated with anidulafungin for two weeks were compared to untreated P. murina-infected mice. Homologs of genes associated with cell remodeling and stress were up-regulated in the anidulafungin-treated Pneumocystis. Down-regulated expression was noted in genes involved in cell replication, genome stability, ribosomal biogenesis, and major surface glycoproteins. These data indicate that anidulafungin-treated P. murina attempt to sexually replicate due to the stressed environment, but are unable because of the inhibition of ß-1,3-D-glucan synthesis. Therefore, it appears that ß-1,3-D-glucan is a requirement for sexual reproduction and that asci production is required in the Pneumocystis life cycle. Identifying additional genes required for reproduction will aid our understanding of the Pneumocystis life cycle and accelerate the discovery of therapeutic options.
Author: Sayson, Steven (University of Cincinnati College of Medicine; The Veterans Affairs Medical Center)Co-Authors: Ashbaugh Alan; Lynch, Keeley (Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, OH, United States; The Veterans Affairs Medical Center, Cincinnati, OH, United States); Linke Michael J. (Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, OH, United States); Tisdale Nikeya L. (Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, OH, United States; The Veterans Affairs Medical Center, Cincinnati, OH, United States); Cox, Jeremy W. (Division of Biomedical Informatics, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, United States; Department of Electrical Engineering and Computing Systems, University of Cincinnati, Cincinnati, OH, United States); Porollo, Alexey (Division of Biomedical Informatics, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, United States); Cushion, Melanie T. (Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, OH, United States; The Veterans Affairs Medical Center, Cincinnati, OH, United States)Presentation Type: Poster
LIST OF ABSTRACTS
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# 1756
Title: Membrane active determinants of Toxoplasma egressToxoplasma gondii destroys the infected cell upon egress, releasing products that stoke the inflammatory response and drive disease progression. Cytolytic egress is controlled by calcium signaling in the parasite, which promotes secretion of micronemal products including perforin-like protein 1 (PLP1) and activates parasite actinomyosin gliding motility. The combination of PLP1-dependent disruption of the parasitophorous vacuole membrane and mechanical force from motility is thought to be main determinants of egress. Nevertheless, how calcium signaling and PLP1-mediated disruption of the PVM are regulated remains poorly understood. Our recent work suggests that a secreted phospholipase termed LCAT contributes to potentiation of calcium signaling for timely secretion of PLP1 and rapid egress. We additionally show that PLP1 activity is regulated by accessibility of specific phospholipid receptors and by pH via the PLP1 C-terminal membrane-binding domain (C-term domain), and that it mainly targets the PVM rather than the host plasma membrane. Crystallographic determination of the PLP1 Cterm domain reveals a novel double layers beta-prism fold with a conspicuous hydrophobic loop that putatively inserts into the target lipid bilayer. The structure also reveals molecular features that potentially mediate phospholipid specificity and pH regulation. Together this work suggests that the secreted membrane active proteins LCAT and PLP1 function upstream and downstream of calcium signaling to foster timely egress from host cells.
Author: Carruthers, Vern (University of Michigan)Presentation Type: Platform
# 1757
Title: Toxoplasma depends on lysosomal consumption of autophagosomes for persistent infectionGlobally, nearly 2 billion people are infected with the intracellular protozoan Toxoplasma gondii. This persistent infection can cause severe disease in immunocompromised people and is epidemiologically linked to major mental illnesses and cognitive impairment. There are currently no options for curing this infection. The lack of effective therapeutics is due partly to a poor understanding of essential pathways that maintain this long-term infection. Although it is known that Toxoplasma replicates slowly within intracellular cysts demarcated with a cyst wall, precisely how it sustains itself and remodels organelles in this niche is unknown. Here we identify a key role for proteolysis within the parasite lysosomal organelle (the vacuolar compartment or VAC) in turnover of autophagosomes and persistence during neural infection. We found that disrupting a VAC-localized cysteine protease compromised VAC digestive function and markedly reduced chronic infection. Death of parasites lacking the VAC protease was preceded by accumulation of undigested autophagosomes in the parasite cytoplasm. Genetic ablation of two genes encoding autophagy proteins abolished development of undigested autophagosomes. These findings suggest an unanticipated function for parasite lysosomal degradation in chronic infection and identify an intrinsic role for autophagy in the T. gondii parasite and its close relatives. The work also identifies a key element of Toxoplasma persistence and suggests that VAC proteolysis is a prospective target for pharmacologic development.
