The gene hxuA encodes a protein which shares high sequence identity with other C. coli proteins annotated as filamentous haemagglutinin. The sequence also contains a region with high similarity to several conserved haemagglutination activity domains. Filamentous haemagglutinin proteins are involved in virulence of several bacterial pathogens, including Bordetella pertussis and Avibacterium (formerly Haemophilus) paragallinarum (Hobb et al., 2002; Inatsuka et al., 2005). The genome of Arcobacter butzleri ATCC 49616 contains the gene hcpA which encodes an iron activated filamentous hemagglutinin (Douidah et al., 2012). BLASTP comparison of this protein with C. coli OR12 HxuA revealed no significant similarity, nor did comparison with the C. concisus HcpA, the HagA protein of A. paragallinarum, or the Bordetella proteins FhaA and FhaB. C. fetus subsp. fetus 82-40 encodes a putative filamentous haemagglutinin of the HcpA family. It may be coincidental that this species is associated with bacteraemic infections, however, it is unknown whether this potential virulence factor is functional in vivo (Miller, 2008). The protein may have a possible role in iron acquisition or metabolism, which is known to be involved in the oxidative stress response (Atack and Kelly, 2009). The function of the putative filamentous haemagglutinin encoded by hxuA in C. coli OR12 remains obscure but the reading frame is notable in that it is phase variable, being intact in 96% of the wild-type sequence reads but appears disrupted in aerotolerant C. coli OR12.

#### Carbonic Anhydrases

Carbonic anhydrase enzymes catalyze the reversible hydration of CO2 to bicarbonate and can be divided into at least six different phylogenetic groups, with varying expressions across kingdoms (Bury-Moné et al., 2008; Al-Haideri et al., 2015). These groups have similar function but differing structures, which are believed to have arisen by convergent evolution (Smith and Ferry, 2000). Strains of C. jejuni are reported to encode a β and a γ carbonic anhydrase, the former class are oligomeric, containing between 2 and 8 monomers and the latter are homotrimers (Smith and Ferry, 2000). However, the putative carbonic anhydrase annotated genes affected in the aerotolerant C. coli OR12 are not similar to those that have been investigated in campylobacters thus far. Whether these represent

a different class of carbonic anhydrases not yet characterized in Campylobacter, or encode a hitherto unknown function requires further investigation.

Eleven other genes contained coding mutations, all of which involved single nucleotide substitutions or changes of up to three amino acids at the C-terminus. None of these involved genes which could be expected to influence aerotolerance, such as those encoding oxidative stress resistance proteins or their regulatory genes. Further investigation of these changes would include protein modeling software to determine whether the changes induce significant structural alterations or cause relocation of the protein.

### CONCLUSION

The C. coli strain OR12, which is a robust colonizer of chickens, was found to be capable of aerobic growth on blood agar. This aerotolerance became further evident after serial passage and was associated with increased peroxide stress resistance but did not influence cellular morphology or motility. Colonization of chickens by aerotolerant C. coli OR12 was lower than the wildtype strain at 3 days after challenge but not by 7 days, suggesting adaptation was occurring. Whole genome sequencing did not reveal any changes in genes known to be related to oxidative stress. Instead, significant mutations were present within genes encoding phosphoglycerate kinase and pyruvate carboxylase, suggesting alterations to cellular metabolism.

#### REFERENCES

#### AUTHOR CONTRIBUTIONS

PO'K and IC designed and executed experiments, analyzed data, prepared and reviewed the manuscript.

#### FUNDING

This work was supported by the Biotechnology and Biological Sciences Research Council [grant number BB/I024585/1], UK. PO'K was supported by Biotechnology and Biological Sciences Research Council Doctoral Training Programme [grant number BB/J014508/1]. The chicken colonization experiment was funded by University of Nottingham Bio Services Unit strategic funding.

#### ACKNOWLEDGMENT

The assistance of Dr. Phillippa Connerton, Nicola Cummings, Geraldine Lafontaine, Antung S. Firleyanti, Lu Liang, and Philip Richards are gratefully acknowledged.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fmicb. 2017.00513/full#supplementary-material

Campylobacter jejuni Bf, an atypical strain able to grow under aerobic conditions. Front. Microbiol. 7:278. doi: 10.3389/fmicb.2016.01002

cycles of free-range and organic chickens. Appl. Environ. Microbiol. 71, 1259–1266. doi: 10.1128/AEM.71.3.1259-1266.2005

of broilers at slaughter. Avian. Dis. 54, 1072–1074. doi: 10.1637/9113-101809- ResNote.1

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2017 O'Kane and Connerton. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Function and Regulation of the C4-Dicarboxylate Transporters in *Campylobacter jejuni*

Marc M. S. M. Wösten1 \*, Chris H. A. van de Lest 2 , Linda van Dijk 1 and Jos P. M. van Putten1

1 Department of Infectious Diseases and Immunology, Utrecht University, Utrecht, Netherlands, 2 Department of Biochemistry and Cell Biology, Utrecht University, Utrecht, Netherlands

C4-dicarboxylates are important molecules for the human pathogen C.jejuni, as they are used as carbon and electron acceptor molecules, as sugars cannot be utilized by this microaerophilic organism. Based on the genome analysis, C. jejuni may possess five different C4–dicarboxylate transporters: DctA, DcuA, DcuB, and two homologs of DcuC. Here, we investigated the regulation and function of various C4–dicarboxylate transporters in C. jejuni. Transcription of the dctA and dcuC homologs is constitutive, while dcuA and dcuB are both directly regulated by the two-component RacR/RacS system in response to limited oxygen availability and the presence of nitrate. The DctA transporter is the only C4-dicarboxylate transporter to allow C. jejuni to grow on C4-carbon sources such as aspartate, fumarate, and succinate at high oxygen levels (10% O2) and is indispensable for the uptake of succinate from the medium under these conditions. Both DcuA and DcuB can sequester aspartate from the medium under low-oxygen conditions (0.3% O2). However, under these conditions, DcuB is the only transporter to secrete succinate to the environment. Under low-oxygen conditions, nitrate prevents the secretion of succinate to the environment and was able to overrule the phenotype of the C4-transporter mutants, indicating that the activity of the aspartate–fumarate–succinate pathway in C. jejuni is strongly reduced by the addition of nitrate in the medium.

#### *Edited by:*

Ian F. Connerton, University of Nottingham, UK

#### *Reviewed by:*

Stuart A. Thompson, Augusta University, USA David Kelly, University of Sheffield, UK

> *\*Correspondence:* Marc M. S. M. Wösten<EMAIL_ADDRESS>
#### *Specialty section:*

This article was submitted to Food Microbiology, a section of the journal Frontiers in Microbiology

*Received:* 27 September 2016 *Accepted:* 24 January 2017 *Published:* 07 February 2017

#### *Citation:*

Wösten MMSM, van de Lest CHA, van Dijk L and van Putten JPM (2017) Function and Regulation of the C4-Dicarboxylate Transporters in Campylobacter jejuni. Front. Microbiol. 8:174. doi: 10.3389/fmicb.2017.00174 Keywords: *Campylobacter jejuni,* C4-dicarboxylates transporters, DctA, Dcu, gene regulation, metabolism, RacRS

### INTRODUCTION

Bacteria utilize C4-dicarboxylates such as fumarate, succinate, malate, and aspartate when sugars or related compounds are not available (Janausch et al., 2002). C4-dicarboxylates serve as carbon and energy source and are oxidized to CO2 in the citric acid cycle under aerobic conditions. Under anaerobic conditions fumarate, malate, and aspartate are taken up into the cell. Malate and aspartate are reduced to fumarate, which is used as electron acceptor in the fumarate respiration pathway where it is converted to succinate. Succinate cannot be further metabolized by most bacteria due to the lack of a functional citric acid cycle under these conditions and is excreted.

The microaerophilic Gram-negative bacterium Campylobacter jejuni is the most common cause of food-borne bacterial gastroenteritis worldwide. Despite the medical and public health importance of Campylobacter infection, it is remarkable that C. jejuni is one of the least understood enteropathogens. C. jejuni possesses a highly branched electron transport chain, which allows both aerobic and anaerobic respiration (Kelly, 2008). Most C. jejuni strains cannot utilize sugars (Parkhill et al., 2000; Pearson et al., 2007; Stahl et al., 2011) and it seems that selected amino acids and C4-dicarboxylates act as primary energy source (Guccione et al., 2008; Zientz et al., 1999). It remains largely unknown how the transport and regulation of C4-dicarboxylates occurs in C. jejuni.

Five C4-dicarboxylate carriers, DctA, DcuAB, DcuC, CitT, and DctPQM, are known to transport C4-dicarboxylates from the periplasm across the inner membrane into bacteria (Janausch et al., 2002). In Escherichia coli DctA is a C4-dicarboxylate/H+ or Na+ cation symporter that catalyses the uptake of C4 dicarboxylates during aerobic growth. During anaerobiosis the transcription of the dctA gene is strongly repressed by the twocomponent ArcBA system. Due to the cAMP-CRP complex glucose can also prevent the transcription of the dctA gene. DcuAB and DcuC have similar functions as they catalyse the exchange, uptake and efflux of C4-dicarboxylates under anaerobic growth conditions (Zientz et al., 1999). DcuB and DcuC are the main transporters for succinate efflux during anaerobic growth (Zientz et al., 1999). While the dcuA gene is expressed constitutively, both DcuB and DcuC are activated by the O2-dependent regulator FNR. Furthermore, DcuB is repressed by nitrate due to the two-component NarXL regulatory system and activated by the two-component DcuSR system in response to presence of fumarate (Overton et al., 2006). CitT is a citrate:succinate antiporter, which is regulated by the two-component CitAB system in response to citrate (Scheu et al., 2012). Finally, the three proteins DctPQM in Rhodobacter capsulatus form a C4-dicarboxylate transporter which is in Pseudomonas aeruginosa dependent on the two-component dctSR system (Forward et al., 1997; Valentini et al., 2011).

C. jejuni contains all the enzymes for a complete oxidative TCA cycle, central to a flexible energy metabolism. Campylobacter possess only the C4-dicarboxylate carriers, DctA and DcuAB, some strains also contain one or two proteins similar to DcuC (Hofreuter et al., 2006). Like in other bacteria under oxygen-limited conditions, the transcription of the C. jejuni dcuA and dcuB genes is upregulated and under these conditions the antiporters are able to transport aspartate and fumarate (Woodall et al., 2005; Guccione et al., 2008). In contrast, all the transcription factors known to regulate the C4-dicarboxylate transporters carriers in other bacteria are lacking in C. jejuni.

Recently, we have shown that the two-component RacR/RacS system of C. jejuni directly represses the operon aspA-dcuAcj0089 under oxygen-limited conditions in the presence of nitrate (van der Stel et al., 2015). In this work, we investigated the function and regulation of all C4-dicarboxylate carriers in C. jejuni.

#### MATERIALS AND METHODS

#### Bacterial Strains, Media, and Growth Conditions

Bacterial strains and plasmids used in this study are listed in **Table 1**. C. jejuni was routinely cultured under microaerophilic conditions (5% O2, 10% CO2, 75% N2)on Blood Agar Base No. 2 (BA) medium containing 5% horse blood or in Heart Infusion broth (HI; Oxoid). Kanamycin (25µg ml−1 ) and/or chloramphenicol (15µg ml−1 ) and/or spectinomycin (30µg ml−1 ) were added when appropriate. E. colistrains were routinely grown at 37◦C in Luria–Bertani (LB) broth or on LB agar plates supplemented with ampicillin (50µg ml−1 ), kanamycin (30µg ml−1 ) or chloramphenicol (34 µg ml−1 ).

### Construction of a *dcuA*, *dcuB*, *dcuC*, *dcuC2* or *dctA* Mutant

To disrupt the dcuA, dcuB, dcuC, dcuC2, or dctA genes, the genes as well as ∼1 kb of the flanking regions were first amplified by PCR using the primer pairs dcuA-F/dcuA-R, dcuB-F/dcuB-R, dcuC-F/dcuC-R, dcuC-2F/dcuC-2R, or dctA-F/dctA-R, respectively. Primers are listed in **Table 2**. The ∼3 kb PCR fragments were ligated into pJET1.2/blunt Cloning Vector resulting into the plasmids pJETdcuA, pJETdcuB, pJETdcuC, pJETdcuC2, and pJETdctA. Inverse PCR was performed on the plasmids pJETdcuA, pJETdcuB, pJETdcuC, and pJETdctA using the primers sets dcuABamHI F/dcuABamHI R, dcuBBamHI F/dcuBBamHI R, dcuCBamHI F/dcuCBamHI R, or dctABamHI F/dctABamHI R, respectively, to delete the dcuA, dcuB, dcuC, and dctA genes present in pJET and to introduce a BamHI restriction site. The pJETdcuA and pJETdctA inverse PCR fragment were ligated to a BamHI fragment containing a chloramphenicol resistance gene of pAV35 resulting in the knock-out constructs pJETdcuA::Cm and pJETdctA::Cm. The pJETdcuB and pJETdcuC inverse PCR fragment were ligated to a BamHI fragment containing the kanamycin resistance gene of pMW2 or spectomycin resistance gene of pNBspec resulting into the knock-out constructs pJETdcuB::Km and pJETdcuC::speC, respectively. Plasmid pNBspec is a pAV35 derivate containing the spectomycin resistance gene of pZW2 (Zhou et al., 2012). pNBspec was constructed by amplifying pAV35 with the primers RBSCATrev and CATstop and the spectomycin resistance gene of pZW2 with the primers RBSspec and Specstop. In a second PCR these two PCR fragments were connected and after self-ligating of the PCR product pNBspec was obtained. To disrupt the dcuC2 gene, plasmid pJETdcuC2 was digested with XmaJI and ligated to an XbaI fragment containing the pAV35 chloramphenicol resistance gene, resulting in the pJETdcuC2::Cm knock-out construct. To mutate the dcuA, dcuB, dcuC, dcuC2, and dctA genes, the knockout constructs pJETdcuA::Cm, pJETdcuB::km, pJETdcuC::speC, pjetDcuC2::Cm, and pJETdctA::Cm were introduced by natural transformation in C. jejuni 81116. Homologous recombinations resulting in double cross-over events were verified by PCR.

