-
저작자표시-비영리-변경금지 2.0 대한민국
이용자는 아래의 조건을 따르는 경우에 한하여 자유롭게
l 이 저작물을 복제, 배포, 전송, 전시, 공연 및 방송할 수 있습니다.
다음과 같은 조건을 따라야 합니다:
l 귀하는, 이 저작물의 재이용이나 배포의 경우, 이 저작물에 적용된 이용허락조건을 명확하게 나타내어야
합니다.
l 저작권자로부터 별도의 허가를 받으면 이러한 조건들은 적용되지 않습니다.
저작권법에 따른 이용자의 권리는 위의 내용에 의하여 영향을 받지 않습니다.
이것은 이용허락규약(Legal Code)을 이해하기 쉽게 요약한 것입니다.
Disclaimer
저작자표시. 귀하는 원저작자를 표시하여야 합니다.
비영리. 귀하는 이 저작물을 영리 목적으로 이용할 수 없습니다.
변경금지. 귀하는 이 저작물을 개작, 변형 또는 가공할 수 없습니다.
http://creativecommons.org/licenses/by-nc-nd/2.0/kr/legalcodehttp://creativecommons.org/licenses/by-nc-nd/2.0/kr/
-
A Dissertation for the Degree of Doctor of Philosophy
Role of Toll-like Receptor 2 and 4 in Host Immune
Response against Acinetobacter baumannii
Acinetobacter baumannii 에 대항하는 숙주면역
반응에서 톨유사 수용체 2 및 4 의 역할
Chang-Hwan Kim, D.V.M.
August 2014
Department of Laboratory Animal Medicine
College of Veterinary Medicine
Graduate School of Seoul National University
-
Role of Toll-like Receptor 2 and 4 in Host Immune
Response against Acinetobacter baumannii
By
Chang-Hwan Kim
A dissertation submitted in partial fulfillment of
the requirement for the degree of
DOCTOR OF PHILOSOPHY
Supervisor: Jae-Hak Park, D.V.M., Ph.D.
June 2014
Dissertation Committee:
Woo, Hee-Jong (인) (Chairman of Committee) Park, Jae-Hak (인)
(Vice chairman of Committee) Chae, Chan-Hee (인) (Committee member)
Hur, Gyeung-Haeng (인) (Committee member) Park, Jong-Hwan (인)
(Committee member)
-
- i -
ABSTRACT
Role of Toll-like Receptor 2 and 4 in Host Immune
Response against Acinetobacter baumannii
(Supervisor: Jaehak Park)
Chang-Hwan Kim
Department of Laboratory Animal Medicine, College of
Veterinary Medicine Graduate School, Seoul National
University
Interest in the genus Acinetobacter, from both the scientific
and public
community, has risen sharply over recent years. Toll–like
receptors (TLRs) are the
most studied pattern recognition receptors (PRRs) and TLR2 and
TLR4 play
important roles in the recognition of bacterial pathogen. TLR2
is a membrane
sensor for bacterial lipoprotein and TLR4 has been identified as
a sensor for LPS,
a major cell wall component of Gram-negative bacteria. In these
studies, we
investigated in vitro and in vivo innate immune mechanism
against Acinetobacter
-
- ii -
baumannii focusing on TLR2 and TLR4.
In the first study, we studied the role of TLR2 and TLR4 on
innate immune
responses of immune cells against A. baumannii. Bone
marrow-derived
macrophages (BMDMs) and bone marrow-derived dendritic cells
(BMDCs) were
isolated from wild type (WT), TLR2- and TLR4-deficient mice and
infected with
A. baumannii. Enzyme-linked immunosorbent assays (ELISAs) were
performed
revealing that the production of interleukin-6 (IL-6) and tumor
necrosis factor-α
(TNF-α) by A. baumannii was impaired in TLR4-deficient
macrophages. In
addition, TLR4 was required for the optimal production of IL-6,
TNF-α, and IL-
12 in BMDCs in response to A. baumannii. However, the absence of
TLR2 did
not affect A. baumannii-induced cytokines production in BMDMs.
Western blot
analysis showed that A. baumannii leads to the activation of
nuclear factor-kappa
B (NF-κB) and mitogen-activated protein kinases (MAPKs) in
macrophages via
TLR4-dependent pathway. mRNA expression of inducible nitric
oxide synthase
(iNOS) and nitric oxide (NO) production was elicited in WT BMDMs
in response
to A. baumannii, which was abolished in TLR4-deficienct cells.
Although TLR4
deficiency did not affect phagocytic activity of macrophages
against A. baumannii,
bacterial killing ability was impaired in TLR4-deficient BMDMs.
In addition, A.
baumannii induced apoptosis in BMDMs via TLR4-independent
pathway.
In the second study, WT, TLR2- and TLR4-deficient mice were
infected
-
- iii -
intranasally with A. baumannii to determine the role of TLR2 and
TLR4 in host
defense against A. baumannii infection. Body weight, pulmonary
bacterial load,
cytokine and chemokine levels in bronchoalveolar lavage fluid
(BALF) and lung
histopathology were examined after infection. Body weight loss
of TLR2-
deficient mice was comparable to WT mice but that of
TLR4-deficient mice was
significantly less than WT mice. Pulmonary bacterial loads of
TLR2-deficient
mice were only increased at 1 day and those of TLR4-deficient
mice were higher
than WT mice at 1, 3 and 5 days after infection. In
TLR2-deficient mice, there
was a significant increase in pulmonary IL-6 and chemokine
(C-X-C motif) ligand
2 (CXCL2) at 1 day after infection. When compared with WT mice,
cytokine and
chemokine concentrations of TLR4-deficient mice were
significantly increased at
day 1 but decreased thereafter. The histopathological features
of lung tissue were
comparable between WT and TLR2-deficient mice but inflammation
was marked
alleviated in TLR4-deficient mice compared with WT mice at 5
days after
infection.
In conclusion, our studies demonstrated that TLR4 was essential
for inducing
innate immune response in immune cells and host against A.
baumannii and TLR2
contributed to the host defense against A. baumannii at an early
stage of infection.
-
- iv -
Key words: Acinetobacter baumannii, toll-like receptor, innate
immunity, bone
marrow derived macrophages, mouse, pneumonia, cytokine.
Student number: 2007-30453
-
- v -
CONTENTS
ABSTRACT
.........................................................................................................i
CONTENTS........................................................................................................
v
LIST OF FIGURES
..........................................................................................vii
ABBREVIATIONS
............................................................................................
ix
LITERATURE
REVIEW...................................................................................
1
Genus Acinetobacter
...........................................................................................
2
Microbiology
.......................................................................................................
3
Epidemiology
......................................................................................................
5
Virulence Factor
.................................................................................................
6
Pathogenesis......................................................................................................
10
Immune Respose against Acinetobacter Infection
........................................... 11
Resistance to antibiotics
...................................................................................
13
Clinical Manifestinations
.................................................................................
14
Detection and Diagnosis
...................................................................................
18
Treatment
.........................................................................................................
20
Toll-like Receptor
.............................................................................................
21
References
.........................................................................................................
30
-
- vi -
CHAPTER I. Essential role of toll-like receptor 4 in
Acinetobacter
baumannii-induced immune responses in immune cells
................................. 48
Introduction
......................................................................................................
49
Materials and Methods
....................................................................................
51
Results
...............................................................................................................
56
Discussion
.........................................................................................................
60
References
.........................................................................................................
73
CHAPTER II. Role of toll-like receptor 2 and 4 in the pulmonary
infection
with Acinetobacter baumannii
..........................................................................
78
Introduction
......................................................................................................
79
Materials and Methods
....................................................................................
82
Results
...............................................................................................................
85
Discussion
.........................................................................................................
88
References
.........................................................................................................
99
GENERAL CONCLUSION
...........................................................................
106
ABSTRACT IN KOREAN
.............................................................................
108
-
- vii -
LIST OF FIGURES
CHAPTER I.
Figure 1. Cytokine production by WT and TLR4-deficient BMDMs in
response to
A. baumannii
......................................................................................................
65
Figure 2. Cytokine production by WT and TLR2-deficient BMDMs in
response to
A. baumannii..
....................................................................................................
67
Figure 3. Cytokine production by A. baumannii in WT and
TLR4-deficient
BMDCs .
............................................................................................................
68
Figure 4. NF-κB and MAPK activation in WT and TLR4-deficient
BMDMs in
response to A. baumannii...
.................................................................................
69
Figure 5. iNOS expression and NO production in WT and
TLR4-deficient
BMDMs infected with A. baumannii
..................................................................
70
Figure 6. Ability of phagocytosis and bacterial killing against
A. baumannii and
induction of apoptosis by A. baumannii in BMDMs
........................................... 71
CHAPTER II.
Figure 1. Body weight changes by A. baumannii infection in WT,
TLR2- and
TLR4-deficient mice
..........................................................................................
93
Figure 2. Bacterial clearance in the lung of mice infected with
A. baumannii. ..... 94
-
- viii -
Figure 3. The production of cytokines and chemokines by A.
baumannii in WT
and TLR2-deficient mice.
...................................................................................
95
Figure 4. The production of cytokines and chemokines by A.
baumannii in WT
and TLR4-deficient mice.
...................................................................................
96
Figure 5. Histopathology in the lung of A. baumannii-infected
mice ................... 98
-
- ix -
ABBREVIATIONS
AP-1 Activator protein-1
BALF Bronchoalveolar lavage fluid
BMDC Bone marrow derived dendritic cell
BMDM Bone marrow derived macrophage
CCL Chemokine (C-C motif) ligand
CFU Colony forming unit
CXCL Chemokine (C-X-C motif) ligand
ELISA Enzyme linked immunosorbent assay
ERK Extracellular signal-regulated kinase
IKK IκB kinase
IL Interleukin
iNOS Inducible nitric oxide synthase
IRAK Interleukin-1 receptor-associated kinase
IRF Interferon regulatory factors
JNK c-Jun N-terminal kinases
LDH Lactate dehydrogenase
LPS Lipopolysaccharide
MAPK Mitogen-activated protein kinase
-
- x -
MDR Multidrug resistant
MOI Multiplicity of infection
MyD88 Myeloid differentiation primary response protein 88
NF-κB Nuclear factor kappa B
NO Nitric oxide
PAMP Pathogen associated molecular pattern
PRR Pattern recognition receptor
TLR Toll-like receptor
TNF-α Tumor necrosis factor alpha
TRIF TIR-domain-containing adapter-inducing interferon
-β
WT Wild type
-
- 1 -
LITERATURE REVIEW
-
- 2 -
Genus Acinetobacter
Bacteria of the genus Acinetobacter have gained increasing
attention over the
past several decades. Acinetobacter baumannii is the most
significant species in
the genus and a major cause of hospital-acquired infection
globally (Munoz-Price
and Weinstein, 2008; Visca et al., 2011). Until now, A.
baumannii strains resistant
to all known antibiotics have been reported (Peleg et al., 2007;
Prashanth and
Badrinath, 2005) and they suddenly cause infections involving
several patients in
a clinical care unit (Fierobe et al., 2001; Poirel et al.,
2003). Acting in synergy
with this emerging resistance profile, some strains have the
ability to survive on
the surfaces of hospital facilities and equipments for weeks,
thus creating the
potential for nosocomial spread (Knapp et al., 2006; Peleg et
al., 2008).
