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Summary: Bacteria have evolved different mechanisms to catabolize carbon sources from nutrient mixtures. They first consume their preferred carbon source, before others are used. Regulatory mechanisms adapt the metabolism accordingly to maximize growth and to outcompete other organisms. The human pathogen Campylobacter jejuni is an asaccharolytic Gram‐negative bacterium that catabolizes amino acids and organic acids for growth. It prefers serine and aspartate as carbon sources, however it lacks all regulators known to be involved in regulating carbon source utilization in other organisms. In which manner C. jejuni adapts its metabolism towards the presence or absence of preferred carbon sources is unknown. In this study, we show with transcriptomic analysis and enzyme assays how C. jejuni adapts its metabolism in response to its preferred carbon sources. In the presence of serine as well as lactate and pyruvate C. jejuni inhibits the utilization of other carbon sources, by repressing the expression of a number of central metabolic enzymes. The regulatory proteins RacR, Cj1000 and CsrA play a role in the regulation of these metabolic enzymes. This metabolism dependent transcriptional repression correlates with an accumulation of intracellular succinate. Hence, we propose a demand‐based catabolite repression mechanism in C. jejuni, depended on intracellular succinate levels. [ABSTRACT FROM AUTHOR]

The generation of a membrane potential (Δ ψ), the major constituent of the proton motive force (pmf), is crucial for ATP synthesis, transport of nutrients and flagellar rotation. Campylobacter jejuni harbors a branched electron transport chain, enabling respiration with different electron donors and acceptors. Here, we demonstrate that a relatively high Δ ψ is only generated in the presence of either formate as electron donor or oxygen as electron acceptor, in combination with an acceptor/donor respectively. We show the necessity of the pmf for motility and growth of C. jejuni. ATP generation is not only accomplished by oxidative phosphorylation via the pmf, but also by substrate-level phosphorylation via the enzyme AckA. In response to a low oxygen tension, C. jejuni increases the transcription and activity of the donor complexes formate dehydrogenase (FdhABC) and hydrogenase (HydABCD) as well as the transcription of the alternative respiratory acceptor complexes. Our findings suggest that in the gut of warm-blooded animals, C. jejuni depends on at least formate or hydrogen as donor (in the anaerobic lumen) or oxygen as acceptor (near the epithelial cells) to generate a pmf that sustains efficient motility and growth for colonization and pathogenesis. [ABSTRACT FROM AUTHOR]

Bacterial flagella assembly is tightly regulated to ensure a timely and sequential production of the various flagellum constituents. In the pathogen Campylobacter jejuni the hierarchy in flagella biosynthesis is largely determined at the transcriptional level through the activity of the alternative sigma factors sigma54 and sigma28. Here, we report that C. jejuni flagellin levels are also controlled at the post-transcriptional level via the thus far poorly-characterized flagellar assembly factor FliW. Analysis of flagellin synthesis in C. jejuni 81116 and a Δ fliW knock-out mutant showed reduced flagellin protein levels in the mutant strain while ectopic expression of FliW resulted in enhanced levels. Real-time RT-PCR revealed relatively minor changes in flaA and flaB mRNA levels for the recombinant and parent strain consistent with post-transcriptional regulation. Purified FliW was found to bind to FlaA and FlaB flagellin as well as to the global post-transcriptional regulator CsrA. Inactivation of CsrA resulted in increased levels of flagellin translation. An in vitro translation assay confirmed the regulatory role of CsrA in flagellin biosynthesis. We propose that competitive reciprocal binding of FliW to flagellins and the RNA binding protein CsrA serves as a feedback mechanism to control the number of cytosolic flagellin copies at the protein level. [ABSTRACT FROM AUTHOR]

Responses to host amyloids and curli amyloid fibrils of Escherichia coli and Salmonella enterica serotype Typhimurium are mediated through Toll-like receptor (TLR) 2. Here we show that TLR2 alone was not sufficient for mediating responses to curli. Instead, transfection experiments with human cervical cancer (HeLa) cells and antibody-mediated inhibition of TLR signalling in human macrophage-like (THP-1) cells suggested that TLR2 interacts with TLR1 to recognize curli amyloid fibrils. TLR1/TLR2 also serves as a receptor for tri-acylated lipoproteins, which are produced by E. coli and other Gram-negative bacteria. Despite the presence of multiple TLR1/TLR2 ligands on intact bacterial cells, an inability to produce curli amyloid fibrils markedly reduced the ability of E. coli to induce TLR2-dependent responses in vitro and in vivo. Collectively, our data suggest that curli amyloid fibrils from enterobacterial biofilms significantly contribute to TLR1/TLR2-mediated host responses against intact bacterial cells. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

