David R. Hendrixson

Identification of Campylobacter jejuni genes involved in commensal colonization of the chick gastrointestinal tract

Campylobacter jejuni is the leading cause of bacterial gastroenteritis in humans in developed countries throughout the world. This bacterium frequently promotes a commensal lifestyle in the gastrointestinal tracts of many animals including birds and consumption or handling of poultry meats is a prevalent source of C. jejuni for infection in humans. To understand how the bacterium promotes commensalism, we used signature‐tagged transposon mutagenesis and identified 29 mutants representing 22 different genes of C. jejuni strain 81–176 involved in colonization of the chick gastrointestinal tract. Among the determinants identified were two adjacent genes, one encoding a methyl‐accepting chemotaxis protein (MCP), presumably required for proper chemotaxis to a specific environmental component, and another gene encoding a putative cytochrome c peroxidase that may function to reduce periplasmic hydrogen peroxide stress during in vivo growth. Deletion of either gene resulted in attenuation for growth throughout the gastrointestinal tract. Further examination of 10 other putative MCPs or MCP‐domain containing proteins of C. jejuni revealed one other required for wild‐type levels of caecal colonization. This study represents one of the first genetic screens focusing on the bacterial requirements necessary for promoting commensalism in a vertebrate host.

Transposon mutagenesis of Campylobacter jejuni identifies a bipartite energy taxis system required for motility

Campylobacter jejuni constitutes the leading cause of bacterial gastroenteritis in the United States and a major cause of diarrhoea worldwide. Little is known about virulence mechanisms in this organism because of the scarcity of suitable genetic tools. We have developed an efficient system of in vitro transposon mutagenesis using a mariner‐based transposon and purified mariner transposase. Through in vitro transposition of C. jejuni chromosomal DNA followed by natural transformation of the transposed DNA, large random transposon mutant libraries consisting of ≈ 16 000 individual mutants were generated. The first genetic screen of C. jejuni using a transposon‐generated mutant library identified 28 mutants defective for flagellar motility, one of the few known virulence determinants of this pathogen. We developed a second genetic system, which allows for the construction of defined chromosomal deletions in C. jejuni, and demonstrated the requirement of σ28 and σ54 for motility. In addition, we show that σ28 is involved in the transcription of flaA and that σ54 is required for transcription of three other flagellar genes, flaB and flgDE. We also identified two previously uncharacterized genes required for motility encoding proteins that we call CetA and CetB, which mediate energy taxis responses. Through our analysis of the Cet proteins, we propose a unique mechanism for sensing energy levels and mediating energy taxis in C. jejuni.

Motility and chemotaxis in Campylobacter and Helicobacter .

Flagellar motility of Campylobacter jejuni and Helicobacter pylori influences host colonization by promoting migration through viscous milieus such as gastrointestinal mucus. This review explores mechanisms C. jejuni and H. pylori employ to control flagellar biosynthesis and chemotactic responses. These microbes tightly control the activities of σ(54) and σ(28) to mediate ordered flagellar gene expression. In addition to phase-variable and posttranslational mechanisms, flagellar biosynthesis is regulated spatially and numerically so that only a certain number of organelles are placed at polar sites. To mediate chemotaxis, C. jejuni and H. pylori combine basic chemotaxis signal transduction components with several accessory proteins. H. pylori is unusual in that it lacks a methylation-based adaptation system and produces multiple CheV coupling proteins. Chemoreceptors in these bacteria contain nonconserved ligand binding domains, with several chemoreceptors matched to environmental signals. Together, these mechanisms allow for swimming motility that is essential for colonization.

Structural diversity of bacterial flagellar motors

The bacterial flagellum is one of natures most amazing and well‐studied nanomachines. Its cell‐wall‐anchored motor uses chemical energy to rotate a microns‐long filament and propel the bacterium towards nutrients and away from toxins. While much is known about flagellar motors from certain model organisms, their diversity across the bacterial kingdom is less well characterized, allowing the occasional misrepresentation of the motor as an invariant, ideal machine. Here, we present an electron cryotomographical survey of flagellar motor architectures throughout the Bacteria. While a conserved structural core was observed in all 11 bacteria imaged, surprisingly novel and divergent structures as well as different symmetries were observed surrounding the core. Correlating the motor structures with the presence and absence of particular motor genes in each organism suggested the locations of five proteins involved in the export apparatus including FliI, whose position below the C‐ring was confirmed by imaging a deletion strain. The combination of conserved and specially‐adapted structures seen here sheds light on how this complex protein nanomachine has evolved to meet the needs of different species.

