Peptidoglycan-Modifying Enzyme Pgp1 Is Required for Helical Cell Shape and Pathogenicity Traits in Campylobacter jejuni

The impact of bacterial morphology on virulence and transmission attributes of pathogens is poorly understood. The prevalent enteric pathogen Campylobacter jejuni displays a helical shape postulated as important for colonization and host interactions. However, this had not previously been demonstrated experimentally. C. jejuni is thus a good organism for exploring the role of factors modulating helical morphology on pathogenesis. We identified an uncharacterized gene, designated pgp1 (peptidoglycan peptidase 1), in a calcofluor white-based screen to explore cell envelope properties important for C. jejuni virulence and stress survival. Bioinformatics showed that Pgp1 is conserved primarily in curved and helical bacteria. Deletion of pgp1 resulted in a striking, rod-shaped morphology, making pgp1 the first C. jejuni gene shown to be involved in maintenance of C. jejuni cell shape. Pgp1 contributes to key pathogenic and cell envelope phenotypes. In comparison to wild type, the rod-shaped pgp1 mutant was deficient in chick colonization by over three orders of magnitude and elicited enhanced secretion of the chemokine IL-8 in epithelial cell infections. Both the pgp1 mutant and a pgp1 overexpressing strain – which similarly produced straight or kinked cells – exhibited biofilm and motility defects. Detailed peptidoglycan analyses via HPLC and mass spectrometry, as well as Pgp1 enzyme assays, confirmed Pgp1 as a novel peptidoglycan DL-carboxypeptidase cleaving monomeric tripeptides to dipeptides. Peptidoglycan from the pgp1 mutant activated the host cell receptor Nod1 to a greater extent than did that of wild type. This work provides the first link between a C. jejuni gene and morphology, peptidoglycan biosynthesis, and key host- and transmission-related characteristics.     

Does Campylobacter jejuni Form Biofilms in Food-Related Environments?

Campylobacter jejuni is one of the most frequent causes of bacterial gastrointestinal food-borne infection worldwide. This species is part of the normal flora of the gastrointestinal tracts of animals used for food production, including poultry, which is regarded as the primary source of human Campylobacter infections. The survival and persistence of C. jejuni in food processing environments, especially in poultry processing plants, represent significant risk factors that contribute to the spread of this pathogen through the food chain. Compared to other food-borne pathogens, C. jejuni is more fastidious in its growth requirements and is very susceptible to various environmental stressors. Biofilm formation is suggested to play a significant role in the survival of C. jejuni in the food production and processing environment. The aims of this minireview were (i) to examine the evidence that C. jejuni forms biofilms and (ii) to establish the extent to which reported and largely laboratory-based studies of C. jejuni biofilms provide evidence for biofilm formation by this pathogen in food processing environments. Overall existing studies do not provide strong evidence for biofilm formation (as usually defined) by most C. jejuni strains in food-related environments under the combined conditions of atmosphere, temperature, and shear that they are likely to encounter. Simple attachment to and survival on surfaces and in existing biofilms of other species are far more likely to contribute to C. jejuni survival in food-related environments based on our current understanding of this species.     

Quantitative Proteomics of Intracellular Campylobacter jejuni Reveals Metabolic Reprogramming

Campylobacter jejuni is the major cause of bacterial food-borne illness in the USA and Europe. An important virulence attribute of this bacterial pathogen is its ability to enter and survive within host cells. Here we show through a quantitative proteomic analysis that upon entry into host cells, C. jejuni undergoes a significant metabolic downshift. Furthermore, our results indicate that intracellular C. jejuni reprograms its respiration, favoring the respiration of fumarate. These results explain the poor ability of C. jejuni obtained from infected cells to grow under standard laboratory conditions and provide the bases for the development of novel anti microbial strategies that would target relevant metabolic pathways.     

The Natural Antimicrobial Carvacrol Inhibits Campylobacter jejuni Motility and Infection of Epithelial Cells

Background

Natural compounds with anti-microbial properties are attractive reagents to reduce the use of conventional antibiotics. Carvacrol, the main constituent of oregano oil, inhibits the growth of a variety of bacterial foodborne pathogens. As concentrations of carvacrol may vary in vivo or when used in animal feed, we here investigated the effect of subinhibitory concentrations of the compound on major virulence traits of the principal bacterial foodborne pathogen Campylobacter jejuni.