An increasing number of bacterial pathogens produce an array of glycoproteins of unknown function. Here we report that Campylobacter jejuni proteins that are modified by the N-linked glycosylation machinery encoded by the pgl locus bind the human Macrophage Galactose-type lectin (MGL). MGL receptor binding was abrogated by EDTA and N-acetylgalactosamine (GalNAc) and was successfully transferred to Escherichia coli by introducing the C. jejuni pgl locus together with a glycan acceptor protein. In addition to glycoproteins, C. jejuni lipooligosaccharide with a terminal GalNAc residue was recognized by MGL. Recombinant E. coli expressing the C. jejuni pgl locus in the absence of a suitable glycan acceptor protein produced altered lipopolysaccharide glycoforms that gained MGL reactivity. Infection assays demonstrated high levels of GalNAc-dependent interaction of the recombinant E. coli with MGL-transfected mammalian cells. In addition, interleukin-6 production by human dendritic cells was enhanced by C. jejuni lacking N-linked glycans compared with wild-type bacteria. Collectively, our results provide evidence that both N-linked glycoproteins and distinct lipooligosaccharide glycoforms of C. jejuni are ligands for the human C-type lectin MGL and that the C. jejuni N-glycosylation machinery can be exploited to target recombinant bacteria to MGL-expressing eukaryotic cells. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Type IV pili (Tfp) are multifunctional surface appendages expressed by many Gram negative species of medical, environmental and industrial importance. The N-terminally localized, so called α-helical spine is the most conserved structural feature of pilin subunits in these organelles. Prevailing models of pilus assembly and structure invariably implicate its importance to membrane trafficking, organelle structure and related functions. Nonetheless, relatively few studies have examined the effects of missense substitutions within this domain. Using Neisseria gonorrhoeae as a model system, we constructed mutants with single and multiple amino acid substitutions localized to this region of the pilin subunit PilE and characterized them with regard to pilin stability, organelle expression and associated phenotypes. The consequences of simultaneous expression of the mutant and wild-type PilE forms were also examined. The findings document for the first time in a defined genetic background the phenomenon of pilin intermolecular complementation in which assembly defective pilin can be rescued into purifiable Tfp by coexpression of wild-type PilE. The results further demonstrate that pilin subunit composition can impact on organelle dynamics mediated by the PilT retraction protein via a process that appears to monitor the efficacy of subunit–subunit interactions. In addition to confirming and extending the evidence for PilE multimerization as an essential component for competence for natural genetic transformation, this work paves the way for detailed studies of Tfp subunit–subunit interactions including self-recognition within the membrane and packing within the pilus polymer. [ABSTRACT FROM AUTHOR]

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 ( phate ensor/ phate response egulator) and the phosphate-regulated genes as the pho regulon of C. jejuni. [ABSTRACT FROM AUTHOR]

Neisseria meningitidis lipopolysaccharide (LPS) has been identified as a major determinant of dendritic cell (DC) function. Here we report that one of a series of meningococcal mutants with defined truncations in the lacto- N-neotetraose outer core of the LPS exhibited unique strong adhesion and internalization properties towards DC. These properties were mediated by interaction of the GlcNAc(β1-3)-Gal(β1-4)-Glc-R oligosaccharide outer core of lgtB LPS with the dendritic-cell-specific ICAM-3 grabbing non-integrin (DC-SIGN) lectin receptor. Activation of DC-SIGN with this novel oligosaccharide ligand skewed T-cell responses driven by DC towards T helper type 1 activity. Thus, the use of lgtB LPS may provide a powerful instrument to selectively induce the desired arm of the immune response and potentially increase vaccine efficacy. [ABSTRACT FROM AUTHOR]