Transcription of σ54-dependent but not σ28-dependent flagellar genes in Campylobacter jejuni is associated with formation of the flagellar secretory apparatus

We performed a genetic analysis of flagellar regulation in Campylobacter jejuni, from which we elucidated key portions of the flagellar transcriptional cascade in this bacterium. For this study, we developed a reporter gene system for C. jejuni involving astA, encoding arylsulphatase, and placed astA under control of the σ54‐regulated flgDE2 promoter in C. jejuni strain 81‐176. The astA reporter fusion combined with transposon mutagenesis allowed us to identify genes in which insertions abolished flgDE2 expression; genes identified were on both the chromosome and the plasmid pVir. Included among the chromosomal genes were genes encoding a putative sensor kinase and the σ54‐dependent transcriptional activator, FlgR. In addition, we identified specific flagellar genes, including flhA, flhB, fliP, fliR and flhF, that are also required for transcription of flgDE2 and are presumably at the beginning of the C. jejuni flagellar transcriptional cascade. Deletion of any of these genes reduced transcription of both flgDE2 and another σ54‐dependent flagellar gene, flaB, encoding a minor flagellin. Transcription of the σ28‐dependent gene flaA, encoding the major flagellin, was largely unaffected in the mutants. Further examination of flaA transcription revealed significant σ28‐independent transcription and only weak repressive activity of the putative anti‐σ28 factor FlgM. Our study suggests that σ54‐dependent transcription of flagellar genes in C. jejuni is linked to the formation of the flagellar secretory apparatus. A key difference in the C. jejuni flagellar transcriptional cascade compared with other bacteria that use σ28 for transcription of flagellar genes is that a mechanism to repress significantly σ28‐dependent transcription of flaA in flagellar assembly mutants is absent in C. jejuni.

Secretion of virulence determinants by the general secretory pathway in Gram-negative pathogens: an evolving story

Secretion of proteins by the general secretory pathway (GSP) is a two-step process requiring the Sec translocase in the inner membrane and a separate substrate-specific secretion apparatus for translocation across the outer membrane. Gram-negative bacteria with pathogenic potential use the GSP to deliver virulence factors into the extracellular environment for interaction with the host. Well-studied examples of virulence determinants using the GSP for secretion include extracellular toxins, pili, curli, autotransporters, and crystaline S-layers. This article reviews our current understanding of the GSP and discusses examples of terminal branches of the GSP which are utilized by factors implicated in bacterial virulence.

The Haemophilus influenzae Hap Serine Protease Promotes Adherence and Microcolony Formation, Potentiated by a Soluble Host Protein

Haemophilus influenzae initiates infection by colonizing the upper respiratory mucosa. The process of colonization involves adherence to epithelium and evasion of host immunity. In this study, we examined the H. influenzae Hap adhesin, which has serine protease activity and undergoes autoproteolytic cleavage and extracellular release in broth. We found that the uncleaved cell-associated form of Hap mediates adherence to cultured epithelial cells and promotes bacterial aggregation and microcolony formation. Adherence and aggregation are augmented by secretory leukocyte protease inhibitor, a natural component of respiratory secretions that inhibits Hap autoproteolysis. These observations suggest a novel paradigm in host-pathogen relations, in which a soluble host protein whose primary function is to protect host epithelium potentiates properties that facilitate bacterial colonization.

Structural determinants of processing and secretion of the Haemophilus influenzae Hap protein