Methods/Principal Findings

Motility assays revealed that subinhibitory concentrations of carvacrol inhibited the motility of C. jejuni without affecting bacterial growth. Immunoblotting and electron microscopy showed that carvacrol-treated C. jejuni still expressed flagella. The loss of motility was not caused by reduced intracellular ATP levels. In vitro infection assays demonstrated that subinhibitory concentrations of carvacrol also abolished C. jejuni invasion of human epithelial cells. Bacterial uptake of invasive Escherichia coli was not blocked by carvacrol. Exposure of C. jejuni to carvacrol prior to infection also inhibited cellular infection, indicating that the inhibition of invasion was likely caused by an effect on the bacteria rather than inhibition of epithelial cell function.

Conclusions/Significance

Bacterial motility and invasion of eukaryotic cells are considered key steps in C. jejuni infection. Our results indicate that subinhibitory concentrations of carvacrol effectively block these virulence traits by interfering with flagella function without disturbing intracellular ATP levels. These results broaden the spectrum of anti-microbial activity of carvacrol and support the potential of the compound for use in novel infection prevention strategies.     

Basolateral Invasion and Trafficking of Campylobacter jejuni in Polarized Epithelial Cells

Campylobacter jejuni is a major cause of bacterial diarrheal disease. Most enteropathogenic bacteria including C. jejuni can invade cultured eukaryotic cells via an actin- and/or microtubule-dependent and an energy-consuming uptake process. Recently, we identified a novel highly efficient C. jejuni invasion pathway that involves bacterial migration into the subcellular space of non-polarized epithelial cells (termed subvasion) followed by invasion from the cell basis. Here we report cellular requirements of this entry mechanism and the subsequent intracellular trafficking route of C. jejuni in polarized islands of Caco-2 intestinal epithelial cells. Advanced microscopy on infected cells revealed that C. jejuni invades the polarized intestinal cells via the subcellular invasion pathway. Remarkably, invasion was not blocked by the inhibitors of microtubule dynamics colchicine or paclitaxel, and was even enhanced after disruption of host cell actin filaments by cytochalasin D. Invasion also continued after dinitrophenol-induced cellular depletion of ATP, whereas this compound effectively inhibited the uptake of invasive Escherichia coli. Confocal microscopy demonstrated that intracellular C. jejuni resided in membrane-bound CD63-positive cellular compartments for up to 24 h. Establishment of a novel luciferase reporter-based bacterial viability assay, developed to overcome the limitations of the classical bacterial recovery assay, demonstrated that a subset of C. jejuni survived intracellularly for up to 48 h. Taken together, our results indicate that C. jejuni is able to actively invade polarized intestinal epithelial cells via a novel actin- and microtubule-independent mechanism and remains metabolically active in the intracellular niche for up to 48 hours.     

Survival of Campylobacter jejuni in Water: Effect of Grazing by the Freshwater Crustacean Daphnia carinata (Cladocera)

Environmental studies of the human-pathogenic bacterium Campylobacter jejuni have focused on linking distributions with potential sources. However, in aquatic ecosystems, the abundance of C. jejuni may also be regulated by predation. We examine the potential for grazing by the freshwater planktonic crustacean Daphnia carinata to reduce the survival of C. jejuni. We use a system for measuring grazing and clearance rates of D. carinata on bacteria and demonstrate that D. carinata can graze C. jejuni cells at a rate of 7% individual⁻¹ h⁻¹ under simulated natural conditions in the presence of an algal food source. We show that passage of C. jejuni through the Daphnia gut and incorporation into fecal material effectively reduces survival of C. jejuni. This is the first evidence to suggest that grazing by planktonic organisms can reduce the abundance of C. jejuni in natural waters. Biomanipulation of planktonic food webs to enhance Daphnia densities offers potential for reducing microbial pathogen densities in drinking water reservoirs and recreational water bodies, thereby reducing the risk of contracting water-borne disease.     

