Benjamin Duqué

Sandrine Rezé

Albert Rossero

Jeanne-Marie Membré

Sandrine Guillou

Nabila Haddad
email: nabila.haddad@oniris-nantes.fr

Campylobacter molecular response to stress Quantification of Campylobacter jejuni gene expression after successive stresses mimicking poultry slaughtering steps

Keywords: RT-qPCR, strain variability, food safety, foodborne pathogen, cell history

Highlights

• C. jejuni molecular response studied after consecutive thermal stresses by RT-qPCR • Similar gene expression patterns shared between two of the three strains tested • Up-regulated genes mainly involved in the heat shock response • Down-regulated genes belonged to metabolic pathways (lipid, amino-acid metabolisms)

• A strain-specific gene expression profile consistent with atypical stress response

Introduction

Campylobacter has been the main cause of bacterial enteritis worldwide in humans for many years [START_REF] Efsa | The European Union One Health 2018 zoonoses report[END_REF]. The number of reported confirmed cases of campylobacteriosis is often under-estimated: it was 246,571 in 2018 with an EU notification rate of 64.1 per 100,000 population [START_REF] Efsa | The European Union One Health 2018 zoonoses report[END_REF]. C. jejuni infection is usually benign, but may also lead to chronic sequelae such as Guillain-Barré syndrome (GBS) and Miller-Fisher syndrome (MFS) [START_REF] Altekruse | Campylobacter jejuni -An emerging foodborne pathogen[END_REF][START_REF] Silva | Campylobacter spp. as a foodborne pathogen: A review[END_REF][START_REF] Who | The global view of Campylobacteriosis[END_REF]. Consumption of animal products, mainly poultry, is a major cause of infection in humans [START_REF] Zhong | Prevalence, genetic diversity and antimicrobial susceptibility of Campylobacter jejuni isolated from retail food in China[END_REF]. Although chicken meat is the main source of this pathogen, other foods have been involved in campylobacteriosis outbreaks such as milk [START_REF] Efsa | The European Union One Health 2018 zoonoses report[END_REF].

Campylobacter are apparently fragile organisms that cannot grow under aerobiosis conditions; they cannot multiply outside the animal host and are highly susceptible to a number of environmental conditions [START_REF] Park | The physiology of Campylobacter species and its relevance to their role as foodborne pathogens[END_REF]. Nevertheless, Campylobacter are paradoxically considered as one of the main foodborne pathogens [START_REF] Solomon | Campylobacter jejuni: a bacterial paradox[END_REF]. During poultry processing, bacteria are submitted to numerous stressful steps assimilated to heat stress, cold stress, acid stress or oxidative stress. To mitigate stressful effects, the pathogen sets up various defense strategies like production of heat shock proteins (e.g. DnaK, GroEL, ClpB) in response to heat or acid stress [START_REF] Konkel | Characterization of the thermal stress response of Campylobacter jejuni[END_REF]Reid et al., 2008a;Reid et al., 2008b;[START_REF] Stintzi | Gene expression profile of Campylobacter jejuni in response to growth temperature variation[END_REF][START_REF] Thies | Cloning and expression of the Campylobacter jejuni lon gene detected by RNA arbitrarily primed PCR[END_REF]Thies et al., 1999a;Thies et al., 1999b), but also enzymes (e.g. AhpC, KatA, SodB) involved in cell detoxification [START_REF] Baillon | An ironregulated alkyl hydroperoxide reductase (AhpC) confers aerotolerance and oxidative stress resistance to the microaerophilic pathogen Campylobacter jejuni[END_REF][START_REF] Grant | Molecular characterization of katA from Campylobacter jejuni and generation of a catalase-deficient mutant of Campylobacter coli by interspecific allelic exchange[END_REF][START_REF] Pesci | Genetic, enzymatic, and pathogenic studies of the iron superoxide dismutase of Campylobacter jejuni[END_REF][START_REF] Van Vliet | Campylobacter jejuni contains two fur homologs: characterization of iron-responsive regulation of peroxide stress defense genes by the PerR repressor[END_REF]. Such mechanisms enable Campylobacter to adapt to environmental conditions.

Unlike other foodborne pathogens, Campylobacter does not grow effectively in the food environment or on food products. However, it can survive under stressful conditions [START_REF] Duqué | Influence of cell history on the subsequent inactivation of Campylobacter jejuni during cold storage under modified atmosphere[END_REF][START_REF] Klančnik | Stress response and pathogenic potential of Campylobacter jejuni cells exposed to starvation[END_REF]Murphy et al., 2003a, b;[START_REF] Oh | Impact of oxidative stress defense on bacterial survival and morphological change in Campylobacter jejuni under aerobic conditions[END_REF]Reid et al., 2008b). Adaptation to stress has been investigated at the transcriptomic level and has determined which genes could be involved.

Most studies addressed the acid, oxidative or heat stress responses (Reid et al., 2008a;Reid et al., 2008b;[START_REF] Stintzi | Gene expression profile of Campylobacter jejuni in response to growth temperature variation[END_REF][START_REF] Varsaki | The acid adaptive tolerance response in Campylobacter jejuni induces a global response, as suggested by proteomics and microarrays[END_REF], in which single stresses were applied independently. Many genes play a role in the stress response, such as genes implied in the heat shock response or in the amino acid or lipid metabolisms [START_REF] Cameron | Hyperosmotic stress response of Campylobacter jejuni[END_REF][START_REF] Guccione | Transcriptome and proteome dynamics in chemostat culture reveal how Campylobacter jejuni modulates metabolism, stress responses and virulence factors upon changes in oxygen availability[END_REF][START_REF] Palyada | Characterization of the oxidative stress stimulon and PerR regulon of Campylobacter jejuni[END_REF][START_REF] Stintzi | Gene expression profile of Campylobacter jejuni in response to growth temperature variation[END_REF]. In order to decipher how this pathogen can survive along the food chain, it seemed relevant to evaluate the impact of various food production and preservation conditions on the stress adaptation potential of this foodborne pathogen. Consequently, we transposed consecutive heat and cold stresses inspired from the poultry slaughtering process to the laboratory. The aim was to assess the impact of successive hot and cold temperature stresses on C. jejuni gene expression and better understand the adaptation capacity of this pathogen.

To our knowledge, the molecular mechanism underlying the adaptive response of C. jejuni resulting from exposure to successive stresses, especially stresses similar to those encountered during the poultry processing steps, has not been studied yet. Exposure to a stress may indeed induce a general stress response enabling bacterial cross-protection from other stress conditions [START_REF] Kim | Regulation of oxidative stress resistance in Campylobacter jejuni, a microaerophilic foodborne pathogen[END_REF]. In a previous study, we evaluated the response of C. jejuni to the application of successive stresses inspired from the poultry slaughtering process at the phenotypic level [START_REF] Duqué | Influence of cell history on the subsequent inactivation of Campylobacter jejuni during cold storage under modified atmosphere[END_REF]: the inactivation of C. jejuni induced by cold storage significantly depended on the previously applied heat stress and differed according to the strain. Our results pointed out the complexity of bacterial behavior and strain variability, and highlighted the need to decipher the molecular mechanisms underlying adaptive responses through gene expression profiling.

The investigation of bacterial adaptive responses requires the use of molecular approaches. In this respect, reverse transcription quantitative PCR (RT-qPCR) is still considered as a standard method for accurate and sensitive measurement of gene expression [START_REF] Nolan | Quantification of mRNA using real-time RT-PCR[END_REF]. In addition to the recommendations about the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) [START_REF] Bustin | MIQE précis: Practical implementation of minimum standard guidelines for fluorescence-based quantitative real-time PCR experiments[END_REF][START_REF] Bustin | The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments[END_REF], stringent quality controls at each critical point throughout the entire RT-qPCR workflow are necessary [START_REF] Desriac | Trustworthy Identification of Resistance Biomarkers of Bacillus weihenstephanensis: Workflow of the Quality Assurance Procedure[END_REF]. Besides, to disentangle experimental variation from true biological variation, an internal control to compensate for experimental errors has to be used [START_REF] Huggett | Real-time RT-PCR normalisation; strategies and considerations[END_REF]. Housekeeping genes are generally used as internal controls. However, finding appropriate internal control genes whose expression remains stable whatever the experimental conditions is still quite a challenge [START_REF] Ritz | Determination of rpoA as the most suitable internal control to study stress response in C. jejuni by RT-qPCR and application to oxidative stress[END_REF].

In the current study, three C. jejuni strains were submitted to successive stresses inspired from those encountered during the poultry slaughtering process. First, an accurate method of RT-qPCR was developed [START_REF] Rezé | Validation de la RT-qPCR en vue d'évaluer l'effet de stress sur l'expression génique de Campylobacter jejuni[END_REF]. Second, this method was used to study the influence of successive hot and cold temperatures on the expression of selected genes to investigate how bacteria modulate gene expression after two successive stresses.