Haemophilus influenzae elaborates a surface protein called Hap, which is associated with the capacity for intimate interaction with cultured epithelial cells. Expression of hap results in the production of three protein species: outer membrane proteins of approximately 155 kDa and 45 kDa and an extracellular protein of approximately 110 kDa. The 155 kDa protein corresponds to full‐length mature Hap (without the signal sequence), and the 110 kDa extracellular protein represents the N‐terminal portion of mature Hap (designated Haps). In the present study, we examined the mechanism of processing and secretion of Hap. Site‐directed mutagenesis suggested that Hap is a serine protease that undergoes autoproteolytic cleavage to generate the 110 kDa extracellular protein and the 45 kDa outer membrane protein. Biochemical analysis confirmed this conclusion and established that cleavage occurs on the bacterial cell surface. Determination of N‐terminal amino acid sequence and mutagenesis studies revealed that the 45 kDa protein corresponds to the C‐terminal portion of Hap, starting at N1037. Analysis of the secondary structure of this protein (designated Hapβ) predicted formation of a β‐barrel with an N‐terminal transmembrane α‐helix followed by 14 transmembrane β‐strands. Additional analysis revealed that the final β‐strand contains an amino acid motif common to other β‐barrel outer membrane proteins. Upon deletion of this entire C‐terminal consensus motif, Hap could no longer be detected in the outer membrane, and secretion of Haps was abolished. Deletion or complete alteration of the final three amino acid residues had a similar but less dramatic effect, suggesting that this terminal tripeptide is particularly important for outer membrane localization and/or stability of the protein. In contrast, isolated point mutations that disrupted the amphipathic nature of the consensus motif or eliminated the C‐terminal tryptophan had no effect on outer membrane localization of Hap or secretion of Haps. These results provide insight into a growing family of Gram‐negative bacterial exoproteins that are secreted by an IgA1 protease‐like mechanism; in addition, they contribute to a better understanding of the structural determinants of targeting of β‐barrel proteins to the bacterial outer membrane.

A phase-variable mechanism controlling the Campylobacter jejuni FlgR response regulator influences commensalism

Phase variation of genes in bacteria enables phenotypic alteration to modulate interactions within a host as conditions change. To promote commensalism in animals and disease in humans, Campylobacter jejuni produces a flagellar organelle for motility. In addition to tight transcriptional regulation of flagellar genes, C. jejuni also controls flagellar biosynthesis by phase variation. In this study, an unusual phase‐variable mechanism controlling production of FlgR, the response regulator of the FlgSR two‐component system required for transcription of σ54‐dependent flagellar genes, is identified. Phase variation of FlgR production is due to loss or gain of a nucleotide in homopolymeric adenine or thymine tracts within flgR. This mechanism occurs during commensalism in poultry to alter the colonization capacity of C. jejuni, presumably by influencing the motility phenotype of the bacterium. These findings provide more understanding into the genetic and colonization strategies C. jejuni employs to achieve commensalism in a natural host. Second, due to the richness of the C. jejuni genome in adenine or thymine residues and the apparent lack of the normal set of mismatch repair enzymes, the results from this study may suggest that the C. jejuni genome is more unstable and variable than previously realized. Furthermore, phase variation of flagellar motility by targeting flgR may be a phenomenon specific to C. jejuni that is absent in other Campylobacter species and contribute to reasons why C. jejuni is more frequently found as a commensal organism in poultry and as the cause of disease in humans.

Human milk lactoferrin is a serine protease that cleaves Haemophilus surface proteins at arginine-rich sites

Lactoferrin is a member of the lactotransferrin family of non‐haem, iron‐binding glycoproteins and is found at high concentrations in all human secretions, where it plays a major role in mucosal defence. In recent work, we observed that lactoferrin has proteolytic activity and attenuates the pathogenic potential of Haemophilus influenzae by cleaving and removing two putative colonization factors, namely the IgA1 protease protein and the Hap adhesin. Experiments with protease inhibitors further suggested that lactoferrin may belong to a serine protease family. In the present study we explored the mechanism of lactoferrin protease activity and discovered that mutation of either Ser259 or Lys73 results in a dramatic decrease in proteolysis. Examination of the crystal structure revealed that these two residues are located in the N‐terminal lobe of the protein, adjacent to a 12–15 Å cleft that separates the N‐lobe and the C‐lobe and that can readily accommodate large polypeptide substrates. In additional work, we found that lactoferrin cleaves IgA1 protease at an arginine‐rich region defined by amino acids 1379–1386 (RRSRRSVR) and digests Hap at an arginine‐rich sequence between amino acids 1016 and 1023 (VRSRRAAR). Based on our results, we conclude that lactoferrin is a serine protease capable of cleaving arginine‐rich sequences. We speculate that Ser259 and Lys73 form a catalytic dyad, reminiscent of a number of bacterial serine proteases. In addition, we speculate that lactoferrin may cleave arginine‐rich sequences in a variety of microbial virulence proteins, contributing to its long‐recognized antimicrobial properties.