Application of the 5′-Nuclease PCR Assay in Evaluation and Development of Methods for Quantitative Detection of Campylobacter jejuni

Campylobacter jejuni is recognized as a leading human food-borne pathogen. Traditional diagnostic testing for C. jejuni is not reliable due to special growth requirements and the possibility that this bacterium can enter a viable but nonculturable state. Nucleic acid-based tests have emerged as a useful alternative to traditional enrichment testing. In this article, we present a 5′-nuclease PCR assay for quantitative detection of C. jejuni and describe its evaluation. A probe including positions 381121 to 381206 of the published C. jejuni strain NCTC 11168 genome sequence was identified. When this probe was applied, the assay was positive for all of the isolates of C. jejuni tested (32 isolates, including the type strain) and negative for all other Campylobacter spp. (11 species tested) and several other bacteria (41 species tested). The total assay could be completed in 3 h with a detection limit of approximately 1 CFU. Quantification was linear over at least 6 log units. Quantitative detection methods are important for both research purposes and further development of C. jejuni detection methods. In this study, we used the assay to investigate to what extent the PCR signals generated by heat-killed bacteria interfere with the detection of viable C. jejuni after exposure at elevated temperatures for up to 5 days. An approach to the reduction of the PCR signal generated by dead bacteria was also investigated by employing externally added DNases to selectively inactivate free DNA and exposed DNA in heat-killed bacteria. The results indicated relatively good discrimination between exposed DNA from dead C. jejuni and protected DNA in living bacteria.     

Characterisation of a Multi-ligand Binding Chemoreceptor CcmL (Tlp3) of Campylobacter jejuni

Campylobacter jejuni is the leading cause of human gastroenteritis worldwide with over 500 million cases annually. Chemotaxis and motility have been identified as important virulence factors associated with C. jejuni colonisation. Group A transducer-like proteins (Tlps) are responsible for sensing the external environment for bacterial movement to or away from a chemical gradient or stimulus. In this study, we have demonstrated Cj1564 (Tlp3) to be a multi-ligand binding chemoreceptor and report direct evidence supporting the involvement of Cj1564 (Tlp3) in the chemotaxis signalling pathway via small molecule arrays, surface plasmon and nuclear magnetic resonance (SPR and NMR) as well as chemotaxis assays of wild type and isogenic mutant strains. A modified nutrient depleted chemotaxis assay was further used to determine positive or negative chemotaxis with specific ligands. Here we demonstrate the ability of Cj1564 to interact with the chemoattractants isoleucine, purine, malic acid and fumaric acid and chemorepellents lysine, glucosamine, succinic acid, arginine and thiamine. An isogenic mutant of cj1564 was shown to have altered phenotypic characteristics of C. jejuni, including loss of curvature in bacterial cell shape, reduced chemotactic motility and an increase in both autoagglutination and biofilm formation. We demonstrate Cj1564 to have a role in invasion as in in vitro assays the tlp3 isogenic mutant has a reduced ability to adhere and invade a cultured epithelial cell line; interestingly however, colonisation ability of avian caeca appears to be unaltered. Additionally, protein-protein interaction studies revealed signal transduction initiation through the scaffolding proteins CheV and CheW in the chemotaxis sensory pathway. This is the first report characterising Cj1564 as a multi-ligand receptor for C. jejuni, we therefore, propose to name this receptor CcmL, Campylobacter chemoreceptor for multiple ligands. In conclusion, this study identifies a novel multifunctional role for the C. jejuni CcmL chemoreceptor and illustrates its involvement in the chemotaxis pathway and subsequent survival of this organism in the host.     

Polyphosphate Kinase 2: A Novel Determinant of Stress Responses and Pathogenesis in Campylobacter jejuni

Background

Inorganic polyphosphate (poly P) plays an important role in stress tolerance and virulence in many bacteria. PPK1 is the principal enzyme involved in poly P synthesis, while PPK2 uses poly P to generate GTP, a signaling molecule that serves as an alternative energy source and a precursor for various physiological processes. Campylobacter jejuni, an important cause of foodborne gastroenteritis in humans, possesses homologs of both ppk1 and ppk2. ppk1 has been previously shown to impact the pathobiology of C. jejuni.

Methodology/Principal Findings

Here, we demonstrate for the first time that the deletion of ppk2 in C. jejuni resulted in a significant decrease in poly P-dependent GTP synthesis, while displaying an increased intracellular ATP:GTP ratio. The Δppk2 mutant exhibited a significant survival defect under osmotic, nutrient, aerobic, and antimicrobial stresses and displayed an enhanced ability to form static biofilms. However, the Δppk2 mutant was not defective in poly P and ppGpp synthesis suggesting that PPK2-mediated stress tolerance is not ppGpp-mediated. Importantly, the Δppk2 mutant was significantly attenuated in invasion and intracellular survival within human intestinal epithelial cells as well as in chicken colonization.

Conclusions/Significance

Taken together, we have highlighted the role of PPK2 as a novel pathogenicity determinant that is critical for C. jejuni survival, adaptation, and persistence in the host environments. PPK2 is absent in humans and animals; therefore, can serve as a novel target for therapeutic intervention of C. jejuni infections.     

