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Contents lists available at ScienceDirect
Microbiological Research
journal homepage: www.elsevier.com/locate/micres
Isogenic mutations in the Moraxella catarrhalis CydDC system
displaypleiotropic phenotypes and reveal the role of a palindrome
sequence in itstranscriptional regulation
Yosra I. Nagy, Manal M.M. Hussein, Yasser M. Ragab, Ahmed S.
Attia⁎
Department of Microbiology and Immunology, Faculty of Pharmacy,
Cairo University, Cairo, 11562, Egypt
A R T I C L E I N F O
Keywords:Moraxella catarrhalisCydDCCysteineRegulationRepeats
A B S T R A C T
Moraxella catarrhalis is becoming an important human respiratory
tract pathogen affecting significant propor-tions from the
population. However, still little is known about its physiology and
molecular regulation. To thisend, the CydDC, which is a
heterodimeric ATP binding cassette transporter that has been shown
to contribute tothe maintenance of the redox homeostasis across the
periplasm in other Gram-negative bacteria, is studied here.Amino
acids multiple sequence alignments indicated that M. catarrhalis
CydC is different from the CydC proteinsof the bacterial species in
which this system has been previously studied. These findings
prompted furtherinterest in studying this system in M. catarrhalis.
Isogenic mutant in the CydDC system showed suppression ingrowth
rate, hypersensitivity to oxidative and reductive stress and
increased accumulation of intracellular cy-steine levels. In
addition, the growth of cydC− mutant exhibited hypersensitivity to
exogenous cysteine; how-ever, it did not display a significant
difference from its wild-type counterpart in the murine pulmonary
clearancemodel. Moreover, a palindrome was detected 94 bp upstream
of the cydD ORF suggesting it might act as apotential regulatory
element. Real-time reverse transcription-PCR analysis showed that
deletion/change in thepalindrome resulted into alterations in the
transcription levels of cydC. A better understanding of such
systemand its regulation helps in developing better ways to combat
M. catarrhalis infections.
1. Introduction
Moraxella catarrhalis is a Gram-negative, human restricted
pathogenpreviously considered as a commensal bacterium of the upper
re-spiratory tract (de Vries et al., 2009; Murphy and
Parameswaran,2009). Nowadays, M. catarrhalis is known to be an
important mucosalpathogen causing many cases of acute otitis media
(OM) in children.After Haemophilus influenzae and Streptococcus
pneumoniae, M. catar-rhalis is considered to be the third common
cause of childhood OM(Wald, 1998; Karalus and Campagnari, 2000;
Murphy et al., 2005;Sillanpaa et al., 2016). In adults, M.
catarrhalis is recognized as thesecond most common bacterial cause
of exacerbations of chronic ob-structive pulmonary disease (COPD)
after H. influenzae (Brooks et al.,2008; Barker et al., 2015). It
is also of concern as a cause of infections inimmunocompromised
patients (Meyer et al., 1995; Funaki et al., 2016).
ATP binding cassette (ABC) transporters constitute one of the
largestfamilies of proteins, that have been widely spread in all
genera of thethree kingdoms of life (Bouige et al., 2002). ABC
transporters arefunctionally diverse and now recognized to
contribute to a wide rangeof essential cellular functions (Dassa
and Bouige, 2001). One of the
most important functions of ABC transporters is playing a key
role inthe maintenance of electrical and chemical concentration
gradientsacross the cell membrane (Rees et al., 2009). In addition,
recent in-terests in the ABC transporters of the emerging pathogen
M. catarrhalishas revealed the crucial role of some ABC
transporters in the virulenceand the survival in the respiratory
tract (Murphy et al., 2016).
All bacteria need to cope with the continuous changes in
environ-mental conditions to achieve an optimum growth rate and
yield.Bacteria respond to stressful conditions mainly by alteration
in geneexpression (Aertsen and Michiels, 2004). Activation or
repression oftenfunctions when transcription factors (TFs) bind to
specific DNA se-quence on the promoter helix. These DNA sequences
are known astranscription factor binding sites (TFBSs). The size of
a single TFBSusually varies between 12 and 30 nt. TFs often
recognize and bind toDNA as homodimers or homomultimeric protein
complexes. Invertedrepeats (palindromes) and direct repeats (tandem
repeats) are the mostcommon structures of TFBSs (Wolberger, 1999;
Rodionov, 2007).
CydDC is a heterodimeric ABC transporter mainly recognized by
itsrequirement for the assembly of a functional cytochrome bd
terminaloxidase in Escherichia coli. In previous studies, the
periplasm of a
http://dx.doi.org/10.1016/j.micres.2017.06.002Received 3 June
2017; Accepted 4 June 2017
⁎ Corresponding author at: Department of Microbiology and
Immunology, Faculty of Pharmacy, Cairo University, Kasr El-Ainy
Street, Room #D404, Cairo, 11562, Egypt.E-mail address:
[email protected] (A.S. Attia).
Microbiological Research 202 (2017) 71–79
Available online 09 June 20170944-5013/ © 2017 Elsevier GmbH.
All rights reserved.