Materials and methods

Bacterial strains and culture conditions

The culture conditions described in [START_REF] Duqué | Influence of cell history on the subsequent inactivation of Campylobacter jejuni during cold storage under modified atmosphere[END_REF] were adopted. To sum up, three strains originally isolated from poultry were used: C. jejuni C09MJLT205, C. jejuni RM1221, and C. jejuni C97anses 640 [START_REF] Fouts | Major structural differences and novel potential virulence mechanisms from the genomes of multiple Campylobacter species[END_REF][START_REF] Guyard-Nicodeme | Prevalence and characterization of Campylobacter jejuni from chicken meat sold in French retail outlets[END_REF][START_REF] Guyard-Nicodeme | Characterization of Campylobacter spp. transferred from naturally contaminated chicken legs to cooked chicken slices via a cutting board[END_REF]. The cultures were stored at -80°C in brain heart infusion (BHI, Biomérieux, Marcy l'Etoile, France) supplemented with 20% (v/v) glycerol. Prior to the experiments, they were cultured on Karmali (Oxoid, Dardilly, France) agar plates at 42°C for 48-72 h under microaerobic conditions. Then, two successive cultures were incubated in Mueller-Hinton broth (MH, Oxoid, Dardilly, France) for 20 h and 18 h, respectively, under microaerobic atmosphere and shaking. Finally, 10-fold dilutions were prepared to obtain the final cultures, which were enumerated.

Induction of stress conditions

The experimental procedure was as described previously, with slight modifications [START_REF] Duqué | Influence of cell history on the subsequent inactivation of Campylobacter jejuni during cold storage under modified atmosphere[END_REF]. Briefly, C. jejuni strains were submitted to stresses inspired from conditions encountered during the poultry slaughtering process and considered as stressful. These steps are scalding and chilling, and are associated with heat and cold stresses.

The experimental design used "strain" and "hot bath temperature" as variables. For each strain, ten mL of the culture at an initial concentration of 8 log10 CFU.mL -1 were transferred into a glass tube and successively submitted to heat and cold. The glass tubes had the following dimensions: 150 mm in length, 18 cm in diameter and 1 mm in thickness. Heat stress consisted in immersing C. jejuni cultures in hot water baths at 46, 51 or 54°C for 4 min. After exposure to heat stress, the tubes were immediately cooled for 5 min in a water bath at 22°C. Cold stress was applied immediately after cooling by immersing the cultures into freezing-cold water containing ethylene glycol (-4°C, for 2 h).

In parallel, 10 mL from the same initial culture were kept as a control unstressed culture.

After each step, viable counts of C. jejuni were enumerated on Columbia sheep blood agar plates (Biomérieux, Marcy l'Etoile, France) and incubated for 48 h at 42°C under microaerobic conditions using a SPIRAL plater (EasySpiral Interscience, Saint Nom, France) to determine the viability loss, expressed in Δlog10, resulting from each step. Besides, a volume of culture was kept after each step for RNA extraction. Each experiment was performed in three independent replicates.

Transcriptomic analysis by RT-qPCR

The molecular response of C. jejuni was investigated using RT-qPCR instead of a global approach like RNA-seq because the final objective of this work will be to correlate the phenotypic response of C. jejuni (inactivation) with its transcriptomic response, using mathematical modeling to identify biomarkers of stress. The search for biomarkers represents a challenge, and this proof of concept was investigated using RT-qPCR as already performed by Desriac et al [START_REF] Desriac | mRNA biomarkers selection based on Partial Least Square algorithm in order to further predict Bacillus weihenstephanensis acid resistance[END_REF][START_REF] Desriac | An integrative approach to identify Bacillus weihenstephanensis resistance biomarkers using gene expression quantification throughout acid inactivation[END_REF][START_REF] Desriac | Prediction of Bacillus weihenstephanensis acid resistance: the use of gene expression patterns to select potential biomarkers[END_REF]. To this end, quantitative data had to be provided using a quantitative method. This was why we chose RT-qPCR rather than RNA-seq, which only provides the relative abundance of reads. Moreover, RT-qPCR is appropriate to study well-known phenotypes and associated genes well described in the literature.

Gene selection

Using RT-qPCR to evaluate the modulation of gene expression requires selecting a limited number of genes to be studied. To compare the transcriptomic response of C. jejuni following one stress versus two consecutive stresses, we chose forty genes already known to be involved in the C. jejuni stress response according to the literature. The selection was based on a decision tree as described in Figure 1. A literature search was first performed on the Web of Science platform by seeking articles with the following keywords in their title and/or their abstract: adaptive AND response One hundred and twenty studies were selected out of the initial batch of 1,096 publications because they showed differential gene expression following stresses or the involvement of a given gene in the stress response. Around 700 genes were considered in these studies, i.e. approximately 43% of the C. jejuni whole genome. To avoid selecting marginally studied genes, only genes studied in at least two different laboratories were kept. This represented 430 genes. All the regulators involved in the C. jejuni stress response (13 genes) were retained because we assumed that their expression would probably change under stress conditions. Then, we stringently selected 27 genes among the 430 genes studied in at least two different laboratories by trying to capture the variability of the metabolic pathways in which they were involved. To do so, the gene classification according to metabolic pathways was retrieved from the Kyoto Encyclopedia of Genes and Genomes (KEGG) database.

Finally, at least one gene from each metabolic pathway was picked up, but genes involved in several metabolic pathways and involved in different stress responses were preferentially selected. Out of 1,600 initially selected genes, 40 were eventually kept for analysis (Table 1).

Only the genes amplified by PCR and therefore present in the three strains were kept. Consequently, two genes -putA and rprB -were excluded from the analysis because they were not amplified in the C. jejuni C09MJLT205 strain and in the RM1221 and C94anses640 strains, respectively. The expression levels of the 38 remaining genes were then analyzed.

Selection of reference genes

Reference genes are needed for normalized expression. They are usually housekeeping genes, and their expression should remain constant whatever the experimental conditions. Reference genes were selected among a pool of 44 genes composed of i) six genes previously described as potential reference genes for RT-qPCR in C. jejuni [START_REF] Ritz | Determination of rpoA as the most suitable internal control to study stress response in C. jejuni by RT-qPCR and application to oxidative stress[END_REF], and ii) the 38 genes selected as potentially involved in the stress response.

Stability of gene expression was assessed as demonstrated by [START_REF] Vandesompele | Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes[END_REF]. The gene expression stability measure (M) was calculated using the geNorm module in qBase+ software (version 3.2, Biogazelle). To determine the optimal number of reference genes, the pairwise variation Vn/n+1 was calculated between the two sequential normalization factors (NFn and NFn+1) for all samples. The software program recommended that V should be less than 0.15.

Primer design

For each gene, primers were designed using Primer3Plus, a web interface developed by [START_REF] Untergasser | Primer3Plus, an enhanced web interface to Primer3[END_REF] based on the genome of strain RM1221 -the only sequenced strain in this study. Pairs with the smallest penalty score were retained, and sequences were tested for specificity using NCBI BLAST software, and compared to all available genomes of C. jejuni species. Oligonucleotide sequences of the targeted genes with BLAST score values greater than 30 were retained, as suggested by [START_REF] Desriac | Trustworthy Identification of Resistance Biomarkers of Bacillus weihenstephanensis: Workflow of the Quality Assurance Procedure[END_REF].

Determination of primer efficiency

DNA was isolated from unstressed C. jejuni RM1221 cultures. Briefly, 1 mL was centrifuged at 10,000 x g for 6 min. Then, the supernatant was removed, and DNA was extracted using a Dneasy® Blood and Tissue Kit (Qiagen, Courtaboeuf, France). Combined lysozyme (15 mg.mL -1 ) and proteinase K (20 mg.mL -1 ) digestion was used for lysis. DNA quantity and purity were measured using an Implen NanoPhotometer and stored at -20°C.

After in silico design, the PCR efficiency (E) of each primer pair was assessed (Table 2). Briefly, after thawing, each DNA suspension was serially diluted tenfold before amplification and quantification. Then, for each primer pair, Ct values were plotted on a logarithmic scale along with corresponding DNA concentrations. Efficiency was calculated from a linear regression curve through the data points, using the following equation: E = -1+10 (-1/slope) . All primer pairs showed satisfactory efficiency values ranging between 97 and 109% with an R² higher than 0.97.

RNA isolation

RNA was isolated from cultures (i) after the heat stress, and (ii) after the cold stress applied following the heat stress. RNA was also extracted from unstressed cultures that remained under optimal conditions (42°C in microaerobiosis) and considered as the control.

Briefly, 30 mL of stressed or unstressed culture were centrifuged at 10,000 x g at 4°C for 4 min. Thirty mL of culture were necessary to have enough RNA to extract (mostly after stress application). Consequently, the centrifugation step was performed before adding RNA protect to resuspend the pellet in a smaller volume of RNA-protect, as performed by Desriac et al. [START_REF] Desriac | mRNA biomarkers selection based on Partial Least Square algorithm in order to further predict Bacillus weihenstephanensis acid resistance[END_REF][START_REF] Desriac | An integrative approach to identify Bacillus weihenstephanensis resistance biomarkers using gene expression quantification throughout acid inactivation[END_REF][START_REF] Desriac | Prediction of Bacillus weihenstephanensis acid resistance: the use of gene expression patterns to select potential biomarkers[END_REF]. After removal of the supernatant, 1 mL of RNA protect (Qiagen, Courtaboeuf, France) was added to suspend the pellet. The suspension was incubated for 5 min at ambient temperature and then centrifuged at 5,000 x g at 4°C for 10 min as recommended by the manufacturer. The supernatant was removed, and the pellet was snap-frozen in liquid nitrogen and stored at -80°C for up to 1 month.

