Unveiling the matrix effect on Bacillus licheniformis and Bacillus subtilis spores heat inactivation between plant-based milk alternatives, bovine milk and culture medium.

This study examined the inactivation of spores of Bacillus licheniformis and Bacillus subtilis in four pea-based milk alternatives, semi-skimmed bovine milk and Brain Heart Infusion (BHI) broth to assess the matrix impact on the thermal inactivation of bacterial spores. Heat inactivation was performed with the method of capillary tubes in temperature range 97-110 °C. A four-parameter non-linear model, including initial level, shoulder duration, inactivation rate and tailing, was fitted to the data obtained. D-values were estimated and secondary ZT-value models were developed for both species. A secondary model for the shoulder length of B. licheniformis in a plant-based milk alternative formulation was built too. Models were validated at a higher temperature, 113.5 °C. D-values in the different matrices ranged between 2.3 and 8.2 min at 97 °C and 0.1-0.3 min at 110 °C for B. licheniformis. D-values for B. subtilis ranged between 3.9 and 6.3 min at 97 °C and 0.2-0.3 min at 110 °C. ZT-values in the different matrices ranged between 7.3 and 8.9 °C and 8.9-10.0 °C for B. licheniformis and B. subtilis, respectively. Significant differences in inactivation parameters were found within the pea-based formulations as well as when compared to bovine milk. Heat resistance was higher in pea-based matrices. Shoulders observed were temperature- and matrix-dependent, while no such trend was found for the tailings. These results provide insights, useful on designing safe thermal processing, limiting spoilage in plant-based milk alternatives and thus, reducing global food waste.

Intestinal Organoids: New Tools to Comprehend the Virulence of Bacterial Foodborne Pathogens.

Foodborne diseases cause high morbidity and mortality worldwide. Understanding the relationships between bacteria and epithelial cells throughout the infection process is essential to setting up preventive and therapeutic solutions. The extensive study of their pathophysiology has mostly been performed on transformed cell cultures that do not fully mirror the complex cell populations, the in vivo architectures, and the genetic profiles of native tissues. Following advances in primary cell culture techniques, organoids have been developed. Such technological breakthroughs have opened a new path in the study of microbial infectious diseases, and thus opened onto new strategies to control foodborne hazards. This review sheds new light on cellular messages from the host-foodborne pathogen crosstalk during in vitro organoid infection by the foodborne pathogenic bacteria with the highest health burden. Finally, future perspectives and current challenges are discussed to provide a better understanding of the potential applications of organoids in the investigation of foodborne infectious diseases.

Quantification of Campylobacter jejuni gene expression after successive stresses mimicking poultry slaughtering steps.

Broiler meat is considered as the most important source of the foodborne pathogen Campylobacter jejuni. Exposure to stress conditions encountered during the slaughtering process may induce bacterial adaptation mechanisms, and enhance or decrease pathogen resistance to subsequent stress. This adaptation may result from changes in bacterial gene expression. This study aims to accurately quantify the expression of selected C. jejuni genes after stresses inspired from the poultry slaughtering process. RT-qPCR was used to quantify gene expression of 44 genes in three strains after successive heat and cold stresses. Main results indicated that 26 genes out of 44 were differentially expressed following the successive thermal stresses. Three clusters of genes were differentially expressed according to the strain and the stress condition. Up-regulated genes mainly included genes involved in the heat shock response, whereas down-regulated genes belonged to metabolic pathways (such as lipid, amino-acid metabolisms). However, four genes were similarly overexpressed in the three strains; they might represent indicators of the thermal stress response at the species scale. Advances in the molecular understanding of the stress response of pathogenic bacteria, such as Campylobacter, in real-life processing conditions will make it possible to identify technological levers and better mitigate the microbial risk.

Quantitative approach to assess the compliance to a performance objective (PO) of Campylobacter jejuni in poultry meat in France.