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mutant with a defect in the CydDC system was found to be more
oxi-dizing than that of the wild-type strain suggesting that CydDC
extrudesa reducing molecule to the periplasm. CydDC is now
recognized as akey participant in the maintenance of the redox
homeostasis across theperiplasm through exporting thiol-containing
redox-active moleculespermitting appropriate disulphide bond
formation (Holyoake et al.,2016).
Interestingly, the M. catarrhalis CydDC was not one of the
ABCtransporters that have been covered by the elegant work
publishedrecently by Murphy and co-workers (Murphy et al., 2016).
Therefore, tothe best of our knowledge the current work would be
the first study toinvestigate the CydDC of M. catarrhalis in great
details. With the in-creasing threat of infections with strains
that show resistance to mul-tiple of the currently used antibiotics
(Hoban et al., 2001; Shaikh et al.,2015; Sillanpaa et al., 2016),
it is crucial to understand more about suchsystem and how it is
regulated in this background. Such investigationcould enable the
design of better therapeutic and/or preventive stra-tegies against
this pathogen.
2. Materials and methods
2.1. Bioinformatics analyses
Protein sequences of the CydC protein of M. catarrhalis
togetherwith its homologs in other bacterial species which were
studied beforewere retrieved from the NCBI database. The% identity
and% similaritywere calculated using the EMBOSS 6.3.1: matcher
Waterman-Eggertlocal alignment of two sequences
(http://www.ebi.ac.uk/Tools/psa/emboss_matcher/index.html) (Rice et
al., 2000). The Clustal Omegasoftware
(http://www.ebi.ac.uk/Tools/msa/clustalo/) (Sievers et al.,2011; Li
et al., 2015) was used to align the protein sequences and
theproduced multiple sequence alignment was used as an input file
tocompute the protein distance matrix using the Distmat software
usingthe kimura protein method
(http://www.bioinformatics.nl/cgi-bin/emboss/distmat).
The Palindrome software program
(http://emboss.bioinformatics.nl/cgi-bin/emboss/palindrome) was
used to detect the position of theinverted repeats in the 400
nucleotides upstream of cydDC. The pro-moter sequence was predicted
(in the 400 nucleotides upstream thestarting codon) using BPROM
(http://www.softberry.com/berry.phtml?topic=bprom&group=programs&subgroup=gfindb)
to findout the position of the repeats relative to the predicted
promoter. Mapswere generated using the Bio-Edit version 7.0.9.0
program software(Hall, 1999).
2.2. Bacterial strains and culture conditions
The strains used in this study are listed in supplementary Table
S1online. M. catarrhalis strains were grown on brain heart infusion
(BHI)or BHI agar (BD Diagnostics, USA) and incubated in a candle
jar(∼ 2.3–3.5% CO2) (Lewis et al., 1974) at 37 °C. When needed,
thesemedia were supplemented with spectinomycin (15 μg ml−1),
kana-mycin (15 μg ml−1), or streptomycin (250 μg ml−1).
2.3. Recombinant DNA techniques
Standard molecular biology and recombinant DNA techniques
wereperformed (Sambrook and Russell, 2001). Restriction
endonucleasesand T4 DNA ligase were obtained from Promega, USA. PCR
was per-formed with ExTaq DNA polymerase (Takara, Japan).The
oligonu-cleotide primers used in this study (listed in
supplementary Table S2online) were designed using NCBI Primer-Blast
tools (Ye et al., 2012).Genomic DNA was extracted using the Wizard
Genomic DNA purifica-tion kit (Promega). PCR products were purified
using the QIAquick PCRpurification kit (Qiagen, Germany). DNA was
purified from agarose gelsusing the QIAquick Gel Extraction kit
(Qiagen).
2.4. Isolation of a streptomycin-resistant O35E mutant
Streptomycin-resistant mutant of O35E was obtained as
previouslydescribed (Attia et al., 2005). The mutated rpsL gene
together with theflanking region (∼3 kb amplicon) were amplified by
PCR using theprimer pair (Rpsl-5′-Rpsl-3′) and were used for
congression experiments(Nester et al., 1963). Briefly, the desired
insert together with the mu-tated rpsL gene (mixed in a ratio 10:1)
were mixed with one or twocolonies of freshly cultured recipient
strain on a BHI plate in an areaapproximately 1 cm in diameter.
Followed by incubation at 37 °C for6 h, the bacterial growth was
then suspended in BHI broth and platedon BHI plates supplemented
with streptomycin.
2.5. Construction of M. catarrhalis ΔcydC deletion mutant
Most of the cydC ORF of M. catarrhalis O35E was replaced
withpromoterless kanamycin resistance cartridge (Menard et al.,
1993).Briefly, using M. catarrhalis O35E chromosomal DNA as a
template, theprimer pair (YN071-YN072) was used to amplify the
region (854 bp)immediately upstream of codon 310 in the cydC ORF
and the primerpair (YN004-YN006) was used to amplify the 1067 bp
immediatelydownstream the ORF. The non-polar kanamycin resistance
cartridge(Menard et al., 1993) was amplified using the primer pair
(AA111-A-A116). Overlap extension PCR (Urban et al., 1997) was
performed withprimer pair (YN015-YN004), using the downstream
fragment togetherwith the kanamycin resistance cassette as a
template. The amplicon(YN071-YN072) and the amplicon of the primer
pair(YN015-YN004)were then digested using BamHI and ligated
together. The ligationproduct was used as a template for PCR
reaction using primer pair(YN071-YN004). The gel-purified ligation
product was then used fortransformation in M. catarrhalis O35E as
previously described in(Pearson et al., 2006). Isolated colonies
which could grow on15 μg ml−1 kanamycin were selected and the
construction of the ΔcydCmutant was confirmed by a series of PCR
reactions using primers withinand outside the mutant construct.