#### RNA Isolation

RNA was extracted from C. jejuni wild-type grown in HI with or without 25 mM serine, aspartate, fumarate or succinate under 10 or 0.3% oxygen concentration at logarithmic (10 h) or stationary phase (20 h). RNA was also extracted from the wild-type, the racR mutant and the racR complemented strain grown in HI with 50 mM of NaNO3 under 0.3% oxygen concentration until

#### TABLE 1 | Bacterial strains and plasmids used in this study.

late logarithmic (log) phase (16 h) using the RNA-BeeTM kit (Tel-Test, Inc.). RNA samples were treated with RNAse-free DNAse I (Fermentas) according to the manufacturer's manual.

#### Real-Time RT-PCR

Real-time RT-PCR analysis was performed as previously described (Wösten et al., 2004). Primers used in this assay are listed in **Table 2**. The calculated threshold cycle (Ct) for each gene amplification was normalized to the Ct of the gyrA gene amplified from the corresponding sample before calculating fold change using the arithmetic formula 2−11Ct (Schmittgen, 2001). Each sample was examined in four replicates and was repeated with at least two independent preparations of RNA. Standard deviations were calculated and are displayed as error bars.

#### Electrophoretic Mobility Shift Assay (EMSA)

Recombinant His-tag labeled RacR was isolated and EMSA were performed as described before (van der Stel et al., 2015). The promoter regions upstream of the genes aspA, dcuB, dcuC, dcuC2, and dctA were amplified by PCR using the primer pairs listed in **Table 2** and C. jejuni 81116 genomic DNA as template. Radioactive labeled PCR products, ∼25 pmol, were incubated with 0 or 50 pmol of recombinant RacR and 25 pmol RacScyto (van der Stel et al., 2015) for 20 min at RT in binding buffer containing 20 mM Tris, pH 7.4, 5 mM MgCl2, 50 mM KCl, 2 mM ATP, 50µg/ml bovine serum albumin, 10µg/ml poly-(dIdC), and 10% glycerol. For competition assays RacR was preincubated for 15 min with 10 times excess of unlabelled PCR fragment. Samples were run on 6% non-denaturing Tris-glycine polyacrylamide gels at 4◦C. After electrophoresis, gels were dried and autoradiographed.

#### Growth Experiments

Growth curves were generated under microaerobic conditions (10% O2, 10% CO2, 70% N2, 10% H2) or under oxygenlimited conditions (0.3% O2, 10% CO2, 79% N2, 10% H2) at 42◦C in a "honeycomb" 10 × 10 well micro-plate using a Bioscreen C MRB (Oy Growth Curves Ab Ltd) computercontrolled incubator placed in the anaerobic chamber (Coy labs, Michigan, United States). Campylobacter precultures grown for 6 h in HI were diluted to OD600nm of 0.01 in fresh HI media containing 25 mM fumarate, 25 mM aspartate, 25 mM serine, or 25 mM succinate with or without 25 mM

#### TABLE 2 | Primers used in this study.

nitrate. The optical density at 600 nm was measured every 15 min. The experiments were repeated at least three times in duplicate.

### High-Performance Liquid Chromatography (HPLC)-MS-MS Analysis

During the growth experiments of C. jejuni at 10% O2, culture samples (50 µl) were taken at 4, 8, 16, and 24 h, under oxygenlimited conditions (0.3% O2) samples were taken at 6, 12, 24, and 36 h. The culture samples were centrifuged at 14,000 rpm for 5 min. The supernatants were diluted in milli-Q water and adjusted to pH 2.4 with formic acid and injected on a Synergi 4u Fusion-RP (150 × 2.0 mm, particle size of 4µm) analytical column (Phenomenex, Utrecht, NL). Elution was performed isocratically with milli-Q (adjusted to pH 2.4 with formic-acid): acetonitrile (9:1 [v/v]) at a flow rate of 0.3 ml/min, and the column effluent was introduced by an atmospheric pressure chemical ionization (APCI) interface, in negative mode with an ionization current of −1µA and a source temperature of 350◦C, into a 2000 QTRAP mass spectrometer (Sciex, Toronto, ON). For maximal sensitivity and for linearity of the response, the mass spectrometer was operated in multiple-reaction monitoring (MRM) mode at unit mass resolution. Peaks were identified by comparison of retention time and mass spectrum with authentic standards. Ion transitions monitored were m/z 115.0/71.0 (fumarate), 117.0/73.0 (succinate), 132.1/88.0 (aspartic acid), and 104.0/74.0 (serine) at collision energies of −12, −15, −20, and −18V, respectively. Simultaneously the four molecules were monitored by singleion monitoring (SIM). Data were analyzed with Analyst software version 1.6.1 (Applied Biosystems).

#### Statistical Analysis

Prism software (GraphPad, San Diego, CA) was used for statistical analysis. Data was expressed as mean ± SD. Results were analyzed by two tailed paired t-test, p < 0.05 was considered statistically significant.

### RESULTS

#### Genome Locations of the *C. jejuni* C4-Dicarboxylate Transporters

Genome analysis revealed that C. jejuni possesses up to five putative C4-dicarboxylate transporters, DctA, DcuA, DcuB, and some strains contain one or two homologs of the DcuC C4 dicarboxylate transporter. C. jejuni 81116 possess five C4 dicarboxylate transporters, which are all located at a different locus in the genome (**Figure 1**). The genes dcuB, dcuC, and dctA are not co-transcribed with other genes located in a single-gene operon, while dcuA is located in one operon together with the aspA gene and the dcuC2 gene is located in one operon with C8J\_1306 and the metC genes.

#### Transcription Regulation of the *C. jejuni* C4-Dicarboxylate Transporters

In a number of bacterial species, the C4-dicarboxylate transporters are regulated by oxygen, the available C4 dicarbolytes and/or by growth phase. To investigate whether this also applies for the C4-dicarboxylate transporters in C. jejuni we performed real-time RT PCR. We first used mRNA isolated from the wild-type 81116 strain grown at 10 or 0.3%

oxygen (**Figure 2A**). Only the transcripts of the dcuA and dcuB genes showed a minor increase of three- to four-fold under oxygen limited conditions. Addition of serine or the C4-carbon sources, aspartate, succinate or fumarate to the culture medium of the wild-type grown until the logarithmic or stationary phase (data not shown) at 0.3 or 10% oxygen (data not shown) did not influence the transcription of the C4-dicarboxylate transporters (**Figure 2B**). Finally, we tested whether the growth phase (by comparing logarithmic vs. stationary phase) influences the transcription of the C4-dicarboxylate genes (**Figure 2C**). Only the transcription of the dcuA gene was regulated by growth phase as a 15-fold higher transcript level was observed at the logarithmic phase compared to the stationary phase. These results indicate that the regulation of C4-dicarboxylate transporters in other bacteria cannot be extrapolated to that of C. jejuni.

## *dcuA* and *dcuB* Are Directly Regulated by *racR*

We previously showed that the two-component regulator RacR regulates the dcuA transporter gene. As dcuC and dcuC2 were not present on the used microarrays slides we performed realtime RT PCR using RNA isolated of the wild-type and RacR mutant grown under RacR inducing conditions (0.3% O2 and 25 mM nitrate). Transcription of dcuC, dcuC2, and dctA genes was not affected by mutation of racR, however like the proven RacR dependent genes dcuA and aspA, a decrease of the dcuB transcription was observed in the racR mutant (**Figure 3A**).

To investigate whether RacR directly regulates the dcuB promoter we performed electrophoretic mobility shift assays (EMSA). As previously shown, phosphorylated RacR was able to bind to the 32P-labeled aspA-dcuA promoter region, however RacR also binds to the dcuB promoter (**Figure 3B**). The band shift disappeared when unlabelled DNA corresponding to the dcuB promoter region was added in excess. As expected from the real-time RT PCR results, the dcuC, dcuC2, and dctA promoter regions are not recognized by the RacR protein. These results show that the dcuA as well as dcuB are directly regulated by the two-component system regulator RacR.

### Dcta Is Active at Elevated Oxygen Levels

To phenotypically address the function of the C4-dicarboxylate transporter in C. jejuni we disrupted the dctA, dcuA, dcuB, dcuC, and dcuC2 genes by substituting large parts of the genes with an antibiotic resistance cassette. Although the transcription of C4 dicarboxylate transporter in C. jejuni is not regulated by oxygen (**Figure 2A**), we investigated whether oxygen has an influence of the activity of these transporters. Hereto growth curves in the presence of 10% O2 were generated of the wild-type and the dctA, dcuA, dcuB, dcuC, and dcuC2 mutants in HI or with the addition of 25 mM serine, aspartate, succinate, or fumarate. No clear growth differences between the strains were observed when they were growing in HI or HI with 25 mM serine (**Figures 4A,B**). However, all strains reached a higher OD when serine was added to the medium, suggesting that the available serine or carbon is a limited compound in HI. When one of the C4-carbon sources, aspartate, succinate, or fumarate were added to the HI medium, the maximum final OD of all strains except for the dctA mutant, was also higher compared to HI alone (**Figures 4C–E**), indicating that carbon availability in HI is a limiting growth factor. The dctA mutant reached a similar maximum final OD in HI as in HI with additional aspartate, succinate or fumarate indicating that the dctA mutant is unable to utilize or to take up these carbon sources from the medium. Based on these results we conclude that the DctA transporter is the only C4-dicarboxylate transporter needed to allow C. jejuni to growth on C4-carbon sources at high oxygen levels.

in HI or HI with 25 mM serine, aspartate, fumarate, or succinate for 10 h. (C) Influence of the growth phase on the transcription of the C4-dicarboxylate

(Continued)

#### FIGURE 2 | Continued

genes as estimated for wild-type bacteria by real-time RT-PCR. Total RNA was extracted from logarithmic (10 h), or stationary (20 h) phase cultures. Fold change relative to the transcription levels was calculated using the arithmetic formula (2−11Ct). The gyrA gene was used as normalization gene. Data of four independent experiments with two independent preparations of RNA are presented as mean values ± standard deviation, \*\*P < 0.01.

#### Main Function of *dctA* Is the Uptake of Succinate from the Medium at Elevated Oxygen Levels

To investigate why the dctA mutant is unable to use aspartate, fumarate, and succinate at 10% O2, we measured concentrations of serine, aspartate, succinate, and fumarate in the culture supernatants of the wild-type and C4-dicarboxylate transporter mutants at 4, 8, 16, and 24 h (**Figure 5A**). Without the addition of extra C4-dicarboxylate compounds HI medium contains no detectable fumarate, 0.4 mM succinate, 1.0 mM aspartate, and 1.2 mM serine. All added carbon sources, serine, succinate, aspartate, and fumarate were completely utilized by the wildtype bacteria within 24 h growth at 10% O2, however serine and fumarate were removed earlier from the media than succinate and aspartate. Fumarate was converted to succinate and then secreted to the medium. Once the fumarate was completely used the secreted succinate was taken up again and utilized by C. jejuni (**Figure 5A**) Similar results were obtained for the dcuA, dcuB, dcuC, and dcuC2 mutants (data not shown). A different result was obtained for the dctA mutant. The dctA mutant was still able to take up serine, to convert fumarate to succinate and, although reduced, to take up aspartate (**Figure 5B**), but was unable to take up succinate from the media and accumulated in the supernatant of media containing excess serine, aspartate, or fumarate. These results indicate that the DctA transporter of C. jejuni is involved in the uptake of aspartate and indispensable is for the uptake of succinate from the media at elevated oxygen conditions.

### Redundancy of C4-Dicarboxylate Transporter Function under Oxygen-Limited Conditions

To investigate which of the C4-dicarboxylate transporters are active under oxygen-limited conditions, we generated growth curves of the wild-type, the dctA, dcuA, dcuB, dcuC, and dcuC2 mutants as well as a double dcuA/dcuB and a triple dcuA/dcuB/dcuC mutant in HI or HI with the addition of 25 mM serine, aspartate, succinate, or fumarate (**Figures 6A–E**). The maximum optical densities reached under these oxygenlimited conditions were all lower compared to the growth curves obtained at 10% O2. No obvious growth differences were observed between the strains when they were grown in HI, HI + serine, or HI + succinate. The maximum OD of all strains was higher in HI + serine compared to HI alone, indicating that serine is utilized by C. jejuni under these conditions (**Figure 6B**). In contrast, succinate is not utilized under the oxygen-limited conditions as the growth curves generated in HI + succinate were

mutant strain. Data of four independent experiments with two independent preparations of RNA are presented as mean ds ± standard deviation, \*P < 0.05, \*\*P < 0.01, \*\*\*P < 0.001. (B) Electrophoretic mobility shift assays with the aspA, dcuB, dcuC, dcuC2, and dctA promoter regions labeled with [γ-32P]ATP and phosphorylated RacR protein. Radioactive labeled promoter regions are marked with \*. RacR was phosphorylated by RacScyto in the presence of ATP. The specificity of the protein-DNA interaction was determined by the addition of a 10-fold excess of unlabelled promoter region DNA.

similar as in HI. Growth defects were observed when fumarate or aspartate was added to the HI medium (**Figures 6C,E**). All single mutants grew less compared to the wild-type in HI + aspartate, especially the dcuB mutant. To investigate whether the C4-carboxylate transporter can replace each other function, we also tested double and triple mutants. A more severe growth defect in HI + aspartate compared to the dcuB mutant was observed for the dcuA/dcuB mutant but not for a dctA/dcuA (data not shown). A similar dcuA/dcuB growth defect was seen for the dcuA/dcuB/dcuC triple mutant, suggesting that dcuC is not involved in the utilization of aspartate. A small reduction in growth yield was observed for the single mutants dcuC and dcuB in HI + fumarate. The growth defect was more obvious in the triple mutant dcuA/dcuB/dcuC, suggesting that both DcuB and DcuC are involved in the utilization of fumarate.

#### Secretion of Succinate Is Dependent on a Functional *dcuB* under Limited Oxygen Conditions

To investigate the role of various active C4-transporters under oxygen-limited conditions we measured at time points 6, 12, 24, and 36 h the serine, aspartate, succinate, and fumarate concentration in the culture supernatants of the wild-type and C4-transporters mutants (**Figure 7**). Serine was used within 12 h by the wild-type and within 24 h by the dcuB mutant, the slowest grower in HI + serine (**Figure 7A**). When the strains were grown on aspartate they secreted succinate to the medium, except for the strains which were also mutated in the dcuB gene (**Figure 7B**). No succinate could be measured in the supernatant of the dcuB mutants showing that DcuB under oxygen-limited conditions transports succinate into the environment. Both the dcuA and dcuB mutants were able to utilize aspartate, however when both genes were mutated no decrease in the aspartate concentration in the medium was observed, indicating that both genes are needed to sequester aspartate from the medium. Growing the strains in excess fumarate revealed that not dcuC, but both dcuA and dcuB might be involved in the uptake of fumarate, as at 12 h the supernatant of these single mutants contain more fumarate than the supernatant of the wild-type (**Figure 7C**). The supernatant of all strains tested grown in excess fumarate contained succinate. However, the supernatant of the dcuB mutant strains contained less succinate, indicating that Campylobacter possesses a dcuindependent fumarate–succinate reductase.

