Campylobacter jejuni survives within epithelial cells by avoiding delivery to lysosomes

Campylobacter jejuni is one of the major causes of infectious diarrhea world-wide, although relatively little is know about its mechanisms of pathogenicity. This bacterium can gain entry into intestinal epithelial cells, which is thought to be important for its ability to persistently infect and cause disease. We found that C. jejuni is able to survive within intestinal epithelial cells. However, recovery of intracellular bacteria required pre-culturing under oxygen-limiting conditions, suggesting that C. jejuni undergoes significant physiological changes within the intracellular environment. We also found that in epithelial cells the C. jejuni-containing vacuole deviates from the canonical endocytic pathway immediately after a unique caveolae-dependent entry pathway, thus avoiding delivery into lysosomes. In contrast, in macrophages, C. jejuni is delivered to lysosomes and consequently is rapidly killed. Taken together, these studies indicate that C. jejuni has evolved specific adaptations to survive within host cells.     

Identification of a fibronectin-binding domain within the Campylobacter jejuni CadF protein

The binding of Campylobacter jejuni to fibronectin (Fn), a component of the extracellular matrix, is mediated by a 37 kDa outer membrane protein termed CadF for Campylobacter adhesion to Fn. Previous studies have indicated that C. jejuni binds to Fn on the basolateral surface of T84 human colonic cells. To further characterize the interaction of the CadF protein with Fn, enzyme-linked immunosorbent assays were performed to identify the Fn-binding domain (Fn-BD). Using overlapping 30-mer and 16-mer peptides derived from translated cadF nucleotide sequence, maximal Fn-binding activity was localized to four amino acids (AA 134-137) consisting of the residues phenylalanine-arginine-leucine-serine (FRLS). A mouse alpha-CadF peptide polyclonal antibody (M alpha-CadF peptide pAb) was generated using FRLS containing peptides and found to react with viable C. jejuni as judged by indirect fluorescent microscopy, suggesting that the FRLS residues are surface-exposed. Binding of CadF to purified Fn and INT 407 human epithelial cells was significantly inhibited with peptides containing the Fn-BD. Moreover, a CadF recombinant variant protein, in which the Phe-Arg-Leu residues (CadF AA 134-136) were altered to Ala-Ala-Gly, exhibited a 91% decrease in Fn-binding activity as compared with the wild-type CadF protein. Collectively, these data indicate that the FRLS residues (CadF AA 134-137) of the C. jejuni CadF protein possess Fn-binding activity.     

Relaxation of DNA supercoiling leads to increased invasion of epithelial cells and protein secretion by Campylobacter jejuni

Invasion of intestinal epithelial cells by Campylobacter jejuni is a critical step during infection of the intestine by this important human pathogen. In this study we investigated the role played by DNA supercoiling in the regulation of invasion of epithelial cells and the mechanism by which this could be mediated. A significant correlation between more relaxed DNA supercoiling and an increased ability of C. jejuni strains to penetrate human epithelial cells was demonstrated. Directly inducing relaxation of DNA supercoiling in C. jejuni was shown to significantly increase invasion of epithelial cells. Mutants in the fibronectin binding proteins CadF and FlpA still displayed an increased invasion after treatment with novobiocin suggesting these proteins were not essential for the observed phenotype. However, a large increase in protein secretion from multiple C. jejuni strains upon relaxation of DNA supercoiling was demonstrated. This increase in protein secretion was not mediated by outer membrane vesicles and appeared to be dependent on an intact flagellar structure. This study identifies relaxation of DNA supercoiling as playing a key role in enhancing C. jejuni pathogenesis during infection of the human intestine and identifies proteins present in a specific invasion associated secretome induced by relaxation of DNA supercoiling.     

Transcellular translocation of Campylobacter jejuni across human polarised epithelial monolayers

The mechanisms whereby Campylobacter jejuni translocates across the host intestinal epithelium are not yet understood and the transepithelial route remains undefined. During C. jejuni translocation, the transmonolayer electrical resistance (TER) across polarised monolayers of Caco-2 cells is not affected and the penetration of [(14)C]inulin across the monolayers does not increase. Over 24 h, however, bacteria damage the monolayer integrity, causing a decrease in the TER. These results support C. jejuni translocation through the cytoplasm of invaded cells (transcellular) rather than via intercellular spaces (paracellular).     