After cell pellets were thawed, RNA extraction was performed using an RNeasy® Mini Kit (Qiagen, Courtaboeuf, France) according to the manufacturer's recommendations. Combined lysozyme (15 mg.ml -1 ) and proteinase K (20 mg.ml -1 ) digestion was used for cell lysis. Contaminant genomic DNA was removed from each sample using an Invitrogen Turbo DNA-free ™ Kit (Ambion, Cambridge, USA). A first incubation of 30 min at 37°C was performed, and the supernatant was extracted. A second incubation of 30 min at 37°C was performed to efficiently remove contaminant genomic DNA.

Quality controls of the extracted RNAs were carried out to validate RNA isolation. First, RNA quantity and purity were measured using an Implen NanoPhotometer. Then, after RNA denaturation at 70°C for 9 min, RNA integrity was assessed by electrophoresis on agarose gel (1.2%). Profiles presenting RNA degradation were automatically discarded. The absence of genomic DNA in the RNA samples was checked by PCR. Amplification was performed using primers 341F and 758R [START_REF] Rodrigues | Comparison of proteomics profiles of Campylobacter jejuni strain Bf under microaerobic and aerobic conditions[END_REF]. The thermo-cycling program consisted of 95°C for 5 min, followed by 30 cycles of 30 s at 95°C, 30 s at 59°C and 30 s at 68°C. The absence of DNA was checked using agarose gel (1.2%) electrophoresis.

Each extraction was performed on three independent replicates.

Reverse Transcription (cDNA synthesis)

As recommended by [START_REF] Stahlberg | Properties of the reverse transcription reaction in mRNA quantification[END_REF], reverse transcription was performed on 200 ng of the total RNA extract using an iScript cDNA synthesis kit (Bio-Rad, Marnes la Coquette, France). The cDNA synthesis program consisted in initial priming at 25°C for 5 min, followed by reverse transcription at 46°C for 20 min, and inactivation at 95°C for 1 min. cDNA was then stored at -20°C.

Quantitative PCR

Amplification and quantification were performed using a CFX connect device (Bio-Rad, Mitry-Mory, France). For each sample, the reaction mixture contained 10 µL of SoAdvanced SYBR Green Supermix (Bio-Rad, Marnes la Coquette, France), 1 µL of each forward and reverse primer, 4 µL of water and 4 µL of cDNA. The program consisted in initial denaturation at 95°C for 3 min, followed by 40 cycles of 15 s at 95°C and 30 s at 60°C.

A sample maximization method was used, as advised by [START_REF] Hellemans | qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data[END_REF]. All samples were assayed in the same run, but genes were analyzed separately. This method avoided the need for an inter-run calibrator (IRC).

Data analysis

Gene expression quantification was performed after each step of the experimental setup (i.e. after heat stress and after successive heat and cold stresses). RT-qPCR results were expressed in relative quantity scaled to the expression of the same gene in the control sample (i.e. unstressed culture) using the delta Ct transformation with efficiency correction. Then, expression levels were normalized relatively to the expression levels of the reference genes. Reference genes were selected among the 44 genes.

The normalization factor (NF) was calculated as the geometric mean of reference gene expression [START_REF] Vandesompele | Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes[END_REF]. After normalization, relative gene expression was log2 transformed for further ANOVA analysis [START_REF] Bengtsson | Gene expression profiling in single cells from the pancreatic islets of Langerhans reveals lognormal distribution of mRNA levels[END_REF].

For each strain, ANOVA analysis (α=5%) using XLSTAT software (version 2018.5) was performed to assess if the application of successive heat and cold stress had a significant impact on the gene expression compared to the gene expression after the heat stress only. Besides, ANOVA analysis was also performed on genes whose absolute relative expression level (in log2) was altered more than 2fold, to assess the impact of the different temperatures of heat stress (Reid et al., 2008a). Fisher's least significant difference test (P<0.05) was applied to determine significant differences in mean log2 fold changes between stresses.

Hierarchical cluster analysis of significantly differentially expressed transcripts was performed using the function heatmap.2 of the gplots package in R. Only genes with an expression level above a 2 log2 fold change or below a -2 log2 fold change were considered (Reid et al., 2008a).

Results

Selection of reference genes

As depicted by [START_REF] Desriac | Trustworthy Identification of Resistance Biomarkers of Bacillus weihenstephanensis: Workflow of the Quality Assurance Procedure[END_REF], appropriate quality procedures and quality controls should be set up and assessed carefully to reliably interpret RT-qPCR results. The selection of good reference genes is one of the numerous critical points in the procedure. A good reference gene should display constant expression whatever the condition, and the number of reference genes has to be adapted according to their stability.

To select genes with the most stable expression, all 44 genes were considered as candidate reference genes, including those selected as targets. Two criteria were adopted to select the reference genes: (i) the M value, calculated for each gene and for each strain (Supplementary Figure S1), and (ii) pairwise variation -Vn/n+1 -, calculated for each strain. The V2/3 pairwise variation values were < 0.15, meaning that the optimal number of reference genes in this experimental situation was 2 (Supplementary Figure S2). Finally, the two most stable genes were retained for each strain (Table 3). Unfortunately, the reference genes were not the same for all three strains.

C. jejuni inactivation following application of successive stresses

C. jejuni encounters several stresses during the poultry slaughtering process. For example, the scalding and chilling steps can be assimilated to heat and cold stresses, respectively. The inactivation generated by these stresses varied from -0.28 ± 0.10 to 0.54 ± 0.04 according to the strain and the stress applied (Table 4). Negative values mean no inactivation due to the uncertainty from enumeration method.

Since bacteria may respond differently and use different mechanisms of defense according to the stress encountered, their gene expression may also differ from one stress to another. Thus, the gene expression was expected to be modified following the application of the second cold stress in comparison with the application of the only first heat stress. This was evaluated in the next section.

Differential gene expression of C. jejuni between heat and cold stress

Gene expression was evaluated both after heat stress alone and after successive application of heat and cold stresses. Analysis of variance was performed per strain to determine if gene expression was altered in a stress-dependent manner. For C97anses640 strain, gene expression was shown to significantly depend on gene, temperature of the heat stress and the application or not of the second stress, i.e. the cold stress (P < 0.001). For the other two strains, the application of the cold stress was shown to affect expression of specific genes only when heat stress had been performed at some temperatures (temperature*coldstress and gene*coldstress significant interactions; P<0.001). It appeared that the application of the second stress altered more expression of some genes when strains had been previously submitted to heat stress at 46°C, rather than at 51°C or at 54°C. Genes whose expression was significantly altered at the different temperatures of the heat stress are listed in supplementary materials (Table S1). However, the difference in expression between both stresses remained very low, generally below 2 log2 fold changes. Among these genes, it is noteworthy that four genes were differentially expressed by all strains after application of 46°C. These are: katA, lysR, pebC and racR. Expression of these genes after the heat stress applied at 46°C with or without subsequent application of the cold stress is described in Figure 2. It appears that heat stress followed by cold stress indeed induced a slight change in expression, but in the same direction (up or downregulation) whatever the strain. Three genes, i.e. katA, lysR, pebC, were downregulated following the heat plus cold stresses whereas racR was upregulated. The highest change in expression between the heat stress at 46°C and the heat plus cold cold stresses was observed for pebC, downregulated from -2 to -3.9 log2 fold changes depending on the strain.

Differential C. jejuni gene expression following the consecutive stresses

Gene expression following heat + cold stresses slightly differed from gene expression following heat stress.

Only genes differentially expressed by more than 2 log2 fold change (in absolute value), were considered (Reid et al., 2008a). Overall, among the 44 selected genes, 26 were differentially expressed in at least one of the three heat stress conditions and in one or more strains, with a maximum log2 fold change of 6.21 and a minimal fold change of -4.17 (Supplementary Table S2).

Consequently, these 26 differentially expressed genes were subjected to hierarchical clustering analysis and were grouped into 3 main clusters designated as A, B, and C (Figure 3).

The four genes belonging to cluster A were globally up-regulated in the three strains, and their overexpression varied between 2 and 6 log2-fold change according to the gene and the strain. However, the expression of all four genes decreased at 54°C in strain C09MJLT205, compared with 46 and 51°C. This cluster mainly included genes associated with bacterial heat shock responses, namely the transcriptional regulator hrcA and several chaperones and co-chaperones considered as heat shock proteins (HSPs) (grpE, dnaK and clpB), in agreement with heat stress (Figure 4A).

Clusters B and C were composed of genes differentially expressed according to the strains (Figure 3). Strain C97anses640 had a rather different pattern of gene expression from that of the other two strains.