### The Availability of Nitrate under Oxygen-Limited Conditions Reduces the Role of the C4-Dicarboxylate Transporters

We showed that RacR directly regulates by inhibition, the dcuA and dcuB genes under limited oxygen and high nitrate conditions. To address the role of the dcu genes under these conditions, we performed growth curves in HI under oxygenlimited conditions with nitrate and aspartate or fumarate (**Figure 8**). The growth rate as well as the maximum optical densities of the wild-type and the mutants were similar under these conditions, suggesting that the C4-dicarboxylate transporters are less active under these conditions.

repeated three times in duplicate.

### Nitrate Prevents the Secretion of Succinate under Oxygen-Limited Conditions

To further address the role of the C4-dicarboxylate transporters under RacR inducing conditions, we measured the aspartate, fumarate, and succinate concentrations in the supernatant of the wild-type and the C4-dicarboxylate mutants at 6, 12, 24, and 36 h. The C4-carbon content in the supernatants of the strains growing under oxygen-limited conditions with nitrate were much more similar compared to the strains grown under oxygen-limited conditions (**Figure 9**). The aspartate uptake in the wild-type, dcuA and dcuB mutants under these conditions were similar, however only half of the aspartate in the supernatant of these cultures was utilized (**Figure 9A**). These results are in accordance with the growth curves and show that the C4-transporters are less active under these conditions. No reduction of the aspartate concentration was seen in the supernatant of the dcuAB mutant, confirming that both DcuA and DcuB are needed to take up aspartate from the medium. The amount of succinate produced by the wild-type under these conditions was nine-fold lower than under oxygen-limited conditions, similar results were seen for

the dctA and dcuC2 mutants. In all other mutants no succinate could be detected in the supernatant. These results show that the activity like the transcription (**Figure 3A**) of aspartate–fumarate– succinate pathway under oxygen-limited condition is strongly reduced when nitrate is available. In the wild-type the utilization of fumarate in the presence of nitrate was similar as without nitrate, however the amount of secreted succinate was two-fold lower when nitrate was present (**Figure 9B**). Similar results were obtained for all other mutants (data not shown), indicating that Campylobacter possesses a dcu nitrate independent fumarate– succinate reductase.

#### DISCUSSION

Dependent on the oxygen concentration, C4-dicarboxylate transporters have been shown to play an important role in the transport of C4-carbon sources such as fumarate, succinate, and aspartate. These carbon sources are even more important for the microaerophilic bacterium C. jejuni as most strains cannot ferment nor oxidize carbohydrates. C. jejuni cannot grow under aerobic or strictly anaerobic oxygen conditions therefore we have attempted to obtain a more complete understanding of the function and regulation of the C4-dicarboxylate transporters in C. jejuni.

Like in other bacteria the C4-dicarboxylate transporter genes of C. jejuni are dispersed over the genome (**Figure 1**). As they all are transcribed from different promoters, they might be regulated differently, as is seen in E. coli, where several transcription factors are involved in the regulation of the C4-dicarboxylate transporters in response to oxygen, C4-dicarboxylate compounds and growth phase (Janausch et al., 2002). Here we showed by real-time RT-qPCR that the oxygen concentration had only a

minor but not significant effect on the transcription of the dcuA and dcuB genes in strain 81116, and that the addition of C4-dicarboxylate compounds in the complex media HI had no influence on the transcription of the C4-dicarboxylate transporter genes (**Figure 2**). Similar results were obtained in another C. jejuni strain 11168 (Woodall et al., 2005). Growth phase of the culture influenced only the transcription of the dcuA gene. Based on these results no transcription factor of C. jejuni has obtained a similar function as the E. coli transcription factors regulating the C4-dicarboxylates, such as FNR, ArcAB, DcuRS,

FIGURE 7 | DcuB secretes succinate into the environment under oxygen-limited conditions. HPLC-MS-MS data of the supernatants of wild-type, dcuA, dcuB, or dcuAB mutant cultures taken after 6, 12, 24, or 36 h of growth at 0.3% O2 with 25 mM serine (A), aspartate (B), or fumarate (C). Data represent the mean and standard error of three independent experiments.

or CRP (Janausch et al., 2002). Apparently, the transcription regulation in response to oxygen, C4-dicarboxylates and growth phase as seen in E. coli is not important for the function of the genes in C. jejuni.

We have shown that both the dcuA and dcuB genes are repressed and directly regulated by the RacRS system in response to low oxygen and nitrate. The dcuA is in most bacteria constitutively expressed and so far C. jejuni is the only organism in which both genes are regulated in a similar manner. The phenotypes observed for the dcuA and dcuB mutants under oxygen-limited conditions (**Figure 6**) were restored by the addition of nitrate to the medium (**Figure 8**), clearly showing that these genes are not needed under these conditions. Regulation of the C4-dicarboxylate genes by nitrate is not uncommon as the dcuB gene in E. coli is repressed by the two-component NarXL system (Overton et al., 2006). The use of nitrate as electron acceptor (Em nitrate/nitrite + 430 mV) is preferred overfumarate (Em fumarate/succinate + 30 mV) enabling fumarate to be used as carbon source instead of electron acceptor under oxygenlimited conditions. This explains the observed reduced secretion of succinate depicted in **Figure 9**.

The secretion of succinate in C. jejuni appeared to be mediated solely by the DcuB C4-transporter (**Figure 7**). In E. coli, the DcuB and DcuC C4-transporters, both regulated by FNR, can act synergistically to secrete succinate under anaerobic conditions (Golby et al., 1999). So far, we were unable to address the function of the two DcuC homologs in C. jejuni 81116 which are not co-regulated with dcuB and are absent in other C. jejuni strains (Parkhill et al., 2000; Hofreuter et al., 2006). Both DcuA and DcuB are involved in the uptake of aspartate as well as fumarate, confirming the data of Guccione et al. (2008). By regulating both DcuA and DcuB, the RacRS system completely controls the

fumarate respiration in response to limited oxygen availability and the presence of nitrate.

represent the mean and standard error of three independent experiments.

Succinate is taken up by the DctA transporter and used as carbon source by C. jejuni when oxygen is not scarce (**Figure 5**). The DctA transporter is also needed to allow C. jejuni to use aspartate and fumarate as carbon source, indicating that the dctA mutant is unable to use or to take up these carbon sources from the medium. In E. coli and Bacillus subtilis, DctA also mediates the uptake of succinate as well as fumarate and aspartate under aerobic conditions (Davies et al., 1999; Asai et al., 2000; Janausch et al., 2002). However, no difference was observed between the C. jejuni wild-type and dctA mutant in the uptake of fumarate, suggesting that C. jejuni DctA is not involved in the uptake of fumarate. When fumarate or aspartate are present in the media, a large amount of energy rich succinate accumulated in the media which could not be re-used by the dctA mutant explaining the reduced growth of dctA mutant under these conditions. Beside the regulation it also appears that the function of C4 dicarboxylate genes differs in C. jejuni.

#### REFERENCES

In our work, we highlighted the regulation and function of various C4-dicarboxylate genes in the microaerophilic bacterium C. jejuni. The DctA transporter is responsible for the uptake of succinate under high oxygen levels. The dcuA and dcuB genes are the only C4-dicarboxylateregulated genes and are dependent on the two-component RacRS system in response to low O2 and high nitrate concentrations. DcuB is the only C4-dicarboxylate/succinate antiporter in C. jejuni which secretes succinate when oxygen levels are low, but is not necessary when nitrate is available.

#### AUTHOR CONTRIBUTIONS

MW designed experiments, wrote the article, and performed growth experiments. JVP wrote the article, CVDL performed the High-Performance Liquid Chromatography analysis, and LVD performed all other experiments.

homologs in diverse gram-negative bacteria. J. Bacteriol. 179, 5482–5493. doi: 10.1128/jb.179.17.5482-5493.1997

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2017 Wösten, van de Lest, van Dijk and van Putten. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# The Campylobacter jejuni Oxidative Stress Regulator RrpB Is Associated with a Genomic Hypervariable Region and Altered Oxidative Stress Resistance

Ozan Gundogdu1 , Daiani T. da Silva1 , Banaz Mohammad1 , Abdi Elmi1 , Brendan W. Wren1 , Arnoud H. M. van Vliet2 and Nick Dorrell1 \*

1 Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, UK, 2 School of Veterinary Medicine, Faculty of Health and Medical Sciences, University of Surrey, Guildford, UK

Edited by: Odile Tresse, Oniris, France

#### Reviewed by:

Mehrdad Mark Tajkarimi, The University of North Carolina at Greensboro, USA Issmat Kassem, American University of Beirut, Lebanon

> \*Correspondence: Nick Dorrell<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Food Microbiology, a section of the journal Frontiers in Microbiology

Received: 26 August 2016 Accepted: 15 December 2016 Published: 26 December 2016

#### Citation:

Gundogdu O, da Silva DT, Mohammad B, Elmi A, Wren BW, van Vliet AHM and Dorrell N (2016) The Campylobacter jejuni Oxidative Stress Regulator RrpB Is Associated with a Genomic Hypervariable Region and Altered Oxidative Stress Resistance. Front. Microbiol. 7:2117. doi: 10.3389/fmicb.2016.02117 Campylobacter jejuni is the leading cause of bacterial foodborne diarrhoeal disease worldwide. Despite the microaerophilic nature of the bacterium, C. jejuni can survive the atmospheric oxygen conditions in the environment. Bacteria that can survive either within a host or in the environment like C. jejuni require variable responses to survive the stresses associated with exposure to different levels of reactive oxygen species. The MarR-type transcriptional regulators RrpA and RrpB have recently been shown to play a role in controlling both the C. jejuni oxidative and aerobic stress responses. Analysis of 3,746 C. jejuni and 486 C. coli genome sequences showed that whilst rrpA is present in over 99% of C. jejuni strains, the presence of rrpB is restricted and appears to correlate with specific MLST clonal complexes (predominantly ST-21 and ST-61). C. coli strains in contrast lack both rrpA and rrpB. In C. jejuni rrpB+ strains, the rrpB gene is located within a variable genomic region containing the IF subtype of the type I Restriction-Modification (hsd) system, whilst this variable genomic region in C. jejuni rrpB− strains contains the IAB subtype hsd system and not the rrpB gene. C. jejuni rrpB− strains exhibit greater resistance to peroxide and aerobic stress than C. jejuni rrpB+ strains. Inactivation of rrpA resulted in increased sensitivity to peroxide stress in rrpB+ strains, but not in rrpB− strains. Mutation of rrpA resulted in reduced killing of Galleria mellonella larvae and enhanced biofilm formation independent of rrpB status. The oxidative and aerobic stress responses of rrpB− and rrpB+ strains suggest adaptation of C. jejuni within different hosts and niches that can be linked to specific MLST clonal complexes.

Keywords: Campylobacter jejuni, oxidative stress response regulation, aerobic stress response regulation, transcription factors, restriction modification system

### INTRODUCTION

Campylobacter jejuni is the leading cause of bacterial foodborne diarrhoeal disease worldwide with an estimated 400 million human infections occurring each year (Ruiz-Palacios, 2007). The predominance of C. jejuni can be attributed to the ability to survive in the environment as well as within avian and mammalian hosts despite the microaerophilic nature of this bacterium

(Byrne et al., 2007). C. jejuni has evolved specific adaptation mechanisms to survive under atmospheric oxygen conditions (Kim et al., 2015). In addition to aerobic stress such as the exposure to increased levels of oxygen under atmospheric conditions, C. jejuni can also encounter stress conditions within the host, specifically oxidative stress in the form of reactive oxygen species (ROS) during in vivo survival (Fang, 2004; Palyada et al., 2009). ROS is a collective term that describes the chemical species generated upon incomplete reduction of oxygen (Imlay, 2003) with examples including the superoxide anion (O2 −), hydrogen peroxide (H2O2) and the hydroxyl radical (•OH) (D'Autreaux and Toledano, 2007). The accumulation of ROS in the bacterial cytoplasm and periplasm leads to damage of nucleic acids, proteins and membrane structures (Atack and Kelly, 2009).

Bacteria that can survive either within a host or in the environment like C. jejuni require variable responses to survive the stresses associated with exposure to different levels of ROS (Kim et al., 2015). Therefore it is not surprising that C. jejuni contains a number of regulatory proteins involved in the oxidative stress response such as PerR (Handley et al., 2015), Fur (van Vliet et al., 2000), and CosR (Hwang et al., 2011). The C. jejuni NCTC 11168 genome also contains two MarR-type transcriptional regulators that have previously been designated as RrpA and RrpB (Gundogdu et al., 2011, 2015). Using C. jejuni 11168H [a hypermotile derivative of the original sequenced strain NCTC 11168 that shows higher levels of cecal colonization in a chick colonization model (Karlyshev et al., 2002; Jones et al., 2004)] we have shown that both RrpA and RrpB play a role in oxidative and aerobic stress responses with auto-regulatory activity typical of MarR-type transcriptional regulators (Gundogdu et al., 2011, 2015). In addition, RrpA has also been shown to bind upstream of katA suggesting that RrpA directly influences the expression of catalase (KatA). Both 11168H rrpA and rrpB mutants exhibited reduced KatA activity. However, a 11168H rrpAB double mutant exhibited higher levels of resistance to hydrogen peroxide oxidative stress, but similar levels of KatA activity compared to the wild-type strain. Neither the 11168H rrpA mutant nor the 11168H rrpB mutant exhibited any significant difference in sensitivity to either cumene hydroperoxide or menadione oxidative stresses, but both mutants exhibited reduced cytotoxicity in the Galleria mellonella model of infection and enhanced biofilm formation. However, the 11168H rrpAB double mutant exhibited wildtype levels of both cytotoxicity in the G. mellonella model of infection and biofilm formation. Together these data indicate a role for both RrpA and RrpB in the C. jejuni oxidative and aerobic stress responses, enhancing bacterial survival both within a host and in the environment, but also prompted further investigations in order to understand the specific roles of RrpA and RrpB.