Author: Carruthers, Vern (University of Michigan)Presentation Type: Poster
# 1758
Title: Glycan triggers of trophozoite development in CryptosporidiumThe apicomplexan parasite Cryptosporidium causes a diarrheal disease that can become chronic and life threatening in malnourished and immunocompromised populations. Characterizing the environmental cues for Cryptosporidium life cycle development could reveal therapeutic targets. Previous work has shown that the mucin-associated glycans galactose (Gal) and n-acetyl galactosamine (GalNAc) play an important role during attachment to and invasion of host cells, and that exposure to host cell mucin secretions or commercial Gal-GalNAc preparations can trigger Cryptosporidium sporozoites to switch to replicative trophozoites. The present study aimed at determining the extent to which trophozoite development is triggered by glycopolymers displaying Gal, GalNAc, or other glycans. We quantified the transformation of banana-shaped sporozoites to rounded trophozoites following treatments with bovine submaxillary mucin (BSM; a glycoprotein rich in Gal-GalNAc), ovalbumin (a glycoprotein lacking Gal-GalNAc), and glycomimetic polymers displaying Gal, GalNAc, glucsose, n-acetyl glucosamine, lactose, n-acetyl lactosamine, rhamnose, and sialyllactose. The effects of glycomimetic polymers were examined in suspension and as density arrays grafted onto a glass surface. Results showed that BSM and glycomimetic polymers displaying Gal or GalNAc were more effective triggers of trophozoite development than ovalbumin or glycomimetics displaying other glycans. The effect of Gal and GalNAc was concentration dependent in suspensions and density dependent on a glass surface. These data support an important role for host glycopolymers displaying Gal and GalNAc in triggering the Cryptosporidium transition from an invasive to a replicative form.
Author: McEvoy, John (North Dakota State University)Co-Authors: Edwinson, Adam (North Dakota State University, Fargo, ND, United States)Presentation Type: Platform
LIST OF ABSTRACTS
34
# 1759
Title: The art of adaptation of Pneumocystis: Insights from studies of the Pneumocystis genomeAs an atypical fungus, Pneumocystis has evolved a distinct lifestyle. While its presence has been reported in all mammals studied to date, each Pneumocystis species can infect only one host species. This organism is found almost exclusively in mammalian lungs; there is no convincing evidence of an environmental reservoir. Its ability to cause life-threatening pneumonia in immunocompromised hosts strongly supports Pneumocystis as a parasitic pathogen, while its widespread colonization of healthy hosts and lack of identified virulence factors suggest it has established a commensal relationship with healthy hosts. Its persistent resistance to in vitro cultivation remains a major challenge. The unusual lifestyle of Pneumocystis has attracted the attention of numerous investigators from around the world. This year marks the 20th anniversary of the Pneumocystis genome initiative; genome studies over the past 2 decades have lead to substantial advances in our understanding of the biology of Pneumocystis. Based on genome analysis of three Pneumocystis species that infect humans, mice and rats, Pneumocystis has adapted in innovative ways as an extracellular fungal symbiont. Its genome has been remarkably reduced in size and content related to various metabolic and biological processes, including the complete loss of the pathway for CO2 hydration and deprotonation, as well as chitin synthesis and degradation, which are ubiquitous in fungi including the intracellular Microsporidia. Pneumocystis has retained the ability to maximally exploit the host for resources necessary for survival, and efficiently avoid attacks by the host innate and acquired immune systems. These findings suggest that Pneumocystis has ingeniously adapted to life in the mammalian hosts, with apparent absolute dependence on the host lung environment for survival. Further work will aim to better understand the biological functions of the Pneumocystis genome and translate the genomic data into new culture methods and better strategies for diagnosis and treatment.