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.     

Molecular Evidence for the Thriving of Campylobacter jejuni ST-4526 in Japan

Campylobacter jejuni is a leading cause of human gastroenteritis worldwide. This study aimed at a better understanding of the genetic diversity of this pathogen disseminated in Japan. We performed multilocus sequence typing (MLST) of Campylobacter jejuni isolated from different sources (100 human, 61 poultry, and 51 cattle isolates) in Japan between 2005 and 2006. This approach identified 62 sequence types (STs) and 19 clonal complexes (CCs), including 11 novel STs. These 62 STs were phylogenetically divided into 6 clusters, partially exhibiting host association. We identified a novel ST (ST-4526) that has never been reported in other countries; a phylogenetic analysis showed that ST-4526 and related STs showed distant lineage from the founder ST, ST-21 within CC-21. Comparative genome analysis was performed to investigate which properties could be responsible for the successful dissemination of ST-4526 in Japan. Results revealed that three representative ST-4526 isolates contained a putative island comprising the region from Cj0737 to Cj0744, which differed between the ST-4526 isolates and the reference strain NCTC11168 (ST-43/CC-21). Amino acid sequence alignment analyses showed that two of three ST-4526 isolates expressed 693aa- filamentous hemagglutination domain protein (FHA), while most of other C. jejuni strains whose genome were sequenced exhibited its truncation. Correspondingly, host cell binding of FHA-positive C. jejuni was greater than that of FHA-truncated strains, and exogenous administration of rFHA protein reduced cell adhesion of FHA-positive bacteria. Biochemical assays showed that this putative protein exhibited a dose-dependent binding affinity to heparan sulfate, indicating its adhesin activity. Moreover, ST-4526 showed increased antibiotic-resistance (nalidixic acid and fluoroquinolones) and a reduced ability for DNA uptake. Taken together, our data suggested that these combined features contributed to the clonal thriving of ST-4526 in Japan.     

Accumulation of Peptidoglycan O-Acetylation Leads to Altered Cell Wall Biochemistry and Negatively Impacts Pathogenesis Factors of Campylobacter jejuni

Campylobacter jejuni is a leading cause of bacterial gastroenteritis in the developed world. Despite its prevalence, its mechanisms of pathogenesis are poorly understood. Peptidoglycan (PG) is important for helical shape, colonization, and host-pathogen interactions in C. jejuni. Therefore, changes in PG greatly impact the physiology of this organism. O-acetylation of peptidoglycan (OAP) is a bacterial phenomenon proposed to be important for proper cell growth, characterized by acetylation of the C6 hydroxyl group of N-acetylmuramic acid in the PG glycan backbone. The OAP gene cluster consists of a PG O-acetyltransferase A (patA) for translocation of acetate into the periplasm, a PG O-acetyltransferase B (patB) for O-acetylation, and an O-acetylpeptidoglycan esterase (ape1) for de-O-acetylation. In this study, reduced OAP in ΔpatA and ΔpatB had minimal impact on C. jejuni growth and fitness under the conditions tested. However, accumulation of OAP in Δape1 resulted in marked differences in PG biochemistry, including O-acetylation, anhydromuropeptide levels, and changes not expected to result directly from Ape1 activity. This suggests that OAP may be a form of substrate level regulation in PG biosynthesis. Ape1 acetylesterase activity was confirmed in vitro using p-nitrophenyl acetate and O-acetylated PG as substrates. In addition, Δape1 exhibited defects in pathogenesis-associated phenotypes, including cell shape, motility, biofilm formation, cell surface hydrophobicity, and sodium deoxycholate sensitivity. Δape1 was also impaired for chick colonization and adhesion, invasion, intracellular survival, and induction of IL-8 production in INT407 cells in vitro. The importance of Ape1 in C. jejuni biology makes it a good candidate as an antimicrobial target.     

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.     

