Michael E. Konkel

Secretion of Virulence Proteins from Campylobacter jejuni Is Dependent on a Functional Flagellar Export Apparatus

Campylobacter jejuni, a gram-negative motile bacterium, secretes a set of proteins termed the Campylobacter invasion antigens (Cia proteins). The purpose of this study was to determine whether the flagellar apparatus serves as the export apparatus for the Cia proteins. Mutations were generated in five genes encoding three structural components of the flagella, the flagellar basal body (flgB and flgC), hook (flgE2), and filament (flaA and flaB) genes, as well as in genes whose products are essential for flagellar protein export (flhB and fliI). While mutations that affected filament assembly were found to be nonmotile (Mot-) and did not secrete Cia proteins (S-), a flaA (flaB+) filament mutant was found to be nonmotile but Cia protein secretion competent (Mot-, S+). Complementation of a flaA flaB double mutant with a shuttle plasmid harboring either the flaA or flaB gene restored Cia protein secretion, suggesting that Cia export requires at least one of the two filament proteins. Infection of INT 407 human intestinal cells with the C. jejuni mutants revealed that maximal invasion of the epithelial cells required motile bacteria that are secretion competent. Collectively, these data suggest that the C. jejuni Cia proteins are secreted from the flagellar export apparatus.

Identification and molecular cloning of a gene encoding a fibronectin‐binding protein (CadF) from Campylobacter jejuni

Campylobacter jejuni, a Gram‐negative bacterium, is a common cause of gastrointestinal disease. By analogy with other enteric pathogens such as Salmonella and Shigella, the ability of C. jejuni to bind to host cells is thought to be essential in the pathogenesis of enteritis. Scanning electron microscopy of infected INT407 cells suggested that C. jejuni bound to a component of the extracellular matrix. Binding assays using immobilized extracellular matrix proteins and soluble fibronectin showed specific and saturable binding of fibronectin to C. jejuni. Ligand immunoblot assays using 125I‐labelled fibronectin revealed specific binding to an outer membrane protein with an apparent molecular mass of 37 kDa. A rabbit antiserum, raised against the gel‐purified protein, reacted with a 37 kDa protein in all C. jejuni isolates (n = 15) as tested by immunoblot analysis. Antibodies present in convalescent serum from C. jejuni‐infected individuals also recognized a 37 kDa protein. The gene encoding the immunoreactive 37 kDa protein was cloned and sequenced. Sequencing of overlapping DNA fragments revealed an open reading frame (ORF) that encodes a protein of 326 amino acids with a calculated molecular mass of 36 872 Da. The deduced amino acid sequence of the ORF exhibited 52% similarity and 28% identity to the root adhesin protein from Pseudomonas fluorescens. Isogenic C. jejuni mutants which lack the 37 kDa outer membrane protein, which we have termed CadF, displayed significantly reduced binding to fibronectin. Biotinylated fibronectin bound to a protein with an apparent molecular mass of 37 kDa in the outer membrane protein extracts from wild‐type C. jejuni as judged by ligand‐binding blots. These results indicate that the binding of C. jejuni to fibronectin is mediated by the 37 kDa outer membrane protein which is conserved among C. jejuni isolates.

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.

Comparative Metagenomics Reveals Host Specific Metavirulomes and Horizontal Gene Transfer Elements in the Chicken Cecum Microbiome