Author: Ma, Liang (National Institutes of Health)Co-Authors: Cisse, Ousmane (National Institutes of Health, Bethesda, MD, United States); Cuomo, Christina (Broad Institute of Harvard and Mas-sachusetts Institute of Technology, Cambridge, MA, United States); Kovacs, Joseph (National Institutes of Health, Bethesda, MD, United States)Presentation Type: Platform
# 1760
Title: The trophic forms of Pneumocystis suppress macrophage cytokine productionPneumocysis species have two identifiable life forms that include an ascus (cyst) that has a fungal cell wall composed of ß-glucans and a trophic form that is devoid of the cell wall. There is a paucity of information how innate immune cells in the lungs recognize and interact with the trophic forms of Pneumocystis. To address this we utilized in vitro assays to stimulate macrophages with either a commercially available ß-glucan preparation, mixed Pneumocystis organisms with approximately 1 cyst per 10 trophic forms, or highly enriched trophic forms. We found that the trophic forms failed to stimulate production of TNF, IL-1ß, or IL-6 and were unable to induce translocation of NF?B to the nucleus. Moreover, the trophic forms were able to suppress secretion of TNF in response to the ß-glucan, curdlan. This suppression was not specific to ligation of ß-glucan receptors as the trophic forms could also suppress TNF production induced by LPS. Experiments are underway to identify the mechanism of the suppressive activity.
Author: Garvy, Beth A. (University of Kentucky)Co-Authors: Hollifield, Melissa (University of Kentucky, Lexington, KY, United States)Presentation Type: Poster
# 1761
Title: A vaccine strategy to prevent Pneumocystis coinfection in a non-human primate model of HIVt has long been the goal in this field to develop an effective vaccine for the prevention of Pneumocystis (Pc) infection and Pc-related pulmonary sequelae in HIV+ individuals and other immunocompromised populations. To achieve these goals, our laboratory has established non-human primate models of PCP in simian immunodeficiency virus (SIV)-infected macaques. We have identified a recombinant protein sub-unit vaccine, KEX1, that induces robust anti-Pc immunity in immune-competent macaques that is durable and prevents Pc pneumonia following SIV-induced immunosuppression and Pc challenge. In clinical studies, we have shown that immunity to KEX1 is associated with decreased risk of PCP in HIV+ individuals. In the present study, we addressed whether vaccination with KEX1 can be effective following immunosuppression (during a phase of HIV infection when CD4 T cells are stabilized above the level at which patients are at risk of PCP. To compensate for immune deficits of HIV infection, development of effective vaccines for immunocompromised populations requires innovative vaccination strategies and/or novel adjuvants. Type I, or invariant natural killer T (iNKT) cells are a subpopulation of lymphocytes bearing the invariant T cell receptor chain (Va24-Ja18, in humans) that recognize glycolipids presented by the non-classical MHC class Ib molecule CD1d (4). When activated, iNKT cells are capable of rapidly expressing a wide array of cytokines and co-stimulatory molecules that can enhance humoral response. The central hypothesis is that effective immunity to KEX1 can be elicited in the context of HIV-induced immune dysregulation and that alternative, CD4-independent vaccination strategies can induce invariant natural killer (iNKT) cell-B cell help for enhance immunity to Pneumocystis.
We have tested the effectiveness of the KEX1 vaccine as a therapeutic immunologic booster in immunocompromised, SIV-infected macaques. SIV-infected rhesus macaques were boosted with KEX1 adjuvanted with the iNKT-ligand α-galactoceramide (α-GC). Vaccination with KEX1+α-GC induced a robust increase in KEX1-specific antibody titers that occurred even in SIV-infected animals with circulating CD4+ T cells <500/μl. KEX1+α-GC vaccinated macaques were protected from PCP compared with mock α-GC immunized animals (P=0.0471). These data support the concept that humoral immunity to Pneumocystis may be effectively augmented via iNKT-related vaccination strategies in persons with diminished CD4+ T cell function.
