Marc M. S. M. Wösten

Identification of the lipopolysaccharide modifications controlled by the Salmonella PmrA/PmrB system mediating resistance to Fe(III) and Al(III)

Iron is an essential metal but can be toxic in excess. While several homeostatic mechanisms prevent oxygen‐dependent killing promoted by Fe(II), little is known about how cells cope with Fe(III), which kills by oxygen‐independent means. Several Gram‐negative bacterial species harbour a regulatory system – termed PmrA/PmrB – that is activated by and required for resistance to Fe(III). We now report the identification of the PmrA‐regulated determinants mediating resistance to Fe(III) and Al(III) in Salmonella enterica serovar Typhimurium. We establish that these determinants remodel two regions of the lipopolysaccharide, decreasing the negative charge of this major constituent of the outer membrane. Remodelling entails the covalent modification of the two phosphates in the lipid A region with phosphoethanolamine and 4‐aminoarabinose, which has been previously implicated in resistance to polymyxin B, as well as dephosphorylation of the Hep(II) phosphate in the core region by the PmrG protein. A mutant lacking the PmrA‐regulated Fe(III) resistance genes bound more Fe(III) than the wild‐type strain and was defective for survival in soil, suggesting that these PmrA‐regulated lipopolysaccharide modifications aid Salmonellas survival and spread in non‐host environments.

Molecular Mechanisms of Campylobacter Infection

Campylobacter jejuni is the principal bacterial foodborne pathogen. A major challenge still is to identify the virulence strategies exploited by C. jejuni. Recent genomics, proteomics, and metabolomics approaches indicate that C. jejuni displays extensive inter- and intrastrain variation. The diverse behavior enables bacterial adaptation to different environmental conditions and directs interactions with the gut mucosa. Here, we report recent progress in understanding the molecular mechanisms and functional consequences of the phenotype diversity. The results suggest that C. jejuni actively penetrates the intestinal mucus layer, secretes proteins mainly via its flagellar apparatus, is engulfed by intestinal cells, and can disrupt the integrity of the epithelial lining. C. jejuni stimulates the proinflammatory pathway and the production of a large repertoire of cytokines, chemokines, and innate effector molecules. Novel experimental infection models suggest that the activation of the innate immune response is important for the development of intestinal pathology.

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.

The Campylobacter jejuni PhosS/PhosR operon represents a non-classical phosphate-sensitive two-component system

The bacterial pathogen Campylobacter jejuni carries several putative two‐component signal transduction systems of unknown function. Here we report that the PhosS (Cj0889) and PhosR (Cj0890) proteins constitute a two‐component system that is activated by phosphate limitation. Microarray analysis, real‐time RT‐PCR, and primer extension experiments indicated that this system regulates 12 genes (including the pstSCAB genes) present in three transcriptional units. Gel shift assays confirmed that recombinant PhosR protein bound DNA fragments containing the promoter regions upstream of these three transcriptional units. Although functionally similar, the PhosS/PhosR does not exhibit sequence homology with the classical PhoBR systems, has a different pho box (5′‐GTTTCNAAAANGTTTC‐3′) recognized by the C. jejuni response regulator, and is not autoregulated. Because of these atypical properties, we designated the Cj0889‐Cj0890 operon as the C. jejuni PhosS/PhosR system (phosphate sensor/phosphate response regulator) and the phosphate‐regulated genes as the pho regulon of C. jejuni.

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.

Altered Linkage of Hydroxyacyl Chains in Lipid A of Campylobacter jejuni Reduces TLR4 Activation and Antimicrobial Resistance

Modification of the lipid A moiety of bacterial lipopolysaccharide influences cell wall properties, endotoxic activity, and bacterial resistance to antimicrobial peptides. Known modifications are variation in the number or length of acyl chains and/or attached phosphoryl groups. Here we identified two genes (gnnA and gnnB) in the major foodborne pathogen Campylobacter jejuni that enable the synthesis of a GlcN3N precursor UDP 2-acetamido-3-amino-2,3-dideoxy-α-d-glucopyranose (UDP-GlcNAc3N) in the lipid A backbone. Mass spectrometry of purified lipooligosaccharide verified that the gene products facilitate the formation of a 2,3-diamino-2,3-dideoxy-d-glucose (GlcN3N) disaccharide lipid A backbone when compared with the β-1′-6-linked d-glucosamine (GlcN) disaccharide observed in Escherichia coli lipid A. Functional assays showed that inactivation of the gnnA or gnnB gene enhanced the TLR4-MD2-mediated NF-κB activation. The mutants also displayed increased susceptibility to killing by the antimicrobial peptides polymyxin B, colistin and the chicken cathelicidin-1. The gnnA and gnnB genes are organized in one operon with hemH, encoding a ferrochelatase catalyzing the last step in heme biosynthesis. These results indicate that lipid A modification resulting in amide-linked acyl chains in the lipid A is an effective mechanism to evade activation of the innate host defense and killing by antimicrobial peptides.