Acinetobacter can cause various kinds of clinical symptoms in
humans.
Although pulmonary diseases are the most common infections
caused by this
organism (Glew et al., 1977), infections involving the
bloodstream, skin and soft
tissue, central nervous system, urinary tract and bone have
emerged as highly
problematic in recent times. The organism commonly targets the
most susceptible
hospitalized patients who are critically ill with skin wounds
and airway problems.
The mortality rate associated with A. baumannii infection in the
intensive care
unit setting can reach 40%.
Despite the great increase of infections caused by multidrug
resistant (MDR)
-
- 3 -
Acinetobacter, there is still a lack of awareness about these
microorganisms
(Doughari et al., 2010). Because of the limited therapeutic
options for MDR
Acinetobacter infections, prevention of transmission among
health care associated
facilities is critical in preventing morbidity. Moreover, there
is also an urgent need
to develop novel therapeutic agents active against multidrug
resistant strains.
Microbiology
The history of the genus Acinetobacter dates back to 1911, when
Beijerinck, a
Dutch microbiologist, described an organism named Micrococcus
calcoaceticus
that was isolated from soil with enrichment medium (Beijerinck,
1911). Over the
subsequent decades, similar organisms were described and
assigned to at least 15
different genera and species. The current genus designation,
Acinetobacter was
initially proposed by Brisou and Pre´vot in 1954 to separate the
nonmotile from
the motile microorganisms within the genus Achromobacter (Brisou
and Prevot,
1954). The name “Acinetobacter” originates from the Greek word
“akinetos”
meaning “unable to move”, as these bacteria are not motile.
The genus Acinetobacter belongs to the family Moraxellaceae and
order
Pseudomonadales. It consists of Gram-negative, strictly aerobic,
non-motile, non-
fastidious, non-fermentative, oxidative-negative,
indole-negative, catalase-
positive bacteria with a DNA G+C content of 39% to 47% (Barbe et
al., 2004;
-
- 4 -
Vallenet et al., 2008). Acinetobacter spp. grow well on solid
media that are
routinely used in clinical microbiology laboratories. The
optimum incubation
temperature is 33-35°C for most strains. They are bacilli with
0.9 to 1.6 um in
diameter and 1.5 to 2.5 um in length in the exponential growth
phase (Peleg et al.,
2008).
The cells of Acinetobacter vary in size and arrangement. They
generally form
smooth and sometimes mucoid colonies on solid media, ranging in
color from
white to pale yellow or grayish white. Some environmental
strains have been
reported to produce a diffusible brown pigment. Several clinical
isolates show
hemolysis on sheep blood agar plates (Peleg et al., 2008).
Although the cell wall
of Acinetobacter is typically Gram-negative bacteria, destaining
is difficult and
may therefore be misidentified as Gram-positive cocci (Marcella
Alsan and
Michael Klompas, 2010).
Based on molecular studies, thirty-two species of Acinetobacter
have now been
recognized. Twenty-two of them have assigned valid names,
whereas other
species are described as a genomic group. Four of the species
(A. calcoaceticus, A.
baumannii, Acinetobacter genomic species 3, and Acinetobacter
genomic species
13TU) are very closely related and difficult to distinguish from
each other by
phenotypic properties. Therefore, it has been proposed to refer
to these species as
the A. baumannii-A.calcoaceticus (ABC) complex (Gerner-Smidt,
1992).
-
- 5 -
Epidemiology
Acinetobacter species are ubiquitous in nature and have been
found in soil,
water, animals and humans. Some strains of Acinetobacter can
survive for weeks
in the environment promoting transmission within the hospital
settings (Doughari
et al., 2011). A. baumannii was recovered from the skin, throat,
rectum and
respiratory tract of humans and account for nearly 80% of
reported Acinetobacter
infections (Eliopoulos et al., 2008). Skin carriage of
Acinetobacter species has
been implicated as a cause of nosocomial outbreaks of infection
(Fournier et al.,
2006). However, an epidemiological study found that most people
are typically
colonized with Acinetobacter species other than A. baumannii
(Seifert et al.,
1997). Although A. baumannii is not a normal inhabitant of human
skin, its DNA
was detected in 21% of 622 lice collected worldwide, suggesting
that A.
baumannii is endemic to human body lice (La Scola and Raoult,
2004).
Pathogenic Acinetobacter infections were encountered in military
personnel
during the wars in Afghanistan and Iraq and was named by the
media as
Iraqibacter (O'Shea, 2012). Between January 2002 and August
2004, multi-drug
resistant ABC was isolated from blood samples of 102 veterans of
Iraq-
Afghanistan combat who were hospitalized in military medical
facilities in Iraq.
Epidemics of ABC in soldiers wounded abroad are primarily
attributable to
nosocomial transmission because strains recovered from healthy
U.S.-based
-
- 6 -
soldiers differ from those recovered from injured soldiers
(Griffith et al., 2006)
and A. baumannii has not routinely been isolated from soil and
water reservoirs in
Iraq (Griffith et al., 2007).
Spread of multidrug resistant A. baumannii can occur on a
national and even
international scale. There are several cases of infection in
many countries (Coelho
et al., 2006; Lolans et al., 2006). Some studies have reported
the epidemiology of
A. baumannii infections in different parts of the world
including Europe, the
United States and South America (Kurcik-Trajkovska, 2009; Siau
et al., 1999).
The movement of personnel, patients, equipments or other shared
products may
cause the monoclonal multi-institutional outbreaks, which
suggests the
importance of rigorous infection control procedures.
Virulence Factor
Acinetobacter was considered to be an organism with low
virulence in the past.
However, the occurrence of fulminant Acinetobacter pneumonia
indicates that
these organisms may sometimes be of high pathogenicity and cause
invasive
disease. The study of more specific virulence mechanisms in
Acinetobacter has
focused on the the lipopolysaccharide (Erridge et al., 2007;
Knapp et al., 2006),
siderophore (Dorsey et al., 2004), quorum sensing (Bhargava et
al., 2011;
González et al., 2001) and outer membrane protein (OMP) function
(Lee et al.,
-
- 7 -
2006; Siroy et al., 2006).
When the genome of A. baumannii was compared to that of the
nonpathogenic
species A. baylyi, 28 gene clusters were unique to A. baumannii,
with 16 of these
clusters having a potential role in virulence. One of the most
interesting of these
was a 133,740-bp island that contained not only transposons and
integrases but
also genes homologous to the Legionella/Coxiella type IV
virulence/secretion
systems. Other relevant genes included those involved in the
pilus biogenesis, cell
envelope and iron uptake and metabolism (Smith et al.,
2007).
Lipopolysaccharide and Capsular Polysaccharide
Lipopolysaccharides found in the outer membrane of Gram-negative
bacteria
are large molecules consisting of a lipid and a polysaccharide
joined by a covalent
bond. The lipopolysaccharide produced by Acinetobacter elicits a
strong immune
response and is responsible for lethal toxicity in laboratory
animals (Pantophlet,
2008). It also induces a positive endotoxin detection test
during Acinetobacter
bloodstream infection in humans.
Acting in synergy with the capsular exopolysaccharide, the
lipopolysaccharide
is involved in resistance to complement system in human serum. A
relationship
has been investigated between Gram-negative bacteria isolated
from bacteremic
patients and their in vitro resistance against the lytic
activity of complement. In
-
- 8 -
experimental models of Gram-negative infections, it has been
demonstrated that
capsular polysaccharide blocks the access of complement to the
microbial cell
wall and prevents the triggering of the alternative pathway of
complement
activation (Goel and Kapil, 2001).
Siderophores
Siderophores are small, high-affinity iron chelating compounds
responsible for
iron uptake in bacteria. Bacteria meet their iron requirement by
binding
exogenous iron using siderophores or hemophores (Lesouhaitier et
al., 2009).
Acinetobacter siderophores are called acinetobactins and are
chiefly made up of
the amine histamine which results from histidin decarboxylation
(Mihara et al.,
2004). In order to thrive in the iron-deficient condition of a
human host,
Acinetobacter spp. secrete acinetobactins around the environment
(Dorsey et al.,
2004).
Quorum Sensing
Quorum sensing has been shown to regulate a wide array of
virulence
mechanisms in many Gram-negative organisms such as P.
aeruginosa. In
Acinetobacter spp., four different quorum sensing signal
molecules capable of
activating N-acylhomoserine-lactone biosensors have been
identified (González et
-
- 9 -
al., 2001). Quorum sensing may be a central mechanism for auto
induction of
multiple virulence factors in an opportunistic pathogen such as
Acinetobacter and
this process should be studied for its clinical implications
(Joly-Guillou, 2005).
Outer Membrane Protein (OMP)
Outer membrane proteins in some Gram-negative bacteria are known
to have
essential roles not only in pathogenesis and adaptation in host
cells but also in
antibiotic resistance. Several OMPs of the OmpA family have been
characterized
in various Acinetobacter strains (Dijkshoorn et al., 2007;
Gordon and Wareham,
2010). The cells of Acinetobacter spp. are surrounded by OmpA, a
protein that
kills host cells (Choi et al., 2008). During an infection, OmpA
binds to eukaryotic
cells and gets translocated into the nucleus where it causes
cell death (Choi et al.,
2008; Dijkshoorn et al., 2007).
Verotoxins
Verotoxin production in Acinetobacter was first identified from
A.
haemolyticus (Grotiuz et al., 2006). The toxins belong to the
RNA N-glycosidases
which directly target the cell ribosome machinery and inhibit
protein synthesis.
Verotoxins can be classified into two antigenic groups, vtx-1
and vtx-2, which
include an important number of genotypic variants. The mechanism
by which A.
-
- 10 -
haemolyticus produces this toxin is not well understood. The
pathogenicity, basic
structure, and chemical components of the toxins are the same as
those of
verotoxins from E. coli and other bacteria (Lambert et al.,
1993).