Traditional typing methods have failed to identify C. jejuni strains from different sources that cause disease in humans (Champion et al., 2005). Human infections are largely attributed to undercooking of poultry products or poor food hygiene practices involving handling of such produce (Sheppard et al., 2009). C. jejuni can survive in many different niches with the organism isolated from avian, animal, human, and environmental sources (Young et al., 2007). However, whole genome phylogenetic analysis of C. jejuni strains using microarrays has identified different clades and subclades linked to the source of the isolate (Champion et al., 2005; Stabler et al., 2013). Previously we identified differences in the distribution of rrpA and rrpB regulators amongst 111 C. jejuni strains with rrpA present in over 95% and rrpB in approximately only 50% of these strains (Gundogdu et al., 2011). A more recent analysis of 270 C. jejuni strains identified nine clusters (C1–C9) based on genotype and multilocus sequence typing (MLST) data and indicated that clusters C1–C6 were dominated by livestock-associated clonal complexes, whilst clusters C7–C9 contained the majority of the water and wildlife associated clonal complexes (Stabler et al., 2013). Using the original data from this study, rrpA was identified in 129/133 (96.99%) strains within the C1–C6 subclades and in 130/137 (94.89%) strains within the C7–C9 subclades. In contrast rrpB was identified in 102/133 (76.67%) strains within the C1–C6 subclades and only in 19/137 (13.87%) strains within the C7–C9 subclades. In total rrpA was identified in 259/270 (95.92%) strains whilst rrpB was identified in only in 121/270 (44.81%) strains (Supplementary Figure 1; Supplementary Table 1).

The discovery of the varied distribution for rrpA and rrpB has led us to further explore the potential reasons as to why certain strains have one or both regulators. To try and understand the reasons for this, we have investigated the presence or absence of rrpA and rrpB in 4,232 C. jejuni and C. coli genome sequences. Analysis of 4,232 Campylobacter genomes showed that whilst rrpA is present in over 99% of C. jejuni strains, the presence of rrpB is restricted and appears to correlate with livestockassociated MLST clonal complexes. Further analysis showed that the presence ofrrpB is linked to a hypervariable region containing the IF subtype of the type I Restriction-Modification (hsd) system, whereas rrpB− strains contain the IAB subtype hsd system. Further investigation of the phenotypes of different C. jejuni rrpB− and rrpB+ strains identified a link between the presence of the MarR-type transcriptional regulator RrpB with the ability of C. jejuni to adapt and survive in different environmental niches. The oxidative and aerobic stress response of rrpB− and rrpB+ strains suggests adaptation of C. jejuni within different hosts and niches that can be linked with specific MLST clonal complexes.

#### MATERIALS AND METHODS

#### Comparative Genomics of rrpA and rrpB within Campylobacter Genomes

A total of 4,232 complete and draft Campylobacter genome sequences (3,746 C. jejuni and 486 C. coli) were obtained from public collections (Jolley and Maiden, 2010; Cody et al., 2013) and are listed in Supplementary Table 2 with accession numbers/pubMLST IDs and assembly status. These genomes were previously used for identification of DNase-genes (Brown et al., 2015), CRISPR repeats and cas genes (Pearson et al., 2015) and the fucose utilization operon (Dwivedi et al., 2016). Genomes

included were between 1.5 and 2.0 Mbp and had at least 5 of the 7 MLST alleles identifiable using BLAST. The genomes were phylogenetically clustered using FFPry feature frequency profiling with a word length of 18 (Van Vliet and Kusters, 2015) and further analyzed by MLST (Pearson et al., 2015). Genomes were provisionally annotated using Prokka (Seemann, 2014) and searched for the presence of the predicted RrpA and RrpB proteins using BLASTP (Supplementary Table 2). Presence of the corresponding genes was also assessed at the DNA level using the MIST program (Kruczkiewicz et al., 2013) and the BLAST+ (v2.28) suite. Both DNA and amino acid comparisons were done using the rrpA genes/RrpA proteins from C. jejuni NCTC 11168, C. jejuni 81116, C. jejuni 414, and C. coli 76639, and the rrpB genes/RrpB proteins of C. jejuni NCTC 11168 and C. coli 2544. Conservation of flanking genes was assessed using the output from the comparative genomics software package Roary (Page et al., 2015) and the provisional Prokka annotation of the 4,232 C. jejuni and C. coli genome sequences (Supplementary Tables 2 and 3).

#### Bacterial Strains and Growth Conditions

Campylobacter jejuni strains (**Table 1**) were grown at 37◦C in a microaerobic chamber (Don Whitley Scientific, United Kingdom) containing 85% N2, 10% CO2 and 5% O2 either on blood agar (BA) plates containing Columbia agar base (Oxoid, United Kingdom) supplemented with 7% (v/v) horse blood (TCS Microbiology, United Kingdom) and Campylobacter Selective Supplement (Oxoid) or in Brucella broth (Oxoid) with shaking at 75 rpm. C. jejuni strains were grown on BA plates for 24 h prior to use in all assays unless otherwise stated. Escherichia coli XL-2 Blue MRF' competent cells (Stratagene, United Kingdom) were used for cloning experiments and were grown at 37◦C in aerobic conditions either on Luria-Bertani (LB) agar plates (Oxoid) or in LB broth (Oxoid) with shaking at 200 rpm. Antibiotics were added at the following concentrations; ampicillin (100 µg/ml), kanamycin (50 µg/ml), and chloramphenicol (50 µg/ml for E. coli studies or 10 µg/ml for C. jejuni studies). All reagents were obtained from Fisher Scientific (United Kingdom) unless otherwise stated.

#### Construction of C. jejuni Mutants

Campylobacter jejuni mutants were constructed as described previously (Gundogdu et al., 2011). Briefly, genes or gene fragments were amplified from C. jejuni genomic DNA using the appropriate gene specific primers (**Table 2**). PCR products were ligated with pGEM-T Easy vector (Promega, United Kingdom) and then transformed into XL-2 Blue MRF' cells. If required, inverse PCR mutagenesis (IPCRM) was performed to introduce a unique BglII site into the cloned gene. A kanamycin cassette (KanR ) was then ligated into the unique BglII site within the cloned gene (Trieu-Cuot et al., 1985; van Vliet et al., 1998). These constructs were electroporated into competent C. jejuni cells and putative clones were confirmed by PCR and sequencing as described previously (Gundogdu et al., 2011).

## Oxidative Stress and Aerobic Growth Assays

Oxidative stress and aerobic growth assays were performed as described previously (Gundogdu et al., 2011, 2015). Briefly, bacterial cells were harvested into 1 ml PBS and diluted to an OD600 of 1. For oxidative stress assays, bacterial cells were exposed to H2O2 at final concentrations of 25, 50, or 100 mM for 15 min, menadione at a final concentration of 100 mM for 60 min and cumene hydroperoxide at 0.05% (w/v) for 15 min, all at 37◦C under microaerobic conditions. Serial dilutions were prepared and 10 µl of the 10−1–10−6 dilutions spotted onto BA plates, incubated for 48 h and colonies counted. For growth curves, 10 ml Brucella broth was pre-incubated in a 30 ml flask at 37◦C under microaerobic conditions for 24 h. Bacterial cells grown on BA plates for 24 h were used to inoculate pre-incubated Brucella broth at an OD600 of 0.1 and grown for up to 24 h at 37◦C under microaerobic and aerobic conditions. OD600 readings were performed at selected time points. In addition bacterial colony forming units (CFUs) were assessed at time point 16 h under microaerobic and aerobic conditions.

### Galleria mellonella Infection Model

Galleria mellonella infection assays were performed as described previously (Champion et al., 2010; Gundogdu et al., 2015). Briefly, G. mellonella larvae (LiveFoods Direct, United Kingdom) were stored at 16◦C on wood chips. Ten larvae for each experiment were infected with a 10 µl inoculum of a 24 h C. jejuni culture diluted to OD600 0.1 by micro-injection (Hamilton, Switzerland) in the right foremost leg, giving an infectious dose of approximately 106 CFU (Champion et al., 2010). Controls were injection with PBS and no injection. Larvae were incubated at 37◦C with survival recorded at 24 h.

### Biofilm Assays

Biofilm assays were performed as described previously (Gundogdu et al., 2015). Briefly, bacterial cells were harvested into Mueller Hinton broth, then inoculated to an OD600 of 0.1 into 10 ml Mueller Hinton broth pre-incubated in a 25 ml flask at 37◦C under microaerobic conditions for 24 h prior to inoculation, then grown for 5 h at 37◦C under microaerobic conditions with shaking at 75 rpm. The OD600 was readjusted to 0.1, then 1 ml of culture was added to a 24 well polystyrene plate (Corning, U.S.A) and incubated at 37◦C under either aerobic or microaerobic conditions stationary for 72 h. The wells were washed twice with PBS, dried for 20 min at 37◦C followed by addition of 1% (w/v) crystal violet (Sigma–Aldrich) for 15 min. The wells were washed three times with PBS, then destained with 10% (v/v) acetic acid / 30% (v/v) methanol. Absorbance (A595) was measured using a SpectraMax M3 microplate reader (Molecular Devices, USA).

#### Statistical Analyses

The data is presented as mean + SD. All experiments were performed with at least three biological replicates. Each biological replicate was performed in three technical replicates. Statistical analyses were performed using Prism software (GraphPad

#### TABLE 1 | Campylobacter jejuni strains used in this study.

fmicb-07-02117 December 23, 2016 Time: 10:54 # 4

(Continued)

#### TABLE 1 | Continued

fmicb-07-02117 December 23, 2016 Time: 10:54 # 5

Software). All statistical analyses were performed comparing two data sets directly assuming a normal distribution using a two-way student's t-test (∗ = p < 0.05, ∗∗ = p < 0.01, ∗∗∗ = p < 0.001).

#### RESULTS

#### Presence of rrpA and rrpB in a Collection of 4,232 C. jejuni and C. coli

To assess the distribution of rrpA and rrpB in C. jejuni and C. coli we utilized a collection of 4,232 Campylobacter genome sequences (3,746 C. jejuni and 486 C. coli) from public databases (Cody et al., 2013; Brown et al., 2015), which were phylogenetically clustered using FFPry feature frequency profiling (Van Vliet and Kusters, 2015; Dwivedi et al., 2016) and further analyzed for MLST sequence type and clonal complex (Pearson et al., 2015). The vast majority of C. jejuni strains contain rrpA whilst the presence of rrpB is more restricted (**Figure 1**; Supplementary Table 2 – Sheet 2). The presence or absence of rrpB appears to correlate with MLST clonal complex, especially ST-21 and ST-61. Most C. coli strains contain neither rrpA nor rrpB, with only 12 C. coli genomes encoding an RrpA ortholog and only a single C. coli genome encoding an RrpB ortholog (Supplementary Table 2). Variation in RrpA and RrpB was observed with isolates from C. jejuni MSLT clonal complex ST-607 having a shorter RrpA protein lacking the N-terminal 27 amino acids, while a proportion of C. jejuni ST-353 isolates have a RrpB protein either lacking the N-terminal 29 amino acids, the C-terminal 29 amino acids or a combination of 29 N-terminal amino acids and 9 C-terminal amino acids (Supplementary Figure 2). There was no apparent link between the truncated versions and source of the isolates (Supplementary Table 2), suggesting these are different forms of RrpA and RrpB circulating within clonal complexes.

Further analysis of the C. jejuni and C. coli genome sequences investigated the distribution of genes flanking rrpA and rrpB

#### TABLE 2 | Oligonucleotide primers used in this study.

fmicb-07-02117 December 23, 2016 Time: 10:54 # 6

FIGURE 1 | Prevalence of the rrpA and rrpB genes among 3,746 Campylobacter jejuni and 486 C. coli genomes. Genomes were phylogenetically clustered using FFPry feature frequency profiling with L = 18 (Van Vliet and Kusters, 2015). The first two rows labeled 'rrpA' and 'rrpB' indicate genomes possessing the respective genes, which are shown in red, while those lacking the pathway are shown in black. The third bar shows the primary combinations of MLST clonal complexes for C. jejuni and C. coli, with red-labeled clonal complexes representing livestock-associated lineages, blue-labeled clonal complexes representing water and wildlife-associated lineages (Stabler et al., 2013), with the exception of the ST-61 clonal complex, which has been described as cattle (livestock)-associated (Kwan et al., 2008; Rotariu et al., 2009). The asterisks at ST-42 and ST-353 indicate that these clonal complexes may have a proportion of livestock-associated isolates. The association of some clonal complexes such as ST-464 was not reported previously and these are in black font. The lowercase letters indicate the approximate position of reference strains 81116 and M1 (a), 81-176 (b), RM1221 (c) and NCTC 11168 (d).

and identified two conserved flanking regions with a more variable central region (**Figure 2**). The upstream flanking region contains rrpA whilst the downstream flanking region contains an arsenic resistance operon, an MCP-gene and a paralyzed flagella gene pflA. The hypervariable central region contains a type I Restriction-Modification (hsd) system. The NCTC 11168 version of this hypervariable central region contains rrpB whilst the 81116 version does not (Supplementary Table 3). The 81116 version of this hypervariable central region is representative of all the other C. jejuni ST strains analyzed in this study. Analysis of the C. coli genome sequences (which have primarily a structure very similar to 81116) has versions mostly without rrpA and rrpB.

### Campylobacter jejuni rrpB− Wild-Type Strains Exhibit Increased Resistance to Oxidative Stress Compared to C. jejuni rrpB+ Wild-Type Strains

Characterization of the role of RrpA and RrpB in the C. jejuni 11168H wild-type strain has shown that both rrpA and rrpB mutants exhibit increased sensitivity to H2O2 when compared to the wild-type strain (Gundogdu et al., 2011, 2015). Based on the observed variation of rrpA and rrpB amongst C. jejuni isolates (Gundogdu et al., 2011), we utilized the Stabler et al. (2013) study to select 25 wild-type strains (**Table 1**) (13 rrpB+ and 12 rrpB− strains) for testing sensitivity toward H2O2 (**Figure 3**). rrpB− strains (**Figure 3B**) displayed significantly greater resistance to H2O2 when compared to rrpB+ strains (**Figure 3A**). The variation in the presence of rrpA and rrpB identified from the microarray data (Champion et al., 2005) for these 25 strains was also confirmed using PCR (data not shown).