Invasion of human epithelial cells by Campylobacter upsaliensis

Few data exist on the interaction of Campylobacter upsaliensis with host cells, and the potential for this emerging enteropathogen to invade epithelial cells has not been explored. We have characterized the ability of C. upsaliensis to invade both cultured epithelial cell lines and primary human small intestinal cells. Epithelial cell lines of intestinal origin appeared to be more susceptible to invasion than non-intestinal-derived cells. Of three bacterial isolates studied, a human clinical isolate, CU1887, entered cells most efficiently. Although there was a trend towards more efficient invasion of Caco-2 cells by C. upsaliensis CU1887 at lower initial inocula, actual numbers of intracellular organisms increased with increasing multiplicity of infection and with prolonged incubation period. Confocal microscopy revealed C. upsaliensis within primary human small intestinal cells. Both Caco-2 and primary cells in non-confluent areas of the infected monolayers were substantially more susceptible to infection than confluent cells. The specific cytoskeletal inhibitors cytochalasin B, cytochalasin D and vinblastine attenuated invasion of Caco-2 cells in a concentration-dependent manner, providing evidence for both microtubule- and microfilament-dependent uptake of C. upsaliensis. Electron microscopy revealed the presence of organisms within Caco-2 cell cytoplasmic vacuoles. C. upsaliensis is capable of invading epithelial cells and appears to interact with host cell cytoskeletal structures in order to gain entry to the intracellular environment. Entry into cultured primary intestinal cells ex vivo provides strong support for the role of host cell invasion during human enteric C. upsaliensis infection.     

The ALPK1 pathway drives the inflammatory response to Campylobacter jejuni in human intestinal epithelial cells

The Gram-negative bacterium Campylobacter jejuni is a major cause of foodborne disease in humans. After infection, C. jejuni rapidly colonizes the mucus layer of the small and large intestine and induces a potent pro-inflammatory response characterized by the production of a large repertoire of cytokines, chemokines, and innate effector molecules, resulting in (bloody) diarrhea. The virulence mechanisms by which C. jejuni causes this intestinal response are still largely unknown. Here we show that C. jejuni releases a potent pro-inflammatory compound into its environment, which activates an NF-κB-mediated pro-inflammatory response including the induction of CXCL8, CXCL2, TNFAIP2 and PTGS2. This response was dependent on a functional ALPK1 receptor and independent of Toll-like Receptor and Nod-like Receptor signaling. Chemical characterization, inactivation of the heptose-biosynthesis pathway by the deletion of the hldE gene and in vitro engineering identified the released factor as the LOS-intermediate ADP-heptose and/or related heptose phosphates. During C. jejuni infection of intestinal cells, the ALPK1-NF-κB axis was potently activated by released heptose metabolites without the need for a type III or type IV injection machinery. Our results classify ADP-heptose and/or related heptose phosphates as a major virulence factor of C. jejuni that may play an important role during Campylobacter infection in humans.     

CapA, an autotransporter protein of Campylobacter jejuni, mediates association with human epithelial cells and colonization of the chicken gut

Two putative autotransporter proteins, CapA and CapB, were identified in silico from the genome sequence of Campylobacter jejuni NCTC11168. The genes encoding each protein contain homopolymeric tracts, suggestive of phase variation mediated by a slipped-strand mispairing mechanism; in each case the gene sequence contained frameshifts at these positions. The C-terminal two-thirds of the two genes, as well as a portion of the predicted signal peptides, were identical; the remaining N-terminal portions were gene specific. Both genes were cloned and expressed; recombinant polypeptides were purified and used to raise rabbit polyclonal monospecific antisera. Using immunoblotting, expression of the ca.116-kDa CapA protein was demonstrated for in vitro-grown cells of strain NCTC11168, for 4 out of 11 recent human fecal isolates, and for 2 out of 8 sequence-typed strains examined. Expression of CapB was not detected for any of the strains tested. Surface localization of CapA was demonstrated by subcellular fractionation and immunogold electron microscopy. Export of CapA was inhibited by globomycin, reinforcing the bioinformatic prediction that the protein is a lipoprotein. A capA insertion mutant had a significantly reduced capacity for association with and invasion of Caco-2 cells and failed to colonize and persist in chickens, indicating that CapA plays a role in host association and colonization by Campylobacter. In view of this demonstrated role, we propose that CapA stands for Campylobacter adhesion protein A.     