Predictive modelling is used in microbiological risk assessment to quantify the growth and inactivation of microorganisms through the use of mathematical models. Campylobacter jejuni is one of the main foodborne pathogens and broiler meat is considered as the most important source of human campylobacteriosis. The purpose of this study was to assess the effects of heating and chilling during the poultry slaughter process on inactivation kinetics of Campylobacter jejuni during chilled storage in order to predict its contamination level prior to preparation and consumption in the consumer's home, and then to assess the compliance to a Performance Objective (PO). Three strains of C. jejuni were submitted to consecutive heat (54 °C for 3 min) and cold (3 °C for 2 h) stresses, mimicking the two main slaughtering steps, i.e. scalding and chilling, by inoculating chicken fillets with three different concentrations (4, 6 and 8 log10 CFU/g). Fillets were then stored at 6 °C during 17 days under the modified atmosphere currently used by food processors (70% O2/30% CO2). For all strains, bacterial log reduction was the lowest when inoculated at 8 log10 CFU/g. One strain showed an enhanced resistance during cold storage after application of stressing steps, suggesting an impact of the cell history on further bacterial resistance. Taking strain variability into account, after six days of storage, predictions showed compliance of ready-to-be-cooked chicken meat with a hypothetical PO of 2.55 log10 CFU/g, value set before the meat enters the consumer's home by the ICMSF (International Commission on Microbiological Specifications for Foods). This study opens the path to assess the compliance to a PO of Campylobacter jejuni in poultry meat and more generally provides inputs to refine microbiological risk assessment by taking into account the cell history and more particularly the impact of stressful steps on the subsequent inactivation at consumer's home.

Influence of cell history on the subsequent inactivation of Campylobacter jejuni during cold storage under modified atmosphere.

Worldwide, Campylobacter infections are the main cause of human bacterial enteritis and broiler meat is considered as the most important source of human campylobacteriosis. Some mitigation strategies have been focused on reduction of Campylobacter at the slaughtering steps. This study aimed to determine the influence of consecutive stresses inspired by slaughtering steps on the subsequent inactivation of Campylobacter jejuni during cold storage under different modified atmospheres. Using a full experimental design, three strains of C. jejuni of poultry origin were submitted to consecutive heat (46°, 50° or 54 °C for 4 min) and cold (-4° or 3 °C for 2 h) stresses by plunging cultures into baths at appropriate temperatures. Cultures were then stored at 6 °C during seven days under modified atmospheres (70% O2/30% CO2 or 50% CO2/50% N2). Inactivation of C. jejuni induced by cold storage was shown to depend significantly (P < 0.0001) upon the heat stress previously applied. It was shown to be the highest under the atmosphere enriched in oxygen, after application of 54 °C. Strain inactivation variability was also quantified. These results show that consecutive stresses influence further inactivation of C. jejuni during storage and consequently the contamination level at consumer's plate.

An adapted in vitro assay to assess Campylobacter jejuni interaction with intestinal epithelial cells: Taking into stimulation with TNFα.

Campylobacter jejuni is the most prevalent foodborne bacterial infection agent. This pathogen seems also involved in inflammatory bowel diseases in which pro-inflammatory cytokines, such as tumor necrosis factor α (TNFα), play a major role. C. jejuni pathogenicity has been extensively studied using in vitro cell culture methods, and more precisely "healthy" cells. In fact, no information is available regarding the behavior of C. jejuni in contact with TNFα-stimulated cells. Therefore, this research was designed to investigate the effect of TNFα on C. jejuni interaction with human intestinal epithelial cells (HT29 and HT29-MTX). To ensure IL-8 production induced by TNFα, human rtTNFα was added to HT29 and HT29-MTX before adhesion and invasion assays. About 108 CFU bacteria of C. jejuni strains cells were added to measure their adherence and invasion abilities using TNFα-stimulated cells versus non stimulated cells. Exposure to TNFα results in IL-8 overproduction by intestinal epithelial cells. In addition, the effect of TNFα pre-treatment on C. jejuni adhesion and internalization into eukaryotic cells is strain-dependent. Indeed, the adhesion/invasion process is affected in <50% of the strains tested when TNFα is added to the intestinal cells. Interestingly, TNFα affects more strains in their ability to adhere to and invade the mucus-secreting HT29-MTX cells. Among the 10 strains tested, the aero-tolerant C. jejuni Bf strain is one of the most virulent. These results suggest that the TNFα signalling pathway could participate in the internalization of C. jejuni in human intestinal cells and can help in understanding the pathogenicity of this microorganism in contact with TNFα-stimulated cells.

Next generation microbiological risk assessment-Potential of omics data for hazard characterisation.