### Mutation of rrpA Results in Increased Sensitivity to Peroxide Stress in Strains 11168H and 81-176 (rrpB+), But Not in Strains 81116 and M1 (rrpB−)

To further investigate the link between oxidative stress resistance and the varied distribution of rrpA and rrpB, rrpA and rrpB single mutants were constructed in the 11168H and 81-176 wild-type strains (rrpB+) and rrpA mutants were also constructed in the 81116 and M1 wild-type strains (rrpB−). The mutants obtained did not have any observable deficiency in motility or growth rate under standard conditions (data not shown). Compared to the respective wild-type strains, both the 11168H and 81- 176 rrpA mutants exhibited an increased sensitivity to 25 mM H2O2, however, the 81116 and M1 rrpA mutants exhibited wildtype levels of survival (**Figure 4**). Neither the 11168H and 81- 176 wild-type strains nor the respective rrpA mutants survived exposure to higher concentrations of H2O2 (50 or 100 mM), however, the 81116 and M1 wild-type strains and the respective rrpA mutants all exhibited only slightly increased sensitivity to these higher concentrations of H2O2 (**Figure 4**). 11168H and 81-176 rrpB mutants exhibited the same increased sensitivity to 25 mM H2O2 as the rrpA mutants (**Figure 5**), but did not survive the higher concentrations of H2O2 (data not shown). Cumene hydroperoxide and menadione stress assays were also performed on the 11168H, 81-176, 81116, M1 wild-type strains and the respective rrpA and rrpB mutants (Supplementary Figure 3). There were no significant differences in sensitivity to either cumene hydroperoxide or menadione between any of the wildtype strains and respective mutants, with the exception that the 81116 rrpA mutant appeared to be more resistant to cumene hydroperoxide stress than the 81116 wild-type strain.

### Mutation of rrpA Reduces Growth under Aerobic Stress Conditions in rrpB+ Strains, But Not in rrpB− Strains

In C. jejuni 11168H, rrpA and rrpB mutants exhibit reduced growth under aerobic stress conditions (Gundogdu et al., 2011, 2015). The ability of rrpA and rrpB mutants to grow under aerobic stress conditions was compared to the respective four wild-type strains. No differences in bacterial growth kinetics (Supplementary Figure 4A) or in CFU counts after 16 h (**Figure 6A**) was observed under microaerobic conditions. However, differences were observed under aerobic conditions. All four rrpA mutants displayed significantly reduced growth kinetics during the logarithmic phase under aerobic conditions (Supplementary Figure 4B). However, compared to the respective wild-type strains, only the 11168H and 81-176 rrpA mutants exhibited a reduction in CFU counts after 16 h, whilst the 81116 and M1 rrpA mutants did not (**Figure 6B**).

### Mutation of rrpA Results in Decreased Cytotoxicity in the Galleria mellonella Larvae Model of Infection

Galleria mellonella larvae have been used as a model to study infection with many different enteric pathogens including C. jejuni (Champion et al., 2009). Insect larvae possess specialized phagocytic cells, termed haemocytes (Bergin et al., 2005; Mylonakis et al., 2007). Haemocytes mimic the functions of phagocytic cells in mammals and are able to degrade bacterial pathogens as well as generate bactericidal compounds such as

superoxide via a respiratory burst (Lavine and Strand, 2002; Bergin et al., 2005). Both 11168H rrpA and rrpB mutants have been shown to exhibit reduced cytotoxicity in this model of infection compared to the wild-type strain (Gundogdu

et al., 2011, 2015). The cytotoxicity of these mutants in G. mellonella larvae compared to the respective wild-type strains was investigated. Infection with either the 11168H or 81-176 rrpA mutants resulted in a statistically significant decrease in cytotoxicity to G. mellonella larvae compared to infection with the respective wild-type strains (**Figure 7**). Infection with the 81116 or M1 rrpA mutants also resulted in a decrease in cytotoxicity to G. mellonella larvae compared to infection with the respective wild-type strains, although this decrease was not statistically significant (**Figure 7**).

### Mutation of rrpA Results in Increased Biofilm Formation

Several studies have demonstrated that C. jejuni exhibits increased biofilm formation under increased oxidative stress conditions (Fields and Thompson, 2008; Reuter et al., 2010). Previously C. jejuni 11168H rrpA and rrpB mutants exhibited an enhanced ability to form biofilms under both aerobic and microaerobic conditions (Gundogdu et al., 2015). All four rrpA mutants exhibited an increase in biofilm formation under both aerobic (**Figure 8A**) and microaerobic (**Figure 8B**) conditions after 72 h compared to the respective wild-type strains, which was statistically significant for the 11168H and 81116 rrpA mutants under aerobic conditions and for the 11168H, 81-176, and M1 rrpA mutants under microaerobic conditions. In addition both the 11168H rrpB mutant (Gundogdu et al., 2015) and 81-176 rrpB mutant (data not shown) exhibit increased biofilm formation under aerobic and microaerobic conditions when compared to the respective wild-type strains.

#### DISCUSSION

Campylobacter jejuni will be exposed to ROS both during colonization or infection of a host and in the environment as well as during the course of normal bacterial metabolism. It remains a conundrum as to how this microaerophilic pathogen is so widely dispersed, highly prevalent and able to survive

in the ambient environment. C. jejuni contains a number of different mechanisms for counteracting the effects of oxidative stress and the control of the C. jejuni oxidative stress response is complex involving multiple inter-linked levels of regulation (Atack and Kelly, 2009). The re-annotation of the C. jejuni NCTC 11168 genome sequence (Gundogdu et al., 2007) identified both RrpA and RrpB as putative MarR-type transcriptional regulators which were subsequently shown to be involved in the C. jejuni peroxide and aerobic stress response (Gundogdu et al., 2011, 2015). All the other regulators of the C. jejuni oxidative stress response, such as PerR, Fur, CosR, CsrA, CprRS, and RacRS, are all conserved (>99.8%) amongst all the C. jejuni and C. coli wild-type strains in this study (van Vliet et al., 1999; Atack and Kelly, 2009; Palyada et al., 2009; Hwang et al., 2012). Analysis of the distribution of rrpA and rrpB in 3,746 C. jejuni and 486 C. coli genomes from public databases confirmed that the vast majority of C. jejuni strains contain rrpA, whilst the presence of rrpB is more restricted, with the distribution linked to MLST clonal complex. The majority of C. jejunistrains that contain both rrpA and rrpB are from ST-21 and ST-61. ST-21 strains are often associated with human infections (Sheppard et al., 2009) and ST-61 strains with infections of livestock (Kwan et al., 2008; Rotariu et al., 2009).

Analysis of the distribution of rrpA and rrpB from the microarray data study of 270 C. jejuni strains (Stabler et al., 2013) identified water and wildlife strains predominantly as rrpB−, whereas livestock-associated strains were predominantly rrpB+ (Supplementary Figure 1; Supplementary Table 1). This distribution of rrpA and rrpB was also reflected in the analysis of the whole genome sequence data (**Figure 1**) where redlabeled clonal complexes represent livestock-associated lineages and blue-labeled clonal complexes represent water and wildlifeassociated lineages. It is, however, not always clear whether each clonal complex is associated with a single host as clonal complexes are regularly isolated from multiple animal species and thus attribution to a single host reservoir is difficult using MLST data alone (Dearlove et al., 2016). One example is the ST-48 complex that has been isolated from humans, cattle and sand from beaches (Dingle et al., 2002). Another example is ST-257 and ST-61 that have been isolated from chicken and ruminants, yet ST-257 is designated as a water and wildlifeassociated lineage, whereas ST-61 is denoted as a livestockassociated lineage (Sheppard et al., 2011). These livestock and water and wildlife descriptions may be somewhat generalized and investigating the properties of individual STs will be more important to understand the source. ST-21, ST-206, and ST-48 may form a "complex group" of related genotypes that are widely distributed, perhaps reflecting the "generalist" ability to colonize a wide range of hosts (Dingle et al., 2002; Sheppard et al., 2011).

The location of rrpA and rrpB close to a type I Restriction-Modification (hsd) system suggests an explanation as to why rrpA is present in almost all C. jejuni strains, whilst the presence of rrpB is restricted and appears to correlate with MLST clonal complexes. Restriction-modification (R-M) systems are ubiquitous in the bacterial world and provide a defense against foreign DNA and bacteriophages (Wilson and Murray, 1991). Foreign DNA is cleaved by endonucleases but host DNA avoids damage due to methylation. R-M systems have been classified into four distinct groups, type I, type II, type III, and type IV. A type I R-M locus was identified in the genome sequence of NCTC 11168 (Cj1549–Cj1553) (Parkhill et al., 2000). The type I enzyme is a bi-functional, multi-subunit complex consisting of HsdR, HsdM, and HsdS. Further analysis of the type I R-M system from 73 C. jejuni strains (including NCTC 11168, 81- 176, and 81116) assigned some hsd systems to the classical type IC family but also identified two additional type I R-M families, termed type IAB and type IF (Miller et al., 2005). 81116 contains a type IAB hsd system, whilst both NCTC 11168 and 81-176 contain a type IF hsd system. This study also found evidence for

extensive rearrangements within the C. jejuni hsd loci of the same family but suggested that the low sequence similarity between the IC, IAB, and IF families would make inter-family recombination events unlikely. The NCTC 11168 version of the hypervariable central region identified in this study, which contains the type IF hsd system and rrpB, represents the majority of ST-21, ST-61, ST-42, and ST-353 strains. 81-176 also contains the type IF hsd system and rrpB. However, the 81116 version of this hypervariable central region, which represents the majority of all the other C. jejuni ST strains analyzed in this study, contains a type IAB hsd system and does not contain rrpB. The lack of recombination between C. jejuni strains containing the type IF hsd system and rrpB with other C. jejuni strains containing a different family of type I hsd system could be the explanation for the restricted distribution of rrpB amongst C. jejuni strains. This region of the C. jejuni genome has previously been identified as hypervariable region 14, one of 16 hypervariable regions (Taboada et al., 2004). The presence of intervening ORFs between hsdR and hsdS (referred to as rlo genes for R-linked ORF) and also between hsdS and hsdM (referred to as mlo genes for M-linked ORFs) are a distinct feature of the C. jejuni hsd loci (Miller et al., 2005). H. pylori ModH from the type III R-M system has been shown to have a regulatory role (Srikhanta et al., 2011) suggesting that R-M systems may also have a role in the regulation of virulence gene expression (Vasu and Nagaraja, 2013). Recently the same hypervariable region in C. coli has been shown to contain a novel streptomycin resistance gene (Olkkola et al., 2016). The genomics-based screening also showed that there are different alleles of RrpA and RrpB (Supplementary Figure 2) leading to a N-terminal truncation, a C-terminal truncation or both. The truncated version of RrpB was predominantly observed in isolates of the ST-353 clonal complex, while truncated versions of RrpA were rare in most clonal complexes, but dominant in ST-607 isolates (Supplementary Table 2). Future studies could utilize these truncated versions to test for the role of the N-terminal and C-terminal regions in functionality of RrpA and RrpB.

Campylobacter jejuni rrpB− strains exhibit a pattern of greater resistance to peroxide stress when compared to rrpB+ strains. This pattern was the same when comparing the 11168H, 81-176, 81116, and M1 rrpA mutants against the respective wild-type strain where rrpA mutants in rrpB− strains were more resistant to peroxide stress compared to rrpA mutants in rrpB+ strains. In addition, when comparing bacterial growth of 11168H, 81- 176, 81116, and M1 rrpA mutants against the respective wildtype strain under aerobic conditions, all four rrpA mutants displayed a reduced growth rate during the logarithmic phase. Both the 11168H and 81-176 rrpA mutants exhibited lower CFUs at 16 h compared to the respective wild-type strains. Even though both 81116 and M1 rrpA mutants also displayed slower growth rates compared to the respective wild-type strains when comparing respective CFU counts at 16 h, these differences were not statistically significant. The general pattern that rrpB+ strains exhibit increased sensitivity to peroxide and aerobic stresses is rather counterintuitive. Possibly having only a single Rrp regulator is more efficient in responding to such stresses. Certainly rrpA is the most conserved amongst C. jejuni strains. Even though the oxidative stress assays were performed on a relatively small number of selected strains, it is interesting to speculate why livestock-associated strains such as NCTC 11168 (ST-21) would be more sensitive to peroxide and aerobic stress, whilst water and wildlife-associated strains such as 81116 (ST-283) and M1 (ST-45) would be more resistance to such stresses is difficult to explain. One possible hypothesis is that the ability to survive aerobic stress is more important for the latter strains. The variation in the presence of rrpA and rrpB between different wildtype strains may play an important role in the ability of C. jejuni to adapt and survive in different environmental niches.

Infection with rrpA mutants from both rrpB+ and rrpB− strains resulted in an increase in survival of G. mellonella larvae compared to infection with the respective wild-type strains. We have previously demonstrated that infection of G. mellonella with the 11168H rrpA mutant leads to increased survival of the G. mellonella larvae (Gundogdu et al., 2015). Here we demonstrate that the 81-176 rrpA mutant (rrpB+) also exhibits a similar statistically significant reduction in larval cytotoxicity. Both 81116 and M1 rrpA mutants (rrpB−) exhibit reduced larval cytotoxicity, but not to a significant degree. This suggests the rrpA mutants (like the rrpB mutants) are more susceptible to the host immune mechanisms resulting in reduced bacterial survival within G. mellonella. This also suggests that RrpA may in fact play a role in the oxidative stress response of rrpB− strains such as 81116 and M1, but at a different level compared to rrpB+ strains, such as 11168H and 81-176. Certainly the enhanced resistance of the 81116 and M1 rrpA mutants to peroxide stress compared to the 11168H and 81-176 rrpA mutants is not reflected by increased virulence in the G. mellonella larvae infection model.