2.6. Repair of the ΔcydC mutation
Several attempts were done to clone the cydDC operon in
thecloning vector pWW102 B (Wang et al., 2006), however they
wereunsuccessful. This could potentially be due to the relatively
large size ofthe fragment and its inclusion of DNA fragment that
encodes a mem-brane bound component. Therefore, to complement the
ΔcydC mutantwe opted to use the chromosomal repair approach (Attia
et al., 2005) asan alternative. Briefly, the gel-purified PCR
product of the amplicon(YN071-YN004) (2718 bp) together with the
mutated rpsL ampliconwere used for transformation in ΔcydC in a
congression experiment(Nair et al., 1993). Isolated colonies, that
could grow on 250 μg ml−1streptomycin and failed to grow on 15 μg
ml−1 kanamycin, were se-lected as potential repaired mutant
(ΔcydC/R).
2.7. Construction of M. catarrhalis Δrpt:spec deletion
mutant
The detected palindrome was replaced by spectinomycin
resistancecartridge using the same approach described above. Primer
pairs(YN055-YN056) and (YN039-YN059) were used to amplify the
flankingregions of the palindrome yielding PCR products with sizes
of 839 and800 bp, respectively. The non-polar spectinomycin
resistant cartridgewas amplified from pSL60-1 (Lukomski et al.,
2000) using the primerpair (YN057-YN058). The overlap extension PCR
was performed usingprimers (YN041-YN058) in which the amplicon
(YN055-YN056) to-gether with that of spectinomycin resistance
cassette were used as atemplate. The amplicon (YN041-YN058) and
amplicon (YN039-YN059)were digested using XmaI and ligated
together. The ligated product wasused as a template for PCR
reaction using primer pair (YN041-YN059);the gel-purified amplicon
was used for transformation in M. catarrhalis
Y.I. Nagy et al. Microbiological Research 202 (2017) 71–79
72
http://www.bioinformatics.nl/cgi-bin/emboss/distmathttp://www.bioinformatics.nl/cgi-bin/emboss/distmathttp://emboss.bioinformatics.nl/cgi-bin/emboss/palindromehttp://emboss.bioinformatics.nl/cgi-bin/emboss/palindromehttp://www.softberry.com/berry.phtml?topic=bprom%26group=programs%26subgroup=gfindbhttp://www.softberry.com/berry.phtml?topic=bprom%26group=programs%26subgroup=gfindb
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O35E. Isolated colonies which could grow on 15 μg ml−1
spectino-mycin BHI agar plates were selected and the construction
of theΔrpt:spec mutant was confirmed by a series of PCR reactions
usingprimers within and outside the mutant construct.
2.8. Construction of M. catarrhalis Δrpt, ΔSrpt and Rn rpt
The overlap extension PCR mixture for each mutagenesis
constructwas performed using primer pair (YN041-YN059) containing
the 2 se-parate amplicons of the flanking regions mixed in equal
proportions.For Δrpt, the flanking regions of the inverted repeat
were amplifiedusing (YN038-YN081) (upstream region; 778 bp) and
(YN080-YN059)(downstream region; 800 bp). For ΔSrpt, the flanking
regions of theinverted repeat were amplified using (YN038-YN084)
(upstream re-gion; 789 bp) and (YN083-YN059) (downstream region;
811 bp). ForRnrpt, the flanking regions of the inverted repeat were
amplified using(YN038-YN090) (upstream region; 806 bp) and
(YN089-YN059)(downstream region; 817 bp) using chromosomal DNA of
M. catarrhalisΔSrpt as template. The purified PCR product of
amplicon (YN041-YN059) and the amplified mutated rpsL region were
transformed intoΔrpt:spec in a series of congression experiments.
Isolated colonies thatcould grow on 250 μg ml−1 streptomycin and
fail to grow on specti-nomycin were confirmed as Δrpt, ΔSrpt or
Rnrpt by a series of PCRreactions using primers within and outside
the mutant construct.Deletions and/or changes in the inverted
repeats were further con-firmed by DNA sequencing.
2.9. Stress susceptibility testing
Susceptibitlity testing to the stress imposed by either H2O2 or
di-thiotheritol (DTT) using the disk diffusion assay previously
described(Hoopman et al., 2011). Breifly, an aliquot of 20 μl of
bacterial sus-pension (OD600 = 1.0) was added to 20 ml of molten
1.5% BHI agar at45 °C and mixed gently for 30 s. An aliquot of 5 ml
of this mixture wasthen added to a plate containing 10 ml
solidified BHI agar and allowedto cool till solidification. A
sterile disk (5 mm diameter) loaded with10 μl of filter sterilized
88 mM H2O2 or specified concentration(100 mM and 250 mM) of DTT was
applied to the top of the solidifiedagar. Plates were then
incubated at 37 °C for 24 h. The final size of thezone of growth
inhibition around each disk represents the mean of fouraxial
measurements for each disk.