Type IV pili (Tfp) play central roles in prokaryotic cell biology and disease pathogenesis. As dynamic filamentous polymers, they undergo rounds of extension and retraction modelled as pilin subunit polymerization and depolymerization events. Currently, the molecular mechanisms and components influencing Tfp dynamics remain poorly understood. UsingNeisseria gonorrhoeaeas a model system, we show that mutants lacking any one of a set of five proteins sharing structural similarity to the pilus subunit are dramatically reduced in Tfp expression and that these defects are suppressed in the absence of the PilT pilus retraction protein. Thus, these molecules are not canonical assembly factors but rather act as effectors of pilus homeostasis by promoting extension/polymerization events in the presence of PilT. Furthermore, localization studies support the conclusion that these molecules form a Tfp-associated complex and influence levels of PilC, the epithelial cell adhesin, in Tfp-enriched shear fractions. This is the first time that the step at which individual pilin-like proteins impact on Tfp expression has been defined. The findings have important implications for understanding Tfp dynamics and fundamental Tfp structure/function relationships. [ABSTRACT FROM AUTHOR]

The ability of bacteria to establish complex communities on surfaces is believed to require both bacterial–substratum and bacterial–bacterial interactions, and type IV pili appear to play a critical but incompletely defined role in both these processes. Using the human pathogen Neisseria gonorrhoeae, spontaneous mutants defective in bacterial self-aggregative behaviour but quantitatively unaltered in pilus fibre expression were isolated by a unique selective scheme. The mutants, carrying single amino acid substitutions within the conserved amino-terminal domain of the pilus fibre subunit, were reduced in the ability to adhere to a human epithelial cell line. Co-expression of the altered alleles in the context of a wild-type pilE gene confirmed that they were dominant negative with respect to aggregation and human cell adherence. Strains expressing two copies of the altered alleles produced twice as much purifiable pili but retained the aggregative and adherence defects. Finally, the defects in aggregative behaviour and adherence of each of the mutants were suppressed by a loss-of-function mutation in the twitching motility gene pilT. The correlations between self-aggregation and the net capacity of the microbial population to adhere efficiently demonstrates the potential significance of bacterial cell–cell interactions to colonization. [ABSTRACT FROM AUTHOR]

The molecular mechanism of the upregulation of Escherichia coli colicin K (Cka) synthesis during stress conditions was studied. Nutrient starvation experiments and the use of relA spoT mutant strains, IPTG-regulated overproduction of ppGpp and lacZ fusions revealed that the stringent response alarmone guanosine 3′,5′-bispyrophosphate (ppGpp) is the main positive effector of Cka synthesis. Comparison of the amounts of protein produced (Western blotting) and specific mRNA (Northern blotting) before and after nutrient starvation demonstrated increases in Cka protein with unaltered specific mRNA levels, suggesting a post-transcriptional regulatory mechanism. Reporter (β-galactosidase) assays using truncated cka of variable length fused to lacZ located the key regulatory region close to the 5′ end of the cka mRNA. Closer analysis of this region indicated the presence of several rare codons, including the leucine-encoding codon CUA. Synonymous exchange of the rare codons with more frequently used ones abolished the regulatory effect of ppGpp. Supplementation of the strain with the plasmid CodonPlus carrying several rare tRNA genes yielded similar results, indicating that codon usage (in particular, the fifth codon for the amino acid leucine) and tRNA availability (i.e. tRNAleu) are the key elements of the regulatory function of ppGpp. We conclude that ppGpp regulates Cka synthesis via a novel post-transcriptional mechanism that is based on rare codon usage and variable cognate tRNA availability. [ABSTRACT FROM AUTHOR]

The expression of type IV pili (Tfp) by Neisseria gonorrhoeae has been shown to be essential for natural genetic transformation at the level of sequence-specific uptake of DNA. All previously characterized mutants defective in this step of transformation either lack Tfp or are altered in the expression of Tfp-associated properties, such as twitching motility, autoagglutination and the ability to bind to human epithelial cells. To examine the basis for this relationship, we identified potential genes encoding polypeptides sharing structural similarities to PilE, the Tfp subunit, within the N. gonorrhoeae genome sequence database. We found that disruption of one such gene, designated comP (for competence-associated prepilin), leads to a severe defect in the capacity to take up DNA in a sequence-specific manner, but does not alter Tfp biogenesis or expression of the Tfp-associated properties of autoagglutination, twitching motility and human epithelial cell adherence. Indirect evidence based on immunodetection suggests that ComP is expressed at very low levels relative to that of PilE. The process of DNA uptake in gonococci, therefore, is now known to require the expression of at least three distinct components: Tfp, the recently identified PilT protein and ComP. [ABSTRACT FROM AUTHOR]