Author: Rabacal, Whitney (University of Georgia)Co-Authors: Schweitzer, Finja; Kling, Heather; Norris, Karen (University of Georgia, Athens, GA, United States)Presentation Type: Platform
LIST OF ABSTRACTS
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# 1762
Title: Molecular characterization of Enterocytozoon bieneusi in wild carnivores in SpainMicrosporidia comprises a diverse group of obligate intracellular parasites that infect all major animal groups from invertebrates to fish to birds and mammals, including domesticated animals and humans. Among Microsporidia, Enterocytozoon bieneusi is the most frequently detected species in humans and animals worldwide bringing into question the possible role of animal reservoirs in the epidemiology of this pathogen. Although E. bieneusi is an emerging zoonotic pathogen for which is has been confirmed low host-specificity with many domestic and wild mammals that could act as a reservoir of infection for humans and other animals, there is only few studies have documented the occurrence of E. bieneusi in wild carnivores worldwide. This study aimed to provide information on presence and molecular characterization of E. bieneusi in wild carnivores from different regions of Spain to evaluate its zoonotic potential. A total of 190 fecal samples from wild carnivores were collected from País Vasco, Asturias, Castilla-La Mancha, Extremadura, and Andalucía in Spain. Eleven carnivore species from five Families were studied by PCR-sequencing of the ITS DNA region. Twenty-five fecal samples (13.2%) from 3 host species (European badger, beech marten, and red fox) were positive for E. bieneusi. Nucleotide sequence analysis of the ITS region revealed a high degree of genetic diversity in E. bieneusi in wild carnivores with a total of 8 distinct genotypes including 4 known genotypes (PtEbIX, S5, S9, and WildBoar3) and 4 novel genotypes (EbCar1-EbCar4). Phylogenetic analysis showed that the four novel genotypes (EbCar1-EbCar4) and three of the four known genotypes identified in this study (S5, S9, and WildBoar3) clustered within the previously designated zoonotic Group 1 while the fourth one (PtEbIX) belongs to a group considered dog specific. Our results demonstrate that human-pathogenic genotypes are present in wild carnivores, corroborating their potential role as a source of human infection and environmental contamination.
Author: Santin, Monica (United States Department of Agriculture)Co-Authors: Calero-Bernal, Rafael (United States Department of Agriculture, Beltsville, MD, United States); Carmena, David (National Centre for Microbiology, Madrid, Spain); Mateo, Marta (Alfonso X El Sabio University, Madrid, Spain); Balseiro, Ana (Centre for Animal Biotechnology (SERIDA), Gijón, Spain); Barral, Marta (Basque Institute of Agricultural Research and Development (NEIKER), Bizkaia, Spain); Lima Barbero, José F. (Institute for Game and Wildlife Research IREC, Cuidad Real, Spain); Habela, Miguel Á. (Extremadura University, Cáceres, Spain)Presentation Type: Platform
# 1763
Title: Enhancing viability of Pneumocystis in suspension culture by the addition of nutritional supplementsPneumocystis is a genus of obligate fungal pathogens that cause lung infection in mammals. Currently, Pneumocystis cannot be maintained ex vivo for more than 7-10 days without declining viability. The need for a continuous in vitro growth system is critical for drug studies, genomic manipulations, and biochemical analyses. Most of the nutrients sequestered from the host or synthesized by Pneumocystis are unknown. To identify potential growth supplements, we employed a new approach by analyzing published comparative genomics studies and unpublished RNA-seq data. The two publications were: “Genome analysis of three Pneumocystis species reveals adaptation mechanisms to life exclusively in mammalian hosts”, PMCID: PMC4764891, and our publication, “Comparative genomics of pneumocystis species suggests the absence of genes for myo -inositol synthesis and reliance on inositol transport and metabolism”, PMID: 25370490. RNA sequence data from our studies of the metatranscriptome of Pneumocystis murina (Pm)-infected mice were the basis for analysis of the genes expressed during infection. Supplements were added to the current RPMI 1640-serum supplemented growth media in vitro with Pneumocystis carinii (Pc) as the organism source. The assays were sampled at days 1, 3, 7, and 14 and ATP content was assessed using Perkin Elmer ATP-liteM kit and measured on a PolarStar Optima (BMG Lab Technologies). Although most of the supplements did not statistically improve ATP levels, some significantly increased growth up to 14 days, including myo-inositol, thiamine, biotin, and acetyl-CoA. The next steps for the supplements include testing in Pm, longer evaluation times, adding supplements in combination with each other and attempting passage of the cultures.