Cluster B harbored two categories of genes. A first pool was mainly overexpressed in strain C97anses640 when temperature increased, with a maximum of 3.1 log2 fold change, while they were little or not differentially expressed in the other two strains. This group included genes encoding three regulators -cbrR, hspR and CJE1780 -, and the chaperone groEL, and nuoL (Figure 4B). The hspR gene encodes a repressor of the previously described operon clpB-grpE-dnaK, which encodes HSPs. The second group of genes was mainly under-expressed in strains C09MJLT205 and RM1221, down to -2.88 log2 fold change, while it remained relatively stable or slightly increased in strain C97anses640. This group was included pebC, rpoB, gyrA, thiC, trpD, ilvC (Figure 4C).

Lastly, cluster C contained eleven genes significantly down-regulated in the three strains from 51°C. Down-regulation varied between -2 and -4 log2 fold change. It seemed to increase as temperature increased beyond 46°C for strain C09MJLT205, and 54°C for the other two strains, suggesting that the regulation threshold could be strain dependent. This cluster included a two-component sensor histidine kinase CJE1361, a fliP gene, and genes belonging to the amino acid metabolism (argF, slyD), the lipid metabolism (kdtA, yciA) and the energy metabolism (atpA). The regulators cosR (Figure 4D) and csrA, and the katA gene -which are involved in the bacterial oxidative stress response -also belonged to this cluster.

Discussion

The objective of the study was to assess the impact of successive hot and cold temperatures on C. jejuni gene expression to better understand the adaptation capacity of this pathogen. In addition, we investigated the strain variability response at the molecular level.

Studying gene expression requires an accurate and validated RT-qPCR method to quantify transcripts. Following the recommendations of [START_REF] Desriac | Trustworthy Identification of Resistance Biomarkers of Bacillus weihenstephanensis: Workflow of the Quality Assurance Procedure[END_REF], each step was validated [START_REF] Rezé | Validation de la RT-qPCR en vue d'évaluer l'effet de stress sur l'expression génique de Campylobacter jejuni[END_REF]. Besides, the use of reference genes is mandatory to normalize the expression of target genes. A suitable reference gene should display stable expression under the experimental conditions of the study. Otherwise, the selection of inappropriate reference genes may bias gene expression quantification [START_REF] Dheda | The implications of using an inappropriate reference gene for real-time reverse transcription PCR data normalization[END_REF][START_REF] Hyytiainen | Importance of RNA stabilization: evaluation of ansB, ggt, and rpoA transcripts in microaerophilic Campylobacter jejuni 81-176[END_REF][START_REF] Udvardi | Eleven golden rules of quantitative RT-PCR[END_REF]. In most cases, reference genes are a priori selected among genes commonly found in the literature, such as housekeeping genes. We did not limit our selection to genes previously identified in the literature, but considered a pool of 44 genes to evaluate the stability of their expression following heat and cold stresses. The ΔΔCt method is largely used to quantify gene expression. However, according to this method, only one reference gene is used to normalize gene expression [START_REF] Livak | Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method[END_REF]). The use of two reference genes increased quantification accuracy. The pairs of reference genes we chose varied according to the strains (the development of universal reference genes is not always feasible [START_REF] Bustin | The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments[END_REF]. Five different reference genes were selected for the three C. jejuni strains: rrs -common to all three strains -, and asd, proS, thiC and dsbI. rrs has been commonly used as a reference gene in several previous works [START_REF] Bronnec | Adhesion, biofilm formation, and genomic features of Campylobacter jejuni Bf, an atypical strain able to grow under aerobic conditions[END_REF][START_REF] Hyytiainen | Importance of RNA stabilization: evaluation of ansB, ggt, and rpoA transcripts in microaerophilic Campylobacter jejuni 81-176[END_REF][START_REF] Turonova | Profiling of Campylobacter jejuni proteome in exponential and stationary phase of growth[END_REF]. asd, proS, and thiC are involved in the amino-acid biosynthesis pathway, and dsbI is part of the C. jejuni redox system required for disulfide bond formation in some proteins. Surprisingly, rpoA and slyD were considered as reference genes for C. jejuni [START_REF] Phongsisay | Expression of the htrB gene is essential for responsiveness of Salmonella typhimurium and Campylobacter jejuni to harsh environments[END_REF][START_REF] Ritz | Determination of rpoA as the most suitable internal control to study stress response in C. jejuni by RT-qPCR and application to oxidative stress[END_REF][START_REF] Stintzi | Gene expression profile of Campylobacter jejuni in response to growth temperature variation[END_REF], but turned out to be pretty bad reference genes in the current study because their expression changed following stress. This highlights the importance of a robust strategy to select reference genes.

The expression of 44 genes was quantified by RT-qPCR in three C. jejuni strains (C09MJLT205, RM1221, and C97anses640) after exposure to stresses inspired by the poultry slaughtering process. In order to mimic conditions that may be encountered by Campylobacter, three steps were successively applied, namely a hot stress assimilated to the scalding step, followed by a short resting time at 22°C assimilated to the time poultry is left at ambient temperature between scalding and chilling, and finally a cold step, assimilated to chilling. These conditions were determined after visiting several slaughterhouses in France [START_REF] Duqué | Influence of cell history on the subsequent inactivation of Campylobacter jejuni during cold storage under modified atmosphere[END_REF].

In addition to transcriptional analyses, the three strains were enumerated following these stresses. The combined heat and cold stresses barely inactivated all three strains. Despite this low inactivation, these stresses influenced C. jejuni gene expression, since 24 genes were differentially expressed under at least one of the temperature conditions and in one or more strains. By selecting genes a priori, we found that 59% of them were differentially expressed in at least one of these stressful conditions, showing that such a selection procedure may be suitable when a limited number of genes is targeted. Global transcriptomic approaches highlighted that around 20% of the genes were differentially expressed after a thermal stress (Riedel et al., 2020a;[START_REF] Stintzi | Gene expression profile of Campylobacter jejuni in response to growth temperature variation[END_REF]. This represents a higher number of coding sequences than in the present study.

Different profiles of strain-dependent gene expression emerged after thermal stress. Genes expressed in the same manner after stress whatever the strain were mainly involved in the heat shock response. Whereas genes differentially expressed according to the strain were involved in the oxidative stress response or the central metabolism. Most of these genes (14/24) were underexpressed in C. jejuni C09MJLT205 and RM1221 strains from 51°C or 54°C, respectively. Besides, strain variability was higher than biological variability (reproducibility of biologically independent replicates) and was consistent with the literature [START_REF] Aryani | Quantifying variability on thermal resistance of Listeria monocytogenes[END_REF][START_REF] Den Besten | Microbial variability in growth and heat resistance of a pathogen and a spoiler: All variabilities are equal but some are more equal than others[END_REF]. This result highlights the reproducibility of the experimental method.

The comparison of the C. jejuni transcriptional response following application of one stress versus two consecutive stresses revealed that only few genes were differentially expressed following heat stress alone versus heat plus cold stresses. Although the transcriptomic response to cold stress was not investigated alone (without pre-adaptation to heat-shock), the comparison between gene expression after heat stress alone (H) and successive heat stress + ambient temperature + cold stress (H+C), seemed to show that the heat stress had a limited pre-adaptation impact on the C. jejuni transcriptional response to cold stress (H ~ H+C). The molecular response to the cold stress is poorly documented in C. jejuni. No gene encoding cold shock proteins has been identified in genomes available [START_REF] Parkhill | The genome sequence of the food-borne pathogen Campylobacter jejuni reveals hypervariable sequences[END_REF], which suggests that this pathogen may have other tolerance mechanisms to respond to cold stress. Studies so far have shown oxidative stress defense, particularly SodB, played an important role in the cold stress tolerance of Campylobacter [START_REF] Garenaux | Role of oxidative stress in C. jejuni inactivation during freeze-thaw treatment[END_REF][START_REF] Stead | Roles of Fe superoxide dismutase and catalase in resistance of Campylobacter coli to freeze-thaw stress[END_REF][START_REF] Stintzi | Gene expression profile of Campylobacter jejuni in response to growth temperature variation[END_REF]. In comparison with heat shock, cold stress applied in the current study induced repression of pebC gene and overexpression of racR in the three C. jejuni strains. Some studies have already investigated the implication of RacR in C. jejuni thermoregulation under elevated temperatures [START_REF] Apel | Characterization of Campylobacter jejuni RacRS reveals roles in the heat shock response, motility, and maintenance of cell length homogeneity[END_REF][START_REF] Brás | A novel Campylobacter jejuni two-component regulatory system important for temperature-dependent growth and colonization[END_REF]. Our work suggests a potential role of this regulator in a wider range of temperatures. Regarding pebC gene, the transition from heat to cold stress led to a dramatically expression decrease by 4 log2 fold changes, which is different from the results obtained by [START_REF] Shi | Proteomic analysis of the cold stress response in Campylobacter jejuni[END_REF] [START_REF] Shi | Proteomic analysis of the cold stress response in Campylobacter jejuni[END_REF]. The pebC gene encodes a component of the amino acid ABC transporter involved in aspartate and glutamate metabolism and required for the microaerobic growth [START_REF] Stahl | Nutrient acquisition and metabolism by Campylobacter jejuni[END_REF]. This drop may be linked to a decrease in glutamate utilization during cold stress, because of its rapid metabolization during stationary phase before stress induction [START_REF] Stahl | Nutrient acquisition and metabolism by Campylobacter jejuni[END_REF].

