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Microbiological Research

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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: ahmed-attia@cu.edu.eg (A.S. Attia).

Microbiological Research 202 (2017) 71–79

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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

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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

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

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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.

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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

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).

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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

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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