According to the World Health Organization estimates in 2015, 600 million people fall ill every year from contaminated food and 420,000 die. Microbial risk assessment (MRA) was developed as a tool to reduce and prevent risks presented by pathogens and/or their toxins. MRA is organized in four steps to analyse information and assist in both designing appropriate control options and implementation of regulatory decisions and programs. Among the four steps, hazard characterisation is performed to establish the probability and severity of a disease outcome, which is determined as function of the dose of toxin and/or pathogen ingested. This dose-response relationship is subject to both variability and uncertainty. The purpose of this review/opinion article is to discuss how Next Generation Omics can impact hazard characterisation and, more precisely, how it can improve our understanding of variability and limit the uncertainty in the dose-response relation. The expansion of omics tools (e.g. genomics, transcriptomics, proteomics and metabolomics) allows for a better understanding of pathogenicity mechanisms and virulence levels of bacterial strains. Detection and identification of virulence genes, comparative genomics, analyses of mRNA and protein levels and the development of biomarkers can help in building a mechanistic dose-response model to predict disease severity. In this respect, systems biology can help to identify critical system characteristics that confer virulence and explain variability between strains. Despite challenges in the integration of omics into risk assessment, some omics methods have already been used by regulatory agencies for hazard identification. Standardized methods, reproducibility and datasets obtained from realistic conditions remain a challenge, and are needed to improve accuracy of hazard characterisation. When these improvements are realized, they will allow the health authorities and government policy makers to prioritize hazards more accurately and thus refine surveillance programs with the collaboration of all stakeholders of the food chain.

Quantification of Campylobacter jejuni contamination on chicken carcasses in France.

Highly prevalent in poultry, Campylobacter is a foodborne pathogen which remains the primary cause of enteritis in humans. Several studies have determined prevalence and contamination level of this pathogen throughout the food chain. However it is generally performed in a deterministic way without considering heterogeneity of contamination level. The purpose of this study was to quantify, using probabilistic tools, the contamination level of Campylobacter spp. on chicken carcasses after air-chilling step in several slaughterhouses in France. From a dataset (530 data) containing censored data (concentration <10CFU/g), several factors were considered, including the month of sampling, the farming method (standard vs certified) and the sampling area (neck vs leg). All probabilistic analyses were performed in R using fitdistrplus, mc2d and nada packages. The uncertainty (i.e. error) generated by the presence of censored data was small (ca 1 log10) in comparison to the variability (i.e. heterogeneity) of contamination level (3 log10 or more), strengthening the probabilistic analysis and facilitating result interpretation. The sampling period and sampling area (neck/leg) had a significant effect on Campylobacter contamination level. More precisely, two "seasons" were distinguished: one from January to May, another one from June to December. During the June-to-December season, the mean Campylobacter concentration was estimated to 2.6 [2.4; 2.8] log10 (CFU/g) and 1.8 [1.5; 2.0] log10 (CFU/g) for neck and leg, respectively. The probability of having >1000CFU/g (higher limit of European microbial criterion) was estimated to 35.3% and 12.6%, for neck and leg, respectively. In contrast, during January-to-May season, the mean contamination level was estimated to 1.0 [0.6; 1.3] log10 (CFU/g) and 0.6 [0.3; 0.9] log10 (CFU/g) for neck and leg, respectively. The probability of having >1000CFU/g was estimated to 13.5% and 2.0% for neck and leg, respectively. An accurate quantification of contamination level enables industrials to better adapt their processing and hygiene practices. These results will also help in refining exposure assessment models.

Biochemical characterization of Campylobacter jejuni PNPase, an exoribonuclease important for bacterial pathogenicity.

Bacteria need to promptly respond to environmental changes. Ribonucleases (RNases) are key factors in the adaptation to new environments by enabling a rapid adjustment in RNA levels. The exoribonuclease polynucleotide phosphorylase (PNPase) is essential for low-temperature cell survival, affects the synthesis of proteins involved in virulence and has an important role in swimming, cell adhesion/invasion ability, and chick colonization in C. jejuni. However, the mechanism of action of this ribonuclease is not yet known. In this work we have characterized the biochemical activity of C. jejuni PNPase. Our results demonstrate that Cj-PNP is a processive 3' to 5' exoribonuclease that degrades single-stranded RNAs. Its activity is regulated according to the temperature and divalent ions. We have also shown that the KH and S1 domains are important for trimerization, RNA binding, and, consequently, for the activity of Cj-PNP. These findings will be helpful to develop new strategies for fighting against C. jejuni and may be extrapolated to other foodborne pathogens.