Campylobacter jejuni forms biofilms (Joshua et al., 2006; Gundogdu et al., 2011) and this may be an important factor in the survival of C. jejuni both within hosts and in the environment. Though our understanding of the specific mechanisms underlying biofilm formation in C. jejuni is still limited (Svensson et al., 2008), C. jejuni lacks the classical two component regulatory systems involved in biofilm formation that are present in other bacteria such as GacSA in Pseudomonas aeruginosa (Parkins et al., 2001). Biofilm formation has been linked to responses to oxidative and aerobic stress as C. jejuni biofilm formation is increased under aerobic conditions (Reuter et al., 2010) and mutation of genes encoding oxidative stress response proteins results in changes in biofilm formation (Oh and Jeon, 2014; Gundogdu et al., 2015). Analysis of biofilm formation demonstrated that rrpA mutants from both rrpB+ and rrpB− strains exhibited an increased biofilm phenotype compared to the respective wild-type strain under both microaerobic and aerobic conditions. Again the enhanced resistance of the 81116 and M1 rrpA mutants to peroxide stress compared to the 11168H and 81-176 rrpA mutants is not reflected by a decrease in biofilm formation.

The basis of C. jejuni survival is dependent upon the ability to sense and respond to the different environments encountered within hosts and in the environment. In this study we identified bioinformatically that over 99% of C. jejuni strains contain rrpA, whilst rrpB is restricted and appears to correlate with livestock-associated MLST clonal complexes. There exists a conserved genetic structure for rrpA, whilst rrpB seems to be

part of a transferable hypervariable region linked with variation in the type I R-M (hsd) system, giving an explanation for the more restricted distribution of rrpB amongst C. jejuni strains. rrpB− strains possess an increased level of resistance to peroxide and aerobic stress compared to rrpB+ strains. So whilst all the other oxidative stress response regulators such as PerR, Fur, CosR, CsrA, CprRS, RacRS, and RrpA appear to be conserved in C. jejuni, variation in the presence of RrpB between different wild-type strains may play an important role for altered oxidative stress responses through the concerted actions of these multiple regulators in this microaerophilic pathogen. This highlights the potential of genetic variation in the natural population in the adaptation to different environmental niches.

#### AUTHOR CONTRIBUTIONS

OG, DdS, BW, and ND designed this study. OG, DdS, BM, and AE performed all the lab-based experimental work. OG, DdS,

### REFERENCES

and AvV performed all the bioinformatic analysis. OG, DdS, BW, AvV, and ND wrote the manuscript.

#### ACKNOWLEDGMENTS

DdS is funded through a Doctorate Scholarship from CNPq in Brazil. We thank Sarah Leir and Naomi Henderson for technical assistance. This publication made use of the PubMLST website (http://pubmlst.org/) developed by Keith Jolley and sited at the University of Oxford. The development of that website was funded by the Wellcome Trust.

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fmicb. 2016.02117/full#supplementary-material

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2016 Gundogdu, da Silva, Mohammad, Elmi, Wren, van Vliet and Dorrell. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Profiling of *Campylobacter jejuni* Proteome in Exponential and Stationary Phase of Growth

Hana Turonova1 , Nabila Haddad2 , Mathieu Hernould2 , Didier Chevret 3 , Jarmila Pazlarova1 and Odile Tresse2 \*

1 Department of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology, University of Chemistry and Technology, Prague, Czechia, 2 SECALIM UMR1014, Institut National de la Recherche Agronomique, Nantes, France, 3 UMR1319 MICALIS, Plateforme d'Analyse Protéomique de Paris Sud-Ouest, Institut National de la Recherche Agronomique, Jouy-en-Josas, France

#### *Edited by:*

David Rodriguez-Lazaro, University of Burgos, Spain

#### *Reviewed by:*

Beatriz Melero, University of Burgos, Spain Alberto Quesada, University of Extremadura, Spain

> *\*Correspondence:* Odile Tresse<EMAIL_ADDRESS>
#### *Specialty section:*

This article was submitted to Food Microbiology, a section of the journal Frontiers in Microbiology

*Received:* 20 January 2017 *Accepted:* 04 May 2017 *Published:* 18 May 2017

#### *Citation:*

Turonova H, Haddad N, Hernould M, Chevret D, Pazlarova J and Tresse O (2017) Profiling of Campylobacter jejuni Proteome in Exponential and Stationary Phase of Growth. Front. Microbiol. 8:913. doi: 10.3389/fmicb.2017.00913 Campylobacter jejuni has been reported as a major cause of bacterial food-borne enteritides in developed countries during the last decade. Despite its fastidious growth requirements, including low level of oxygen and high level of CO2, this pathogen is able to persist in the environment without permanent loss of its viability and virulence. As C. jejuni is not able to multiply outside a host, the cells spend significant amount of time in stationary phase of growth. The entry into the stationary phase is often correlated to resistance to various stresses in bacteria. The switching between exponential and stationary phases is frequently mediated by the regulator sigma S (RpoS). However, this factor is absent in C. jejuni and molecular mechanisms responsible for transition of cells to the stationary phase remain elusive. In this work, proteomic profiles of cells from exponential and stationary phases were compared using 2-D electrophoresis (2DE) fingerprinting combined with mass spectrometry analysis and qRT-PCR. The identified proteins, whose expression differed between the two phases, are mostly involved in protein biosynthesis, carbon metabolism, stress response and motility. Altered expression was observed also in the pleiotropic regulator CosR that was over-expressed during stationary phase. A shift between transcript and protein level evolution of CosR throughout the growth of C. jejuni was observed using qRT-PCR and (2DE). From these data, we hypothesized that CosR could undergo a negative autoregulation in stationary phase. A consensus sequence resulting from promoter sequence alignment of genes potentially regulated by CosR, including its own upstream region, among C. jejuni strains is proposed. To verify experimentally the potential autoregulation of CosR at the DNA level, electrophoretic mobility shift assay was performed with DNA fragments of CosR promoter region and rCosR. Different migration pattern of the promoter fragments indicates the binding capacity of CosR, suggesting its auto-regulation potential.

Keywords: foodborne pathogen, *Campylobacter jejuni*, growth, exponential phase, stationary phase, CosR, regulation

## INTRODUCTION

Campylobacter jejuni is continuously reported as the main cause of bacterial food-borne infections in developed countries (EFSA and ECDC, 2015). The disease caused by this pathogen, namely campylobacteriosis, is triggered mainly by consumption of contaminated food or water, although a direct transmission from infected animals to human hosts can occasionally occur (Bronowski et al., 2014). It manifests as an acute inflammatory diarrhea with symptoms common to other bacterial enteritides abdominal pain, fever and watery diarrhea often accompanied with the presence of blood and leukocytes in stool (Blaser and Engberg, 2008). In most cases, campylobacteriosis is selflimiting and does not require specific therapy, however severe autoimmune disorders, such as Guillain-Barré and Miller-Fisher syndromes (Salloway et al., 1996; Nachamkin, 2002), reactive arthritis (Pope et al., 2007), and inflammatory bowel disease (Rodriguez et al., 2006) may appear. These late-onset complications, together with long convalescence time and high occurrence of campylobacteriosis, are the reasons why the disease is ranked as an infection with one of the highest annual burden (Batz et al., 2012; Gibney et al., 2014; Mangen et al., 2015). Although various strategies have been adopted by member states of EU in order to decrease numbers of campylobacteriosis (Lin, 2009; Saxena et al., 2013), the prevalence of this disease remains very high. It is therefore necessary to identify genetic and environmental factors affecting the persistence of C. jejuni in the environment, in order to develop new methods mitigating the campylobacteriosis cases.

As a pathogen with fastidious growth requirements, C. jejuni puzzles scientists with its ability to withstand broad range of stresses encountered during its lifecycle. In other pathogens represented by Escherichia coli, Salmonella spp., Shigella spp., and Vibrio spp., general stress response is regulated by sigma factor RpoS that is also responsible for switching the growth to stationary phase (Duval et al., 2015). The entry to the stationary phase requires cooperation of the sigma factor and many other regulators (Llorens et al., 2010). When switching to the stationary phase, growth rate of cells dramatically decreases as a result of reduced protein synthesis. The overall role of RpoS is to ensure adaptation and resistance of the cells to challenging environments. It directly regulates 10% of E. coli genes (Weber et al., 2005) that play role in morphological changes of the cells, resistance to broad range of stresses (oxidative and osmotic stress, heat shock, pH changes, etc.), virulence, metabolic processes, and the GASP (growth advantage in stationary phase) phenotype (Martinez-Garcia et al., 2001; Raiger-Iustman and Ruiz, 2008).

Unlike other Gram-negative bacteria, C. jejuni lacks several stress response genes, including the sigma factor RpoS (Parkhill et al., 2000; Garenaux et al., 2008). Despite its small genome, this strictly microaerobic pathogen had to develop other mechanisms allowing its survival in stressful conditions, such as lack of nutrients in aquatic environments, or high concentration of oxygen when exposed to air or to an oxidative attack of macrophages. The molecular mechanisms responsible for its survival in food, persistence in the environment and virulence have not yet been fully understood. Similarly, no information concerning the transition of C. jejuni cells from exponential to stationary phase are available nowadays. Functional replacement of the sigma factor RpoS has not yet been described and the molecular mechanisms facilitating a cellular switch from exponential to stationary phase remain unknown. Therefore, in this work, proteomic profiles of the cells from exponential and early stationary phase of growth were compared using a 2- D electrophoresis (2DE) and quantitative real-time PCR (qRT-PCR), in order to contribute to better understanding of the molecular changes occurring during cellular transition from exponential to stationary phase.

### MATERIALS AND METHODS

### Bacterial Strains and Growth Conditions

In this work, C. jejuni strain 81-176 isolated from a raw milk outbreak (Korlath et al., 1985) was used. Cells were resuscitated from a stock on Karmali agar plates (Oxoid) at 42◦C for 48 hours (h) in stainless steel jars filled with gas mixture containing 5% O2, 10% CO2, and 85% N2 (microaerobic atmosphere). Grown cells were subcultured microaerobically in BHI (Merck) for 48 h at 42◦C and 110 rpm, and then used for preparation of final suspension. The final suspension was cultivated at 42◦C, with shaking at 110 rpm in microaerobic atmosphere for 7 h to harvest proteins in exponential phase and for 18 h for stationary phase. All experiments were performed in 3 biological replicates.

### Protein Isolation Purification

Proteins were extracted and purified as described previously (Bieche et al., 2012; Haddad et al., 2012). Briefly, cells were harvested by centrifugation at 7000 g, 4◦C for 20 min and washed consecutively with 200 mM glycine (Sigma-Aldrich) and 100 mM Tris-HCl pH 7.0 (Sigma-Aldrich). Pellets were resuspended in 10 ml of a 10 mM Tris-HCl pH 7.0 and the cells were disrupted by series of 6 × 30 s sonication at 20 kHz with 6 min intervals on ice (Vibracell 72434, Bioblock Scientific). Cell debris was removed by two consecutive centrifugations at 10 000 g, 4◦C for 20 min. Then, cytoplasmic proteins were separated from membrane fractions by ultracentrifugation at 188 000 g for 1 h at 4◦C. The cytoplasmic protein fraction in the supernatant was treated with protease inhibitor cocktail tablets COMPLETE (Roche Diagnostics) and nuclease solution with final concentrations of 18 mg/ml RNAse and 9 mg/ml DNAse (Sigma-Aldrich). Protein samples were dialyzed using cellulose membrane tubing with a cut-off at 12 kDa (Sigma-Aldrich) against MilliQ water at 4◦C with shaking for 3 days by refreshing the dialysis bath each day. Total protein concentration was determined using the Micro BCATM Protein Assay Kit (Perbio-Science).

#### Two-Dimensional Gel Electrophoresis

A quantity of 100 µg of proteins isolated after 7 and 18 h of cultivation was concentrated using Concentrator 5301 (Eppendorf, France) at room temperature. Samples were mixed with rehydration buffer containing 6 M urea, 2 M thiourea, 4% CHAPS, 0,4% dithiothreitol (DTT), and 2% Bio-Lyse 3/10 Ampholyte (Bio-Rad) and few grains of bromophenol blue (BB). Turonova et al. Switch in C. jejuni Growth

Proteins were then loaded into 17 cm IPG strips (pH 4-7, Bio-Rad) by active rehydration at 50 V for 12 h. After that, the isoelectric focusing (IEF) was performed using the Bio-Rad IEF program as follows: from 50 to 250 V for 3 h, from 250 to 6,000 V for 3 h and at 6,000 V until reaching 54,000 Vh. Strips were then equilibrated in migration buffer containing 6 M urea, 50 mM Tris-HCl (pH 8.8), 2% SDS, 30% glycerol, 2% DTT, 4% iodoacetamide, and few grains of BB. The second dimension was performed using SDS-PAGE with 12% acrylamide gels. The IPG strips were immobilized to the gel using 1% low-melting point agarose (Bio-Rad). The migration ran at 40 mA/gel using Protean II xi cell (Bio-Rad) at 14◦C, until the bromophenol blue reached the base of the gels. Proteins in gels were finally silver stained and scanned with a GS-800 densitometer operated with the QuantityOne R software (Bio-Rad). Three independent experiments were performed, with two technical replicates for each of them.

#### Statistical Analysis and Protein Identification

The image analysis was performed using the Progenesis Samespots R software (NonLinear Dynamics). For the statistical analysis of the results, three independent experiments were performed, each with two technical replicates. Differences between the two conditions from the independent experiments and replicates were validated by Principal Component Analysis (PCA) and differences among matched spot intensities were statistically validated by ANOVA test at a 5% significance level. Lower and higher abundant proteins were taken into account only if the mean difference in spot intensities passed the threshold of 1.5. Predominant proteins whose amount differed significantly between the phases (q- and p-value ≤ 0.05, power ≥ 0.8, fold difference of spot intensities ≥ 1.5) were excised from BioSafe colloidal Coomassie blue (Bio-Rad) stained gels containing in total 700µg of proteins. The excised proteins were analyzed after trypsin digestion with a mass spectrometer MALDI-TOF Voyager DE super STR (Applied Biosystems) at the PAPPSO platform of the INRA Center in Jouy-en-Josas (France). Gel plugs were first washed twice with 50% (v/v) acetonitrile, 25 mM ammonium carbonate in water, and then dried in a vacuum speed concentrator. Aliquot (10µl) of a trypsin solution (Promega, 12.5 ng/µl in 50 mM NH4HCO3) was added to each sample and digestion was performed for 6 h at 37◦C. A 1µl aliquot of each supernatant was spotted directly onto the MALDI plate then dried at room temperature before adding 1µl of the matrix solution (α-cyano-4-hydroxycinnamic acid, 3 mg/ml in 50% (v/v) acetonitrile 0.1% (v/v) trifluororacetic acid). Mass spectra were acquired on a Voyager-DE-STR (Applied Biosystems, Framingham, MA), equipped with a nitrogen laser (Laser Science, Franklin, MA). Tryptic autodigestion ion peaks, (M + H)+ = 2211.104 and 842.509 Da, were used for internal calibration of spectra. The peptide mass lists were used for protein identification in a C. jejuni, strain 81-176, amino acid sequence database (UniprotKB, 09.09.2010), using the Protein Prospector v 3.2.1 software set with the following parameters: mass tolerance < 20 ppm, four peptides required to match, one missed cleavage, oxidized methionine and carbamidomethylated cysteine, respectively as possible and fixed modifications. Identification was evaluated with regard to sequence coverage, Mowse score, and compatibility between theoretical and experimental molecular weight and isoelectric point. Genes of proteins significantly over-expressed in one of the growth phases were then chosen for qRT-PCR experiments to verify their expression on translational level.