FlaC, a protein of Campylobacter jejuni TGH9011 (ATCC43431) secreted through the flagellar apparatus, binds epithelial cells and influences cell invasion

Type III secretion systems identified in bacterial pathogens of animals and plants transpose effectors and toxins directly into the cytosol of host cells or into the extracellular milieu. Proteins of the type III secretion apparatus are conserved among diverse and distantly related bacteria. Many type III apparatus proteins have homologues in the flagellar export apparatus, supporting the notion that type III secretion systems evolved from the flagellar export apparatus. No type III secretion apparatus genes have been found in the complete genomic sequence of Campylobacter jejuni NCTC11168. In this study, we report the characterization of a protein designated FlaC of C. jejuni TGH9011. FlaC is homologous to the N- and C-terminus of the C. jejuni flagellin proteins, FlaA and FlaB, but lacks the central portion of these proteins. flaC null mutants form a morphologically normal flagellum and are highly motile. In wild-type C. jejuni cultures, FlaC is found predominantly in the extracellular milieu as a secreted protein. Null mutants of the flagellar basal rod gene (flgF) and hook gene (flgE) do not secrete FlaC, suggesting that a functional flagellar export apparatus is required for FlaC secretion. During C. jejuni infection in vitro, secreted FlaC and purified recombinant FlaC bind to HEp-2 cells. Invasion of HEp-2 cells by flaC null mutants was reduced to a level of 14% compared with wild type, suggesting that FlaC plays an important role in cell invasion.     

Bacterial secreted proteins are required for the internalization of Campylobacter jejuni into cultured mammalian cells

Presented here is the first evidence that Campylobacter jejuni secrete proteins upon co-cultivation with host cells and in INT 407 cell-conditioned medium. A C. jejuni gene designated ciaB for Campylobacter invasion antigen B was identified, using a differential screening technique, which is required for this secretion process and the efficient entry of this bacterium into a host cell. The C. jejuni ciaB gene encodes a protein of 610 amino acids with a calculated molecular mass of 73 154 Da. The deduced amino acid sequence of the CiaB protein shares similarity with type III secreted proteins associated with the invasion of host cells from other more extensively characterized bacterial pathogens. In vitro binding and internalization assays revealed that the binding of C. jejuni ciaB null mutants was indistinguishable from that of the parental isolate, whereas a significant reduction was noted in internalization. Confocal microscopic examination of C. jejuni-infected cells revealed that CiaB was translocated into the cytoplasm of the host cells. Culturing C. jejuni with INT 407 cells or in INT 407-conditioned medium resulted in the secretion of at least eight proteins, ranging in size from 12.8 to 108 kDa, into the culture medium. C. jejuni ciaB null mutants were deficient in the secretion of all eight proteins, indicating that CiaB is required for the secretion process. The identification of the C. jejuni ciaB gene represents a significant advance in understanding the molecular mechanism of C. jejuni internalization and the pathogenesis of C. jejuni-mediated enteritis.     

Active migration into the subcellular space precedes Campylobacter jejuni invasion of epithelial cells

The bacterial pathogen Campylobacter jejuni invades mucosal cells via largely undefined and rather inefficient (0.01–2 bacteria per cell) mechanisms. Here we report a novel, highly efficient C. jejuni infection pathway resulting in 10–15 intracellular bacteria per cell within 3 h of infection. Electron microscopy, pulse–chase infection assays and time-lapse multiphoton laser confocal microscopy demonstrated that the mechanism involved active and rapid migration of the pathogen into the subcellular space (termed 'subvasion'), followed by bacterial entry ('invasion') at the cell basis. Efficient subvasion was maximal after repeated rounds of selection for the subvasive phenotype. Targeted mutagenesis indicated that the CadF, JlpA or PEB1 adhesins were not required. Dissection of the selected and parental phenotypes by SDS-PAGE yielded comparable capsule polysaccharide and lipooligosaccharide profiles. Proteomics revealed reduced amounts of the chemotaxis protein CheW for the subvasive phenotype. Swarming assays confirmed that the selected phenotype exhibited altered migration behaviour. Introduction of a plasmid carrying chemotaxis genes into the subvasive strain yielded wild-type subvasion levels and migration behaviour. These results indicate that alterations in the bacterial migration machinery enable C. jejuni to actively penetrate the subcellular space and gain access to the cell interior with unprecedented efficiency.     

The HtrA protease of Campylobacter jejuni is required for heat and oxygen tolerance and for optimal interaction with human epithelial cells

Campylobacter jejuni is a predominant cause of food-borne bacterial gastroenteritis in the developed world. We have investigated the importance of a homologue of the periplasmic HtrA protease in C. jejuni stress tolerance. A C. jejuni htrA mutant was constructed and compared to the parental strain, and we found that growth of the mutant was severely impaired both at 44 degrees C and in the presence of the tRNA analogue puromycin. Under both conditions, the level of misfolded protein is known to increase, and we propose that the heat-sensitive phenotype of the htrA mutant is caused by an accumulation of misfolded protein in the periplasm. Interestingly, we observed that the level of the molecular chaperones DnaK and ClpB was increased in the htrA mutant, suggesting that accumulation of non-native proteins in the periplasm induces the expression of cytoplasmic chaperones. While lack of HtrA reduces the oxygen tolerance of C. jejuni, the htrA mutant was not sensitive to compounds that increase the formation of oxygen radicals, such as paraquat, cumene hydroperoxide, and H2O2. Using tissue cultures of human epithelial cells (INT407), we found that the htrA mutant adhered to and invaded human epithelial cells with a decreased frequency compared to the wild-type strain. This defect may be a consequence of the observed altered morphology of the htrA mutant. Thus, our results suggest that in C. jejuni, HtrA is important for growth during stressful conditions and has an impact on virulence.     