To asses the stress susceptibity imposed by cysteine on the
growth ofM. catarahlis a growth curve assay was conducted.
Bacterial cells fromfresh plates were collected and resuspended in
BHI broth. The OD600 ofeach suspension was adjusted to 1.0, then
diluted 1:50 using BHI broth.When required; cysteine was introduced
into the culture media in afinal concentration of 5 mM. The
cultures were incubated in a shakingincubator at 37 °C and 180 rpm.
At specified time points, the OD600 of100 μl aliquots from each
culture was measured using Synergy2 mi-croplate reader (Biotek
Instruments, USA). Growth curves were con-structed by plotting
absorbance at 600 nm vs. time.
Finally, to asses the role of the CydC in controlling the levels
ofcysteine to accomdate with the stress imposed by this moiety
themethod described by Yamada and co-workers (Yamada et al.,
2006)was adopted with minor modification. Frist, subcellular
fractions wereprepared according to the method described by Ize and
co-workers (Izeet al., 2014). Then a standard curve (3.75 μM–0.48
mM cysteine HCl)was prepared and used to quantify the cysteine
levels in the previouslyprepared cytoplasmic fractions. An aliquot
of 100 μl of the cytoplasmicfractions was treated with 100 μl
ninhydrin reagent. Samples wereheated in a boiling water bath for
10 min, then rapidly cooled on ice.Then, they were diluted to 400
μl using absolute ethanol and left for30 min at room temperature.
Two hundred μl of the reaction productswere transferred to a 96
well plate and measured at 561 nm.
2.10. RNA isolation and cDNA preparation for real-time
reversetranscription-PCR (RT-PCR) analysis
Mid-logarithmic cells (OD600 ∼ 0.7) were used for RNA
isolationusing the RNeasy mini kit (Qiagen). For cDNA preparation,
0.25 μg ofthe extracted RNA was treated with the QuantiTect reverse
transcrip-tion kit (Qiagen) according to manufacturer’s
instructions.Oligonucleotide primer pairs were designed for use in
real-time RT-PCR(supplementary Table S2 online) by IDT primer Quest
(http://www.idtdna.com/Primerquest/Home/Index). Real time PCR was
per-formed in a Rotor-Gene Q (Qiagen) using the Kapa SYBR Fast qPCR
kit(Kapa Biosystems, USA). Equal aliquots of cDNA (2.5 μl), were
used astemplates in the amplification reactions. The 16S rRNA was
chosen as anormalizer of the cDNA loading in each PCR. Normalized
transcriptslevel of the wild-type sample was used as the
calibrator. The foldchange in the levels of the transcripts was
determined using the ΔΔCtmethod (Livak and Schmittgen, 2001).
2.11. Murine pulmonary clearance model
The animal infections were carried out as described by Smidt
andcoworkers (Smidt et al., 2013). Briefly, three groups (n = 5) of
six toeight weeks old female BALB/C mice were infected intranasally
by in-jecting 40 μl of bacterial suspension (∼5 × 106 CFU) using a
micro-pipette, under anesthesia. Six hours post-inoculation, mice
were eu-thanized by an overdose of the anesthesia, followed by
cervicaldislocation. The lungs were excised, homogenized, serially
diluted, andplated. Plates were incubated for 48 h then subjected
to colony counts.All procedures involving the use of animals were
approved by the Re-search Ethics Committee in the Faculty of
Pharmacy, Cairo University.
3. Results
3.1. The M. catarrhalis CydC is different on the amino acid
level from allthe previously studied homologs
Performing a genome mining approach in the annotated
genomesequence of the M. catarrhalis strain O35E looking for ABC
transporters,the CydDC system was identified. Upon looking how
related is the M.catarrhalis CydC to its homologs in the bacterial
species that have beenpreviously studied, namely; Bacillus
subtilis, Staphylococcus aureus,Brucella abortus, Mycobacterium
tuberculosis, Escherichia coli, Shigellaflexneri, and Salmonella
enterica, the protein distance matrix indicatedthat it is distantly
related from them (Fig. 1a). This finding is reflectedin the
protein alignment presented in Fig. S1 online and the% identityand
similarity were calculated and presented in Table S3 online.
Thesefindings, together with the fact that the CydDC system was not
amongthe ABC transporters studied before in M. catarrhalis tract
(Murphyet al., 2016), prompted more interest in studying this
system in thisbackground.
3.2. The cydD ORF is preceded with a palindromic repeat
Analysis of the DNA sequence upstream of the cydD ORF
indicatedthe presence of an inverted repeat consisting of 11
nucleotides andseparated by 6 nucleotides
(aaacaccgccaGCGATTtggcggtgttt) (Fig. 1b).The repeats started 94
nucleotides upstream of the translational startpoint. Upon
analysing the region for potential promoter elements, itwas found
that the inverted repeat is located upstream of the predicted−10
and −35 regions. This finding indicated that this repeat might
beinvolved in the regulation of the expression of CydDC system in
M.catarrhalis.