The lipopolysaccharides of the pathogenic Neisseria species are subject to structural variation owing to a combination of intrinsic changes in iipopolysaccharide (LPS) biosynthesis and external modification of the LPS molecule with sialic acid. This variation appears to control bacterial behaviour by altering their ability to interact with human cells and to evade host Immune defences. This interconversion of LPS phenotypes, which is also observed during the natural infection, is probably due to environmental regulation of LPS biosynthesis superimposed on spontaneous changes in the DNA of distinct LPS loci. LPS variation may be a common strategy of mucosal pathogens to colonize and persist within the human host. [ABSTRACT FROM AUTHOR]

Here we report the cloning and expression, in Escherichia coli, of PCR-amplified DNA encoding the 63-kDa stress-inducible protein of Neisseria gonorrhoeae strains VP1 and PID2, Neisseria meningitidis 2996 and the commensal Neisseria flavescens. DNA sequence analysis revealed in all cases one open reading frame of 541-544 amino acids corresponding to a protein of approximately 57 000 Da. The various neisserial proteins were >98% identical at the amino acid level and showed extensive homology with proteins belonging to the Hsp60 heat-shock-protein family. We constructed defined glutathione S-transferase fusion polypeptides of the gonococcal Hsp60 homologue to locate antigenic domains on the recombinant protein. Variation in the immuno-reactivity of two monoclonal antibodies recognizing a conserved and a neisseria-unique antigenic Hsp60 determinant, respectively, could thus be deduced to result from single amino acid substitutions. Analysis of the antibody response in patients' sera demonstrated reactivity with the same fusion polypeptides in six out of nine sera, indicating that neisserial Hsp60 is expressed during the natural infection and that distinct domains on the protein are immunodominant in vivo. [ABSTRACT FROM AUTHOR]

We have investigated the function of the lsi-1 gene of Neisseria gonorrhoeae previously implicated in lipopolysaccharide (LPS)-inner-core biosynthesis (Petricoin et al., 1991). Disruption of the gene in gonococcal strain MS11 resulted in the production of LPS that migrated faster than that from an isogenic galE mutant, typical for a mutation that influences the inner-core region. Complementation of a panel of Salmonella typhimurium mutants with defined defects in rfa loci demonstrated conclusively that the lsi-1 gene of MS11 is functionally homologous to the rfaF gene, which encodes heptosyltransferase II in both E. coli and S. typhimurium. Comparison of deduced amino acid sequences of the gonococcal and the Salmonella RfaF demonstrated 70% similarity, including 47% identical amino acid residues. Immunochemical analysis of the LPS using monoclonal antibodies directed against chemically defined inner-core glycoconjugates revealed that the gonococcal and Salmonella Rd2-Chemotypes were antigenically similar, further extending the genetic and functional homology. Infection experiments in vitro demonstrated that the lsi-1 mutant could not invade human Chang epithelial cells despite expression of a genetically defined invasion-promoting gonococcal opacity protein. These data imply that the LPS phenotype is a critical factor for gonococcal invasiveness. [ABSTRACT FROM AUTHOR]

Haemophilus infuenzae infections are preceded by airway colonization, a process facilitated by fimbriae. Here, we identified the complete fimbrial gene cluster of H. influenzae type b. HifA forms the major subunit. HifB, a peripiasmic chaperone, and HifC, an outer membrane usher, are typical assembly genes; their inactivation abolished fimbriae formation. HifD and HifE are putative minor subunits, both participating in fimbriae biogenesis, inactivation of either one drastically reduced fimbriae expression. HifD represents a novel type of fimbrial subunit with lipoprotein characteristics, pointing to a membrane-associated function of HifD. Transcription of all fimbrial genes is coregulated through two clustered promoters. The flanking of the fimbrial gene cluster by repetitive extragenic palindromic sequences together with a partial duplication of an adjacent unrelated operon indicated that the cluster was once inserted in the H. influenzae genome as a mobile virulence unit. [ABSTRACT FROM AUTHOR]