#### RNA Isolation and Reverse Transcription

Cells were harvested from cultures cultivated for 4, 7, 12, 16, 18, and 24 h by centrifugation at 3,300 g at 4◦C for 6 min, pellets were homogenized with 1 ml of lysis solution Extract-All (Eurobio) according to manufacturer's guidelines. After that, 200µl of chloroform were added to the suspension allowing separation of cell components. Water phase containing RNA was removed, precipitated with 500µl of isopropanol and dissolved again in 50µl of RNase-free water after washing in 75% cool ethanol. Samples were then treated with DNase I (Sigma-Aldrich) and purified using RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. Possible DNA contamination was detected by PCR detecting house-keeping gene flaA (Nachamkin et al., 1993). The integrity of RNA was verified using the Experion System (Bio-Rad). Its concentration and purity was measured using NanoDrop 2,000 (Thermo Scientific).

For reverse transcription 100 ng of RNA were mixed with 0.5µl of 1µM Random Hexamer Primer (Promega) and 4.5µl of RNase-free water. This mixture was incubated 5 min at 70◦C followed by 5 min on ice. After that 15µl of Master Mix were added to mixture (5µl of 5 × RT buffer, 1µl 25 mM dNTP and 1µl of M-MLV H− reverse transcriptase; Promega) and it was incubated 10 min at room temperature, 50 min at 48◦C and finally 15 min at 70◦C to stop the reaction.

#### Quantitative PCR (q-PCR)

Quantitative real-time PCR assay was performed using the AB7300 Realtime PCR system with SYBR Green-I Master Mix (Applied Biosystems). Primers (Eurobio) were designed with the on-line Primer3 software (www.simgene.com; **Table 1**) and their quality was checked both virtually, using FastPCR software, and by PCR. The efficiency of the primer pairs was calculated with the formula E = [10(1/−s)] × 100, where "s" is the slope of the standard curve. The efficiency of designed primers ranged from 101.27 to 105.38%.

The composition of q-PCR mix and amplification program were the same as described by Bieche et al. (2012) Amplification efficiency was determined from standard curve of serially diluted cDNA. The expression level of examined genes was calculated with AB 7,300 software against endogenous control gene rrs to obtain relative quantity.

#### Purification of Recombinant CosR

In order to produce sufficient amount of His6-tagged recombinant CosR (rCosR), restriction enzymes sites NdeI and BamHI were added to cosR sequence during its amplification using specific primers NtagCosR-NdeI (5'-GCACACATA TGAGAATTTTAGTTATAGAAG-3′ ) and NtagCosR-BamHI

#### TABLE 1 | Primers used in the experiments.

(5′ -TACAGGATCCAAGGTGCAAAATTGTTA-3′ ), to obtain NdeI-CosR-BamHI amplicons. These amplicons were inserted using NdeI and BamHI enzymes and subcloned into plasmid pET-15b (Novagen, France) to obtain a N-terminal His6 tagged protein under the control of T7 promoter. The resulting plasmid was transformed to competent E. coli expression strain BL21(DE3) (Novagen, France). Overexpression of rCosR was obtained from an overnight culture at 28◦C in LB supplemented with 50 µg/ml of ampicillin, and 0.125 mM isopropyl-β-D-1 thiogalactopyranoside (IPTG). Cells were then harvested by centrifugation at 5,000 g for 10 min, washed with TE buffer (Sigma-Aldrich, France) and resuspended for 30 min in a lysis buffer containing 50 mM TrisHCl, pH 7.5, 300 mM NaCl, 5 mM β-mercaptoethanol and 1 mg/ml of lysozyme. The lysate was centrifuged and rCosR was purified using Ni-NTA agarose columns (Qiagen, France) according to the manufacturer's instructions. Columns were equilibrated using lysis buffer before protein binding and then washed sequentially with buffer W1 (lysis solution with 1% glycerol, 0.5% triton and 20 mM imidazole) and buffer W2 (lysis buffer with 20 mM imidazole). The recombinant protein was eluted using 400 mM imidazole and 6 M urea and purified by dialysis according to the manufacturer's instructions. Aliquots of rCosR were stored at −80◦C.

#### Bioinformatic Analysis of the CosR DNA-Binding Box

A bioinformatic analysis was performed to specify the consensus sequence of the CosR DNA-binding box proposed by Hwang et al. (2011, 2012). For this purpose, sequences of 5′ 400 nt upstream regions of 7 genes previously reported as binding CosR were retrieved from the platform MicroScope (Microbial Genome Annotation & Analysis Platform; http:// www.genoscope.cns.fr/agc/microscope/home/). To inspect the conservation of the CosR binding box, sequences of nine different fully sequenced C. jejuni strains were used in the analysis (**Supplementary Table 1**). Resulting consensus sequence logos were created using the WebLogo platform (http://weblogo.threeplusone.com; Crooks et al., 2004).

#### Electrophoretic Mobility Shift Assay (EMSA)

EMSA was carried out to identify putative CosR binding sites. DNA fragments covering promoter regulatory regions or gene internal regions of cosR and katA were PCR amplified using designed primers (**Table 1**). To demonstrate the DNA-binding properties of CosR, concentration range of purified recombinant protein CosR-His6 (1–10µmol/l) were mixed with 2µmol of purified PCR fragments in a total volume of 20µl containing 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 5 mM MgCl2, 10 mM KCl, 10µg/ml sheared salmon sperm DNA, and 10% glycerol. Sonicated salmon sperm DNA was applied as a nonspecific competitor. All samples were incubated at room temperature for 30 min following by the separation of the complexes using electrophoresis on a native 6% polyacrylamide gel in 0.5 × TBE.

#### RESULTS

Considering the lack of information concerning the transition of C. jejuni from exponential to stationary phase, we compared the proteomic profiles of cells harvested after 7 and 18 h of microaerobic cultivation at 42◦C.

The comparison of the 2-DE proteomic profiles revealed numerous differences between the two phases (**Figure 1**). In total, 42 spots were identified as 24 different proteins with 11 proteins more abundant in the exponential phase (17 spots), 10 proteins more abundant in the stationary phase (12 spots) and 3 proteins whose isoforms were overexpressed in both exponential and stationary phase (13 spots; **Table 2**). The identified proteins are involved mostly in metabolism (AcnB, BioB, FumC, HisD, MetK, and OorA), general and oxidative stress response (BetA, ClpB, CosR, DnaK, GroEL, Rbr, and TrxB), translation (FusA, RplL, Tig, Tsf, Tuf, and TypA), and motility (CheW and FlaA).

Subsequently, 10 differently expressed proteins were selected for qRT-PCR analysis according to their potential to play role in the phase switch. The selected proteins participate in pathways important for the survival of C. jejuni, such as carbon metabolism (AcnB, FumC, and OorA), general and oxidative stress response (BetA, DnaK, Rbr, and TrxB), motility (FlaA, CheW) and gene expression regulation represented by the essential twocomponent regulator CosR. When comparing the level of the transcripts, 6 genes were more transcribed in exponential phase (betA, cheW, cosR, fumC, oorA, and trxB), while 3 genes were more transcribed in stationary phase (acnB, flaA, and rbr). There was no difference in the expression of dnaK (**Figure 2**). In most of the cases, the level of transcription corresponded with the level of protein expression. However, in three cases opposite trends were found between the 2-DE and qRT-PCR data, where the proteins were found to be overexpressed in the stationary phase, while the amount of mRNA was higher in the exponential phase. Those include OorA subunit of enzyme involved in carbon metabolism, CheW protein regulating chemotaxis, and regulator CosR.

As the pleiotropic regulator CosR is essential for C. jejuni growth and regulates several pathways, CosR was selected for further analysis. To evaluate the changes of cosR transcript level throughout the growth of C. jejuni, total mRNA was extracted from the cells at different time-points of their cultivation and cosR specific transcript level was analyzed using qRT-PCR. The amount of cosR transcripts evolved during the growth of the strain 81-176 with an overall fold difference of 5.3. The highest level detected in cells was reached at 7 h of cultivation during the exponential phase and declined constantly afterwards in the stationary phase (**Figure 3**).

The comparison of the data obtained by qRT-PCR with the 4.0-fold protein increase in the early stationary phase led us to a hypothesis that CosR could undergo a negative autoregulation

overexpressed proteins. A total of 700µg of cytosolic proteins were separated on IPG strip (pH 4-7) followed by second separation by SDS-PAGE in 12% acrylamide gel stained by Coomassie blue.

#### TABLE 2 | Identification of spots differently expressed in exponential phase (7 h) and early stationary phase (16 h) of *C. jejuni* growth.

The fold changes are normalized on the level of protein abundance detected in exponential phase.

aPositive values indicate a fold change for higher protein abundance in stationary phase. Negative values indicate a fold change for lower protein abundance in stationary phase. bEach protein was identified with a mass tolerance < 20 ppm using at least four peptides

cMP/PC: Number of matched proteins/protein coverage

by binding to its own promoter region. The hypothesis was strengthened by identification of the CosR box described by Hwang et al. (2011, 2012) in C. jejuni 81-176 genome upstream from the cosR gene. Interestingly, the sequences of the CosRbinding boxes of 4 genes responsible for oxidative stress response are conserved among the analysed strains (**Figure 4A**) with the consensus sequence 5′ -wwhhwwwwAAAwTTAdwwTTT-3 ′ which is similar to the one described by Hwang et al. (2012). To further refine the consensus sequence of the CosR box, 400-nt upstream sequences of the 6 genes previously described as binding CosR (ahpC, cmeA dps, katA, luxS, and sodB) by Hwang et al. (2011, 2012) and the one of CosR itself were compared among 9 fully sequenced C. jejuni strains (**Supplementary Table 1**). The refine CosR box resulted in the consensus sequence 5′ -wdnnhdwnwhwwTTwnhhTTd-3′ (**Figure 4B**). In order to verify whether CosR can bind to its own promoter region, EMSA was performed (**Figure 5**). Regions containing the promoter region of cosR and an internal part of cosR gene were amplified and subjected to a concentration range of purified recombinant rCosR. Promoter region of katA from NCTC 11168 was used as a positive control validating the EMSA assays, as rCosR was previously shown to be able to bind to this DNA promoter region (Hwang et al., 2011). A mobility shift was observed when the DNA fragments of cosR and katA promoters

FIGURE 4 | Promoter sequence alignment. (A) Consensus sequence logo from upstream sequence of the promoter region of genes responsible for ROS scavenging (ahpC, cosR, katA and sodB). (B) Consensus sequence logo of CosR-binding box of the upstream sequences of genes with CosR-binding capacity (ahpC, cmeA, cosR, dps, katA, luxS, and sodB). Both logos are based on sequences from 9 strains listed in Supplementary Table 1. D–A, T or G; H–A, T or C; N–A, T, C or G; W–A or T.

were used, while no motility shift was observed for the internal DNA region of cosR, indicating the potential capacity of CosR to bind to its own promoter.

#### DISCUSSION

Campylobacter jejuni is repeatedly reported as the major cause of food-borne bacterial enteritides worldwide, causing severe economic and sociological burden. This generally stress-sensitive bacterium, that supposedly cannot multiply outside a host, is able to survive harsh conditions encountered in the environment (Whiley et al., 2013). Despite the concern of this pathogen, relatively little is known about the molecular mechanisms responsible for its ability to persist in meat processing facilities, on carcasses and in the food environment. Enhanced bacterial resistance to various stresses is often connected to the transition of the cells into different phases of growth. For example in the case of E. coli, cells in stationary phase were found to be more resistant to high hydrostatic pressure, mild heat, oxidative and osmotic stress, and chlorine disinfectants (Kaur et al., 1998; Benito et al., 1999; Cherchi and Gu, 2011). However, Campylobacter cells were reported as generally more resistant in exponential phase of growth, with dynamic emergence of resistant subpopulations in the stationary phase (Kelly et al., 2001, 2003). Molecular mechanismsresponsible for the transition between the growth phases have not yet been fully described. Unlike other bacteria, C. jejuni lacks sigma factor RpoS regulating the switch from one phase to another (Parkhill et al., 2000). According to several authors, post-transcriptional regulator CsrA could contribute to modulation of gene expression in C. jejuni in response to growth phase. Indeed, in E. coli and S. enterica Thyphimurium, CsrA participates in the regulation of genes inducible in stationary phase (Romeo, 1998; Lawhon et al., 2003). Similar data were recently reported also for C. jejuni 81-176 where CsrA strongly influences expression of proteins in stationary phase and might therefore compensate the lack of RpoS (Fields et al., 2016). Another potential regulator that might participate in the transition could be the stringent response regulator spoT, as it was previously reported as important for survival of cells in stationary phase (Gaynor et al., 2005). As regulations during phase transition in C. jejuni remain unexplained at the molecular level, proteomic fingerprinting of cells isolated from exponential and stationary phase were compared in this work, to identify the main protein changes occurring between the two main growth phases of C. jejuni 81-176.