Virulence Conferring Enzymes
Cell surface enzymes facilitate the adhesion of bacterial cells
to host cells. For
example, the urease activity of Acinetobacter promotes
colonization of the mouse
stomach (Costa et al., 2006). Other virulence conferring enzymes
secreted by the
bacteria include esterases, certain amino-peptidases, and acid
phosphatases
(Rathinavelu et al., 2003; Towner, 2006). Two copies of the
phospholipase C gene
with 50% identity to that of Pseudomonas are found in A.
baumannii. It is
assumed that these lipases serve a similar function as a
hydrolytic enzyme
(Vallenet et al., 2008).
Pathogenesis
An infection caused by Acinetobacter spp. results if the host
first line of
defense is compromised. For example, chronic gastritis in
gastrointestinal
infections with A. lwoffıi and H. pylori is induced when the
normal tissue
architecture of the gastric epithelium is altered. Infections
with A. lwoffıi induce
production of pro-inflammatory cytokines and increase gastrin
levels. Persistent
-
- 11 -
inflammation including the activation of antigen presenting
cells and release of
pro-inflammatory molecules involve in acid secretion and changes
in the number
of gastric epithelial cells. This can lead to gastritis, peptic
ulcers, and gastric
cancer (Richet and Pierre Edouard Fournier, 2006).
Acinetobacter poses little risk to healthy people. However,
people who have
weakened immune systems, chronic lung disease, or diabetes may
be more
susceptible to infections with Acinetobacter. Interpreting the
significance of A.
baumannii isolates from skin, pharynx, GI tract, urethra,
conjunctiva, and the
vagina must be performed carefully, as these organisms can
colonize both healthy
and devitalized tissues in these areas. Most infections occur in
tissues with a high
fluid content, such as the respiratory tract, peritoneal fluid,
and the urinary tract.
Nosocomial infection caused by Acinetobacter spp. is very common
and risk
factors include length of hospital stay, surgery, treatment with
broad-spectrum
antibiotics, indwelling catheters, mechanical ventilation, and
breaches in infection
control practices.
Immune Response against Acinetobacter Infection
Several studies have described the innate immune response to A.
baumannii and
the importance of TLR signaling (Erridge et al., 2007; Knapp et
al., 2006). In a
mouse pneumonia model, TLR4 gene-deficient mice had increased
bacterial
-
- 12 -
counts, increased bacteremia, impaired cytokine and chemokine
responses, and
delayed onset of lung inflammation compared to wild-type mice.
A. baumannii
LPS was identified as the major immunostimulatory factor. This
was further
illustrated by the attenuated effects of A. baumannii on mice
deficient in CD14, an
important molecule that enables LPS binding to TLR4 (Knapp et
al., 2006).
These findings were confirmed using human cells, but in contrast
to the mouse
model, TLR2 was also identified as an important signaling
pathway (Erridge et al.,
2007). Authors demonstrated the potent endotoxic potential of A.
baumannii LPS,
which stimulated the proinflammatory cytokines interleukin-8 and
tumor necrosis
factor alpha equally to the stimulation by E. coli LPS at
similar concentrations
(Erridge et al., 2007). These studies suggest that A. baumannii
endotoxin may
incite a strong inflammatory response during infection. Nod like
receptors (NLRs)
such as Nod1 and Nod2 also contribute to host immune response
against A.
baumannii infection (Bist et al., 2014).
Humoral immune responses have also been described for
Acinetobacter
infection, with antibodies being targeted toward
iron-repressible OMPs and the O
polysaccharide component of LPS (Smith and Alpar, 1991). A study
showed that
mouse-derived monoclonal antibodies directed at A. baumannii
OMPs expressed
in an iron depleted environment have bactericidal and opsonizing
activity. These
antibodies were also able to block siderophore-mediated iron
uptake (Goel and
-
- 13 -
Kapil, 2001).
Resistance to antibiotics
The concerning features of A. baumannii are its prodigious
ability to avoid
desiccation and develop resistance to all current antibiotic
classes. Although there
are significant differences in the antimicrobial susceptibility
profile of A.
baumannii, the overall trend is increasing resistance since the
1970s (Wadl et al.,
2010). Resistance to antibiotics has hindered therapeutic
management, causing
growing concern worldwide (Grotiuz et al., 2006; Perez et al.,
2007).
Mechanisms of resistance to antibiotics by Acinetobacter spp.
vary with species,
type of antibiotic, and geographical location (Jain and
Danziger, 2004). A.
baumannii eludes antibiotics by several ways such as efflux
pumps, mutations in
porins, mutations in antibiotic targets, and antibiotic-altering
enzymes (Jain and
Danziger, 2004; Vila et al., 2002). β-lactam antibiotics are
inactivated by the
production of β-lactamases, alterations of penicillin-binding
proteins and
decreased permeability of the outer membrane to β-lactams
(Poirel et al., 2003).
Resistance to cephalosporins is induced by chromosomally
encoded
cephalosporinases and by cell impermeability and aminoglycosides
via
aminoglycoside-modifying enzymes. Quinolones are inactivated by
altering the
target enzymes DNA gyrase and topoisomerase IV through
chromosomal
-
- 14 -
mutations, a decrease in permeability and increase in the active
efflux of the drug
by the microbial cell.
Resistance to antibiotics is transferred via plasmids and
transposons among
Acinetobacter. Plasmids are DNA elements that carry the
antibiotic and heavy
metal resistance conferring genes capable of autonomous
replication. On the other
hand, transposons are sequences of DNA that can move themselves
to new
positions within the genome of a bacterium or any other
prokaryotic cell. These
elements are often present in resistant bacteria and have been
reported in clinical
isolates of Acinetobacter (Gallego and Towner, 2001). Plasmids
and transposons
are easily transferred between bacteria via the process of
genetic transformation.
Gene transfers in Acinetobacter spp. also occur via conjugation
and transduction.
Conjugation in Acinetobacter involves a wide host range and
chromosomal
transfer, while transduction involves a large number of
bacteriophages with a
restricted host range (Rathinavelu et al., 2003).
Clinical Manifestations
As agents of nosocomial bloodstream infections, A. baumannii
spp. are ranked
9th after S. aureus, E. coli, Klebsiella spp. P. aerugenosa, C.
albicans,
Enterococci, Serratia and Enterobacter. They are the second most
commonly
isolated nonfermenters in human specimens (Oberoi et al., 2009)
after
-
- 15 -
Pseudomonas aeruginosa. The incidence of infection is on the
rise and mortality
rates are quite high (Vallenet et al., 2008; Wisplinghoff et
al., 2004).
Acinetobacter spp. cause a wide range of health care associated
infections such
as ventilator-associated pneumonia, bloodstream infections,
urinary tract
infections, meningitis, wound infections, and ventriculitis.
They can also cause
infections in the community and predominant community-acquired
infections are
pneumonia, meningitis, and bacteremia (Falagas et al.,
2007).
Hospital-Acquired Pneumonia
Prior to the 1970s, Acinetobacter infections were mostly
post-surgical urinary
tract infections and Acinetobacter spp. were isolated primarily
from patients
hospitalized in surgical or medical wards. However, the
significant improvement
in resuscitation techniques during the last several decades has
changed the types
of infections caused by Acinetobacter. Today, the most important
role of these
bacteria is as a cause of nosocomial pneumonia, particularly
following the use of
mechanical ventilatory procedures.
Nosocomial pneumonias tend to be multilobar and develop later in
the hospital
stay and can be complicated by effusions and bronchopleural
fistulas (Lolans et
al., 2006). Using data from the National Nosocomial Infections
Surveillance
System, over 410,000 bacterial isolates were analyzed to
determine the
-
- 16 -
epidemiology of Gram-negative bacilli in ICUs. Although the
percentage of
pneumonia caused by Gram-negative bacilli was constant during
the study period,
the proportion of ICU pneumonias attributable to Acinetobacter
species increased
from 4% in 1986 to 7% in 2003 (Gaynes et al., 2005).
Community-Acquired Pneumonia
Community-acquired pneumonia due to A. baumannii has been
described for
tropical regions of Australia and Asia (Anstey et al., 2002;
Leung et al., 2006).
Acute pneumonia is the most frequent community-acquired
infection involving
Acinetobacter. The disease most typically occurs during the
rainy season and may
sometimes require admission to an ICU (Anstey et al., 2002). It
is characterized
by a fulminant clinical course, secondary bloodstream infection,
and mortality rate
of 40 to 60% (Leung et al., 2006). Patients with acute pneumonia
generally have a
history of alcohol abuse, diabetes, cancer and bronchopulmonary
disease.
Bloodstream Infection
Generally, bacteremia caused by Acinetobacter has been described
in tropical
and ⁄ or developing countries such as New Guinea, Thailand and
Australia
(Anstey et al., 2002; Wang et al., 2002). Several cases have
been reported in
temperate countries such as Spain, France and the USA (Salas et
al., 2003). Cases
-
- 17 -
have been shown to be more prevalent in warm and humid months,
even in
temperate regions (McDonald et al., 1999).
Sources of bloodstream infection were not described in the
previous studies but
are typically related or attributed to underlying pneumonia,
UTI, or wound
infection (Seifert et al., 1995). Risk-factors have been defined
in many studies and
are essentially the same as those identified for other
opportunistic bacteria (Blot et
al., 2003).
Traumatic Battlefield and Other Wounds
Acinetobacter is a major pathogen in traumatic wounds and burns.
It was first
noted to be a significant pathogen among the war victims in the
Korean conflict.
This was confirmed in the Vietnam War where it was the most
common Gram-
negative bacillus isolated from traumatic lower extremity
infections and the
second most common organism isolated from the blood (Tong,
1972). Returning
soldiers from the Iraq and Afghanistan battlefields also have
Acinetobacter
infections (Scott et al., 2007).
A. baumannii may occasionally cause skin/soft tissue infections
outside of the
military population. The organism caused 2.1% of ICU-acquired
skin/soft tissue
infections in one assessment (Weinstein et al., 2005). It is a
well-known pathogen
in burn units and may be difficult to eradicate from such
patients (Trottier et al.,
-
- 18 -
2007).
Urinary Tract Infection (UTI)
A. baumannii is an occasional cause of UTI, being responsible
for 1.6% of
ICU-acquired UTIs (Weinstein et al., 2005). Typically, the
organism is associated
with catheter-associated infection or colonization.
Genitourinary infections have
been typically reported in patients with other risk factors for
infection such as
nephrolithiasis or indwelling catheters (Lolans et al.,
2006).