Author: Ficker, Lauren (University of Cincinnati)Presentation Type: Poster
# 1764
Title: Cryptosporidium infecting North American and European cricetids We undertook a study on Cryptosporidium spp. in wild cricetid rodents. Fecal samples were collected from meadow voles (Microtus pennsylvanicus), southern red-backed voles (Myodes gapperi), woodland voles (Microtus pinetorum), muskrats (Ondatra zibethicus), and Peromyscus spp. mice in North America, and from bank voles (Myodes glareolus) and common voles (Microtus arvalis) in Europe. Isolates were characterized by sequence and phylogenetic analyses of the small subunit ribosomal RNA (SSU) and actin genes. Overall, 33.2% (362/1089) of cricetids tested positive for Cryptosporidium, with a greater prevalence in cricetids from North America (50.7%; 302/596) than Europe (12.1%; 60/493). Principal Coordinate analysis separated SSU sequences into three major groups (G1-G3), each represented by sequences from North American and European cricetids. A maximum likelihood tree of SSU sequences had low bootstrap support and showed G1 to be more heterogeneous than G2 or G3. Actin and concatenated actin-SSU trees, which were better resolved and had higher bootstrap support than the SSU phylogeny, showed that closely related cricetid hosts in Europe and North America are infected with closely related Cryptosporidium genotypes. Cricetids were not major reservoirs of human pathogenic Cryptosporidium spp.
Author: McEvoy, John (North Dakota State University)Co-Authors: Clark, Mark (North Dakota State University, Fargo, ND, United States); Horcicková, Michaela (Biology Centre of Czech Academy of Sciences, Ceské Budejovice, Czech Republic); Stenger, Brianna (North Dakota State University, Fargo, ND, United States); Kvac, Martin (Biology Centre CAS, Ceske Budejovice, Czech Republic); Condlová, Šárka (Biology Centre of Czech Academy of Sciences, Ceské Budejovice, Czech Repub-lic); Khan, Eakalak (North Dakota State University, Fargo, ND, United States); Widmer, Giovanni (Tufts University Cummings School of Veterinary Medicine, North Grafton, MA, United States); Stanko, Michal (Slovak Academy of Sciences, Košice, Slovakia); Sak, Bohumil (Biology Centre CAS, Institute of Parasitology, Ceske Budejovice, Czech Republic)Presentation Type: Poster
LIST OF ABSTRACTS
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# 1765
Title: Diagnosis and subtype analysis of Blastocystis isolates from symptomatic and asymptomatic patients in Lower Silesia, Poland
Blastocystis hominis, an unicellular eukaryotic protozoan, is one of the most common parasite found in human faeces. Blastocystis have been isolated from humans and many species of animals. In humans consists of at least 9 genetic subtypes. Little is known about life cycle, transmission and mechanisms of pathogenesis for this parasite. Blastocystis is characterized by high morphological diversity with vacuolar, granular, amoeboid and cyst forms, varying in size from 2 to 200 µm. The differentiation in morphology and cell size makes it difficult to define universal diagnostic standards. The clinical symptoms of B. hominis infection are nonspecific and include nausea, abdominal pain, cramps, bloating, vomiting and diarrhea. Some studies indicate the potential role of Blastocystis in irritable bowel syndrome (IBS).The aim of this study was to determine the prevalence of B. hominis in stool specimens in two group of patients living in Lower Silesia, Poland. 72 patients suffered from various diseases of the digestive system and 40 healthy people were enrolled (totally 112 people). Stool specimens were analysed using xenic in vitro culture (XIVC) with a modified Jones’ medium and molecular methods (PCR). Gene fragment of SSU-rRNA was amplified with forward primer RD5 (5’-ATCTGGTTGATCCTGCCAGT-3’) and reverse primer BhRDr (5’-GAGCTTTTTAACTGCAACAACG-3’).B. hominis was found in 34 (30%) study participants. Infection rates for Blastocystis were 27 (37,5%) and 7 (17,5%) in symptomatic and asymptomatic patients, respectively. Molecular subtyping revealed subtypes: ST1, ST2, ST3, ST4 and ST7, predominantly of ST3. This preliminary study shows high prevalence of B. hominis patients suffered from diseases of the digestive system and indicates that proper diagnosis and identification is necessary to the concept of proper treatment.