Profiling of Campylobacter jejuni Proteome in Exponential and Stationary Phase of Growth.

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.

Methods for Proteome Analysis of Campylobacter jejuni Using 2-D Electrophoresis.

This chapter describes protocols used for two-dimensional electrophoretic analysis of the proteome or subproteome of Campylobacter jejuni, a major human food-borne pathogen. The following protocols, adapted to Campylobacter strains, include all the steps from cultivation to gel-support protein separation.

Use of the potential probiotic strain Lactobacillus salivarius SMXD51 to control Campylobacter jejuni in broilers.

Campylobacteriosis is the most frequently reported zoonotic disease in humans in the EU since 2005. As chicken meat is the main source of contamination, reducing the level of Campylobacter in broiler chicken will lower the risk to consumers. The aim of this project was to evaluate the ability of Lactobacillus salivarius SMXD51 to control Campylobacter jejuni in broilers and to investigate the mechanisms that could be involved. Thirty broilers artificially contaminated with C. jejuni were treated by oral gavage with MRS broth or a bacterial suspension (107CFU) of Lb. salivarius SMXD51 (SMXD51) in MRS broth. At 14 and 35days of age, Campylobacter and Lb. salivarius loads were assessed in cecal contents. The impact of the treatment on the avian gut microbiota at day 35 was also evaluated. At day 14, the comparison between the control and treated groups showed a significant reduction (P<0.05) of 0.82 log. After 35days, a significant reduction (P<0.001) of 2.81 log in Campylobacter loads was observed and 73% of chickens treated with the culture exhibited Campylobacter loads below 7log10CFU/g. Taxonomic analysis revealed that SMXD51 treatment induced significant changes (P<0.05) in a limited number of bacterial genera of the avian gut microbiota and partially limited the impact of Campylobacter on Anaerotruncus sp. decrease and Subdoligranulum sp. increase. Thus, SMXD51 exhibits an anti-Campylobacter activity in vivo and can partially prevent the impact of Campylobacter on the avian gut microbiota.

Comparison of Proteomics Profiles of Campylobacter jejuni Strain Bf under Microaerobic and Aerobic Conditions.

Campylobacter jejuni accounts for one of the leading causes of foodborne bacterial enteritis in humans. Despite being considered an obligate microaerobic microorganism, C. jejuni is regularly exposed to oxidative stress. However, its adaptive strategies to survive the atmospheric oxygen level during transmission to humans remain unclear. Recently, the clinical C. jejuni strain Bf was singled out for its unexpected ability to grow under ambient atmosphere. Here, we aimed to understand better the biological mechanisms underlying its atypical aerotolerance trait using two-dimensional protein electrophoresis, gene expression, and enzymatic activities. Forty-seven proteins were identified with a significantly different abundance between cultivation under microaerobic and aerobic conditions. The over-expressed proteins in aerobiosis belonged mainly to the oxidative stress response, enzymes of the tricarboxylic acid cycle, iron uptake, and regulation, and amino acid uptake when compared to microaerobic conditions. The higher abundance of proteins related to oxidative stress was correlated to dramatically higher transcript levels of the corresponding encoding genes in aerobic conditions compared to microaerobic conditions. In addition, a higher catalase-equivalent activity in strain Bf was observed. Despite the restricted catabolic capacities of C. jejuni, this study reveals that strain Bf is equipped to withstand oxidative stress. This ability could contribute to emergence and persistence of particular strains of C. jejuni throughout food processing or macrophage attack during human infection.

Adhesion, Biofilm Formation, and Genomic Features of Campylobacter jejuni Bf, an Atypical Strain Able to Grow under Aerobic Conditions.