Background The complex microbiome of the ceca of chickens plays an important role in nutrient utilization, growth and well-being of these animals. Since we have a very limited understanding of the capabilities of most species present in the cecum, we investigated the role of the microbiome by comparative analyses of both the microbial community structure and functional gene content using random sample pyrosequencing. The overall goal of this study was to characterize the chicken cecal microbiome using a pathogen-free chicken and one that had been challenged with Campylobacter jejuni. Methodology/Principal Findings Comparative metagenomic pyrosequencing was used to generate 55,364,266 bases of random sampled pyrosequence data from two chicken cecal samples. SSU rDNA gene tags and environmental gene tags (EGTs) were identified using SEED subsystems-based annotations. The distribution of phylotypes and EGTs detected within each cecal sample were primarily from the Firmicutes, Bacteroidetes and Proteobacteria, consistent with previous SSU rDNA libraries of the chicken cecum. Carbohydrate metabolism and virulence genes are major components of the EGT content of both of these microbiomes. A comparison of the twelve major pathways in the SEED Virulence Subsystem (metavirulome) represented in the chicken cecum, mouse cecum and human fecal microbiomes showed that the metavirulomes differed between these microbiomes and the metavirulomes clustered by host environment. The chicken cecum microbiomes had the broadest range of EGTs within the SEED Conjugative Transposon Subsystem, however the mouse cecum microbiomes showed a greater abundance of EGTs in this subsystem. Gene assemblies (32 contigs) from one microbiome sample were predominately from the Bacteroidetes, and seven of these showed sequence similarity to transposases, whereas the remaining sequences were most similar to those from catabolic gene families. Conclusion/Significance This analysis has demonstrated that mobile DNA elements are a major functional component of cecal microbiomes, thus contributing to horizontal gene transfer and functional microbiome evolution. Moreover, the metavirulomes of these microbiomes appear to associate by host environment. These data have implications for defining core and variable microbiome content in a host species. Furthermore, this suggests that the evolution of host specific metavirulomes is a contributing factor in disease resistance to zoonotic pathogens.

The Absence of Cecal Colonization of Chicks by a Mutant of Campylobacter jejuni not Expressing Bacterial Fibronectin-Binding Protein

Campylobacter jejuni is a common cause of human gastrointestinal illness throughout the world. Infections with C. jejuni and Campylobacter coli are frequently acquired by eating undercooked chicken. The ability of C. jejuni to become established in the gastrointestinal tract of chickens is believed to involve binding of the bacterium to the gastrointestinal surface. A 37-kD outer membrane protein, termed CadF, has been described that facilitates the binding of Campylobacter to fibronectin. This study was conducted to determine whether the CadF protein is required for C. jejuni to colonize the cecum of newly hatched chicks. Day-of-hatch chicks were orally challenged with C. jejuni F38011, a human clinical isolate, or challenged with a mutant in which the cadF gene was disrupted via homologous recombination with a suicide vector. This method of mutagenesis targets a predetermined DNA sequence and does not produce random mutations in unrelated genes. The parental C. jejuni F38011 readily colonized the cecum of newly hatched chicks. In contrast, the cadF mutant was not recovered from any of 60 chicks challenged, indicating that disruption of the cadF gene renders C. jejuni incapable of colonizing the cecum. CadF protein appears to be required for the colonization of newly hatched leghorn chickens.

Secretion of the Virulence-Associated Campylobacter Invasion Antigens from Campylobacter jejuni Requires a Stimulatory Signal

Campylobacter jejuni are a common cause of human diarrheal illness. Previous work has demonstrated that C. jejuni synthesize a novel set of proteins upon coculturing with epithelial cells, some of which are secreted. The secreted proteins have been collectively referred to as Campylobacter invasion antigens (Cia proteins). Metabolic labeling experiments revealed that Cia protein synthesis and secretion are separable and that secretion is the rate-limiting step of these processes. Additional work indicated that Cia protein synthesis is induced in response to bile salts and various eukaryotic host cell components. Host cell components also can induce Cia protein secretion. Culturing C. jejuni on plates supplemented with the bile salt deoxycholate retarded the inhibitory effect of chloramphenicol on C. jejuni invasion, as judged by the gentamicin-protection assay. These data suggest that the coordinate expression of the genes encoding the Cia proteins is subject to environmental regulation.