Following the successive heat and cold stresses, the gene expression profiles of each strain were compared by hierarchical clustering analysis. Two strains -C09MJLT205 and RM1221 -had a rather similar profile compared with the third one (C97anses640). The 24 genes differentially expressed following the two stresses were mainly categorized into three different groups: i) genes involved in the general stress response, ii) regulators, and iii) genes involved in the central metabolism.

Among the 24 differentially expressed genes, four genes -the transcriptional regulator hrcA and the chaperone-encoding genes clpB, dnaK, grpE -were up-regulated by thermal stress whatever the strain. This suggests that these genes can be considered as biomarkers of sublethal thermal stress in C. jejuni. The up-regulation of these genes was consistent with several results demonstrating the involvement of these HSPs in response to heat, acid or oxidative stresses in C. jejuni but also in C. coli and C. lari [START_REF] Andersen | Diverse roles for HspR in Campylobacter jejuni revealed by the proteome, transcriptome and phenotypic characterization of an hspR mutant[END_REF][START_REF] Cameron | Hyperosmotic stress response of Campylobacter jejuni[END_REF][START_REF] Flint | Phenotypic screening of a targeted mutant library reveals Campylobacter jejuni defenses against oxidative stress[END_REF][START_REF] Holmes | The hrcA and hspR regulons of Campylobacter jejuni[END_REF][START_REF] Konkel | Characterization of the thermal stress response of Campylobacter jejuni[END_REF][START_REF] Palyada | Characterization of the oxidative stress stimulon and PerR regulon of Campylobacter jejuni[END_REF]Reid et al., 2008a;Reid et al., 2008b;Riedel et al., 2020a;Riedel et al., 2020b;[START_REF] Stintzi | Gene expression profile of Campylobacter jejuni in response to growth temperature variation[END_REF][START_REF] Sulaeman | Enhanced adhesion of Campylobacter jejuni to abiotic surfaces is mediated by membrane proteins in oxygen-enriched conditions[END_REF]. They act by repairing and preventing damage caused by the accumulation of unfolded proteins [START_REF] Stintzi | Gene expression profile of Campylobacter jejuni in response to growth temperature variation[END_REF]. Several heat shock proteins (e.g., DnaK) also play a crucial role under optimal physiological conditions by assisting in the proper folding of newly synthesized proteins [START_REF] Stintzi | Gene expression profile of Campylobacter jejuni in response to growth temperature variation[END_REF]. However, other genes were expected to be differentially expressed following thermal stress. For example, hspR -encoding a transcriptional regulator -and the chaperone-encoding gene groEL, both involved in the C. jejuni heat stress response, were not significantly over-expressed in any of the three strains.

The heatmap analysis also highlighted that three transcripts were significantly less abundant in the three strains at the highest temperature. These genes included the two regulators cosR and csrA, which are involved in the C. jejuni oxidative stress response [START_REF] Fields | Campylobacter jejuni CsrA mediates oxidative stress responses, biofilm formation, and host cell invasion[END_REF][START_REF] Garénaux | Role of the Cj1371 periplasmic protein and the Cj0355c two-component regulator in the Campylobacter jejuni NCTC 11168 response to oxidative stress caused by paraquat[END_REF]Hwang et al., 2011b). The differential expression of genes involved in the oxidative stress response was no surprise because C. jejuni were exposed to atmospheric oxygen in addition to heat stress. Furthermore, oxidative stress is related to the C. jejuni response to temperature stress. This pathogen is indeed more susceptible to oxidative stress at high temperatures (42°C) than at low temperatures (4°C), suggesting that temperature affects oxidative stress resistance in C. jejuni [START_REF] Garenaux | Survival of Campylobacter jejuni strains from different origins under oxidative stress conditions: effect of temperature[END_REF]. In Escherichia coli, increasing heat increases the generation of reactive oxygen species (ROS), which leads to an oxidative stress response [START_REF] Marcén | Oxidative stress in E. coli cells upon exposure to heat treatments[END_REF]. Interestingly, the expression levels of genes involved in the oxidative stress response, namely cosR, csrA and katA, were lower with increased temperature. This result can be linked to oxygen solubility in water, which decreases as temperature increases. This may have resulted in a lower oxygen concentration in contact with C. jejuni cells, and in turn lower expression of these genes at 54°C than at 46°C. CosR (Campylobacter oxidative stress regulator) is an OmpR-type response regulator involved in the control of oxidative stress resistance in C. jejuni (Hwang et al., 2011a;Hwang et al., 2011b;[START_REF] Hwang | Transcriptional regulation of the CmeABC multidrug efflux pump and the KatA catalase by CosR in Campylobacter jejuni[END_REF]. Autoregulation of CosR could also be influenced by the post-transcriptional regulator CsrA [START_REF] Fields | Campylobacter jejuni CsrA regulates metabolic and virulence associated proteins and is necessary for mouse colonization[END_REF]. CosR may respond selectively to superoxide stress (compared to peroxide stress), as superoxide is the first toxic by-product of the oxygen reduction cycle (Hwang et al., 2011b). Resistance of C. jejuni to superoxide stress (and to aerobiosis conditions) may ensue from reduced CosR protein levels, which would derepress sodB. SodB is the only enzyme able to detoxify superoxide ions in C. jejuni. In addition, decreased cosR expression may lead to reduced katA and ahpC gene expression, which cannot contribute to superoxide detoxification [START_REF] Hwang | Transcriptional regulation of the CmeABC multidrug efflux pump and the KatA catalase by CosR in Campylobacter jejuni[END_REF]. In the present study, cosR and katA gene expression decreased at the highest temperature in the three strains, in line with the literature (Hwang et al., 2011a;Hwang et al., 2011b;[START_REF] Hwang | Transcriptional regulation of the CmeABC multidrug efflux pump and the KatA catalase by CosR in Campylobacter jejuni[END_REF]. However, sodB and ahpC expression was not significantly affected. It should be recalled that CosR is not the only regulator of ROS detoxification genes. Regulators PerR and Fur also play a role [START_REF] Butcher | Refined analysis of the Campylobacter jejuni iron-dependent/independent Fur-and PerR-transcriptomes[END_REF][START_REF] Kim | Regulation of perR Expression by Iron and PerR in Campylobacter jejuni[END_REF][START_REF] Palyada | Characterization of the oxidative stress stimulon and PerR regulon of Campylobacter jejuni[END_REF][START_REF] Van Vliet | Campylobacter jejuni contains two fur homologs: characterization of iron-responsive regulation of peroxide stress defense genes by the PerR repressor[END_REF]. Therefore, these findings underline the complexity of the regulation of gene expression and an interaction between coregulators and the environment.

Eleven genes implied in the lipid, amino acid and energy metabolisms were down-regulated in the two C09MJLT205 and RM1221 strains from 51°C and at 54°C, respectively. Among them, four genes were involved in amino-acid transport and metabolism (slyD, argF, trpD, ilvC), in line with previous studies dealing with the effect of cold or heat stresses on Campylobacter [START_REF] Guccione | Transcriptome and proteome dynamics in chemostat culture reveal how Campylobacter jejuni modulates metabolism, stress responses and virulence factors upon changes in oxygen availability[END_REF][START_REF] Shi | Proteomic analysis of the cold stress response in Campylobacter jejuni[END_REF]. Furthermore, the down-regulation of csrA in the three strains at the highest temperature could explain the low level of transcripts involved in the amino acid metabolism (ilvC, argF), the energy metabolism (atpA) and the stress response (katA), as shown by [START_REF] Fields | Campylobacter jejuni CsrA regulates metabolic and virulence associated proteins and is necessary for mouse colonization[END_REF] in csrA mutants. CsrA appears to regulate a number of cellular processes, including transcription and translation, the nucleotide / amino acid metabolism, DNA repair, energy homeostasis, etc. [START_REF] Fields | Campylobacter jejuni CsrA regulates metabolic and virulence associated proteins and is necessary for mouse colonization[END_REF].