3.3. Construction of a ΔcydC mutant and other related
strains
PCR ligations followed by homologous recombination were used
to
Y.I. Nagy et al. Microbiological Research 202 (2017) 71–79
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Fig. 1. M. catarrhalis CydDC is different from previously
studied homologs and is preceded with a potential genetic
regulatory element. [a] A protein distance matrix of the M.
catarrhalisCydC and those of other previously studied bacterial
species. The distance matrix was generated from the multiple
sequence alignment of the CydC protein sequences using the
kimuraprotein method. It is showing the evolution distances,
between M. catarrhalis and different bacteria. The distances are
expressed in terms of the number of substitutions per 100
aminoacids with ignoring the gaps and only exact matches and
ambiguity codes contribute to the match score. The distance matrix
was generated using Distmat software. [b] Schematicdiagram showing
the position and the sequence of the detected inverted repeats
relative to the predicted promoter region to the ORF ofcydD inM.
catarrhalis. Bio-Edit version 7.0.9.0 (IbisBiosciences, United
States) was used for map generation.
Fig. 2. The construction of a series of CydDC-related strains.
Schematic diagrams showing the binding sites of primers used in the
generation and the confirmation of the M. catarrhalisCydDC-related
constructs [a] ΔcydC, [b] repair of ΔcydC construct (ΔcydC/R), [c]
Δrpt:spec, and [d] Δrpt, ΔSrpt and Rnrpt constructs. Bio-Edit
version 7.0.9.0 (Ibis Biosciences, UnitedStates) was used for maps
generation.
Y.I. Nagy et al. Microbiological Research 202 (2017) 71–79
74
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construct the ΔcydC mutant replacing the second half of the cydC
ORFwith a non-polar kanamycin resistance cassette (Fig. 2a). This
mutantwas complemented by transforming it with a full copy of the
wild-typeORF. This repair (ΔcydC/R) was confirmed using PCR by
showing aproduct with the size of 1585 bp using primer pair (YN018,
A primerthat binds within the transformed construct-YN012, A primer
that bindsdownstream the primer used to get the transformed
construct) (Fig. 2b).To study the role of the inverted repeat
upstream of the cydD ORF inregulating the system, a series of
strains was constructed. First, therepeat region was replaced by a
non-polar spectinomycin resistancecassette (Fig. 2c). This strain
was used then as a target for transfor-mation reactions with PCR
constructs that has no repeat (Δrpt), onlyone repeat (ΔSrpt), and
two repeats with one of them being randomized(Rnrpt). The
construction of these strains was confirmed using a seriesof PCR
reactions as described in the methods section and the bindingsites
of the primers used are indicated in Fig. 2d. Final confirmation
wasobtained by DNA sequencing.
3.4. Knocking-out the CydDC system affects growth rate
Upon comparing the growth patterns of O35E to other
constructs(ΔcydC, ΔcydC/R, Δrpt, ΔSrpt and Rnrpt), no significant
differenceswere observed in the constructs (ΔcydC/R, Δrpt, ΔSrpt
and Rnrpt) whencompared to O35E (Fig. 3a). On the other hand, the
growth pattern ofΔcydC was the most affected. It was found that the
absorbance at600 nm of ΔcydC was significantly suppressed with p
values< 0.05 at4, 5, 6, 7, 8, 9, and 10 h post inoculation when
compared to the wild-type strain (Fig. 3b).
3.5. Isogenic mutation in the cydC results in elevation of the
cytoplasmiccontent of cysteine
The intracellular cysteine content of the wild-type strain and
theother constructs was measured. In O35E, the level of
intracellular cy-steine recorded 10.92 μM ± 1.82 (Fig. 4). This
level was elevated by∼3 folds in ΔcydC (31.33 μM ± 6.64). However,
there was no sig-nificant difference in the intracellular cysteine
level in any of ΔcydC/R,Δrpt, ΔSrpt, and Rnrpt when compared to the
wild-type (Fig. 4).
3.6. The cydC− mutant showed increased sensitivity to oxidative
andreductive stress
TheM. catarrhalis ΔcydCmutant exhibited hypersensitivity to
killingby hydrogen peroxide (H2O2) as determined by the disk
diffusion assay(Fig. 5a). When sensitivity to different
concentrations of dithiothreitol(DTT) (100 mM and 250 mM) was
examined, the ΔcydC showed thelargest zone of inhibition at both
concentrations of DTT (Fig. 5b). Incontrast, no significant
differences were observed in H2O2 nor DTTsensitivity among the
wild-type strain and the other tested constructs(Fig. 5b). Upon the
introduction of exogenous cysteine to the culturemedia, the
wild-type strain showed a modest, yet significant, decrease
in absorbance at 600 nm at 3, 4, 5 and 6 h post inoculation
(Fig. 5c). Onthe contrary, the ΔcydC exhibited a remarkable
repression in A600 at alltime points (Fig. 5d). Finally, the
repaired ΔcydC mutant exhibited thewild-type phenotype in the
presence of exogenous cysteine. The mu-tants in which the
nucleotide repeat was altered showed no significantchange in growth
rate in the presence of exogenous cysteine (Fig. S2).