Meningitis
In addition to pneumonia and bacteremia, intracranial infections
with A.
baumannii can occur. Nosocomial postneurosurgical meningitis is
an increasingly
important entity. Meningitis with A. baumannii is generally
described in patients
following neurosurgical procedures and head trauma (Metan et
al., 2007).
Detection and Diagnosis
Infection or colonization with Acinetobacter is usually
diagnosed by culturing
clinical samples and samples from the environment. The most
common
environmental samples include wastewater, soil, vegetables, and
meat. The most
frequent clinical samples are blood, cerebrospinal fluid,
wounds, pus, urine,
-
- 19 -
respiratory secretions, and catheter tips. Microbiologic
cultures can be processed
by standard methods on routine media. A wide range of media has
been employed
in cultivating organisms from different sources. For routine
clinical and laboratory
investigations, traditional culture media such as nutrient agar,
tryptic soy agar and
Luria Bertani agar are used. Bauman’s Enrichment Medium is most
commonly
used for environmental screening (Guardabassi et al., 1999).
Biochemical typing methods include the use of colorimetric
systems which are
antibody-based agglutination tests (Chen et al., 2008).
Serological identification
has been attempted with the analysis of capsular type and
lipopolysaccharide
(Russo et al., 2010) molecules as well as protein profiles for
taxonomy and
epidemiological investigations. A new molecular identification
and typing method
has been developed for detection of Acinetobacter strains which
has led to the
successful identification and outbreak management of the disease
(Ecker et al.,
2006). The most important of these are polymerase chain reaction
(Grotiuz et al.,
2006), PFGE, RAPD-PCR DNA fingerprinting (Peleg et al., 2007),
fluorescent in
situ hybridization (Vanbroekhoven et al., 2004), and 16S rRNA
gene restriction
analysis. A recent diagnostic method, the microsphere based
array technique, was
reported to have high specificity and can discriminate between
Acinetobacter
species. This technique combines an allele specific primer
extension assay and
microsphere hybridization (Lin et al., 2008).
-
- 20 -
Other methods introduced in the epidemiological investigation of
outbreaks
caused by Acinetobacter spp. include biotyping, phage typing,
cell envelope
protein typing, plasmid typing, ribotyping, restriction fragment
length
polymorphisms and arbitrarily primed PCR.
Treatment
Treatment of Acinetobacter infections should be individualized
according to
results of susceptibility testing. For effective treatment of
Acinetobacter infections,
combination therapy is usually required. Antibiotic-susceptible
Acinetobacter
isolates have usually been treated with β-lactams,
broad-spectrum cephalosporins,
β-lactam:β-lactamase inhibitor combinations or carbapenems.
These agents are
used alone or in combination with an aminoglycoside (A Evans et
al., 2013).
Antibiotic choices may be limited in cases of infections caused
by multidrug-
resistant isolates. Carbapenems are often considered first-line
agents in the
treatment of resistant A. baumannii. However, carbapenem
resistant Acinetobacter
is increasingly reported (Jain and Danziger, 2004). Resistance
to the carbapenem
class of antibiotics complicates the treatment of
multidrug-resistant Acinetobacter
infections. Many multidrug-resistant isolates remain susceptible
to sulbactam. It
retain activity against A. baumannii in the setting of
carbapenem resistance and
has been shown to be efficacious in treating
ventilator-associated pneumonia
-
- 21 -
(Wood et al., 2002).
The emergence of multidrug-resistant Acinetobacter strains has
brought the
old antibiotic polymyxins back into clinical use. These
antibiotics disrupt bacterial
cytoplasmic membranes, causing leakage of cytoplasmic contents.
Clinicians
discontinued using this antibiotic in the 1970s due to several
side effects in the
kidneys and neurons. Intravenous colistin has greater activity
when combined
with rifampin (Motaouakkil et al., 2006). Inhaled colistin is
occasionally
employed for ventilator-associated pneumonia although treatment
is sometimes
limited by bronchospasm. Acinetobacter isolates resistant to
colistin and
polymyxin have also been reported (Giamarellos-Bourboulis et
al., 2001).
The new glycycline antibiotic tigecycline has an in vitro
activity against some
strains of multidrug-resistant A. baumannii. However, in vivo
resistance has been
reported to occur within a matter of weeks if not already
present prior to initiation
of therapy.
Toll-like Receptor
General introduction
Innate immunity is considered to act as a sentinel for the
immune system and is
promptly activated after recognition of the diverse repertoire
of microbial
pathogens. Innate immune cells express various PRRs that
recognize signature
-
- 22 -
molecules of pathogens. These signature molecules, which are
known as
pathogen-associated molecular patterns (PAMPs), are considered
to be an
indispensable component for the survival of the pathogen (Akira
et al., 2006;
Beutler, 2009). Up to now, several classes of PRRs such as TLRs,
Nucleotide-
binding oligomerization domain (NOD)-like receptor (NLRs) and
Retinoic acid-
inducible gene (RIG)-I-like receptors (RLRs) have been
identified. These PRRs
recognize various PAMPs in diverse cell compartments and trigger
the release of
inflammatory cytokines and type I interferons for host defense
(Akira et al., 2006;
Beutler, 2009). In addition to the elimination of pathogens, the
innate immune
responses are also important to develop pathogen-specific
adaptive immunity,
which is mediated by B and T cells.
Structure and localization
TLRs are type I integral membrane glycoproteins and consist of a
triple domain
structure. The extracellular N-terminal domain is composed of
16–28 leucine-rich
repeats and is in charge of the interaction with PAMPs from
pathogens. The
intracellular C-terminal domain is known as the Toll/IL-1
receptor (TIR) domain,
which shows homology with that of the IL-1 receptor (Akira et
al., 2006; Beutler,
2009). TIR domain is required for the interaction and
recruitment of various
adaptor molecules including myeloid differentiation primary
response protein 88
-
- 23 -
(MyD88) and TIR-domain-containing adapter-inducing interferon-β
(TRIF) to
activate the downstream signaling pathway. After association
with their respective
agonist/antagonist ligands, these complexes form heterodimers
such as TLR1–
TLR2, TLR4–MD2 or a homodimer such as TLR3–TLR3 and form a
characteristic structure. This structure is essential for ligand
binding and initiation
of downstream signaling pathway (Liu et al., 2008; Park et al.,
2009). TLRs are
expressed in the distinct cellular compartments. TLR1, TLR2,
TLR4, TLR5,
TLR6 and TLR11 are expressed on the cell surface whereas TLR3,
TLR7, TLR8
and TLR9 are expressed in intracellular vesicles such as the
endosome and
endoplamic reticulum.
TLR 1, TLR2 and TLR6
TLR2 recognizes a variety of microbial components like
lipoproteins/lipopeptides from various pathogens, peptidoglycan
and lipoteichoic
acid from Gram-positive bacteria, lipoarabinomannan from
mycobacteria, and
zymosan from fungi (Akira et al., 2006; Takeda and Akira, 2005).
It also
identifies LPS preparations from some Gram-negative bacteria
such as
Porphyromonas gingivalis, Leptospira interrogans and
Helicobacter pylori
(Hirschfeld et al., 2001; Werts et al., 2001). These LPS are
structurally different
from the typical LPS of Gram-negative bacteria recognized by
TLR4 especially in
-
- 24 -
the number of acyl chains in the lipid A component (Smith Jr et
al., 2003). The
fact that TLR2 recognizes components from a variety of microbial
pathogens has
been demonstrated by several studies. The mechanism can be
explained by the
fact that TLR2 functionally associate with other TLRs such as
TLR1 and TLR6 to
discriminate between the specific patterns of pathogens.
TLR 3
Double-stranded RNA (dsRNA) is produced by most viruses during
their
replication and induces the synthesis of type I interferons. The
involvement of
TLR3 in the dsRNA has been observed in TLR3-deficient mice which
show an
impairment in their response to dsRNA (Alexopoulou et al.,
2001). Thus, TLR3 is
implicated in the recognition of dsRNA, thereby detecting viral
infection.
TLR 4
Lipopolysaccharide is a major component of the outer membrane of
Gram-
negative bacteria and shows potent immuno-stimulatory activity.
TLR4 is an
essential receptor for LPS recognition (Hoshino et al., 1999).
In addition, the
response to LPS requires several additional molecules such as
LPS-binding
protein (LBP) and CD14, which was demonstrated by inflammatory
cells and
knockout mice (da Silva Correia et al., 2001; Nagai et al.,
2002). In addition to
-
- 25 -
LPS, TLR4 is implicated in the recognition of several ligands
such as taxol (Byrd-
Leifer et al., 2001) and endogenous ligands including
fibronectins, heparan sulfate
and fibrinogen (Triantafilou and Triantafilou, 2004; Zheng et
al., 2009).
TLR 5
TLR5 has been shown to recognize an evolutionarily conserved
domain of
flagellin through close physical interaction between TLR5 and
flagellin (Smith et
al., 2003). TLR5 is expressed on the basolateral, but not the
apical side of
intestinal epithelial cells (Gewirtz et al., 2001). TLR5
expression is also observed
in the intestinal endothelial cells of the subepithelial
compartment (Maaser et al.,
2004). In addition, flagellin activates lung epithelial cells to
induce inflammatory
cytokine production (Hawn et al., 2003). These findings indicate
the important
role of TLR5 in microbial recognition at the mucosal
surface.
TLR 7 and TLR 8
TLR7 and TLR8 are structurally highly conserved proteins, and
recognize the
same ligand in some cases. Mouse TLR7, human TLR7 and human
TLR8, but not
murine TLR8, recognizes imidazoquinoline compounds which are
clinically used
for treatment of genital warts caused by the infection of human
papillomavirus
(Jurk et al., 2002). TLR7 and human TLR8 recognize guanosine or
uridine-rich
-
- 26 -
single-stranded RNA (ssRNA) from viruses such as human
immunodeficiency
virus, vesicular stomatitis virus and influenza virus (Heil et
al., 2004; Lund et al.,
2004). ssRNA is also produced within the host, but usually the
host-derived
ssRNA is not detected by TLR7 or TLR8. This may be due to the
fact that TLR7
and TLR8 are expressed in the endosome, and host-derived ssRNA
is not
delivered to this site.
TLR 9
TLR9 is essential for the recognition of the CpG motif of
bacterial and viral
DNA and TLR9 knockout mice do not show any response to CpG DNA
(Hemmi
et al., 2000). There are at least two types of CpG DNA which are
recognized by
TLR9, CpG-A and CpG-B (Hemmi et al., 2003). The first to be
identified is CpG
-B DNA. It is conventional and a potent inducer of inflammatory
cytokines such
as IL-12 and TNF-α. The second type, CpG-A DNA, is structurally
different from
conventional CpG DNA in that it has a greater ability to induce
IFN-a production
from plasmacytoid dendritic cells (Verthelyi et al., 2001).