Genes encoding cell surface components were also down-regulated, such as yciA involved in the lipid metabolism, fliP in flagellum biosynthesis, and kdtA encoding for lipid A of the lipooligosaccharide (LOS). C. jejuni LOS consists of a lipid A moiety, an inner core composed of a conserved trisaccharide, and a strain-variable outer core consisting of various sugars [START_REF] Karlyshev | The Campylobacter jejuni glycome[END_REF]. This structure is important for many pathogens to bypass the host immune defenses. The effect of temperature on LOS length is strain-dependent [START_REF] Semchenko | Temperaturedependent phenotypic variation of Campylobacter jejuni lipooligosaccharides[END_REF], suggesting a role in the pathogen's adaptive mechanisms or stress response. kdtA can also be down-regulated under acid or oxidative stress conditions [START_REF] Guccione | Transcriptome and proteome dynamics in chemostat culture reveal how Campylobacter jejuni modulates metabolism, stress responses and virulence factors upon changes in oxygen availability[END_REF][START_REF] Kaakoush | Insights into the molecular basis of the microaerophily of three Campylobacterales: a comparative study[END_REF][START_REF] Palyada | Characterization of the oxidative stress stimulon and PerR regulon of Campylobacter jejuni[END_REF]Reid et al., 2008a), but not under heat stress conditions [START_REF] Stintzi | Gene expression profile of Campylobacter jejuni in response to growth temperature variation[END_REF]. In addition, cell envelope proteins can be down-regulated following cold shock [START_REF] Shi | Proteomic analysis of the cold stress response in Campylobacter jejuni[END_REF][START_REF] Stintzi | Investigation of the Campylobacter jejuni cold-shock response by global transcript profiling[END_REF]. These findings could suggest a remodeling of the membrane and cell wall structures in response to stress. The cell membrane composition is generally modified by bacteria under unfavorable environments such as heat or cold stress in order to maintain membrane integrity against stressful conditions [START_REF] Hughes | The response of Campylobacter jejuni to low temperature differs from that of Escherichia coli[END_REF][START_REF] Siliakus | Adaptations of archaeal and bacterial membranes to variations in temperature, pH and pressure[END_REF]. The rapid chilling of C. jejuni cultures, analogous to poultry carcass chilling, would result in a rapid and large-scale shutdown of cellular processes [START_REF] Hughes | The response of Campylobacter jejuni to low temperature differs from that of Escherichia coli[END_REF].

Interestingly, two strains, C09MJLT205 and RM1221, had rather similar transcriptomic profiles compared with the third one, C97anses640, in agreement with the results of a previous study. Indeed, C. jejuni C97anses640 previously submitted to successive heat and cold stresses was less resistant than strains C09MJLT205 and RM1221 to a subsequent stressing step of refrigerated storage under a modified atmosphere [START_REF] Duqué | Influence of cell history on the subsequent inactivation of Campylobacter jejuni during cold storage under modified atmosphere[END_REF]. These results suggest a possible correlation between stress-induced gene expression and future bacterial behavior.

Conclusion

Consecutive thermal stresses inspired from the broiler slaughtering process induced a limited inactivation of C. jejuni populations, but altered the molecular response of the pathogen at the transcriptomic level. Among the 44 genes under study, 24 were differentially expressed following the two stresses and belonged to three different groups: i) genes involved in the general stress response, ii) regulators, and iii) genes involved in the central metabolism. Our results pointed out transcriptomic variability of C. jejuni strains exposed to stresses based on the poultry slaughtering process. Indeed, thermal stress application induced strain-dependent gene expression patterns, with one strain exhibiting a profile significantly different from the two other strains. Genes involved in the heat shock response were mainly overexpressed after stress application in the same manner no matter the strain. In contrast, genes implicated in the oxidative stress response or the central metabolism were differentially expressed according to the strain. For example, it appeared that among the differentially expressed genes, some genes involved in oxidative response like CbrR, CJE1780 and nuoL, were mainly more expressed in the C97anses640 than in the two other strains whereas genes involved in amino acid metabolism, lipid metabolism and energy metabolism were less down regulated in the atypical strain. These results suggest that the adaptative stress response in bacteria is strain-dependent. Since the atypical C97anses640 strain was also shown to behave differently according to stress, these results pave the way for further investigations on bacterial adaptation.

Further work is indeed needed to consider the possible correlation between the altered gene expression patterns highlighted in the current work and enhanced C. jejuni resistance to a subsequent stress. If this is confirmed, these genes could be used as biomarkers to improve the prediction of bacterial fitness and adaptation because biomarkers are a promising avenue for next-generation predictive models. Campylobacter molecular response to stress Table 3. Reference genes selected and associated M value for each C. jejuni strain. In bold, the two genes selected as internal reference for each strain, according to their M-value (< 0.5).

C. jejuni strain

Table 2 .

 2 Primer pairs sequences for C. jejuni genes used in this study 824

	Genes	Forward	Reverse	Amplicon size (pb)	Efficiency (%)	R²
	Control of RNA samples
	341F/758R	CCTACGGGAGGCAGCAG	CTACCAGGGTATCTAATCC	439	/	/
	Potential reference genes
	rpoA	CGAGCTTGCTTTGATGAGTG	AGTTCCCACAGGAAAACCTA	80	100	1.00
	rrs	AAGGGCCATGATGACTTGAC	AGCGCAACCCACGTATTTAG	107	100	1.00
	ilvC	GCATGCAGAACGCAAAAATA	TGATCCAAGGCATCATAGCA	109		1.00
	gyrA	GTTATTATAGGTCGTGCTTT	CTATGAGGTGGGATGTTTGT	92		1.00
	slyD	TACGATGAAAATGCCGTTCA	TTCGCCAAAAAGCTCCATAC	84		1.00
	thiC	TTATCTTTGGGCGATGCTTT	CATCCCAAGCCCTTTGAGTA	109		1.00
	Target genes
	hrcA	GGGCTTAAGGTAGATGCGCA	TCATGCCGCCTCCTTTATGT	111		0.97
	groEL	AGTAATCGGTGGTGGTGCAG	CTTTCAACGATAGCTGCGCC	96	109	1.00
	atpA	CGTGAAGCTTATCCAGGCGA	GCAATGCCGTCAAAGAACCA	103		1.00
	clpX	AGCTGCAGATGGAGATGTGC	AAGCTTGTTGCACACCCTCT	131		1.00
	dnaK	CACGCGGTATGCCACAAATC	CCTCGCTAAGTCCGCTTGAA	132		1.00
	sodB	CAAAACTTCAAATGGGGGCGT	ACAGCCACAGCCTGTACTTG	97		1.00
	katA	CAGGCGCAAAAGGACCTTTG	TTCCCTTGGCATGAACGGTT	105		1.00
	perR	CCTTCAATCTCTTTAGCGACGG	CCACATTTGGTGCAAACAACA	146		1.00
	cbrR	CTTGCCAGATGCCCCAAATG	GCCTTATCACCACTAGCCGT	90		1.00
	CJE1780	TCCAACAAGCAAAGCAGTGC	AGAGATTGTTTTCTGTGGGAAGT	136		1.00
	cmeR (tetR)	TCCAATTGGCAAGATGTCTATCT	TCAATCAACCAGAAGCTGTAGC	81		1.00
	cosR	GGGAAGCAGAAACGGTATCCT	TGCCATCTGGGAAGAACCTG	141		1.00
	fur	CCATTTCTTTTGGTTCAGCAGGT	GCAATCAAGGCTTGCTGTCT	136		1.00
	hspR	AGAGCCAAGTAGAACCGATGG	ACTCCAGCAAGATTGATTCCCA	111		1.00
	lysR	CGCTCTCAAACGCAGAATTCA	AGCTGGATCAAGTTCTGCTTCA	93		1.00
	putA	GCTAGTGGTGCAGCTATGGT	TTCGCACCTAAGGGCATAGG	83	106	1.00
	racR	TTGTGGGGCTTCAAATCGGT	TCAACTCTTTTTGTGCGACGA	106		1.00
	rprA	CGCTCAAATTCCACCCAAGG	TGCCCCATTTGCTCATAGCT	89		0..98
	rprB	CGAGATTTGTTGCAAGGGACTAA CTTTGCGTTTGATCAGTTTTGC	139		1.00
	fliP	TTTGCGTCCCCATAGCTTGT	TGCGCTCGCTCCAAGTATAG	92		1.00
	dsbl	AAGCCCTACTGCTGTCATGA	GCTGGGGACAAAGAGAAGCA	116		1.00
	pebC	GGTTTCTGGATCAAGGGCTGA	AGCAACCCTTTCAGGTGGAC	112		1.00
	htrA	TGCCGTTATTTCCACCACCA	CAGGTGGAGCTTTGGTGGAT	84	103	1.00
	ahpC	GAACAGTTCGCCATGCTGTG	TCGCCCTTATTCCATCCTGC	133		0.99
	grpE	TGAAAAACATGGGGTGGCTCT	AGCACTTGAACCACTTCACCA	117		1.00
	hupB	TCTTTTTGCCGCTTGCTTTTG	GCTAGAGTACCAAGCACAGGA	112		1.00
	proS	GGCTATTGGTGGAAGTGGCT	GCAGCTTCAACATTAGCCGC	108		1.00
	asd	GCATTCAGGAACCACCAAAGG	GCAGGTGGAAGTGTGAGTGA	120		1.00
	sdhA	GGTAGATCAAGACGCGGCTT	AAACGCTGAGCTACTCCACC	117		1.00

Table 4 .

 4 Mean inactivation (expressed in Δlog CFU.mL -1 ) induced by heat stress only, cold stress only and cumulated stresses, with respective standard deviation, of the three strains of C. jejuni (C97anses40, C09MJLT205 and RM121) as a function of the heat stress temperature (54, 51 and 46°C).