Examination of Campylobacter jejuni Putative Adhesins Leads to the Identification of a New Protein, Designated FlpA, Required for Chicken Colonization

ABSTRACT Campylobacter jejuni colonization of chickens is presumably dependent upon multiple surface-exposed proteins termed adhesins. Putative C. jejuni adhesins include CadF, CapA, JlpA, major outer membrane protein, PEB1, Cj1279c, and Cj1349c. We examined the genetic relatedness of 97 C. jejuni isolates recovered from human, poultry, bovine, porcine, ovine, and canine sources by multilocus sequence typing (MLST) and examined their profile of putative adhesin-encoding genes by dot blot hybridization. To assess the individual contribution of each protein in bacterium-host cell adherence, the C. jejuni genes encoding the putative adhesins were disrupted by insertional mutagenesis. The phenotype of each mutant was judged by performing in vitro cell adherence assays with chicken LMH hepatocellular carcinoma epithelial cells and in vivo colonization assays with broiler chicks. MLST analysis indicated that the C. jejuni isolates utilized in this study were genetically diverse. Dot blot hybridization revealed that the C. jejuni genes encoding the putative adhesins, with the exception of capA, were conserved among the isolates. The C. jejuni CadF, CapA, Cj1279c, and Cj1349c proteins were found to play a significant role in the bacteriums in vitro adherence to chicken epithelial cells, while CadF, PEB1, and Cj1279c were determined to play a significant role in the bacteriums in vivo colonization of broiler chicks. Collectively, the data indicate that Cj1279c is a novel adhesin. Because Cj1279c harbors fibronectin type III domains, we designated the protein FlpA, for fibronectin-like protein A.

Fibronectin-Facilitated Invasion of T84 Eukaryotic Cells by Campylobacter jejuni Occurs Preferentially at the Basolateral Cell Surface

ABSTRACT Previous studies have indicated that the ability to bind to fibronectin is a key feature in successful cell invasion by Campylobacter jejuni. Given the spatial distribution of fibronectin and the architecture of the epithelium, this suggests the possibility that C. jejuni cell invasion might preferentially occur at the basolateral cell surface. To test this hypothesis, we examined the interaction of C. jejuni with T84 human colonic cells. When grown under the appropriate conditions, T84 cells form a polarized cell monolayer. C. jejuni translocation of a T84 cell monolayer appeared to occur via a paracellular (extracellular) route as opposed to a transcellular (intracellular) route based on the finding that a C. jejuni noninvasive mutant translocated as efficiently as its isogenic parent. Additional studies revealed that two distinct C. jejuni wild-type isolates could compete with one another for host cell receptors, whereas a C. jejuni fibronectin-binding-deficient mutant could not compete with a wild-type isolate for host cell receptors. Further, C. jejuni adherence and internalization were significantly inhibited by antifibronectin antibodies but only when cells were first treated with EGTA to expose basolateral cell surfaces. Together, these results support the theory that C. jejuni invasion occurs preferentially at the basolateral surface of eukaryotic cells.

Culture of Campylobacter jejuni with Sodium Deoxycholate Induces Virulence Gene Expression

Campylobacter jejuni, a spiral-shaped gram-negative bacterium, is a leading bacterial cause of human food-borne illness. Acute disease is associated with C. jejuni invasion of the intestinal epithelium. Further, maximal host cell invasion requires the secretion of proteins termed Campylobacter invasion antigens (Cia). As bile acids are known to alter the pathogenic behavior of other gastrointestinal pathogens, we hypothesized that the virulence potential of Campylobacter may be triggered by the bile acid deoxycholate (DOC). In support of this hypothesis, culturing C. jejuni with a physiologically relevant concentration of DOC significantly altered the kinetics of cell invasion, as shown by gentamicin protection assays. In contrast to C. jejuni harvested from Mueller-Hinton (MH) agar plates, C. jejuni harvested from MH agar plates supplemented with DOC secreted the Cia proteins, as judged by metabolic labeling experiments. DOC was also found to induce the expression of the ciaB gene, as determined by beta-galactosidase reporter, real-time reverse transcription-PCR, and microarray analyses. Microarray analysis further revealed that DOC induced the expression of virulence genes (ciaB, cmeABC, dccR, and tlyA). In summary, we demonstrated that it is possible to enhance the pathogenic behavior of C. jejuni by modifying the culture conditions. These results provide a foundation for identifying genes expressed by C. jejuni in response to in vivo-like culture conditions.

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 α‐CadF peptide polyclonal antibody (M α‐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.