3.7. Deletion/change in the inverted repeat affect the cydC
transcriptionlevels
Quantitative real time RT-PCR analysis showed that there was
asignificant decrease in the levels of transcription of the cydC
betweenO35E and both ΔSrpt and Rnrpt (Fig. 6). The transcription
level of cydCshowed by ΔSrpt was significantly lower than that
showed by Rnrpt.Meanwhile, no significant difference was observed
between O35E andΔrpt.
3.8. Knocking out the CydC does not affect the murine pulmonary
clearanceof M. catarrhalis
Mice were capable of clearing the wild-type, ΔcydC, and
ΔcydC/Rfrom their lungs almost to the same extent after 6 h
following a nasalchallenge. All three strains showed about ∼1.5 log
reduction in thebacterial burden inoculated in the mice (Fig. 7).
This finding indicatesthat the CydC does not play a role in
resisting the bacterial pulmonaryclearance using this model.
Fig. 3. CydC is required for normal growth of M.catarrhalis.
Comparison of the growth patterns of M.catarrhalis O35E and the
CydDC-related constructs.Growth curves were constructed by plotting
absor-bance at 600 nm vs. time. The data presented is themean of
three indepndent experiments and the errorbars represent the
standard error. [b] Comparison ofthe absorbance at 600 nm (A600) of
M. catarrhalisO35E and ΔcydC at different time points. Data
wereanalyzed using paired Student’s t-test. The timepoints at which
A600 is significantly repressed aremarked as follows; the*
indicates p value
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4. Discussion
The CydDC system plays a crucial role in the normal
bacterialphysiology and pathogenicity (Shi et al., 2005; Truong et
al., 2014).Previous studies have revealed that in E. coli, the
genes encoding thecytochrome bd quinol oxidase respiratory complex
confers resistance tonitric oxide, a toxic radical produced by the
immune system (Masonet al., 2009). They are expressed maximally
during aerobic stationaryphase or in low oxygen environment (Cotter
et al., 1990; Poole andCook, 2000). In addition to the structural
genes, cydA and cydB, whichencode for the functional subunits of
cytochrome bd oxidase, two ad-ditional genes cydD and cydC are
required for the assembly of a func-tional cytochrome bd in E.coli
(Shepherd, 2015). Isogenic mutation incydDC has pleiotropic
phenotypes. Besides being hypersensitive to ni-trosative stress
(Holyoake et al., 2016), mutants lacking a functional
CydDC exhibit growth defect by being unable to exit aerobically
fromthe stationary phase at 37 °C, hypersensitivity to high
temperature,increased sensitivity to H2O2, and benzyl penicillin
hypersensitivity(Pittman et al., 2002). In addition, the mutation
in the cydD in otherorganisms, such as Shigella flexneri and
Brucella abortus, resulted in at-tenuation of intracellular
survival and virulence (Way et al., 1999;Endley et al., 2001).
Bioinformatic analyses of the M. catarrhalis system revealed
inter-esting findings that warranted the need to further
investigation of thissystem in more details. The CydC is different
from the previously stu-died ones with relatively modest
percentages of identity and similarity.In addition, the promoter
region of the cydD harbors a potentiallyregulatory element.
In the present work, mutant with defective CydC (ΔcydC) was
foundto be viable but with an impaired rate of growth revealing the
criticalrole of this system in the normal physiology of M.
catarrhalis O35E. The
Fig. 5. M. catarrhalis CydDC confers resistance toexogenous
oxidative and reductive stress. Using thedisk diffusion method, the
final size of the zone ofgrowth inhibition around each disk is the
mean offour axial measurements. [a] Diameters of thegrowth
inhibition zones of the wild-type and CydDC-related constructs
around 88 mM H2O2 discs [b]Diameters of the growth inhibition zones
of the wild-type and CydDC-related constructs around 100 mMDTT
discs. Values shown are the means of three in-dependent
experiments. Error bars represent stan-dard error. Data were
analysed using one wayANOVA and Dunnett’s Multiple comparison test.
Theconstructs at which the zone diameter is sig-nificantly
increased compared to wild-type aremarked with * and the respective
p value is indicatedon the figure. [c] and [d] Comparison of the
absor-bance at 600 nm (A600) in absence and presence ofcysteine of
M. catarrhalis O35E and the ΔcydC, re-spectively. Cysteine was
added in a final con-centration 5 mM. Values are the means of three
in-dependent experiments. Error bars represent thestandard error.
Data were analyzed using pairedStudent’s t-test. The time points at
which A600 issignificantly repressed are marked as follows; the
*indicates p value< 0.05, the ** indicates pvalue< 0.01 and
the *** indicates value< 0.001.
Fig. 6. Deletion/change in inverted repeats affects the
transcription levels of cydC in M.catarrhalis. The transcription
levels of thecydC gene were measured using quantitative RT-PCR.