		Gene	M value
		asd	0.16
	C09MJLT205	dsbI proS	0.475 0.16
		thiC	0.255
		rrs	0,485
		asd	0.41
	RM1221	dsbI	0.735
		proS	0.790
		thiC	0.865
		rrs	0,410
		asd	0,525
	C97anses640	dsbI	0.30
		proS	0.375
		thiC	0.30
		rrs	0,565

Negative values mean no inactivation due to the uncertainty from enumeration method.

Acknowledgments

We especially thank the Regions Pays de La Loire and Bretagne for their financial support in the framework of the Biomics project. BD obtained a PhD-fellowship grant from the Région Pays de la Loire.

Conflict of Interest

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. Author Contributions J-M.M., S.G. and N.H. conceived the original idea. B.D., J-M.M., S.G. and N.H. conceived and planned the experiments. B.D., S.R., and A.R. carried out the experiments. B.D., S.R., S.G. and N.H. contributed to the interpretation of the results. B.J., S.G. and N.H. took the lead in writing the manuscript. All authors provided critical feedback and helped shape the research, analysis and manuscript.

Campylobacter molecular response to stress Regulation/regulator hrcA CJE0848 heat-inducible transcriptional repressor X X X X [START_REF] Cameron | Hyperosmotic stress response of Campylobacter jejuni[END_REF][START_REF] Flint | Phenotypic screening of a targeted mutant library reveals Campylobacter jejuni defenses against oxidative stress[END_REF][START_REF] Holmes | The hrcA and hspR regulons of Campylobacter jejuni[END_REF][START_REF] Palyada | Characterization of the oxidative stress stimulon and PerR regulon of Campylobacter jejuni[END_REF]Reid et al., 2008b;[START_REF] Stintzi | Gene expression profile of Campylobacter jejuni in response to growth temperature variation[END_REF] perR CJE0367 peroxide stress response regulator X X [START_REF] Burgess | The response of foodborne pathogens to osmotic and desiccation stresses in the food chain[END_REF][START_REF] Palyada | Characterization of the oxidative stress stimulon and PerR regulon of Campylobacter jejuni[END_REF]Reid et al., 2008b) cbrR CJE0746 two-component response regulator X X [START_REF] Cameron | Hyperosmotic stress response of Campylobacter jejuni[END_REF][START_REF] Kaakoush | Insights into the molecular basis of the microaerophily of three Campylobacterales: a comparative study[END_REF] Fur CJE0449 fur ferric uptake regulator X [START_REF] Askoura | The Campylobacter jejuni ferric uptake regulator promotes acid survival and cross-protection against oxidative stress[END_REF][START_REF] Burgess | The response of foodborne pathogens to osmotic and desiccation stresses in the food chain[END_REF][START_REF] Butcher | Refined analysis of the Campylobacter jejuni iron-dependent/independent Fur-and PerR-transcriptomes[END_REF][START_REF] Palyada | Characterization of the oxidative stress stimulon and PerR regulon of Campylobacter jejuni[END_REF][START_REF] Van Vliet | Campylobacter jejuni contains two fur homologs: characterization of iron-responsive regulation of peroxide stress defense genes by the PerR repressor[END_REF] hspR CJE1365 heat shock transcriptional regulator X X X [START_REF] Askoura | Investigating the influence of iron on Campylobacter jejuni transcriptome in response to acid stress[END_REF][START_REF] Cameron | Hyperosmotic stress response of Campylobacter jejuni[END_REF][START_REF] Holmes | The hrcA and hspR regulons of Campylobacter jejuni[END_REF][START_REF] Stintzi | Gene expression profile of Campylobacter jejuni in response to growth temperature variation[END_REF] lysR CJE1080 transcriptional regulator X [START_REF] Dufour | Inactivation of the LysR regulator Cj1000 of Campylobacter jejuni affects host colonization and respiration[END_REF] CJE1780 CJE1780 two-component regulator X X [START_REF] Guccione | Transcriptome and proteome dynamics in chemostat culture reveal how Campylobacter jejuni modulates metabolism, stress responses and virulence factors upon changes in oxygen availability[END_REF]Reid et al., 2008b) cmeR (tetR) CJE0417 transcriptional regulator X X [START_REF] Butcher | Refined analysis of the Campylobacter jejuni iron-dependent/independent Fur-and PerR-transcriptomes[END_REF][START_REF] Guccione | Transcriptome and proteome dynamics in chemostat culture reveal how Campylobacter jejuni modulates metabolism, stress responses and virulence factors upon changes in oxygen availability[END_REF]Reid et al., 2008b) cosR CJE0404 two-component regulator X X [START_REF] Garenaux | Role of oxidative stress in C. jejuni inactivation during freeze-thaw treatment[END_REF]Hwang et al., 2011b;[START_REF] Varsaki | The acid adaptive tolerance response in Campylobacter jejuni induces a global response, as suggested by proteomics and microarrays[END_REF] racR CJE1397 two-component regulator X X X [START_REF] Apel | Characterization of Campylobacter jejuni RacRS reveals roles in the heat shock response, motility, and maintenance of cell length homogeneity[END_REF][START_REF] Brás | A novel Campylobacter jejuni two-component regulatory system important for temperature-dependent growth and colonization[END_REF] rprA Cj1546 transcriptional regulator X [START_REF] Gundogdu | The Campylobacter jejuni MarR-like transcriptional regulators RrpA and RrpB both influence bacterial responses to oxidative and aerobic stresses[END_REF] rprB Cj1556 transcriptional regulator X [START_REF] Gundogdu | The Campylobacter jejuni MarR-like transcriptional regulators RrpA and RrpB both influence bacterial responses to oxidative and aerobic stresses[END_REF] csrA CJE1246 carbon storage regulator X [START_REF] Fields | Campylobacter jejuni CsrA mediates oxidative stress responses, biofilm formation, and host cell invasion[END_REF] Involvement in one stress response clpX CJE0324 ATP-dependent protease ATP-binding subunit X [START_REF] Cohn | Contribution of conserved ATP-dependent proteases of Campylobacter jejuni to stress tolerance and virulence[END_REF] Involvement in two stress responses atpA CJE0100 Synthase subunit alpha X X [START_REF] Cameron | Hyperosmotic stress response of Campylobacter jejuni[END_REF][START_REF] Kaakoush | Insights into the molecular basis of the microaerophily of three Campylobacterales: a comparative study[END_REF] fliP CJE0907 flagellar biosynthesis protein X X [START_REF] Butcher | Refined analysis of the Campylobacter jejuni iron-dependent/independent Fur-and PerR-transcriptomes[END_REF][START_REF] Palyada | Characterization of the oxidative stress stimulon and PerR regulon of Campylobacter jejuni[END_REF] CJE1361 CJE1361 two-component sensor histidine kinase X X [START_REF] Butcher | Refined analysis of the Campylobacter jejuni iron-dependent/independent Fur-and PerR-transcriptomes[END_REF][START_REF] Kaakoush | Insights into the molecular basis of the microaerophily of three Campylobacterales: a comparative study[END_REF][START_REF] Palyada | Characterization of the oxidative stress stimulon and PerR regulon of Campylobacter jejuni[END_REF][START_REF] Varsaki | The acid adaptive tolerance response in Campylobacter jejuni induces a global response, as suggested by proteomics and microarrays[END_REF] putA CJE1676 proline dehydrogenase /delta-1-pyrroline-5carboxylate dehydrogenase X X [START_REF] Guccione | Transcriptome and proteome dynamics in chemostat culture reveal how Campylobacter jejuni modulates metabolism, stress responses and virulence factors upon changes in oxygen availability[END_REF]Reid et al., 2008b) dsbl CJE0017 disulfite bond formation protein X X [START_REF] Butcher | Refined analysis of the Campylobacter jejuni iron-dependent/independent Fur-and PerR-transcriptomes[END_REF][START_REF] Guccione | Transcriptome and proteome dynamics in chemostat culture reveal how Campylobacter jejuni modulates metabolism, stress responses and virulence factors upon changes in oxygen availability[END_REF]Reid et al., 2008b) pebC CJE1000 amino acid ABC transporter ATP-binding X X [START_REF] Butcher | Refined analysis of the Campylobacter jejuni iron-dependent/independent Fur-and PerR-transcriptomes[END_REF][START_REF] Guccione | Transcriptome and proteome dynamics in chemostat culture reveal how Campylobacter jejuni modulates metabolism, stress responses and virulence factors upon changes in oxygen availability[END_REF]Reid et al., 2008b) Campylobacter molecular response to stress [START_REF] Baillon | An ironregulated alkyl hydroperoxide reductase (AhpC) confers aerotolerance and oxidative stress resistance to the microaerophilic pathogen Campylobacter jejuni[END_REF][START_REF] Birk | Acid stress response and protein induction in Campylobacter jejuni isolates with different acid tolerance[END_REF][START_REF] Kaakoush | Insights into the molecular basis of the microaerophily of three Campylobacterales: a comparative study[END_REF][START_REF] Oh | Impact of oxidative stress defense on bacterial survival and morphological change in Campylobacter