TLR 10
Human TLR10 has been identified as a member that is closely
related to TLR1
and TLR6. The ligand of TLR10 remains unclear.
-
- 27 -
TLR 11
TLR11 has been shown to be expressed in bladder epithelial cells
in mice,
where they have been shown to mediate resistance to infection by
uropathogenic
bacteria (Zhang et al., 2004). Mice deficient in TLR11 are
highly susceptible to
uropathogenic bacterial infection.
TLR 12
TLR12, which is similar to TLR11, recognizes Toxoplasma gondii
profilin
(TgPRF). It is critical for the innate immune response to T.
gondii and may
promote host resistance by triggering pDC and NK cell function
(Koblansky et al.,
2013).
TLR 13
TLR13 is an endosomal TLR expressed in mice and its role and
ligand remain
unclear. Recently, some groups have identified 23S ribosomal RNA
as a ligand
for TLR13 (Hochrein and Kirschning, 2013; Li and Chen, 2012).
Humans lack
TLR13 and probably rely on other pathogen receptors to detect
pathogenic
bacterial infection.
-
- 28 -
TLR signaling
Recognition of microbial components by TLRs facilitates
dimerization of TLRs.
Dimerization of TLRs triggers the activation of signaling
pathways, which
originate from a cytoplasmic Toll-like receptor (TIR) domain. In
the signaling
pathways downstream of the TIR domain, a TIR domain-containing
adaptor,
MyD88, was first shown to be essential for induction of
inflammatory cytokines
such as IL-12 and TNF-α through all TLRs except for TLR3
(Hayashi et al., 2001;
Schnare et al., 2000). However, activation of specific TLRs
leads to slightly
different patterns of gene expression profiles. For example,
activation of TLR3
and TLR4 signaling pathways results in induction of type I
interferons. Thus,
individual TLR signaling pathways are divergent, and there are
MyD88-
dependent and MyD88-independent pathways (Akira et al.,
2006).
MyD88- dependent signaling
MyD88, harboring a C-terminal TIR domain and an N-terminal death
domain,
associates with the TIR domain of TLRs. Upon stimulation, MyD88
recruits
IRAK-4 to TLRs through interaction of the death domains of both
molecules, and
facilitates IRAK-4-mediated phosphorylation of IRAK-1. Activated
IRAK-1 then
associates with TRAF6, leading to the activation of two distinct
signaling
pathways. One leads to activation of AP-1 transcription factors
through activation
-
- 29 -
of MAP kinases. Another pathway activates the TAK1/TAB complex,
which
enhances activity of the IκB kinase (IKK) complex. Once
activated, IKKβ of the
IKK complex induces phosphorylation and subsequent degradation
of IκB, which
leads to nuclear translocation of the transcription factor NF-κB
(Akira and Takeda,
2004). MyD88-deficient mice do not show production of
inflammatory cytokines
such as TNF-α and IL-12p40 in response to all TLR ligands
(Hayashi et al., 2001).
TRIF-dependent (MyD88-independent) signaling
In TLR4 ligand–stimulated MyD88-deficient macrophages,
activation of NF-
κB was observed with delayed kinetics, leading to identification
of a MyD88-
independent pathway (Kawai et al., 1999). This pathway
originates from TLR3
and TLR4, and induces type I IFNs via activation of IRF3. TRIF
is essential for
TLR3- and TLR4-mediated IRF3 activation, whereas TRIF-related
adaptor
molecule (TRAM) is involved in IRF3 activation via TLR4 alone
(Fitzgerald et al.,
2003). TRIF interacts with receptor-interacting protein 1
(RIP1), which leads to
TRIF-dependent NF-κB activation (Meylan et al., 2004). TRIF also
interacts with
TRAF3, which bridges to TBK1 and IKKi/IKKε (Häcker et al., 2006;
Oganesyan
et al., 2006)
-
- 30 -
References
A Evans, B., Hamouda, A., and GB Amyes, S. (2013). The rise of
carbapenem-
resistant Acinetobacter baumannii. Curr Pharm Des 19:
223-238.
Akira, S., and Takeda, K. (2004). Toll-like receptor signalling.
Nat Rev Immunol
4: 499-511.
Akira, S., Uematsu, S., and Takeuchi, O. (2006). Pathogen
recognition and innate
immunity. Cell 124: 783-801.
Alexopoulou, L., Holt, A.C., Medzhitov, R., and Flavell, R.A.
(2001). Recognition
of double-stranded RNA and activation of NF-κB by Toll-like
receptor 3. Nature
413: 732-738.
Anstey, N.M., Currie, B.J., Hassell, M., Palmer, D., Dwyer, B.,
and Seifert, H.
(2002). Community-acquired bacteremic Acinetobacter pneumonia in
tropical
Australia is caused by diverse strains of Acinetobacter
baumannii, with carriage in
the throat in at-risk groups. J Clin Microbiol 40: 685-686.
Barbe, V., Vallenet, D., Fonknechten, N., Kreimeyer, A., Oztas,
S., Labarre, L.,
Cruveiller, S., Robert, C., Duprat, S., and Wincker, P. (2004).
Unique features
revealed by the genome sequence of Acinetobacter sp. ADP1, a
versatile and
naturally transformation competent bacterium. Nucleic Acids Res
32: 5766-5779.
Beijerinck, M.W. (1911). Pigmenten als oxydatieproducten gevormd
door
bacterien. Versl Koninklijke Akad Wetensch 19: 1092-1103.
-
- 31 -
Beutler, B.A. (2009). TLRs and innate immunity. Blood 113:
1399-1407.
Bhargava, N., Sharma, P., and Capalash, N. (2011). Quorum
sensing in
Acinetobacter: an emerging pathogen. Crit Rev Microbiol 36:
349-360.
Bist, P., Dikshit, N., Koh, T.H., Mortellaro, A., Tan, T.T., and
Sukumaran, B.
(2014). The Nod1, Nod2, and Rip2 Axis Contributes to Host Immune
Defense
against Intracellular Acinetobacter baumannii Infection. Infect
Immun 82: 1112-
1122.
Blot, S., Vandewoude, K., and Colardyn, F. (2003). Nosocomial
bacteremia
involving Acinetobacter baumannii in critically ill patients: a
matched cohort
study. Intensive Care Med 29: 471-475.
Brisou, J., and Prevot, A.R. (1954). Studies on bacterial
taxonomy. X. The
revision of species under Acromobacter group. In Annales de
l'Institut Pasteur, p.
722.
Byrd-Leifer, C.A., Block, E.F., Takeda, K., Akira, S., and Ding,
A. (2001). The
role of MyD88 and TLR4 in the LPS-mimetic activity of Taxol. Eur
J Immunol 31:
2448-2457.
Chen, T.-L., Siu, L.-K., Lee, Y.-T., Chen, C.-P., Huang, L.-Y.,
Wu, R.C.-C., Cho,
W.-L., and Fung, C.-P. (2008). Acinetobacter baylyi as a
pathogen for
opportunistic infection. J Clin Microbiol 46: 2938-2944.
Choi, C., Lee, J., Lee, Y., Park, T., and Lee, J. (2008).
Acinetobacter baumannii
-
- 32 -
invades epithelial cells and outer membrane protein A mediates
interactions with
epithelial cells. BMC microbiol 8: 216.
Coelho, J.M., Turton, J.F., Kaufmann, M.E., Glover, J.,
Woodford, N., Warner, M.,
Palepou, M.-F., Pike, R., Pitt, T.L., and Patel, B.C. (2006).
Occurrence of
carbapenem-resistant Acinetobacter baumannii clones at multiple
hospitals in
London and Southeast England. J Clin Microbiol 44:
3623-3627.
Costa, G.F.d.M., Tognim, M.C.B., Cardoso, C.L., Carrara-Marrone,
F.E., and
Garcia, L.B. (2006). Preliminary evaluation of adherence on
abiotic and cellular
surfaces of Acinetobacter baumannii strains isolated from
catheter tips. Braz J
Infect Dis 10: 346-351.
da Silva Correia, J., Soldau, K., Christen, U., Tobias, P.S.,
and Ulevitch, R.J.
(2001). Lipopolysaccharide is in close proximity to each of the
proteins in its
membrane receptor complex transfer from CD14 to TLR4 and MD-2. J
Biol
Chem 276: 21129-21135.
Dijkshoorn, L., Nemec, A., and Seifert, H. (2007). An increasing
threat in
hospitals: multidrug-resistant Acinetobacter baumannii. Nat Rev
Microbiol 5:
939-951.
Dorsey, C.W., Tomaras, A.P., Connerly, P.L., Tolmasky, M.E.,
Crosa, J.H., and
Actis, L.A. (2004). The siderophore-mediated iron acquisition
systems of
Acinetobacter baumannii ATCC 19606 and Vibrio anguillarum 775
are
structurally and functionally related. Microbiology 150:
3657-3667.
-
- 33 -
Doughari, H.J., Ndakidemi, P.A., Human, I.S., and Benade, S.
(2011). The
Ecology, Biology and Pathogenesis of Acinetobacter spp.: An
Overview. Microbes
Environ 26: 101-112.
Doughari, J., Ndakidemi, P., Human, I., and Benade, S. (2010).
Verocytotoxic
diarrhogenic bacteria and food and water contamination in
developing countries: a
challenge to the scientific and health community. RIF 1:
202-210.
Ecker, J.A., Massire, C., Hall, T.A., Ranken, R., Pennella,
T.-T.D., Ivy, C.A., Blyn,
L.B., Hofstadler, S.A., Endy, T.P., and Scott, P.T. (2006).
Identification of
Acinetobacter species and genotyping of Acinetobacter baumannii
by multilocus
PCR and mass spectrometry. J Clin Microbiol 44: 2921-2932.
Eliopoulos, G.M., Maragakis, L.L., and Perl, T.M. (2008).
Acinetobacter
baumannii: epidemiology, antimicrobial resistance, and treatment
options. Clin
Infect Dis 46: 1254-1263.
Erridge, C., Moncayo-Nieto, O.L., Morgan, R., Young, M., and
Poxton, I.R.
(2007). Acinetobacter baumannii lipopolysaccharides are potent
stimulators of
human monocyte activation via Toll-like receptor 4 signalling. J
Med Microbiol
56: 165-171.
Fierobe, L., Lucet, J.-C., Decré, D., Muller-Serieys, C.,
Deleuze, A., Joly-Guillou,
M.-L., Mantz, J., and Desmonts, J.-M. (2001). An outbreak of
imipenem-resistant
Acinetobacter baumannii in critically ill surgical patients.