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 DNase Encoded by Integrated Element CJIE1 Inhibits Natural Transformation of Campylobacter jejuni

The species Campylobacter jejuni is considered naturally competent for DNA uptake and displays strong genetic diversity. Nevertheless, nonnaturally transformable strains and several relatively stable clonal lineages exist. In the present study, the molecular mechanism responsible for the nonnatural transformability of a subset of C. jejuni strains was investigated. Comparative genome hybridization indicated that C. jejuni Mu-like prophage integrated element 1 (CJIE1) was more abundant in nonnaturally transformable C. jejuni strains than in naturally transformable strains. Analysis of CJIE1 indicated the presence of dns (CJE0256), which is annotated as a gene encoding an extracellular DNase. DNase assays using a defined dns mutant and a dns-negative strain expressing Dns from a plasmid indicated that Dns is an endogenous DNase. The DNA-hydrolyzing activity directly correlated with the natural transformability of the knockout mutant and the dns-negative strain expressing Dns from a plasmid. Analysis of a broader set of strains indicated that the majority of nonnaturally transformable strains expressed DNase activity, while all naturally competent strains lacked this activity. The inhibition of natural transformation in C. jejuni via endogenous DNase activity may contribute to the formation of stable lineages in the C. jejuni population.

Temperature‐dependent FlgM/FliA complex formation regulates Campylobacter jejuni flagella length

Regulation of the biosynthesis of the flagellar filament in bacteria containing multiple flagellin genes is not well understood. The major food‐borne pathogen Campylobacter jejuni possesses on both poles a flagellum that consists of two different flagellin subunits, FlaA and FlaB. Here we identify the protein Cj1464 as a regulator of C. jejuni flagellin biosynthesis. The protein shares characteristics of the FlgM family of anti‐σ factor proteins: it represses transcription of σ28‐dependent genes, forms a complex with σ factor FliA, and is secreted through the flagellar filament. However, unlike other FlgM proteins, the interaction of C. jejuni FlgM with FliA is regulated by temperature and the protein does not inhibit FliA activity during the formation of the hook‐basal body complex (HBB). Instead, C. jejuni FlgM limits the length of the flagellar filament by suppressing the synthesis of both the σ28‐ and the σ54‐dependent flagellins. The main function of the C. jejuni FlgM therefore is not to silence σ28‐dependent genes until the HBB is completed, but to prevent unlimited elongation of the flagellum, which otherwise leads to reduced bacterial motility.

The Cyclic AMP-Cyclic AMP Receptor Protein Complex Regulates Activity of the traJ Promoter of the Escherichia coli Conjugative Plasmid pRK100

The TraJ protein is a central activator of F-like plasmid conjugal transfer. In a search for regulators of traJ expression, we studied the possible regulatory role of the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex in traJ transcription using a traJ-lacZ reporter system. A comparison of the enzyme activities in the wild-type Escherichia coli strain MC4100 with those in cya and crp mutants indicated that disruption of the formation of the cAMP-CRP complex negatively influenced the activity of the traJ promoter of the F-like plasmid pRK100. The defect in the cya mutant was partially restored by addition of exogenous cAMP. Competitive reverse transcription-PCR performed with RNA isolated from the wild-type and mutant strains showed that the cAMP-CRP complex exerted its effect at the level of transcription. Electrophoretic mobility shift assays with purified CRP demonstrated that there was direct binding of CRP to the traJ promoter region. DNase I footprint experiments mapped the CRP binding site around position -67.5 upstream of the putative traJ promoter. Targeted mutagenesis of the traJ promoter region confirmed the location of the CRP binding site. Consistent with the demonstrated regulation of TraJ by the cAMP-CRP complex, mutants with defects in cya or crp exhibited reduced conjugal transfer from pRK100.