jejuni under aerobic conditions[END_REF][START_REF] Palyada | Characterization of the oxidative stress stimulon and PerR regulon of Campylobacter jejuni[END_REF]Reid et al., 2008b;[START_REF] Varsaki | The acid adaptive tolerance response in Campylobacter jejuni induces a global response, as suggested by proteomics and microarrays[END_REF] trpD CJE0395 anthranilate synthase subunit II X X [START_REF] Guccione | Transcriptome and proteome dynamics in chemostat culture reveal how Campylobacter jejuni modulates metabolism, stress responses and virulence factors upon changes in oxygen availability[END_REF]Reid et al., 2008b) yidC CJE1038 membrane protein insertase X X [START_REF] Guccione | Transcriptome and proteome dynamics in chemostat culture reveal how Campylobacter jejuni modulates metabolism, stress responses and virulence factors upon changes in oxygen availability[END_REF]Reid et al., 2008b) hupB CJE0991 NA-binding protein X X [START_REF] Guccione | Transcriptome and proteome dynamics in chemostat culture reveal how Campylobacter jejuni modulates metabolism, stress responses and virulence factors upon changes in oxygen availability[END_REF][START_REF] Shi | Proteomic analysis of the cold stress response in Campylobacter jejuni[END_REF] proS CJE0647 proline--tRNA ligase X X [START_REF] Shi | Proteomic analysis of the cold stress response in Campylobacter jejuni[END_REF][START_REF] Varsaki | The acid adaptive tolerance response in Campylobacter jejuni induces a global response, as suggested by proteomics and microarrays[END_REF] asd CJE1167 aspartate-semialdehyde dehydrogenase X X [START_REF] Butcher | Refined analysis of the Campylobacter jejuni iron-dependent/independent Fur-and PerR-transcriptomes[END_REF][START_REF] Guccione | Transcriptome and proteome dynamics in chemostat culture reveal how Campylobacter jejuni modulates metabolism, stress responses and virulence factors upon changes in oxygen availability[END_REF][START_REF] Shi | Proteomic analysis of the cold stress response in Campylobacter jejuni[END_REF] Involvement in ≥ 3 stress responses grpE CJE0849 heat shock protein X X X X [START_REF] Askoura | Investigating the influence of iron on Campylobacter jejuni transcriptome in response to acid stress[END_REF][START_REF] Cameron | Hyperosmotic stress response of Campylobacter jejuni[END_REF][START_REF] Flint | Phenotypic screening of a targeted mutant library reveals Campylobacter jejuni defenses against oxidative stress[END_REF][START_REF] Palyada | Characterization of the oxidative stress stimulon and PerR regulon of Campylobacter jejuni[END_REF]Reid et al., 2008b) groEL CJE1356 Chaperone protein X X X [START_REF] Askoura | Investigating the influence of iron on Campylobacter jejuni transcriptome in response to acid stress[END_REF][START_REF] Cameron | Hyperosmotic stress response of Campylobacter jejuni[END_REF]Reid et al., 2008b;[START_REF] Stintzi | Gene expression profile of Campylobacter jejuni in response to growth temperature variation[END_REF][START_REF] Sulaeman | Enhanced adhesion of Campylobacter jejuni to abiotic surfaces is mediated by membrane proteins in oxygen-enriched conditions[END_REF] dnaK CJE0850 Chaperone protein X X X X [START_REF] Cameron | Hyperosmotic stress response of Campylobacter jejuni[END_REF][START_REF] Flint | Phenotypic screening of a targeted mutant library reveals Campylobacter jejuni defenses against oxidative stress[END_REF]Reid et al., 2008b;[START_REF] Stintzi | Gene expression profile of Campylobacter jejuni in response to growth temperature variation[END_REF][START_REF] Sulaeman | Enhanced adhesion of Campylobacter jejuni to abiotic surfaces is mediated by membrane proteins in oxygen-enriched conditions[END_REF] katA CJE1576 catalase A X X [START_REF] Cameron | Hyperosmotic stress response of Campylobacter jejuni[END_REF][START_REF] Oh | Impact of oxidative stress defense on bacterial survival and morphological change in Campylobacter jejuni under aerobic conditions[END_REF][START_REF] Palyada | Characterization of the oxidative stress stimulon and PerR regulon of Campylobacter jejuni[END_REF]Reid et al., 2008b;[START_REF] Varsaki | The acid adaptive tolerance response in Campylobacter jejuni induces a global response, as suggested by proteomics and microarrays[END_REF] argF CJE1074 delta-aminolevulinic acid dehydratase X X X [START_REF] Guccione | Transcriptome and proteome dynamics in chemostat culture reveal how Campylobacter jejuni modulates metabolism, stress responses and virulence factors upon changes in oxygen availability[END_REF][START_REF] Shi | Proteomic analysis of the cold stress response in Campylobacter jejuni[END_REF][START_REF] Varsaki | The acid adaptive tolerance response in Campylobacter jejuni induces a global response, as suggested by proteomics and microarrays[END_REF] rpoB CJE0528 DNA-directed RNA polymerase subunit beta X X X [START_REF] Guccione | Transcriptome and proteome dynamics in chemostat culture reveal how Campylobacter jejuni modulates metabolism, stress responses and virulence factors upon changes in oxygen availability[END_REF]Reid et al., 2008b;[START_REF] Shi | Proteomic analysis of the cold stress response in Campylobacter jejuni[END_REF] yciA CJE0993 acyl-CoA thioesterase X X X [START_REF] Butcher | Refined analysis of the Campylobacter jejuni iron-dependent/independent Fur-and PerR-transcriptomes[END_REF][START_REF] Guccione | Transcriptome and proteome dynamics in chemostat culture reveal how Campylobacter jejuni modulates metabolism, stress responses and virulence factors upon changes in oxygen availability[END_REF]Reid et al., 2008b) kdtA CJE0807 3-deoxy-D-mannooctulosonic acid transferase X X X [START_REF] Kaakoush | Insights into the molecular basis of the microaerophily of three Campylobacterales: a comparative study[END_REF][START_REF] Palyada | Characterization of the oxidative stress stimulon and PerR regulon of Campylobacter jejuni[END_REF]Reid et al., 2008b;[START_REF] Stintzi | Gene expression profile of Campylobacter jejuni in response to growth temperature variation[END_REF] clpB CJE0616 Chaperone protein X X X [START_REF] Cameron | Hyperosmotic stress response of Campylobacter jejuni[END_REF]Reid et al., 2008b;[START_REF] Stintzi | Gene expression profile of Campylobacter jejuni in response to growth temperature variation[END_REF] sodB CJE0164 superoxide dismutase [START_REF] Cameron | Hyperosmotic stress response of Campylobacter jejuni[END_REF][START_REF] Kaakoush | Insights into the molecular basis of the microaerophily of three Campylobacterales: a comparative study[END_REF][START_REF] Oh | Impact of oxidative stress defense on bacterial survival and morphological change in Campylobacter jejuni under aerobic conditions[END_REF][START_REF] Stintzi | Gene expression profile of Campylobacter jejuni in response to growth temperature variation[END_REF] htrA CJE1363 serine protease X X X X [START_REF] Baek | Different contributions of HtrA protease and chaperone activities to Campylobacter jejuni stress tolerance and physiology[END_REF][START_REF] Boehm | Campylobacter jejuni serine protease HtrA plays an important role in heat tolerance, oxygen resistance, host cell adhesion, invasion, and transmigration[END_REF][START_REF] Brondsted | The HtrA protease of Campylobacter jejuni is required for heat and oxygen tolerance and for optimal interaction with human epithelial cells[END_REF][START_REF] Cameron | Hyperosmotic stress response of Campylobacter jejuni[END_REF]Reid et al., 2008b;[START_REF] Sulaeman | Enhanced adhesion of Campylobacter jejuni to abiotic surfaces is mediated by membrane proteins in oxygen-enriched conditions[END_REF] sdhA CJE0488 succinate dehydrogenase X X X [START_REF] Palyada | Characterization of the oxidative stress stimulon and PerR regulon of Campylobacter jejuni[END_REF]Reid et al., 2008b;[START_REF] Stintzi | Gene expression profile of Campylobacter jejuni in response to growth temperature variation[END_REF] Campylobacter molecular response to stress [START_REF] Phongsisay | Expression of the htrB gene is essential for responsiveness of Salmonella typhimurium and Campylobacter jejuni to harsh environments[END_REF][START_REF] Trigui | Phenotypic and transcriptomic responses of Campylobacter jejuni suspended in an artificial freshwater medium[END_REF] nuoL CJE1739 NADH-quinone oxidoreductase subunit L X X X [START_REF] Cameron | Hyperosmotic stress response of Campylobacter jejuni[END_REF][START_REF] Kaakoush | Insights into the molecular basis of the microaerophily of three Campylobacterales: a comparative study[END_REF][START_REF] Varsaki | The acid adaptive tolerance response in Campylobacter jejuni induces a global response, as suggested by proteomics and microarrays[END_REF] 822

List of figures

Figure 1. Decision tree for the selection of target genes according to the literature analysis.