Lipopolysaccharide is an essential component of the outer membrane of Gram-negative bacteria and an important virulence factor of many pathogens, such as Neisseria gonorrhoeae. We have cloned the gonococcal galE gene which was found to be located in the gonococcal homologue of the meningococcal capsule gene complex region D. Sequence alignment indicated extensive homology with the Escherichia coli and Salmonella GalE proteins. Mutants with insertions in the galE gene were used as a tool to characterize the structure and function of gonococcal lipopolysaccharide. They displayed deep rough phenotypes, end chemical analysis confirmed the loss of galactose from the mutant lipopolysaccharide. Functional analysis indicated that the terminal oligosaccharides contain galactose and that these are lost in galE mutants. The importance of these oligosaccharides in gonococcal biology is clear from the fact that they contain the epitopes that are the targets for killing by normal human serum, and the acceptor site for sialic acid, which acts to protect the gonococcus from this killing. Furthermore, infection experiments in vitro indicate that the galE mutants exhibit unaltered intergonococcal adhesion as well as adhesion to, and invasion of, epithelial cells. [ABSTRACT FROM AUTHOR]

Three out of 10 Helicobacter pylori clinical isolates were found to be naturally competent for genetic transformation to streptomycin resistance by chromosomal DNA extracted from a spontaneous strepto-mycin-resistant H. pylori mutant. The frequency of transformation varied between 5 x 10-4 and 4 x 10-6, depending on the H. pylori isolate used. Transposon shuttle mutagenesis based on this natural competence was established using the flagellin gene flaA as the target. The cloned flaA gene was interrupted by insertion of TnMax1, a mini-Tn1721 transposon carrying a modified chloramphenicol-acetyltransferase gene, the catGC cassette. Natural transformation of competent H. pylori strains with plasmid constructs harbouring a catGC-activated flaA gene resulted in chloramphenicol-resistant transformants at an average frequency of 4 x 10-5. Southern hybridization experiments confirmed the replacement of the chromosomal H. pylori flaA gene by the cat-inactivated cloned gene copy via homologous recombination resulting in allelic exchange. Phenotypic characterization of the mutants demonstrated the absence of flagella under the electron microscope and the loss of bacterial motility. Immunoblots of cell lysates of the H. pylori mutants with an antiserum raised against the C-terminal portion of recombinant H. pylori major flageitin (FlaA) confirmed the absence of the 54 kDa FlaA protein. This efficient transposon shuttle mutagenesis procedure for H. pylori based on natural competence opens up new possibilities for the genetic assessment of putative H. pylori virulence determinants. [ABSTRACT FROM AUTHOR]

Pili confer the initial ability of Neisseria gonorrhoeae to bind to epithelial cells. Pilin (PilE), the major pilus subunit, and a minor protein termed PilC, reportedly essential for pilus biogenesis, undergo intra-strain phase and structural variation. We demonstrate here that at least two different adherence properties are associated with the gonococcal pili: one is specific for erythrocytes, which is virtually unaffected by PilE variation, and another is specific for epithelial cells, and is modulated in response to the variation of PilE. Based on this finding, mutants of a recA- strain were selected that had lost the ability to bind to human cornea epithelial cells (A-) but retained the ability to form pili (P+) and to agglutinate human erythrocytes (H+). The adherence-negative mutants failed to produce detectable levels of PilC1 or PilC2 proteins, representing pilC phase variants generated in the absence of RecA. The A- pilC phase variants were indistinguishable from their A+ parents and spontaneous A+ revertants with regard to the amount of PilE produced and its electrophoretic mobility, the degrees of piliation and haemagglutination, and the pilE nucleotide sequence. These data demonstrate a central role for PilC in pilus-mediated adherence of N. gonorrhoeae to human epithelial cells and further indicate that neither PilC1 nor PilC2 is obligatory for the assembly of gonococcal pili. [ABSTRACT FROM AUTHOR]