Infect Control Hosp
Epidemiol 22: 35-40.
-
- 34 -
Fitzgerald, K.A., Rowe, D.C., Barnes, B.J., Caffrey, D.R.,
Visintin, A., Latz, E.,
Monks, B., Pitha, P.M., and Golenbock, D.T. (2003). LPS-TLR4
signaling to IRF-
3/7 and NF-κB involves the toll adapters TRAM and TRIF. J Exp
Med 198: 1043-
1055.
Fournier, P.E., Richet, H., and Weinstein, R.A. (2006). The
epidemiology and
control of Acinetobacter baumannii in health care facilities.
Clin Infect Dis 42:
692-699.
Gallego, L., and Towner, K.J. (2001). Carriage of class 1
integrons and antibiotic
resistance in clinical isolates of Acinetobacter baumannii from
northern Spain. J
Med Microbiol 50: 71-77.
Gaynes, R., Edwards, J.R., and System, T.N.N.I.S. (2005).
Overview of
nosocomial infections caused by gram-negative bacilli. Clin
Infect Dis: 848-854.
Gerner-Smidt, P. (1992). Ribotyping of the Acinetobacter
calcoaceticus-
Acinetobacter baumannii complex. J Clin Microbiol 30:
2680-2685.
Gewirtz, A.T., Navas, T.A., Lyons, S., Godowski, P.J., and
Madara, J.L. (2001).
Cutting edge: bacterial flagellin activates basolaterally
expressed TLR5 to induce
epithelial proinflammatory gene expression. J Immunol 167:
1882-1885.
Giamarellos-Bourboulis, E.J., Xirouchaki, E., and Giamarellou,
H. (2001).
Interactions of colistin and rifampin on multidrug-resistant
Acinetobacter
baumannii. Diagn Microbiol Infect Dis 40: 117-120.
-
- 35 -
Glew, R.H., Moellering JR, R.C., and Kunz, L.J. (1977).
Infections with
Acinetobacter calcoaceticus (Herellea vaginicola): clinical and
laboratory studies.
Medicine (Baltimore) 56: 79-98.
Goel, V.K., and Kapil, A. (2001). Monoclonal antibodies against
the iron regulated
outer membrane proteins of Acinetobacter baumannii are
bactericidal. BMC
Microbiol 1: 16.
González, R.H., Nusblat, A., and Nudel, B. (2001). Detection and
characterization
of quorum sensing signal molecules in Acinetobacter strains.
Microbiol Res 155:
271-277.
Gordon, N.C., and Wareham, D.W. (2010). Multidrug-resistant
Acinetobacter
baumannii: mechanisms of virulence and resistance. Int J
Antimicrob Agents 35:
219-226.
Griffith, M.E., Ceremuga, J.M., Ellis, M.W., Guymon, C.H.,
Hospenthal, D.R.,
and Murray, C.K. (2006). Acinetobacter skin colonization of US
Army soldiers.
Infect Control Hosp Epidemiol 27: 659-661.
Griffith, M.E., Lazarus, D.R., Mann, P.B., Boger, J.A.,
Hospenthal, D.R., and
Murray, C.K. (2007). Acinetobacter skin carriage among US army
soldiers
deployed in Iraq. Infect Control Hosp Epidemiol 28: 720-722.
Grotiuz, G., Sirok, A., Gadea, P., Varela, G., and Schelotto, F.
(2006). Shiga toxin
2-producing Acinetobacter haemolyticus associated with a case of
bloody diarrhea.
J Clin Microbiol 44: 3838-3841.
-
- 36 -
Guardabassi, L., Dalsgaard, A., and Olsen, J. (1999). Phenotypic
characterization
and antibiotic resistance of Acinetobacter spp. isolated from
aquatic sources. J
Appl Microbiol 87: 659-667.
Häcker, H., Redecke, V., Blagoev, B., Kratchmarova, I., Hsu,
L.-C., Wang, G.G.,
Kamps, M.P., Raz, E., Wagner, H., and Häcker, G. (2006).
Specificity in Toll-like
receptor signalling through distinct effector functions of TRAF3
and TRAF6.
Nature 439: 204-207.
Hawn, T.R., Verbon, A., Lettinga, K.D., Zhao, L.P., Li, S.S.,
Laws, R.J., Skerrett,
S.J., Beutler, B., Schroeder, L., and Nachman, A. (2003). A
common dominant
TLR5 stop codon polymorphism abolishes flagellin signaling and
is associated
with susceptibility to legionnaires' disease. J Exp Med 198:
1563-1572.
Hayashi, F., Smith, K.D., Ozinsky, A., Hawn, T.R., Eugene, C.Y.,
Goodlett, D.R.,
Eng, J.K., Akira, S., Underhill, D.M., and Aderem, A. (2001).
The innate immune
response to bacterial flagellin is mediated by Toll-like
receptor 5. Nature 410:
1099-1103.
Heil, F., Hemmi, H., Hochrein, H., Ampenberger, F., Kirschning,
C., Akira, S.,
Lipford, G., Wagner, H., and Bauer, S. (2004). Species-specific
recognition of
single-stranded RNA via toll-like receptor 7 and 8. Science 303:
1526-1529.
Hemmi, H., Kaisho, T., Takeda, K., and Akira, S. (2003). The
roles of Toll-like
receptor 9, MyD88, and DNA-dependent protein kinase catalytic
subunit in the
effects of two distinct CpG DNAs on dendritic cell subsets. J
Immunol 170: 3059-
3064.
-
- 37 -
Hemmi, H., Takeuchi, O., Kawai, T., Kaisho, T., Sato, S., Sanjo,
H., Matsumoto,
M., Hoshino, K., Wagner, H., and Takeda, K. (2000). A Toll-like
receptor
recognizes bacterial DNA. Nature 408: 740-745.
Hirschfeld, M., Weis, J.J., Toshchakov, V., Salkowski, C.A.,
Cody, M.J., Ward,
D.C., Qureshi, N., Michalek, S.M., and Vogel, S.N. (2001).
Signaling by toll-like
receptor 2 and 4 agonists results in differential gene
expression in murine
macrophages. Infect Immun 69: 1477-1482.
Hochrein, H., and Kirschning, C.J. (2013). Bacteria evade immune
recognition via
TLR13 and binding of their 23S rRNA by MLS antibiotics by the
same
mechanisms. Oncoimmunology 2(3): e23141.
Hoshino, K., Takeuchi, O., Kawai, T., Sanjo, H., Ogawa, T.,
Takeda, Y., Takeda,
K., and Akira, S. (1999). Cutting edge: Toll-like receptor 4
(TLR4)-deficient mice
are hyporesponsive to lipopolysaccharide: evidence for TLR4 as
the Lps gene
product. J Immunol 162: 3749-3752.
Jain, R., and Danziger, L.H. (2004). Multidrug-resistant
Acinetobacter infections:
an emerging challenge to clinicians. Ann Pharmacother 38:
1449-1459.
Joly-Guillou, M.L. (2005). Clinical impact and pathogenicity of
Acinetobacter.
Clin Microbiol Infect 11: 868-873.
Jurk, M., Heil, F., Vollmer, J., Schetter, C., Krieg, A.M.,
Wagner, H., Lipford, G.,
and Bauer, S. (2002). Human TLR7 or TLR8 independently confer
responsiveness
to the antiviral compound R-848. Nat Immunol 3: 499-499.
-
- 38 -
Kawai, T., Adachi, O., Ogawa, T., Takeda, K., and Akira, S.
(1999).
Unresponsiveness of MyD88-deficient mice to endotoxin. Immunity
11: 115-122.
Knapp, S., Wieland, C.W., Florquin, S., Pantophlet, R.,
Dijkshoorn, L.,
Tshimbalanga, N., Akira, S., and van der Poll, T. (2006).
Differential Roles of
CD14 and Toll-like Receptors 4and 2 in Murine Acinetobacter
Pneumonia. Am J
Respir Crit Care Med 173: 122-129.
Koblansky, A.A., Jankovic, D., Oh, H., Hieny, S., Sungnak, W.,
Mathur, R.,
Hayden, M.S., Akira, S., Sher, A., and Ghosh, S. (2013).
Recognition of Profilin
by Toll-like Receptor 12 Is Critical for Host Resistance to
Toxoplasma gondii.
Immunity 38: 119-130.
Kurcik-Trajkovska, B. (2009). Acinetobacter spp.-A Serious Enemy
Threatening
Hospitals Worldwide. Maced J Med Sci 2: 157-162.
La Scola, B., and Raoult, D. (2004). Acinetobacter baumannii in
human body
louse. Emerg Infect Dis 10: 1671.
Lambert, T., Gerbaud, G., Galimand, M., and Courvalin, P.
(1993).
Characterization of Acinetobacter haemolyticus aac (6')-Ig gene
encoding an
aminoglycoside 6'-N-acetyltransferase which modifies amikacin.
Antimicrob
Agents Chemother 37: 2093-2100.
Lee, J.C., Koerten, H., Van den Broek, P., Beekhuizen, H.,
Wolterbeek, R., Van
den Barselaar, M., Van der Reijden, T., Van der Meer, J., Van de
Gevel, J., and
Dijkshoorn, L. (2006). Adherence of Acinetobacter baumannii
strains to human
-
- 39 -
bronchial epithelial cells. Res Microbiol 157: 360-366.
Lesouhaitier, O., Veron, W., Chapalain, A., Madi, A., Blier,
A.-S., Dagorn, A.,
Connil, N., Chevalier, S., Orange, N., and Feuilloley, M.
(2009). Gram-negative
bacterial sensors for eukaryotic signal molecules. Sensors 9:
6967-6990.
Leung, W.-S., Chu, C.-M., Tsang, K.-Y., Lo, F.-H., Lo, K.-F.,
and Ho, P.-L. (2006).
Fulminant community-acquired Acinetobacter baumannii pneumonia
as a distinct
clinical syndrome. Chest 129: 102-109.
Li, X.-D., and Chen, Z.J. (2012). Sequence specific detection of
bacterial 23S
ribosomal RNA by TLR13. Elife 1: e00102.
Lin, Y.-C., Sheng, W.-H., Chang, S.-C., Wang, J.-T., Chen,
Y.-C., Wu, R.-J., Hsia,
K.-C., and Li, S.-Y. (2008). Application of a microsphere-based
array for rapid
identification of Acinetobacter spp. with distinct antimicrobial
susceptibilities. J
Clin Microbiol 46: 612-617.