Importance of Polyphosphate Kinase 1 for Campylobacter jejuni Viable-but-Nonculturable Cell Formation,Natural Transformation,and Antimicrobial Resistance

Campylobacter jejuni, a gram-negative, microaerophilic bacterium, is a predominant cause of bacterial gastroenteritis in humans. Although considered fragile and fastidious and lacking many classical stress response mechanisms, C. jejuni exhibits a remarkable capacity for survival and adaptation, successfully infecting humans and persisting in the environment. Consequently, understanding the physiological and genetic properties that allow C. jejuni to survive and adapt to various stress conditions is crucial for therapeutic interventions. Of importance is polyphosphate (poly-P) kinase 1 (PPK1), which is a key enzyme mediating the synthesis of poly-P, an essential molecule for survival, mediating stress responses, host colonization, and virulence in many bacteria. Therefore, we investigated the role of PPK1 in C. jejuni pathogenesis, stress survival, and adaptation. Our findings demonstrate that a C. jejuni Δppk1 mutant was deficient in poly-P accumulation, which was associated with a decreased ability to form viable-but-nonculturable cells under acid stress. The Δppk1 mutant also showed a decreased frequency of natural transformation and an increased susceptibility to various antimicrobials. Furthermore, the Δppk1 mutant was characterized by a dose-dependent deficiency in chicken colonization. Complementation of the Δppk1 mutant with the wild-type copy of ppk1 restored the deficient phenotypes to levels similar to those of the wild type. Our results suggest that poly-P plays an important role in stress survival and adaptation and might contribute to genome plasticity and the spread and development of antimicrobial resistance in C. jejuni. These findings highlight the potential of PPK1 as a novel target for therapeutic interventions.Campylobacter jejuni, a gram-negative, microaerophilic bacterium, occurs as a commensal among the intestinal microflora of various animals, especially chickens and cattle (6, 73). However, C. jejuni can infect human hosts, invading the intestinal mucosa and causing watery and/or bloody diarrhea (9). C. jejuni is transmitted to humans primarily through the consumption of contaminated chicken products, raw milk, or water (2, 3). Currently, C. jejuni is considered a leading bacterial cause of human food-borne gastroenteritis (3, 61) and has also been associated with a plethora of symptoms, including acute neuromuscular paralysis (Guillain-Barré syndrome) (26). Since an appropriate vaccine for human campylobacteriosis has yet to be introduced, it has been suggested that C. jejuni infections might be alternatively controlled by reducing colonization in food animals (73). Consequently, determining the physiological and genetic properties that allow the survival of C. jejuni and its colonization of animal hosts, pathogenicity, and adaptation to various stresses is of critical importance.The mechanisms underlying C. jejuni adaptation and survival under stresses imposed by its environment and host are not well understood. High variability between different C. jejuni strains and the unavailability of appropriate genetic tools and animal models have contributed to the lack of knowledge regarding its stress tolerance and pathogenicity. However, it is suggested that the capacity of C. jejuni to form viable-but-nonculturable (VBNC) cells under stress (14) and its readiness for natural transformation (68) and acquiring resistance to antibiotics (39) are among the strategies that promote stress adaptation and survival. Although little is known about the genetics underlying these processes, recent advances in C. jejuni genomics show that this bacterium carries several important genes that might play key roles in mediating stress adaptation and survival. Of particular interest are genes encoding polyphosphate (poly-P) kinases, ppk1 (CJJ81176_1361) and ppk2 (CJJ81176_0633), that were predicted to be involved in the metabolism of poly-P (22, 25, 47), an intracellular granule that impacts several physiological properties in many bacterial species, including pathogenicity, host colonization, adaptation to different environments, and survival (28, 31, 46).Poly-P kinase 1 (PPK1) is encoded by ppk1, which mediates the synthesis of all or most of the poly-P in the cell (33), while ppk2 encodes an enzyme (PPK2) that synthesizes GTP from poly-P (27). Both ppk genes have been associated with the metabolism of poly-P, which consists of phosphate residues that are linked by high-energy phosphoanhydride bonds and is widely distributed in bacterial species (60). Previous reports showed that poly-P plays important roles in bacterial survival and stress tolerance, including ATP production (8), entry of DNA through membrane channels (13, 54), capsule composition (67), maintaining nutritional requirements during starvation (34), motility, biofilm formation, and resistance to oxidative, osmotic, heat, acid and alkaline stresses, and stationary-phase survival (28, 31, 46, 48, 50, 52, 65). Because of their importance in many bacterial species, it is not surprising to assume a role for PPK and poly-P in C. jejuni survival, colonization, and stress tolerance (8).Interestingly, PPK1 has been shown to be important for C. jejuni stress responses and pathogenicity (10). However, the role of ppk1 in key metabolic and physiological responses of C. jejuni still needs further analysis. For instance, it has been proposed that during starvation, poly-P might act as a reservoir for phosphorus and energy (7). Subsequently, poly-P would be crucial for maintaining viability/metabolism in stressed cells. This has been observed in H. pylori, where the occurrence of poly-P correlated with culturability and structurally intact cells (45). Poly-P-containing nonculturable H. pylori showed a capacity for ATP and mRNA synthesis after a nutrient stimulus (45). Consequently, poly-P might be an important factor for the formation of VBNC cells by stressed bacteria, including C. jejuni. Furthermore, natural transformation is perhaps one of the most important mechanisms in the adaptation of C. jejuni, and poly-P has been reported to play a role in the entry of DNA through membrane channels (13, 54). It follows that poly-P might be important for natural transformation, adaptation, and acquisition of antibiotic resistance genes in C. jejuni. Poly-P can further impact the survival and adaptation in C. jejuni by modulating antibiotic resistance properties. For example, poly-P interacted with Escherichia coli ribosomes (42), which are known targets of several antibiotics. These observations suggest that ppk1 might be linked to important physiology and functions such as VBNC cell formation, natural transformation, and antimicrobial resistance in C. jejuni. Therefore, in the present study, we determined the contribution of PPK1 to C. jejuni stress responses and adaptation, including the ability to form VBNC cells under acid stress, natural transformation, and antimicrobial resistance. Furthermore, we assessed the impact of ppk1 deletion on in vivo chicken colonization. Our findings highlight the importance of PPK1 in C. jejuni survival, adaptation to different environmental stresses, and in vivo colonization. These findings also indicate the suitability of PPK1 as a potential target for controlling the proliferation of this pathogen.     