Anion-independent iron coordination by the Campylobacter jejuni ferric binding protein

Campylobacter jejuni, the leading cause of human gastroenteritis, expresses a ferric binding protein (cFbpA) that in many pathogenic bacteria functions to acquire iron as part of their virulence repertoire. Recombinant cFbpA is isolated with ferric iron bound from Escherichia coli. The crystal structure of cFbpA reveals unprecedented iron coordination by only five protein ligands. The histidine and one tyrosine are derived from the N-terminal domain, whereas the three remaining tyrosine ligands are from the C-terminal domain. Surprisingly, a synergistic anion present in all other characterized ferric transport proteins is not observed in the cFbpA iron-binding site, suggesting a novel role for this protein in iron uptake. Furthermore, cFbpA is shown to bind iron with high affinity similar to Neisserial FbpA and exhibits an unusual preference for ferrous iron (oxidized subsequently to the ferric form) or ferric iron chelated by oxalate. Sequence and structure analyses reveal that cFbpA is a member of a new class of ferric binding proteins that includes homologs from invasive and intracellular bacteria as well as cyanobacteria. Overall, six classes are defined based on clustering within the tree and by their putative iron coordination. The absence of a synergistic anion in the iron coordination sphere of cFbpA also suggests an alternative model of evolution for FbpA homologs involving an early iron-binding ancestor instead of a requirement for a preexisting anion-binding ancestor.     

Environmental survival mechanisms of the foodborne pathogen Campylobacter jejuni

Campylobacter spp. continue to be the greatest cause of bacterial gastrointestinal infections in humans worldwide. They encounter many stresses in the host intestinal tract, on foods and in the environment. However, in common with other enteric bacteria, they have developed survival mechanisms to overcome these stresses. Many of the survival mechanisms used by Campylobacter spp. differ from those used by other bacteria, such as Escherichia coli and Salmonella spp. This review summarizes the mechanisms by which Campylobacter spp. adapt to stress conditions and thereby increase their ability to survive on food and in the environment.     

The plasminogen-like molecule apically secreted by epithelial thyroid cells is sulfated

Plasminogen (Pl), a circulating protease synthesized in the liver, is also present in several tissues. In the thyroid gland a Pl-like protease was found in the apical lumen where it is involved, through its proteolytic activity, in luminal degradation of thyroglobulin (Tg). Here, we showed for the first time that the Pl-like protease apically secreted by epithelial thyroid cells is sulfated, both on tyrosine residue(s) and on oligosaccharide side chains. The Pl molecule is composed of a large N-terminal moiety made of five distinct Kringle domains (K1-K5) separated by small peptidic fragments, and of a C-terminal domain with serine protease activity. Using a software tool able to predict tyrosine sulfation sites in protein sequences we localized the potential tyrosine sulfation sites of Pl. Then, we became aware that, whatever the species considered, at least three of the four potential tyrosine sulfation sites of Pl were located on Kringle sites, and more precisely, for K1, on the highly conserved binding domain of K1. We determined with the same software tool which potential sulfation sites were the most likely to be really sulfated. We hypothesize that the sulfation of these sites modulates the binding properties of Pl.     

Outer membrane porin protein of Campylobacter jejuni

Abstract Protein e, a 43-kDa protein from the outer membrane of Campylobacter jejuni UA580, was purified and reconstituted into lipid bilayer membranes. It was shown to form small channels with a single channel conductance of 8.82 nS in 1M KCl. Zero current potential measurements demonstrated that the channel was approx. 10-fold selective for K⁺ over Cl⁻ ions. A porin with a similar single channel conductance was observed in fractions from the outer membrane of Campylobacter fetus UA60.     