Campylobacter jejuni is the leading cause of bacterial enteritis in Europe. Human campylobacteriosis cases are frequently associated to the consumption of contaminated poultry meat. To survive under environmental conditions encountered along the food chain, i.e., from poultry digestive tract its natural reservoir to the consumer's plate, this pathogen has developed adaptation mechanisms. Among those, biofilm lifestyle has been suggested as a strategy to survive in the food environment and under atmospheric conditions. Recently, the clinical isolate C. jejuni Bf has been shown to survive and grow under aerobic conditions, a property that may help this strain to better survive along the food chain. The aim of this study was to evaluate the adhesion capacity of C. jejuni Bf and its ability to develop a biofilm. C. jejuni Bf can adhere to abiotic surfaces and to human epithelial cells, and can develop biofilm under both microaerobiosis and aerobiosis. These two conditions have no influence on this strain, unlike results obtained with the reference strain C. jejuni 81-176, which harbors only planktonic cells under aerobic conditions. Compared to 81-176, the biofilm of C. jejuni Bf is more homogenous and cell motility at the bottom of biofilm was not modified whatever the atmosphere used. C. jejuni Bf whole genome sequence did not reveal any gene unique to this strain, suggesting that its unusual property does not result from acquisition of new genetic material. Nevertheless some genetic particularities seem to be shared only between Bf and few others strains. Among the main features of C. jejuni Bf genome we noticed (i) a complete type VI secretion system important in pathogenicity and environmental adaptation; (ii) a mutation in the oorD gene involved in oxygen metabolism; and (iii) the presence of an uncommon insertion of a 72 amino acid coding sequence upstream from dnaK, which is involved in stress resistance. Therefore, the atypical behavior of this strain under aerobic atmosphere may result from the combination of insertions and mutations. In addition, the comparison of mRNA transcript levels of several genes targeted through genome analysis suggests the modification of regulatory processes in this strain.

Recent Advances in Screening of Anti-Campylobacter Activity in Probiotics for Use in Poultry.

Campylobacteriosis is the most common cause of bacterial gastroenteritis worldwide. Campylobacter species involved in this infection usually include the thermotolerant species Campylobacter jejuni. The major reservoir for C. jejuni leading to human infections is commercial broiler chickens. Poultry flocks are frequently colonized by C. jejuni without any apparent symptoms. Risk assessment analyses have identified the handling and consumption of poultry meat as one of the most important sources of human campylobacteriosis, so elimination of Campylobacter in the poultry reservoir is a crucial step in the control of this foodborne infection. To date, the use of probiotics has demonstrated promising results to reduce Campylobacter colonization. This review provides recent insights into methods used for probiotic screening to reduce the prevalence and colonization of Campylobacter at the farm level. Different eukaryotic epithelial cell lines are employed to screen probiotics with an anti-Campylobacter activity and yield useful information about the inhibition mechanism involved. These in vitro virulence models involve only human intestinal or cervical cell lines whereas the use of avian cell lines could be a preliminary step to investigate mechanisms of C. jejuni colonization in poultry in the presence of probiotics. In addition, in vivo trials to evaluate the effect of probiotics on Campylobacter colonization are conducted, taking into account the complexity introduced by the host, the feed, and the microbiota. However, the heterogeneity of the protocols used and the short time duration of the experiments lead to results that are difficult to compare and draw conclusions at the slaughter-age of broilers. Nevertheless, the combined approach using complementary in vitro and in vivo tools (cell cultures and animal experiments) leads to a better characterization of probiotic strains and could be employed to assess reduced Campylobacter spp. colonization in chickens if some parameters are optimized.

Draft Genome Sequence of Campylobacter jejuni Bf, an Atypical Strain Able To Grow under Aerobiosis.

In this study, we describe the draft genome sequence of aCampylobacter jejuniclinical isolate issued from a French patient suffering from severe campylobacteriosis. This atypical strain is characterized by an unusual resistance to oxygen and the ability to grow under an aerobic atmosphere, a characteristic as-of-yet unique to this species.

Description of Campylobacter jejuni Bf, an atypical aero-tolerant strain.