Fold change was calculated using the ΔΔCt method. The data
presented is the meanof three independent experiments (each one was
done in duplicate), and the error barsrepresent the standard error.
Data were analysed using paired Student’s t-test. The *marks the
constructs at which the fold change is significantly repressed with
p va-lues< 0.05.
Fig. 7. Knocking out the CydC exhibits no effect on the
pulmonary clearance of M. cat-arrhalis. Mice were infected
intranasally with approximately 5 × 106 CFUs of strainO35E,
ΔcydC/R, and ΔcydC. Then, 6 h after infection, lungs were
harvested, homo-genized, serially diluted and plated. The bars span
the difference between the minimumand maximum readings. The
horizontal bar represents the mean of the log10 CFU.Statistical
analysis was performed by applying analysis of variance
(ANOVA).
Y.I. Nagy et al. Microbiological Research 202 (2017) 71–79
76
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normal growth rate, showed by the ΔcydC/R, confirms that the
ob-served decrease in growth, exhibited by ΔcydC, was due to the
loss of afunctional CydC. L-cysteine is an essential component of
many proteins,however, the molecule itself exhibits cellular
toxicity, even at lowconcentrations, by acting as threonine
deaminase inhibitor (an enzymeinvolved in L-isoleucine
biosynthesis) (Harris, 1981). Thus, the in-tracellular level of
L-cysteine is under tight control to maintain the L-cysteine
concentrations below the toxicity threshold (Sorensen andPedersen,
1991). Previous studies have reported that the CydDC
systemparticipates in the export of cysteine from cytoplasm
(Holyoake et al.,2015). In the current study, the different degrees
of sensitivity to exo-genous cysteine showed by different
constructs represent a reflection oftheir inability to remove
cysteine from their cellular compartments.This postulate is
supported by the elevated intracellular levels of cy-steine in the
ΔcydC compared to the wild-type strain. This elevation wasabolished
in the repaired mutant ΔcydC/R (Fig. 3). In addition, theremarkable
increase in sensitivity to killing by exogenous H2O2 andDTT showed
by ΔcydC can be attributed to the decrease in its ability toexport
cysteine to the periplasmic compartment. Indeed; the presenceof
L-cysteine in the bacterial periplasm confers a protection to
thebacterial cell against the toxic effect of H2O2. L-cysteine
exhibits itsdetoxification activity in the periplasm by acting as
H2O2 scavengerbefore penetrating the cytoplasm in which the
sulfhydryl group of L-cysteine reacts with H2O2 to yield H2O and
L-cystine (Ohtsu et al.,2010).
Previous studies have revealed the important role played by
theCydDC system in bacterial virulence. In S. flexneri, the authors
attrib-uted the decrease in virulence in case of cydC mutant to the
lack offunctional cytochrome bd-I (Way et al., 1999). On the other
hand, in B.abortus, mutant with defective cytochrome bd showed
survival up to 8weeks within a mouse model compared to only 3 weeks
in cydCmutant,emphasizing the role played by the CydDC system in
facilitating thesurvival in the host environment rather than being
involved in thesynthesis of bd-type oxidases (Truong et al., 2014).
So, to investigatethe impact of the CydDC system on the resistance
of M. catarrhalis topulmonary clearance, the murine model was
adopted in this study.However, no significant differences were
observed among the testedstrains. This can be attributed to the
relatively high level of protectionagainst oxidative stress
exhibited by M. catarrhalis. Wild-type strains ofM. catarrhalis
retain a well conserved group of factors for dealing withoxidative
stress due to the environment in which it normally
colonizes(nasopharyngeal mucosa and lungs of adult patients with
COPD)(Hoopman et al., 2011). Accordingly, the role of the CydC in
combatingclearance in the mice might have been compensated by other
oxidativestress resistance mechanisms in the in vivo model. Another
explanationcould be that the significant very slow growth rate
exhibited by theΔcydC mutant might have offered it more resistance
to killing as seen inother situations (Lewis, 2001; Claudi et al.,
2014). Interestingly, thestudy conducted by Murphy and co-workers
investigating the role ofABC transporters in the virulence of theM.
catarrhalis, showed that only6 out of 14 mutants tested showed
significantly faster clearance frommurine lungs compared to
wild-type when tested in a similar model(Murphy et al., 2016). The
CydDC system was not among the testedABC transporters in that study
and here we demonstrate that the ΔcydCmutant is behaving in a
similar way to the other eight mutants de-scribed by Murphy and
co-workers.
The transcription of cydDC in E. coli was reported to be
activated bythe fumarate and nitrate reductases regulator (FNR) and
the nitrate/nitrite response regulator (NarL) under anaerobic
growth conditions inthe presence of alternative electron acceptors
such as nitrate/nitrite(Cook et al., 1997). Meanwhile, an
anaerobically induced small reg-ulatory RNA (fnrS) was identified
to play a significant role as cydDCrepressor at the
post-transcription level (Boysen et al., 2010). In B.subtilis, the
genetic arrangement of cydABCD is polycistronic, the ex-pression of
such operon is under control of multiple regulators in-cluding
CcpA, Rex and ResD. Such regulators bind to a specific DNA
sequences in the promoter region affecting the expression of the
cy-dABCD operon (Schau et al., 2004; Puri-Taneja et al., 2007). In
Myco-bacterium smegmatis, a 10-bp inverted repeat was required for
themaximal expression of the cydD and the regulation of its
expression isunder the control of unknown regulator (Aung et al.,
2014).