The first group of identified proteins is involved in cellular stress response. This group contains mostly proteins participating in oxidative stress and protein folding. Interestingly, there is no clear pattern of phase-depending overexpression of these proteins. However, two out of three chaperons were overexpressed in exponential phase, suggesting higher abundance of misfolded and aggregated proteins in the exponential phase, probably caused by the request of functional de novo proteins for rapid biosynthesis of cellular material during cell multiplication. The overexpression of stress-related proteins is not surprising, as toxic metabolites accumulate during growth in batch cultivation. The presence of toxic metabolites together with starvation occurring in the stationary phase could be the reasons why the cells overexpressed proteins involved in the stress response. The overexpression of stress response proteins, reported previously by Wright et al. (2009), could speed up the adaptation process of C. jejuni when changing the environment. Such adaptation would also require adjustment of metabolism reflecting the changes in the availability of nutrients during the cultivation of the cells. Indeed, in this work, we also observed altered expression of several proteins playing role in C. jejuni metabolism, mostly of those involved in the citric acid cycle (TCA). Asaccharolytic bacterium C. jejuni uses TCA as the main carbon source through gluconeogenesis (Velayudhan and Kelly, 2002). Unlike other bacteria, the TCA cycle of C. jejuni involves enzymes containing oxygen-sensitive Fe-S clusters, such as Oor enzymes (Atack and Kelly, 2009) that are rapidly inactivated after exposure to ambient air (Kendall et al., 2014). Higher abundance of OorA observed in the stationary phase could be explained by successful detoxification of ROS with the help of TrxB and BetA which are among the overexpressed protein in the exponential phase.

Another group of proteins with altered expression between the two growth phases is involved in protein biosynthesis. The proteins overexpressed in the exponential phase were mostly elongation factors responsible for correct synthesis of new peptides. The proteins whose abundance is higher in the stationary phase play role in binding of tRNA to ribosomes and their translocation from A to P site. These results suggest high importance of protein biosynthesis in C. jejuni regardless of the physiological state of the cells, although they are in contrast with the decline of ribosomal proteins in the stationary phase reported previously (Wright et al., 2009).

Interestingly, the cells in stationary phase overexpressed proteins involved in motility (flagellar protein FlaA) and chemotaxis (regulator CheW). Similar overexpression was already reported both for cells in stationary phase (Wosten et al., 2004; Wright et al., 2009; Fields et al., 2016) and in biofilms (Kalmokoff et al., 2006). The overexpression of FlaA in stationary phase is mediated by CsrA (Fields et al., 2016) and could be explained by multifunctionality of C. jejuni flagella. Besides their role in motility, flagellum apparatus works as a type III secretion system (Konkel et al., 2004) and it was found to be crucial for C. jejuni adhesion and biofilm formation (Svensson et al., 2014). In our case the protein regulating chemotaxis was also overexpressed, suggesting that the chemotaxis-driven motility is enhanced in stationary phase, facilitating the migration of cells toward nutrient-rich environment.

The transcript analysis revealed that for most of the tested genes, the level of the transcription was correlated to the variation in protein abundance, indicating a regulation of the corresponding proteins at the transcriptional level. However, in three cases (CosR, CheW, and OorA) the level of transcripts was higher in the exponential phase while the protein abundance was significantly lower as compared to the amount in the stationary phase. Such difference could be explained by posttrancriptional or post-translation modifications (Maier et al., 2009). The translation can be delayed when Shine-Dalgarno sequence of mRNA sequence is not completely complementary to the rRNA sequence (Maier et al., 2009) or when the mRNA undergoes conformational change, e.g., by binding of small metabolites (Nahvi et al., 2004). Another reason can be the involvement of short regulatory sRNA that is influencing stability of mRNA and translation process by targeting or blocking mRNA binding to ribosomes (Maier et al., 2009). In addition, overall number of available ribosomes and half-life of translated proteins also play role in final mRNA-protein ratio. Among the proteins presenting an opposite trend between transcript level and protein amount, only CosR is identified as a DNA regulator. This recently discovered orphan two component system (TCS), whose sequence is conserved among Campylobacter and Helicobacter species, affects transcription of 93 different genes, including 17 genes essential for C. jejuni viability (Hwang et al., 2012). It is involved in the regulation pathway of ROS detoxification (Hwang et al., 2011), efflux pump expression (Hwang et al., 2012), and also influences biofilm formation and maturation of C. jejuni (Oh and Jeon, 2014; Turonova et al., 2015). The cosR gene is present in all C. jejuni strains (Gundogdu et al., 2016). It is an essential gene for C. jejuni with the absence of a consensus catalytic aspartic acid replaced by an asparagine. It shares 60% amino acid identity with Hp1043, a response-regulator element of TCS from Helicobacter pylori (Beier and Frank, 2000; Schar et al., 2005; Stahl and Stintzi, 2011). The interchange of C. jejuni cosR with homologous gene from H. pylori does not impair its functionality, indicating a probable conserved function of the gene throughout strains and closely related bacterial genera (Muller et al., 2007). As proteomics analyses using time-point profiling are restricted, dynamics of transcript levels and protein abundance of CosR were explored.

The results indicated a maximum transcribed amount of cosR after 7 h of cultivation in exponential phase followed by a decline up to 24 h of cultivation, while the abundance of the protein was 4-times higher at the early stationary phase. The transient upregulation of the transcript level and the protein amount during the growth led us to a hypothesis that CosR could potentially undergo negative autoregulation. Negative autoregulation, or negative autogenous regulation, is one of the three common motifs used for bacterial regulation of transcription (Shen-Orr et al., 2002), where transcription factor negatively regulates its own transcription (Savageau, 1974). This type of autoregulation has been previously detected in E. coli, where it concerns over 40% of all transcription factors, including global regulators Crp, Hns, Ihf, and Lrp (Shen-Orr et al., 2002). The kinetics of transcription is generally slow, faster kinetics can be achieved by shortening the lifetime of a product by degradation, which is inconvenient due to metabolic cost (Rosenfeld et al., 2002). As it was proven both mathematically and experimentally, negative autoregulation speeds up the transcription response (Rosenfeld et al., 2002; Shen-Orr et al., 2002). However, autoregulation requires active binding of the transcription factor to the promoter region of the operon. Previous studies of binding of CosR–DNA sequences in upstream regions of some genes revealed the presence of a specific CosRbinding box (Hwang et al., 2011, 2012). The resulting consensus sequence of the box was estimated from sequences of 5 genes in C. jejuni NCTC 11168. The authors considered some of the nucleotides to be identical although they were not present in all cases (4/5 for positions 3, 9, 11, 14, 20, and 21). The only identical nucleotides present in all 5 examined regions were on positions 5, 10, 15, and 19. Our analysis show lower level of similarity of the CosR boxes. When we compare the sequences of genes responsible for ROS scavenging (ahpC, cosR, katA, and sodB), the CosR-binding box is conserved and similar to the one reported by Hwang et al. (2012). This consensus sequence was refined using 63 sequences from upstream promoter regions of all genes previously described to bind CosR and the own promoter region of CosR. The resulting consensus sequence indicated a well conserved motif at the end of the consensus sequence which might be valuable for further analyses of the CosR regulon. To detect whether CosR could potentially bind to its own promoter, DNA shift mobility analysis was used. The data indicated a shift mobility of CosR in presence of its own promoter region sequence, which indicates that CosR is also able to bind its own promoter region. Therefore, it might have a potential to undergo a negative autoregulation. The autoregulation of CosR could be also influenced by the post-transcriptional regulator CsrA, which was recently reported to affect CosR expression (Fields et al., 2016). However, due to broad spectrum of genes under CrsA regulation it is not clear whether the observed difference in CosR expression is caused by direct or indirect regulation. Further analyses are therefore required to decipher a potential role of CsrA in cosR expression. Taken together, these

#### REFERENCES

data suggest a potential binding of CosR to its own promoter, although additional analyses are required to confirm the in vivo protein-DNA complexation. Future analyses should focus on this potential complexation of CosR with its own promoter and its impact on gene expression.

In summary, our proteomic data show that the transition from exponential to stationary phase is accompanied with numerous changes in the gene expression. The main functional groups of differently expressed proteins involve those playing role in metabolism, stress response, translation, and motility, suggesting active adaptation of the cells to the environment. Our transcriptomic and proteomic analyses also pinpointed the dynamics of the CosR expression, leading to the hypothesis of negative autoregulation of this pleiotropic regulator. Altogether, we outlined mechanisms that C. jejuni could use during the growth.

#### AUTHOR CONTRIBUTIONS

OT conceived the study. OT, NH, MH, and HT designed the experiments. HT and MH performed the experiments, DC performed the protein identification. HT and MH analyzed the results and HT prepared the draft manuscript. NH, JP, and OT contributed to the manuscript.

#### ACKNOWLEDGMENTS

The work was financially supported by Secalim (INRA/Oniris, Nantes, France) and the grant of Czech Ministry of Youth and Education No. 7AMB15FR012–"Project Barrande." Authors would also like to thank Sandrine Rezé, Laurent Marché their technical support.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fmicb. 2017.00913/full#supplementary-material

Supplementary Table 1 | CosR-binding box sequences of genes used in the bioinformatic analysis.

hydrostatic pressure, mild heat, and other stresses. Appl. Environ. Microbiol. 65, 1564–1569.

and stress tolerance of Campylobacter jejuni. PLoS ONE 9:e106063. doi: 10.1371/journal.pone.0106063

Wright, J. A., Grant, A. J., Hurd, D., Harrison, M., Guccione, E. J., Kelly, D. J., et al. (2009). Metabolite and transcriptome analysis of Campylobacter jejuni in vitro growth reveals a stationary-phase physiological switch. Microbiology 155, 80–94. doi: 10.1099/mic.0. 021790-0

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The reviewer BM and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.

Copyright © 2017 Turonova, Haddad, Hernould, Chevret, Pazlarova and Tresse. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Cj0440c Affects Flagella Formation and In Vivo Colonization of Erythromycin-Susceptible and -Resistant Campylobacter jejuni

Haihong Hao1,2† , Xia Fang1,2† , Jing Han3 , Steven L. Foley3 , Yulian Wang1,2 , Guyue Cheng1,2, Xu Wang1,2, Lingli Huang1,2, Menghong Dai1,2, Zhenli Liu1,2 and Zonghui Yuan1,2 \*

1 National Reference Laboratory of Veterinary Drug Residues and MOA Key Laboratory for Detection of Veterinary Drug Residues, Huazhong Agricultural University, Wuhan, China, 2 MOA Laboratory for Risk Assessment of Quality and Safety of Livestock and Poultry Products, Huazhong Agricultural University, Wuhan, China, 3 Division of Microbiology, National Center for Toxicological Research, US Food and Drug Administration, Jefferson, AR, USA

#### Edited by:

Avelino Alvarez-Ordóñez, Universidad de León, Spain

#### Reviewed by:

Hongxia Wang, University of Alabama at Birmingham, USA Odile Tresse, Oniris, France

> \*Correspondence: Zonghui Yuan<EMAIL_ADDRESS>
†These authors have contributed equally to this work.

#### Specialty section:

This article was submitted to Food Microbiology, a section of the journal Frontiers in Microbiology

Received: 01 August 2016 Accepted: 07 April 2017 Published: 25 April 2017

#### Citation:

Hao H, Fang X, Han J, Foley SL, Wang Y, Cheng G, Wang X, Huang L, Dai M, Liu Z and Yuan Z (2017) Cj0440c Affects Flagella Formation and In Vivo Colonization of Erythromycin-Susceptible and -Resistant Campylobacter jejuni. Front. Microbiol. 8:729. doi: 10.3389/fmicb.2017.00729 Campylobacter jejuni is one of the most common foodborne pathogen worldwide. A putative transcriptional regulator, Cj0440c, was up-regulated in the erythromycinresistant C. jejuni, however, the precise role of Cj0440c is yet to be determined. The aim of this study was to determine the biological functions of Cj0440c. The Cj0440c isogenic mutants were constructed from erythromycin-susceptible C. jejuni NCTC 11168 (S) and -resistant C. jejuni 68-ER (R), designating as SM and RM, respectively. The isogenic Cj0440c mutants (SM and RM) and parental strains (S and R) were subjected to microarray and qRT-PCR analysis to examine the transcriptional profile changes contributed by Cj0440c. The antimicrobial susceptibility, flagellar morphology, in vitro growth and in vivo colonization in chickens were carried out to analyze the biological function of Cj0440c. The results showed that 17 genes were down-regulated in SM compared to S, while 9 genes were down-regulated in RM compared to R. The genes with transcriptional change were mainly involved in flagella biosynthesis and assembly. Using transmission electron microscopy, we found that the filaments were impaired in SM and lost in RM. The chicken colonization experiments showed that Cj0440c mutants (SM and RM) had reduced colonization ability in chickens when compared with corresponding parental strains (S and R). In conclusion, Cj0440c regulates flagella biosynthesis and assembly, and consequently affect the in vivo colonization of erythromycin-susceptible and -resistant C. jejuni.

Keywords: Campylobacter jejuni, Cj0440c, flagella, colonization, erythromycin resistance

## INTRODUCTION

Campylobacter jejuni has been recognized as one of the most important pathogens, which can cause infectious diarrhea and severe forms of disease such as Guillain-Barre Syndrome or Miller Fischer Syndrome (Samuel et al., 2004; Hughes and Cornblath, 2005; Riddle et al., 2006). The CDC estimated that in 2009 the number of Campylobacter infection was 13.02 per 100,000 people (Silva et al., 2011). The cost of human Campylobacteriosis in the United States is estimated at \$1.3 to 6.8 billion dollars annually (Scharff, 2012; Epps et al., 2013). Macrolides (e.g., erythromycin) are

the most important drugs of choice for clinical treatment of Campylobacter infections (Gibreel et al., 2005). Unfortunately, macrolides-resistant Campylobacter have emerged and impose a global public health concerns (Gibreel and Taylor, 2006; ECDC et al., 2009). In earlier study we demonstrated that the transcription level of Cj0440c was increased in high-level erythromycin-resistant C. jejuni (Hao et al., 2013).

Bioinformatic analyses suggested that Cj0440c is a putative transcriptional regulator and encodes a TENA/THI-4 family protein, however, the molecular function of this family is yet to be determined. The gene Cj0440c is located downstream of the Cj0437–Cj0439 operon (mfr, methylmenaquinol:fumarate reductase) which plays an important role in the susceptibility to hydrogen peroxide (H2O2) (Parkhill et al., 2000; Weingarten et al., 2009; Kassem et al., 2012) and upstream of Cj0441 (acpP, acyl carrier protein) which is a universal and highly conserved acyl donor for synthesis of fatty acid, endotoxin and acylated homoserine lactones for the quorum sensing in C. jejuni (Byers and Gong, 2007). Both the downstream and upstream genes of Cj0440c were essential for the growth, survival, colonization and pathogenesis in Campylobacter. Although Cj0440c is located on the opposite DNA coding strand, it may divergently transcribed with its up-and-downstream genes, and likely to act as a transcriptional regulator and play an important role in gene regulation and the biological function in C. jejuni. The biological functions of Cj0440c in C. jejuni are largely unknown.