Liu, L., Botos, I., Wang, Y., Leonard, J.N., Shiloach, J.,
Segal, D.M., and Davies,
D.R. (2008). Structural basis of toll-like receptor 3 signaling
with double-stranded
RNA. Science 320: 379-381.
Lolans, K., Rice, T.W., Munoz-Price, L.S., and Quinn, J.P.
(2006). Multicity
outbreak of carbapenem-resistant Acinetobacter baumannii
isolates producing the
carbapenemase OXA-40. Antimicrob Agents Chemother 50:
2941-2945.
Lund, J.M., Alexopoulou, L., Sato, A., Karow, M., Adams, N.C.,
Gale, N.W.,
-
- 40 -
Iwasaki, A., and Flavell, R.A. (2004). Recognition of
single-stranded RNA viruses
by Toll-like receptor 7. Proc Natl Acad Sci USA 101:
5598-5603.
Maaser, C., Heidemann, J., von Eiff, C., Lugering, A., Spahn,
T.W., Binion, D.G.,
Domschke, W., Lugering, N., and Kucharzik, T. (2004). Human
intestinal
microvascular endothelial cells express Toll-like receptor 5: a
binding partner for
bacterial flagellin. J Immunol 172: 5056-5062.
Marcella Alsan, M.D., and Michael Klompas, M.D. (2010).
Acinetobacter
baumannii: an emerging and important pathogen. JCOM 17.
McDonald, L.C., Banerjee, S.N., and Jarvis, W.R. (1999).
Seasonal variation of
Acinetobacter infections: 1987–1996. Clin Infect Dis 29:
1133-1137.
Metan, G., Alp, E., Aygen, B., and Sumerkan, B. (2007).
Acinetobacter baumannii
meningitis in post-neurosurgical patients: clinical outcome and
impact of
carbapenem resistance. J Antimicrob Chemother 60: 197-199.
Meylan, E., Burns, K., Hofmann, K., Blancheteau, V., Martinon,
F., Kelliher, M.,
and Tschopp, J. (2004). RIP1 is an essential mediator of
Toll-like receptor 3–
induced NF-κB activation. Nat Immunol 5: 503-507.
Mihara, K., Tanabe, T., Yamakawa, Y., Funahashi, T., Nakao, H.,
Narimatsu, S.,
and Yamamoto, S. (2004). Identification and transcriptional
organization of a gene
cluster involved in biosynthesis and transport of acinetobactin,
a siderophore
produced by Acinetobacter baumannii ATCC 19606T. Microbiology
150: 2587-
2597.
-
- 41 -
Motaouakkil, S., Charra, B., Hachimi, A., Nejmi, H., Benslama,
A., Elmdaghri, N.,
Belabbes, H., and Benbachir, M. (2006). Colistin and rifampicin
in the treatment
of nosocomial infections from multiresistant Acinetobacter
baumannii. J Infect 53:
274-278.
Munoz-Price, L.S., and Weinstein, R.A. (2008). Acinetobacter
infection. N Engl J
Med 358: 1271-1281.
Nagai, Y., Akashi, S., Nagafuku, M., Ogata, M., Iwakura, Y.,
Akira, S., Kitamura,
T., Kosugi, A., Kimoto, M., and Miyake, K. (2002). Essential
role of MD-2 in
LPS responsiveness and TLR4 distribution. Nat Immunol 3:
667-672.
O'Shea, M. (2012). Acinetobacter in modern warfare. Int J
Antimicrob Agents 39:
363-375.
Oberoi, A., Aggarwal, A., and Lal, M. (2009). A decade of an
underestimated
nosocomial pathogen-Acinetobacter in a tertiary care hospital in
Punjab. JK Sci 11:
24-26.
Oganesyan, G., Saha, S.K., Guo, B., He, J.Q., Shahangian, A.,
Zarnegar, B., Perry,
A., and Cheng, G. (2006). Critical role of TRAF3 in the
Toll-like receptor-
dependent and-independent antiviral response. Nature 439:
208-211.
Pantophlet, R. (2008). Lipopolysaccharides of Acinetobacter.
Acinetobacter
Molecular Biology.
Park, B.S., Song, D.H., Kim, H.M., Choi, B.S., Lee, H., and Lee,
J.O. (2009). The
-
- 42 -
structural basis of lipopolysaccharide recognition by the
TLR4–MD-2 complex.
Nature 458: 1191-1195.
Peleg, A.Y., Potoski, B.A., Rea, R., Adams, J., Sethi, J.,
Capitano, B., Husain, S.,
Kwak, E.J., Bhat, S.V., and Paterson, D.L. (2007). Acinetobacter
baumannii
bloodstream infection while receiving tigecycline: a cautionary
report. J
Antimicrob Chemother 59: 128-131.
Peleg, A.Y., Seifert, H., and Paterson, D.L. (2008).
Acinetobacter baumannii:
emergence of a successful pathogen. Clin Microbiol Rev 21:
538-582.
Perez, F., Hujer, A.M., Hujer, K.M., Decker, B.K., Rather, P.N.,
and Bonomo, R.A.
(2007). Global challenge of multidrug-resistant Acinetobacter
baumannii.
Antimicrob Agents Chemother 51: 3471-3484.
Poirel, L., Menuteau, O., Agoli, N., Cattoen, C., and Nordmann,
P. (2003).
Outbreak of extended-spectrum β-lactamase VEB-1-producing
isolates of
Acinetobacter baumannii in a French hospital. J Clin Microbiol
41: 3542-3547.
Prashanth, K., and Badrinath, S. (2005). Epidemiological
investigation of
nosocomial Acinetobacter infections using arbitrarily primed PCR
& pulse field
gel electrophoresis. Indian J Med Res 122: 408.
Rathinavelu, S., Zavros, Y., and Merchant, J.L. (2003).
Acinetobacter lwoffii
infection and gastritis. Microbes Infect 5: 651-657.
Richet, H., and Pierre Edouard Fournier, M. (2006). Nosocomial
infections caused
-
- 43 -
by Acinetobacter baumannii: a major threat worldwide. Infect
Control Hosp
Epidemiol 27: 645-646.
Russo, T.A., Luke, N.R., Beanan, J.M., Olson, R., Sauberan,
S.L., MacDonald, U.,
Schultz, L.W., Umland, T.C., and Campagnari, A.A. (2010). The K1
capsular
polysaccharide of Acinetobacter baumannii strain 307-0294 is a
major virulence
factor. Infect Immun 78: 3993-4000.
Salas, C.J., Cabezas, F.T., Alvarez-Ossorio, G.d.S.R., and Díez,
G.F. (2003).
Community-acquired Acinetobacter baumannii pneumonia. Rev Clin
Esp 203:
284.
Schnare, M., Takeda, K., Akira, S., and Medzhitov, R. (2000).
Recognition of
CpG DNA is mediated by signaling pathways dependent on the
adaptor protein
MyD88. Curr Biol 10: 1139-1142.
Scott, P., Deye, G., Srinivasan, A., Murray, C., Moran, K.,
Hulten, E., Fishbain, J.,
Craft, D., Riddell, S., and Lindler, L. (2007). An outbreak of
multidrug-resistant
Acinetobacter baumannii-calcoaceticus complex infection in the
US military
health care system associated with military operations in Iraq.
Clin Infect Dis 44:
1577.
Seifert, H., Dijkshoorn, L., Gerner-Smidt, P., Pelzer, N.,
Tjernberg, I., and
Vaneechoutte, M. (1997). Distribution of Acinetobacter species
on human skin:
comparison of phenotypic and genotypic identification methods. J
Clin Microbiol
35: 2819-2825.
-
- 44 -
Seifert, H., Strate, A., and Pulverer, G. (1995). Nosocomial
bacteremia due to
Acinetobacter baumannii: clinical features, epidemiology, and
predictors of
mortality. Medicine (Baltimore) 74: 340-349.
Siau, H., Yuen, K.-Y., Ho, P.-L., Wong, S.S., and Woo, P.C.
(1999). Acinetobacter
bacteremia in Hong Kong: prospective study and review. Clin
Infect Dis 28: 26-30.
Siroy, A., Cosette, P., Seyer, D., Lemaître-Guillier, C.,
Vallenet, D., Van
Dorsselaer, A., Boyer-Mariotte, S., Jouenne, T., and Dé, E.
(2006). Global
comparison of the membrane subproteomes between a
multidrug-resistant
Acinetobacter baumannii strain and a reference strain. J
Proteome Res 5: 3385-
3398.
Smith, A., and Alpar, K. (1991). Immune response to
Acinetobacter calcoaceticus
infection in man. J Med Microbiol 34: 83-88.
Smith Jr, M.F., Mitchell, A., Li, G., Ding, S., Fitzmaurice,
A.M., Ryan, K., Crowe,
S., and Goldberg, J.B. (2003). Toll-like receptor (TLR) 2 and
TLR5, but not TLR4,
are required for Helicobacter pylori-induced NF-κB activation
and chemokine
expression by epithelial cells. J Biol Chem 278:
32552-32560.
Smith, K.D., Andersen-Nissen, E., Hayashi, F., Strobe, K.,
Bergman, M.A.,
Barrett, S.L.R., Cookson, B.T., and Aderem, A. (2003). Toll-like
receptor 5
recognizes a conserved site on flagellin required for
protofilament formation and
bacterial motility. Nat Immunol 4: 1247-1253.
Smith, M.G., Gianoulis, T.A., Pukatzki, S., Mekalanos, J.J.,
Ornston, L.N.,
-
- 45 -
Gerstein, M., and Snyder, M. (2007). New insights into
Acinetobacter baumannii
pathogenesis revealed by high-density pyrosequencing and
transposon
mutagenesis. Genes Dev 21: 601-614.
Takeda, K., and Akira, S. (2005). Toll-like receptors in innate
immunity. Int
Immunol 17: 1-14.
Tong, M.J. (1972). Septic complications of war wounds. JAMA 219:
1044-1047.
Towner, K. (2006). The genus acinetobacter. Prokaryotes 6:
746-758.
Triantafilou, M., and Triantafilou, K. (2004). Heat-shock
protein 70 and heat-
shock protein 90 associate with Toll-like receptor 4 in response
to bacterial
lipopolysaccharide. Biochem Soc Trans 32: 636-639.
Trottier, V., Segura, P.G., Namias, N., King, D., Pizano, L.R.,
and Schulman, C.I.
(2007). Outcomes of Acinetobacter baumannii infection in
critically ill burned
patients. J Burn Care Res 28: 248-254.
Vallenet, D., Nordmann, P., Barbe, V., Poirel, L., Mangenot, S.,
Bataille, E.,
Dossat, C., Gas, S., Kreimeyer, A., and Lenoble, P. (2008).
