Nancy M. C. Bleumink-Pluym

Functional analysis of a Campylobacter jejuni alkaline phosphatase secreted via the Tat export machinery

Bacterial alkaline phosphatases (PhoA) hydrolyse phosphate-containing substrates to provide the preferred phosphorus source inorganic phosphate (P(i)). Campylobacter jejuni does not contain a typical PhoA homologue but contains a phosphatase that is regulated by the two-component system PhosS/PhosR. Here we describe the characterization of the enzyme, its secretion pathway and its function in the bacteriums biology. Phosphatase assays showed that the enzyme utilizes exclusively phosphomonoesters as a substrate, requires Ca(2+) for its activity, and displays maximum activity at a pH of 10. Gene disruption revealed that it is the sole alkaline phosphatase in C. jejuni. The protein contained a twin-arginine motif (RR) at its N terminus, typical of substrates of the Tat secretion system. Substitution of the twin-arginine residues showed that they are essential for enzyme activity. C. jejuni genome analysis indicated the presence of four ubiquitously expressed Tat components that may form a functional Tat secretion system as well as 11 putative Tat substrates, including the alkaline phosphatase (PhoA(Cj)) and the nitrate reductase NapA. Inactivation of tatC caused defects in both PhoA(Cj) and NapA activity as well as a reduction in bacterial growth that were all restored by complementation in trans with an intact tatC copy. The atypical overall features of the PhoA(Cj) compared to Escherichia coli PhoA support the existence in prokaryotes of a separate group of Tat-dependent alkaline phosphatases, classified as the PhoX family.

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.

Identification of a Functional Type VI Secretion System in Campylobacter jejuni Conferring Capsule Polysaccharide Sensitive Cytotoxicity

The pathogen Campylobacter jejuni is the principal cause of bacterial food-borne infections. The mechanism(s) that contribute to bacterial survival and disease are still poorly understood. In other bacterial species, type VI secretion systems (T6SS) are increasingly recognized to contribute to bacterial pathogenesis by toxic effects on host cells or competing bacterial species. Here we report the presence of a functional Type VI secretion system in C. jejuni. Proteome and genetic analyses revealed that C. jejuni strain 108 contains a 17-kb T6SS gene cluster consisting of 13 T6SS-conserved genes, including the T6SS hallmark genes hcp and vgrG. The cluster lacks an ortholog of the ClpV ATPase considered important for T6SS function. The sequence and organization of the C. jejuni T6SS genes resemble those of the T6SS located on the HHGI1 pathogenicity island of Helicobacter hepaticus. The C. jejuni T6SS is integrated into the earlier acquired Campylobacter integrated element CJIE3 and is present in about 10% of C. jejuni isolates including several isolates derived from patients with the rare clinical feature of C. jejuni bacteremia. Targeted mutagenesis of C. jejuni T6SS genes revealed T6SS-dependent secretion of the Hcp needle protein into the culture supernatant. Infection assays provided evidence that the C. jejuni T6SS confers contact-dependent cytotoxicity towards red blood cells but not macrophages. This trait was observed only in a capsule-deficient bacterial phenotype. The unique C. jejuni T6SS phenotype of capsule-sensitive contact-mediated hemolysis represents a novel evolutionary pathway of T6SS in bacteria and expands the repertoire of virulence properties associated with T6SS.

A functional Campylobacter jejuni maf4 gene results in novel glycoforms on flagellin and altered autoagglutination behaviour

Flagellin of Campylobacter jejuni is extensively modified with (derivatives of) pseudaminic acid. The flagellar glycosylation locus contains several genes with homopolymeric G-tracts prone to slipped-strand mispairing, some of which belong to the maf gene family. We investigated the function of the putative phase-variable maf4 gene of C. jejuni strain 108. A constructed maf4 mutant displayed unaltered flagella assembly and bacterial motility. 2D-PAGE analysis revealed that the flagellin of strain 108 migrated at a more acidic pI than the protein of the Maf4 mutant. MS-MS in combination with high-resolution matrix-assisted laser desorption/ionization Fourier transform ion cyclotron MS (MALDI-FT-ICR-MS) on flagellin-derived glycopeptides showed that the flagellins of the mutant lacked two previously unidentified modifications of pseudaminic acid. These glycoforms carried additional CO(2) and C(2)H(2)O(2) groups, consistent with the more acidic pI of the wild-type flagellin. Phenotypically, the maf4 mutant displayed strongly delayed bacterial autoagglutination. Collectively, our results suggest that the presence of a functional Maf4 expands the flagellin glycan repertoire with novel glycoforms of pseudaminic acid and, in the event of phase variation, alters the population behaviour of C. jejuni.

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.