CadF expression in Campylobacter jejuni strains incubated under low-temperature water microcosm conditions which induce the viable but non-culturable (VBNC) state

Campylobacter jejuni is a major gastrointestinal pathogen that colonizes host mucosa via interactions with extracellular matrix proteins such as fibronectin. The aim of this work was to study in vitro the adhesive properties of C. jejuni ATCC 33291 and C. jejuni 241 strains, in both culturable and viable but non-culturable (VBNC) forms. To this end, the expression of the outer-membrane protein CadF, which mediates C. jejuni binding to fibronectin, was evaluated. VBNC bacteria were obtained after 46–48 days of incubation in freshwater at 4 °C. In both cellular forms, the expression of the cadF gene, assessed at different time points by RT-PCR, was at high levels until the third week of VBNC induction, while the intensity of the signal declined during the last stage of incubation. CadF protein expression by the two C. jejuni strains was analysed using 2-dimensional electrophoresis and mass spectrometry; the results indicated that the protein, although at low levels, is also present in the VBNC state. Adhesion assays with culturable and VBNC cells, evaluated on Caco-2 monolayers, showed that non-culturable bacteria retain their ability to adhere to intestinal cells, though at a reduced rate. Our results demonstrate that the C. jejuni VBNC population maintains an ability to adhere and this may thus have an important role in the pathogenicity of this microorganism.     

Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model

The interactions of bacterial pathogens with host cells have been investigated extensively using in vitro cell culture methods. However as such cell culture assays are performed under aerobic conditions, these in vitro models may not accurately represent the in vivo environment in which the host-pathogen interactions take place. We have developed an in vitro model of infection that permits the coculture of bacteria and host cells under different medium and gas conditions. The Vertical Diffusion Chamber (VDC) model mimics the conditions in the human intestine where bacteria will be under conditions of very low oxygen whilst tissue will be supplied with oxygen from the blood stream. Placing polarized intestinal epithelial cell (IEC) monolayers grown in Snapwell inserts into a VDC creates separate apical and basolateral compartments. The basolateral compartment is filled with cell culture medium, sealed and perfused with oxygen whilst the apical compartment is filled with broth, kept open and incubated under microaerobic conditions. Both Caco-2 and T84 IECs can be maintained in the VDC under these conditions without any apparent detrimental effects on cell survival or monolayer integrity. Coculturing experiments performed with different C. jejuni wild-type strains and different IEC lines in the VDC model with microaerobic conditions in the apical compartment reproducibly result in an increase in the number of interacting (almost 10-fold) and intracellular (almost 100-fold) bacteria compared to aerobic culture conditions¹. The environment created in the VDC model more closely mimics the environment encountered by C. jejuni in the human intestine and highlights the importance of performing in vitro infection assays under conditions that more closely mimic the in vivo reality. We propose that use of the VDC model will allow new interpretations of the interactions between bacterial pathogens and host cells.