Comparative analysis
of Acinetobacters: three genomes for three lifestyles. PloS one
3: e1805.
Vanbroekhoven, K., Ryngaert, A., Wattiau, P., Mot, R., and
Springael, D. (2004).
Acinetobacter diversity in environmental samples assessed by 16S
rRNA gene
PCR–DGGE fingerprinting. FEMS Microbiol Ecol 50: 37-50.
-
- 46 -
Verthelyi, D., Ishii, K.J., Gursel, M., Takeshita, F., and
Klinman, D.M. (2001).
Human peripheral blood cells differentially recognize and
respond to two distinct
CPG motifs. J Immunol 166: 2372-2377.
Vila, J., Ribera, A., Marco, F., Ruiz, J., Mensa, J., Chaves,
J., Hernandez, G., and
De Anta, M.T.J. (2002). Activity of clinafloxacin, compared with
six other
quinolones, against Acinetobacter baumannii clinical isolates. J
Antimicrob
Chemother 49: 471-477.
Visca, P., Seifert, H., and Towner, K.J. (2011). Acinetobacter
infection–an
emerging threat to human health. IUBMB life 63: 1048-1054.
Wadl, M., Heckenbach, K., Noll, I., Ziesing, S., Pfister, W.,
Beer, J., Schubert, S.,
and Eckmanns, T. (2010). Increasing occurrence of
multidrug-resistance in
Acinetobacter baumannii isolates from four German University
Hospitals, 2002–
2006. Infection 38: 47-51.
Wang, J.-T., McDonald, L.C., Chang, S.-C., and Ho, M. (2002).
Community-
acquired Acinetobacter baumannii bacteremia in adult patients in
Taiwan. J Clin
Microbiol 40: 1526-1529.
Weinstein, R.A., Gaynes, R., and Edwards, J.R. (2005). Overview
of nosocomial
infections caused by gram-negative bacilli. Clin Infect Dis 41:
848-854.
Werts, C., Tapping, R.I., Mathison, J.C., Chuang, T.-H.,
Kravchenko, V., Saint
Girons, I., Haake, D.A., Godowski, P.J., Hayashi, F., and
Ozinsky, A. (2001).
Leptospiral lipopolysaccharide activates cells through a
TLR2-dependent
-
- 47 -
mechanism. Nat Immunol 2: 346-352.
Wisplinghoff, H., Bischoff, T., Tallent, S.M., Seifert, H.,
Wenzel, R.P., and
Edmond, M.B. (2004). Nosocomial bloodstream infections in US
hospitals:
analysis of 24,179 cases from a prospective nationwide
surveillance study. Clin
Infect Dis 39: 309-317.
Wood, G.C., Scott, D.H., Martin, A.C., Timothy, C.F., and
Bradley, A.B. (2002).
Comparison of ampicillin-sulbactam and imipenem-cilastatin for
the treatment of
Acinetobacter ventilator-associated pneumonia. Clin Infect Dis
34: 1425-1430.
Zhang, D., Zhang, G., Hayden, M.S., Greenblatt, M.B., Bussey,
C., Flavell, R.A.,
and Ghosh, S. (2004). A toll-like receptor that prevents
infection by uropathogenic
bacteria. Science 303: 1522-1526.
Zheng, L., Riehl, T.E., and Stenson, W.F. (2009). Regulation of
colonic epithelial
repair in mice by Toll-like receptors and hyaluronic acid.
Gastroenterology 137:
2041-2051.
-
- 48 -
CHAPTER I
Essential role of toll-like receptor 4 in Acinetobacter
baumannii-induced immune responses in immune cells
-
- 49 -
Introduction
Microbial molecules are sensed by PRRs on host cells including
macrophage,
dendritic cells, and epithelial cells, leading to the activation
of host innate
immunity (Creagh and O'Neill, 2006; Kawai and Akira, 2009). TLRs
are a group
of PRRs and play a critical role in the innate immune system.
TLRs recognize
various microbial molecules, so-called as PAMPs such as LPS,
lipoprotein,
flagellin, and viral nucleic acids at the cell surface or
endosomal membrane (Akira
et al., 2006). Signal transduction from TLRs is usually
classified into two
pathways depending on the adaptor molecules; MyD88-dependent and
MyD88-
independent (TRIF-dependent) pathway. MyD88 is an adapter
molecule that
triggers inflammatory signals commonly utilized by various TLRs
with the
exception of TLR3. Recruitment of MyD88 leads to the activation
of NF-κB and
MAPKs to regulate the pro-inflammatory cytokines genes. On the
while, TRIF is
recruited to TLR3 and TLR4 and activates an alternative pathway
that triggers the
activation of NF-κB, MAPKs, and IRF3. These signaling cascades
lead to the
production of proinflammatory cytokines, type I interferons,
chemokines, and
antimicrobial peptides to remove the invading pathogens (Kawai
and Akira, 2006;
Kumar et al., 2009).
A. baumannii is an aerobic, non-motile, Gram-negative
coccobacillus that can
survive long period time in the environment such as soil and
water. Over the last
-
- 50 -
several decades, it has emerged as a significant nosocomial
pathogen worldwide,
especially in patient with weakened immune systems (Doughari et
al., 2011;
Towner, 2009). A. baumannii can cause a variety of clinical
infections including
pneumonia, bloodstream infection, skin and soft tissue
infection, urinary tract
infection, and meningitis (Peleg et al., 2008). The treatment of
these infections has
become increasingly difficult due to the emergence of resistant
strains to all
known antibiotics (Dijkshoorn et al., 2007; Fournier et al.,
2006). Despite the
growing clinical importance of this organism, the immune
mechanisms that
regulate infection are not understood well.
Recognition of bacterial LPS by TLR4 on immune cells such as
macrophages
is thought to be the key factor determining the outcome of
infection with Gram-
negative bacteria. To understand the role of TLR4 on innate
immunity of immune
cells against A. baumannii, we examined the production of
proinflammatory
cytokines and nitric oxide, the activation of NF-κB and MAPKs,
and ability of
bacterial killing in macrophages or dendritic cells from WT and
TLR4-deficient
mice. We demonstrate here that TLR4 is a crucial factor for
optimal induction of
immune responses in immune cells against A. baumannii.
-
- 51 -
Materials and Methods
Mice
TLR2- and TLR4-deficient mice on C57BL/6 background were
purchased from
the Jackson Laboratories (Bar Harbor, ME, USA). WT C57BL/6 mice
were from
Koatech (Pyeongtaek, Korea). Animal studies were approved and
followed by the
regulations of the Institutional Animal Care and Use Committee
in Konyang
University.
Reagents and bacterial culture
Ultrapure LPS from E. coli O111:B4 and poly I:C were purchased
from
InvivoGen (San Diego, CA, USA). A. baumannii strain KCCM 35453
(ATCC
15150) were purchased from Korean Culture Center of
Microorganisms (Seoul,
Korea). For bacterial preparation, single colonies were
inoculated into 5 ml of
Luria Bertani (LB) medium and grown overnight at 37℃ in the
shaking incubator.
A 1:5 dilution of the culture was allowed to grow additional 2
hours at 37℃ with
shaking to A600 = 0.6, which corresponds to ~109 CFU/ml. After
twice wash with
phosphate buffered saline (PBS; pH 7.4), bacteria were diluted
to the desired
concentration with PBS or media and used in subsequent
experiments.
-
- 52 -
Preparation and stimulation of murine macrophages and dendritic
cells
BMDMs and BMDCs were prepared as previously described (Celada et
al.,
1984; Lutz et al., 1999), and finally cultured in 48-well plates
at a concentration
of 2×105 cells/well or in 6-well plates at a concentration of
2×106 cells/well and
incubated in a 5% CO2 incubator at 37℃. The day after plating,
cells were left
untreated, treated with reagents or infected with A. baumannii
at different MOI.
After 1 h, extracellular bacterial growth was inhibited by
gentamicin treatment
and culture supernatant was collected indicated times after
infection for further
analysis.
Measurement of cytokines and NO
The concentration of IL-6 and TNF-α in culture supernatants were
determined
by a commercial ELISA kit (R&D System, Minneapolis, MN,
USA). NO synthase
activity in the supernatant of cultured cells was assayed for
nitrite accumulation
by the Griess reaction (Green et al., 1982).
RNA extraction and reverse transcription-polymerase chain
reaction (RT-
PCR)
BMDMs were infected with A. baumannii at MOI 1/10 and
extracellular
bacteria were removed by the addition of gentamicin 60 min after
infection. Total
-
- 53 -
RNA was extracted from each cell using easy-BLUE (Intron
biotechnology,
Daejeon, Korea) according to the manufacturer’s instruction. One
microgram of
total RNA was reverse transcribed into cDNA, and PCR was
performed using the
Power cDNA Synthesis Kit (Intron biotechnology) and One-step
RT-PCR with
AccuPower® HotStart PCR PreMix (Bioneer, Daejeon, KOREA). The
following
primer sets were used.
mouse iNOS, F:5’-GAGATTGGAGTTCGAGACTTCTGTG-3’
R:5’-TGGCTAGTGCTTCAGACTTC-3’
mouse GAPDH, F:5’-GTGGAGATTGTTGCCATCAACG-3’
R:5’-CAGTGGATGCAGGGATGATGTTCTG-3’
The PCR conditions consisted of 1 cycle of 94℃ for 5 min; 35
cycles of 94℃
for 30 sec, 56-60℃ for 30 sec, and 72℃ for 30 sec; and 1 cycle
of 72℃ for 10
min. PCR products were then electrophoresed on a 1.5 % agarose
gel and
visualized using a gel documentation system.
Immunoblotting
The cells were lysed in buffer containing 1% Nonidet-P40
supplemented with
complete protease inhibitor 'cocktail' (Roche, Mannheim,
Germany) and 2 mM
dithiothreitol. Lysates were separated by 10% SDS-PAGE,
transferred to
nitrocellulose membranes by electro-blotting. Membranes were
immunoblotted
-
- 54 -
with primary antibodies such as regular- or phospho-IκB-α, p38,
ERK, JNK, and
caspase-3 (Cell signaling Technology, Beverly, MA, USA).
Monoclonal anti-β-
actin antibody was from Sigma-Aldrich (St. Louis, MO, USA).
After
immunoblotting with secondary antibodies, proteins were detected
with enhanced
chemiluminescence (ECL) reagent (Intron Biotechnology, Seongnam,
Korea).
Phagocytic activity and bacterial killing ability of
macrophages
To determine the ability of phagocytosis and bacterial killing
of macrophag