BACKGROUND: Campylobacter jejuni is a leading cause of bacterial enteritis worldwide. This microaerophilic bacterium can survive in aerobic environments, suggesting it has protective mechanisms against oxidative stress. The clinical C. jejuni Bf strain is characterized by an increased resistance to oxygen. This study aimed to characterize the behavior of the clinical C. jejuni Bf strain under an aerobic atmosphere and in response to ROS-promoter agents. METHODS: Growth was studied in both aerobic and microaerobic conditions using classic cultivable methods. Electronic microscopy and mreB gene expression were used to evaluate the morphology of this strain under aerobic conditions. The survival under oxidative stress was tested in the presence of different concentrations of hydrogen peroxide (H2O2) and paraquat (PQ). RESULTS: The results showed that C. jejuni Bf strain can grow aerobically, unlike other strains of C. jejuni tested. Cells of C. jejuni Bf exposed to oxidative stress presented changes in morphology and the gene mreB, responsible for maintaining the bacillary cell morphology, was down-expressed. In aerobically acclimated conditions, C. jejuni Bf exhibited a higher survival rate of 52 % in the presence of H2O2 (1 mM) compared to the reference strain NCTC 11168. Concentrations above 1 mM PQ were lethal for the reference strain but not for C. jejuni Bf. CONCLUSIONS: Taken together, these data highlight the resistance to oxidative stress conditions of C. jejuni Bf, indicating that this microorganism seems more adapted to survival in hostile environmental conditions.

Influence of measurement and control of microaerobic gaseous atmospheres in methods for Campylobacter growth studies.

Campylobacter is the leading cause of bacterial enteritis in the world. For this reason, this pathogen is widely studied. As a microaerophilic and capnophilic microorganism, this foodborne pathogen requires an atmosphere with reduced oxygen (O2) and elevated carbon dioxide (CO2) concentrations for its optimal growth in vitro. According to the procedure for Campylobacter spp. isolation and cultivation from food products and environmental samples, European and American standards recommend gas proportions of 5% O2 and 10% CO2, complemented with nitrogen (N2). However, in the literature, the reported proportion of O2 for microaerobic growth conditions of Campylobacter spp. can range from 2.5% to 15% and the reason for this variation is usually not explained. The use of different gas generating systems and media to detect and to grow Campylobacter from foodstuff and the lack of information about gas producing systems are the main sources of the loss of consistancy between data. In this review, the relevance, strengths and weaknesses of these methods and their impact on Campylobacter biology are discussed. In conclusion the minimum information concerning microaerobic gaseous atmospheres are suggested in order to better harmonize data obtained from research studies for a better understanding of Campylobacter features.

The RNase R from Campylobacter jejuni has unique features and is involved in the first steps of infection.

Bacterial pathogens must adapt/respond rapidly to changing environmental conditions. Ribonucleases (RNases) can be crucial factors contributing to the fast adaptation of RNA levels to different environmental demands. It has been demonstrated that the exoribonuclease polynucleotide phosphorylase (PNPase) facilitates survival of Campylobacter jejuni in low temperatures and favors swimming, chick colonization, and cell adhesion/invasion. However, little is known about the mechanism of action of other ribonucleases in this microorganism. Members of the RNB family of enzymes have been shown to be involved in virulence of several pathogens. We have searched C. jejuni genome for homologues and found one candidate that displayed properties more similar to RNase R (Cj-RNR). We show here that Cj-RNR is important for the first steps of infection, the adhesion and invasion of C. jejuni to eukaryotic cells. Moreover, Cj-RNR proved to be active in a wide range of conditions. The results obtained lead us to conclude that Cj-RNR has an important role in the biology of this foodborne pathogen.

Characterization of the biochemical properties of Campylobacter jejuni RNase III.

Campylobacter jejuni is a foodborne bacterial pathogen, which is now considered as a leading cause of human bacterial gastroenteritis. The information regarding ribonucleases in C. jejuni is very scarce but there are hints that they can be instrumental in virulence mechanisms. Namely, PNPase (polynucleotide phosphorylase) was shown to allow survival of C. jejuni in refrigerated conditions, to facilitate bacterial swimming, cell adhesion, colonization and invasion. In several microorganisms PNPase synthesis is auto-controlled in an RNase III (ribonuclease III)-dependent mechanism. Thereby, we have cloned, overexpressed, purified and characterized Cj-RNase III (C. jejuni RNase III). We have demonstrated that Cj-RNase III is able to complement an Escherichia coli rnc-deficient strain in 30S rRNA processing and PNPase regulation. Cj-RNase III was shown to be active in an unexpectedly large range of conditions, and Mn2+ seems to be its preferred co-factor, contrarily to what was described for other RNase III orthologues. The results lead us to speculate that Cj-RNase III may have an important role under a Mn2+-rich environment. Mutational analysis strengthened the function of some residues in the catalytic mechanism of action of RNase III, which was shown to be conserved.