M. catarrhalis has many examples demonstrating its ability to
reg-ulate gene expression via nucleotides repeats, either
homopolymeric orheteropolymeric (Lafontaine et al., 2001;
Mollenkvist et al., 2003; Attiaand Hansen, 2006; Wang et al., 2007;
Blakeway et al., 2014). In ad-dition, the occurrence of palindromes
at the control regions (promoters,terminators and replication
origins) suggests their regulatory functionby acting as TFBS
(Pearson et al., 1996; Ishihama, 2012). Hence, thelocation of the
detected inverted repeat relative to the predicted pro-moter region
(94 bp upstream of the cydD ORF) suggests that this pa-lindrome
might act as a potential regulatory element affecting
thetranscription levels of cydDC. Comparing the detected palindrome
toother previously detected it looks average. For instance, the M.
smeg-matis cydDC operon is controlled with a 10 bp inverted repeat
which arenot separated by any sequence and it is located at
position −61 bprelative to the transcriptional start site (Aung et
al., 2014). Ad-ditionally, the E. coli acrR–acrAB (ABC transporter)
is regulated via apalindrome that consists of 10 bp and separated
with 4 nucleotidestheAcrR (Su et al., 2007).
The decrease in the transcription levels of the cydC showed by
ΔSrptand Rnrpt together with the no effect on the transcription
levels ex-hibited by Δrpt suggest that, it is not the presence of
the repeats that isessential for regular transcription of the
system, rather it is the dimer-ization. Upon deleting one repeat or
even just mutating its sequence aputative transcriptional factor
would not be able to bind properly to thepromoter and drive
adequate transcription. Identification of this puta-tive
transcriptional factor and determining how it exactly regulates
theCydDC system is of a great interest for future studies.
5. Conclusion
This study is the first to report cydC− phenotypes in M.
catarrhalis.Our findings indicate that the cydC plays a crucial
role in controlling thecysteine level in the periplasmic
compartment conferring internal re-sistance of the bacterium to
exogenous oxidative and reductive stresses.In addition, the
detected palindrome upstream of the cydDC operonparticipates in the
regulation of expression of such system. Taking allthis together
suggests that better understanding of the CydDC system inM.
catarrhalis can help the development of novel therapeutics to
combatinfections caused by this emerging pathogen.
Funding
This research did not receive any specific grant from
fundingagencies in the public, commercial, or not-for-profit
sectors. This workwas mainly self-funded by the authors and in part
through the CairoUniversity funding system for assistant
lecturers.
Competing financial interests
The authors declare no competing financial interests.
Author contribution
All authors have contributed to the conception and design of
thestudy. Y.I.N and A.S.A contributed to the acquisition, analysis,
and in-terpretation of the data. All authors have contributed to
the writing andrevising of the manuscript.
Acknowledgements
The authors would like to thank Dr. Eric J. Hansen of the
University
Y.I. Nagy et al. Microbiological Research 202 (2017) 71–79
77
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of Texas Southwestern Medical Centre for providing the
wild-typestrain O35E. Also, we would like to thank Dr. Ramy K. Aziz
of theFaculty of Pharmacy, Cairo University for his assistance in
the bioin-formatics work.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
theonline version, at
http://dx.doi.org/10.1016/j.micres.2017.06.002.
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Isogenic mutations in the Moraxella catarrhalis CydDC system
display pleiotropic phenotypes and reveal the role of a palindrome
sequence in its transcriptional regulationIntroductionMaterials and
methodsBioinformatics analysesBacterial strains and culture
conditionsRecombinant DNA techniquesIsolation of a
streptomycin-resistant O35E mutantConstruction of M. catarrhalis
ΔcydC deletion mutantRepair of the ΔcydC mutationConstruction of M.
catarrhalis Δrpt:spec deletion mutantConstruction of M. catarrhalis
Δrpt, ΔSrpt and Rn rptStress susceptibility testingRNA isolation
and cDNA preparation for real-time reverse transcription-PCR
(RT-PCR) analysisMurine pulmonary clearance model
ResultsThe M. catarrhalis CydC is different on the amino acid
level from all the previously studied homologsThe cydD ORF is
preceded with a palindromic repeatConstruction of a ΔcydC mutant
and other related strainsKnocking-out the CydDC system affects
growth rateIsogenic mutation in the cydC results in elevation of
the cytoplasmic content of cysteineThe cydC− mutant showed
increased sensitivity to oxidative and reductive
stressDeletion/change in the inverted repeat affect the cydC
transcription levelsKnocking out the CydC does not affect the
murine pulmonary clearance of M. catarrhalis
DiscussionConclusionFundingCompeting financial interestsAuthor
contributionAcknowledgementsSupplementary dataReferences