PorA protein of Campylobacter jejuni is not a cytotoxin mediating inflammatory diarrhoea

Campylobacter jejuni is a major food-borne pathogen and a leading cause of diarrhoea. A cytotoxin is most likely involved in the pathogenesis of inflammatory diarrhoea due to C. jejuni. A 45-kDa outer membrane protein encoded by the porA gene was reported to exhibit cytotoxic activity for cultured mammalian cells in vitro. We cloned and expressed the porA gene in Escherichia coli BL21 codon plus RIL strain using the fusion vector pGEX-4T-1. The fusion protein solubilised in urea in denatured form or solubilised in Empigen BB in native form or their thrombin-cleaved products did not exhibit cytotoxic activity for Chinese hamster ovary (CHO) cells. The urea-solubilised fusion protein did not induce fluid accumulation in the rabbit ileal loop assay. All 76 clinical isolates of Campylobacter spp. tested were positive for porA by PCR, but only 13 isolates were positive for cytotoxin on CHO cells. Both cytotoxin-positive as well as cytotoxin-negative strains expressed PorA as determined by immunoblot analysis. These findings show that the porA gene product of C. jejuni is not a cytotoxin mediating inflammatory diarrhoea.     

Minicell formation by Campylobacter jejuni

Campylobacter jejuni sheds its flagella and varying proportions of the poles of the cell late in the growth cycle, resulting in the production of very small flagellated structures 0.1 to 0.3 microM in diameter. Electron microscopy revealed that these structures were minicells possessing outer membrane, cytoplasmic membrane, flagellar basal complex, and polar membrane; nucleoplasms were not seen. The initial event in the formation of these minicells involved a constriction of the cytoplasmic membrane, segregating the polar regions of the cell. The peptidoglycan layer of the cell wall was not visible, but was presumed to lyse at the separation site of minicell formation, and to reform or remain intact along the main length of the cell because the rods did not spheroplast. Finally, rupture and resealing of the outer membrane component of the wall resulted in the release of fully enclosed minicells and nonflagellated rods.     

Campylobacter jejuni infection within a laboratory animal production unit

A conventional laboratory animal production unit in which rats, mice, guineapigs and rabbits were bred in one building and cats maintained in a separate, but adjacent area was examined for the presence of intestinal thermophilic Campylobacter spp. Campylobacter jejuni was recovered from 18.84% of 552 animals. The infection rate was highest amongst the cats (51.7%), with rats being the second most commonly infected (23.2%), whereas only 7.7% of guineapigs and a single rabbit (1%) were positive. Campylobacter-like organisms were cultured from 10% of the mice, but these bacteria failed to grow on subsequent subculturing. By using bacterial restriction endonuclease DNA analysis (BRENDA), a single type of C. jejuni was identified from all isolates recovered from the rats, guineapigs and a rabbit, suggesting a common source of infection. In contrast, there were 5 different BRENDA patterns derived from cat isolates. No isolates of C. jejuni were obtained from humans working within the unit or from animal bedding or the immediate environment, although it was suggested that the organism may have entered and spread within the unit from sawdust.     

Lipopolysaccharide-binding protein is vectorially secreted and transported by cultured intestinal epithelial cells and is present in the intestinal mucus of mice

Lipopolysaccharide-binding protein (LBP) is an important modulator of the host's response to endotoxin. In a previous study, we found evidence for the synthesis of LBP by intestinal epithelial cells. In this study, we explored the polarity of LBP secretion by these cells. Polarized monolayers of Caco-2 cells were used as intestinal mucosa model. Cells were stimulated apically or basally with cytokines, and LBP secretion was analyzed. Furthermore, the presence of LBP in intestinal mucus of healthy and endotoxemic mice was studied using a mucus-sampling technique. The constitutive unipolar LBP secretion from the apical cell surface was markedly enhanced when cells were exposed to cytokines at their apical surface. However, bioactive LBP was secreted from both cell surfaces after basolateral stimulation of cells. Cytokines also influenced the secretion of the acute phase proteins serum amyloid A, apoA-I, and apoB from both surfaces of Caco-2 cells. Furthermore, transport of exogenous LBP from the basolateral to the apical cell surface was demonstrated. In line with these in vitro data, the presence of LBP in intestinal mucus was strongly enhanced in mice after a challenge with endotoxin. The results indicate that LBP is present at the mucosal surface of the intestine, a phenomenon for which secretion and transport of LBP by intestinal epithelial cells may be responsible.     

The adhesion of clinical isolates of Campylobacter jejuni to intestinal epithelial cells in vitro

The adhesive activity of C. jejuni isolated from feces of children with Campylobacter infection was studied with the use of a newly developed model. 47 clinical isolates were analyzed; of these, 91% were found to be enteroadhesive to a variable degree. As the result of in vitro studies, Campylobacter were found to have much greater tropism to colonic cells and epithelial cells of Peyer's patches in comparison with the epithelial cells of the small intestine. The correlation between the degree of adhesive activity and the severity of the course of Campylobacter infection in children.