Phylogenetic position of Taylorella equigenitalis determined by analysis of amplified 16S ribosomal DNA sequences

The 16S ribosomal DNA sequence of Taylorella equigenitalis (formerly Haemophilus equigenitalis), the causative organism of contagious equine metritis, was determined. A phylogenetic analysis of this sequence revealed a phylogenetic position of T. equigenitalis in the β subclass of the class Proteobacteria apart from the position of Haemophilus influenzae, which belongs to the γ subclass of Proteobacteria. A close phylogenetic relationship among T. equigenitalis, Alcaligenes xylosoxidans, and Bordetella bronchiseptica was detected; Spirillum volutans and Chromobacterium fluviatile (Iodobacter fluviatile) were in the same group but slightly removed. This relationship is surprising in view of the considerable differences in the G+C contents of the genomes of these bacteria.

Campylobacter jejuni is highly susceptible to killing by chicken host defense peptide cathelicidin-2 and suppresses intestinal cathelicidin-2 expression in young broilers.

Little is known about the interactions of chicken host defense peptides (HDPs) with Campylobacter jejuni in young chicks. To examine the role of the chicken HDP, cathelicidin-2 (CATH-2) in host-pathogen interactions we challenged 4-day-old Ross 308 broilers with a chicken-derived C. jejuni isolate (WS356) and used the chicken pathogen Salmonella enterica Enteritidis phage type 4 (FGT1) as a reference. Immunohistochemical staining was used to localize CATH-2, C. jejuni and Salmonella enteritidis. Intestinal CATH-2 mRNA expression levels were determined by quantitative PCR. Antibacterial activities of CATH-2 peptide against C. jejuni and S. enteritidis isolates were assessed in colony count assays. In contrast to S. enteritidis, C. jejuni was not seen to attach to intestinal epithelium and C. jejuni challenge did not result in recruitment of CATH-2 containing heterophils to the small intestinal lamina propria. Minimal inhibitory concentrations found for CATH-2 peptide against human- and chicken-derived C. jejuni isolates were similar (0.6-2.5 μM) and much lower than for S. enteritidis (20 μM). Compared to wild-type C. jejuni 81116, the lipooligosaccharide (LOS)-deficient 81116ΔwaaF mutant was much more susceptible to CATH-2. Interestingly, CATH-2 mRNA expression levels in the small intestine were significantly lower 48 h p.i. in C. jejuni-challenged chicks. These findings indicate that human clinical and chicken-derived C. jejuni are equally highly susceptible to chicken CATH-2 peptide and that C. jejuni uses LOS to protect itself to some extent against HDPs. Moreover, suppression of intestinal CATH-2 expression levels may be part of the C. jejuni immune evasion strategy.

Inflammasome Activation by Campylobacter jejuni

The Gram-negative pathogen Campylobacter jejuni is the most common cause of bacterial foodborne disease worldwide. The mechanisms that lead to bacterial invasion of eukaryotic cells and massive intestinal inflammation are still unknown. In this study, we report that C. jejuni infection of mouse macrophages induces upregulation of pro–IL-1β transcript and secretion of IL-1β without eliciting cell death. Immunoblotting indicated cleavage of caspase-1 and IL-1β in infected cells. In bone marrow–derived macrophages from different knockout mice, IL-1β secretion was found to require NLRP3, ASC, and caspase-1/11 but not NLRC4. In contrast to NLRP3 activation by ATP, C. jejuni activation did not require priming of these macrophages. C. jejuni also activated the NLRP3 inflammasome in human macrophages as indicated by the presence of ASC foci and caspase-1–positive cells. Analysis of a vast array of C. jejuni mutants with defects in capsule formation, LPS biosynthesis, chemotaxis, flagella synthesis and flagellin (-like) secretion, type 6 secretion system needle protein, or cytolethal distending toxin revealed a direct correlation between the number of intracellular bacteria and NLRP3 inflammasome activation. The C. jejuni invasion–related activation of the NLRP3 inflammasome without cytotoxicity and even in nonprimed cells extends the known repertoire of bacterial inflammasome activation and likely contributes to C. jejuni–induced intestinal inflammation.

Campylobacter DNA Is Present in Circulating Myelomonocytic Cells of Healthy Persons and in Persons with Guillain-Barré Syndrome

Campylobacter jejuni is the prime cause of foodborne bacterial gastroenteritis. An important complication of C. jejuni enteritis is Guillain-Barré syndrome (GBS), an immune-mediated disorder of the peripheral nerve. The presence of C. jejuni DNA in peripheral blood mononuclear cells (PBMC) of patients with GBS, patients with C. jejuni enteritis, and healthy subjects was studied. Two target genes, the flagellin and the ceuE genes, were used for polymerase chain reaction (PCR) identification of Campylobacter species in DNA extracted from PBMC. Approximately 30% of the healthy subjects and 50% of the patients with GBS had PBMC containing C. jejuni DNA as verified by Southern blot analysis or sequencing of the PCR products. Cell sorting revealed that Campylobacter DNA was present in CD14(+) and CD33(+) populations, indicating that cells from the myelomonocytic lineage are the Campylobacter DNA-carrying cells. These findings show that Campylobacter DNA is present in blood cells of healthy humans, although viable bacteria could not be demonstrated.