Author: Wesolowska, Maria (Wroclaw Medical University)Co-Authors: Janicki, Przemyslaw; Fraczkowski, Martek; Kicia, Marta; Kopacz, Zaneta; Poniewierka, Elzbieta; Szetela, Bartosz (Wroclaw Medical Univeristy, Wroclaw, Poland); Salamatin, Ruslan (Medical University of Warsaw, Warsaw, Poland)Presentation Type: Poster
# 1766
Title: Ibrutinib reduces the inflammatory response of alveolar macrophages to P. murinaPneumocystis pneumonia (PCP) remains the most prevalent opportunistic infection in patients infected with human immunodeficiency virus. PCP is also prevalent in patients with immune systems debilitated due to solid organ transplantation, chemotherapy for cancer, or autoimmune disease. In the immunocompetent host, innate immune responses shape the adaptive immune response leading to clearance of the organism. In the absence of an adaptive immune response, the organism is not eliminated and the infection progresses to PCP. Recent evidence correlating P. jirovecii as co-morbidity agents in respiratory conditions like chronic obstructive pulmonary disease, in association with anti-TNF therapies, or with lung cancers indicates a broadening of the population susceptible to PCP. Patients in these categories fare worse than those with HIV. Treatment of chronic lymphocytic leukemia (CLL) patients with the Bruton’s tyrosine kinase inhibitor ibrutinib increases susceptibility to PCP. We have recently shown treatment of immunocompetent mice with Ibrutinib promotes enhanced P. murina colonization and that treatment of immunosuppressed mice with Ibrutinib promotes P. murina infection. Treatment of immunocompetent mice with Ibrutinib also reduced B cell numbers in the spleen. We performed a series of in vitro experiments to determine if Ibrutinib also acts on the alveolar macrophages, the major effector cells in the host response to Pneumocystis. Alveolar macrophages were isolated from the lungs of mice and treated with Ibrutinib for 1 hour. The cells were then incubated with P. murinafor 2 hours. MIP-2 and TNF-a were then measured in the supernatants by ELISA. In vitro Ibrutinib treatment of alveolar macrophages inhibited stimulation of MIP-2 and TNF-a by P. murina. These results demonstrate that the mouse model may be used to study the mechanism of action of increased susceptibility to PCP caused by ibrutinib and suggest that the effect of ibrutinib on alveolar macrophages may be related to the increased susceptibility.
Author: Linke, Michael (University of Cincinnati/Cincinnati VAMC)Co-Authors: Cushion, Melanie T. (University of Cincinnati/Cincinnati VAMC, Cincinnati, OH, United States); Ashbaugh, Alan (University of Cincin-nati, Cincinnati, OH, United States)Presentation Type: Platform
LIST OF ABSTRACTS
37
# 1767
Title: Functional genomics of the mouse model of Pneumocystis pneumoniaPneumocystis species are obligate opportunistic fungal pathogens causing Pneumocystis pneumonia (PCP) in immunocompromised mammalian hosts. From previous genome analyses, it is known that these parasitic fungi lack many metabolic pathways. To gain insights in functional genomics of pneumocystosis, RNA-seq was conducted at the moderate and heavy stages of PCP in immunosuppressed mice. The corresponding gene expression profiles of the host, pathogen and lung microbiome were analyzed to reveal host-pathogen interactions. In this study, we present the results of profiling P. murina genes. The entire genome of the fungus appears to be actively transcribed in both groups of animals. No statistically differentially expressed fungal genes were found between the groups suggesting that the pathogen is active and has no dominating forms, trophic versus asci, throughout the course of infection. In the search for enzymes critical in nutrient acquisition, corresponding expressed genes were mapped to metabolic pathways. Highly expressed genes were found from folate biosynthesis, nicotinate/nicotinamide metabolism, and lipoic acid metabolism. Following these observations, supplementation of the culture media with quinolinate and lipoate was assessed to improve viability of the isolated fungi. Moderate increases in viability of the organisms were observed in the short term, but further optimization needs to be conducted for the development of long-term culture media using these new ingredients.
Author: Porollo, Alexey (Cincinnati Childrens Hospital and Medical Center)Co-Authors: Porollo, Aleksey (Center for Autoimmune Genomics and Etiology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, United States; Division of Biomedical Informatics, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, United States); Cox, Jeremy W. (Center for Autoimmune Genomics and Etiology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, United States; Department of Electrical Engineering and Computing Systems, University of Cincinnati, Cincinnati, OH, United States); Sesterhenn, Thomas M.; Collins, Margaret S.; Ficker, Lauren; Nikeya, Tisdale (The Veterans Affairs Medical Center, Cincinnati, OH, United States; Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, OH, United States); Salomonis, Nathan (Division of Biomedical Informatics, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, United States); Cushion, Melanie T. (The Veterans Affairs Medical Center, Cincinnati, OH, United States; Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, OH, United States) Presentation Type: Platform
LIST OF ABSTRACTS
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