The signaling pathway of Campylobacter jejuni-induced Cdc42 activation: Role of fibronectin, integrin beta1, tyrosine kinases and guanine exchange factor Vav2

<p>Abstract</p> <p>Background</p> <p>Host cell invasion by the foodborne pathogen <it>Campylobacter jejuni </it>is considered as one of the primary reasons of gut tissue damage, however, mechanisms and key factors involved in this process are widely unclear. It was reported that small Rho GTPases, including Cdc42, are activated and play a role during invasion, but the involved signaling cascades remained unknown. Here we utilised knockout cell lines derived from fibronectin^-/-, integrin-beta1^-/-, focal adhesion kinase (FAK)^-/-and Src/Yes/Fyn^-/-deficient mice, and wild-type control cells, to investigate <it>C. jejuni</it>-induced mechanisms leading to Cdc42 activation and bacterial uptake.</p> <p>Results</p> <p>Using high-resolution scanning electron microscopy, GTPase pulldowns, G-Lisa and gentamicin protection assays we found that each studied host factor is necessary for induction of Cdc42-GTP and efficient invasion. Interestingly, filopodia formation and associated membrane dynamics linked to invasion were only seen during infection of wild-type but not in knockout cells. Infection of cells stably expressing integrin-beta1 variants with well-known defects in fibronectin fibril formation or FAK signaling also exhibited severe deficiencies in Cdc42 activation and bacterial invasion. We further demonstrated that infection of wild-type cells induces increasing amounts of phosphorylated FAK and growth factor receptors (EGFR and PDGFR) during the course of infection, correlating with accumulating Cdc42-GTP levels and <it>C. jejuni </it>invasion over time. In studies using pharmacological inhibitors, silencing RNA (siRNA) and dominant-negative expression constructs, EGFR, PDGFR and PI3-kinase appeared to represent other crucial components upstream of Cdc42 and invasion. siRNA and the use of Vav1/2^-/-knockout cells further showed that the guanine exchange factor Vav2 is required for Cdc42 activation and maximal bacterial invasion. Overexpression of certain mutant constructs indicated that Vav2 is a linker molecule between Cdc42 and activated EGFR/PDGFR/PI3-kinase. Using <it>C. jejuni </it>mutant strains we further demonstrated that the fibronectin-binding protein CadF and intact flagella are involved in Cdc42-GTP induction, indicating that the bacteria may directly target the fibronectin/integrin complex for inducing signaling leading to its host cell entry.</p> <p>Conclusion</p> <p>Collectively, our findings led us propose that <it>C. jejuni </it>infection triggers a novel fibronectin→integrin-beta1→FAK/Src→EGFR/PDGFR→PI3-kinase→Vav2 signaling cascade, which plays a crucial role for Cdc42 GTPase activity associated with filopodia formation and enhances bacterial invasion.</p

Comparative proteomics reveals differences in host-pathogen interaction between infectious and commensal relationship with Campylobacter jejuni

Campylobacter jejuni is the leading food-borne poisoning in industrialized countries. While the bacteria causes disease in humans, it merely colonizes the gut in poultry or pigs, where seems to establish a commensal relationship. Until now, few studies have been conducted to elucidate the relationship between C. jejuni and its different hosts. In this work, a comparative proteomics approach was used to identify the underlying mechanisms involved in the divergent outcome following C. jejuni infection in human and porcine host. Human (INT-407) and porcine (IPEC-1) intestinal cell lines were infected by C. jejuni for 3 h (T3h) and 24 h (T24h). C. jejuni infection prompted an intense inflammatory response at T3h in human intestinal cells, mainly characterized by expression of proteins involved in cell spreading, cell migration and promotion of reactive oxygen species (ROS). Proteomic analysis evidenced significantly regulated biofunctions in human cells related with engulfment and endocytosis, and supported by canonical pathways associated to infection such as caveolar- and clathrin-mediated endocytosis signaling. In porcine IPEC-1 cells, inflammatory response as well as signaling pathways that control cellular functions such as cell migration, endocytosis and cell cycle progression resulted downregulated. These differences in the host response to infection were supported by the different pattern of adhesion and invasion proteins expressed by C. jejuni in human and porcine cells. No marked differences in expression of virulence factors involved in adaptive response and iron acquisition functions were observed. Therefore, the results of this study suggest that both host and pathogen factors are responsible for commensal or infectious character of C. jejuni in different hosts.Peer reviewedVeterinary Pathobiolog

Plakophilin 1 stimulates translation by promoting eIF4A1 activity

p120 armadillo protein plakophilin 1 binds to eukaryotic translation factor eIF4A1, recruiting it into cap-binding complexes and stimulating translation

Identification of hyperinvasive Campylobacter jejuni strains isolated from poultry and human clinical sources

Campylobacter jejuni causes gastroenteritis with a variety of symptoms in humans. In the absence of a suitable animal model, in vitro models have been used to study virulence traits such as invasion and toxin production. In this study, 113 C. jejuni isolates from poultry and poultry-related (n=74) environments as well as isolates from human cases (n=39) of campylobacteriosis and bacteraemia were tested for invasiveness using INT 407 cells. The method was sufficiently reproducible to observe a spectrum of invasiveness amongst strains. As a result, strains were classified as low, high and hyper-invasive. The majority of strains (poultry and human) were low invaders (82 % and 88 %, respectively). High invasion was found for 5 % of human strains and 11 % of poultry-related isolates. However, only 1 % of poultry strains were classified as hyperinvasive compared to 13 % of human isolates (P=0.0182). Of those isolates derived from the blood of bacteraemic patients, 20 % were hyperinvasive, though this correlation was not statistically significant. An attempt was made to correlate invasiveness with the presence of seven genes previously reported to be associated with virulence. Most of these genes did not correlate with invasiveness, but gene cj0486 was weakly over-represented, and a negative correlation was observed for the gene ciaB. This trend was stronger when the two genes were analysed together, thus ciaB– cj0486+ was over-represented in high and hyperinvasive strains, with low invaders more commonly found to lack these genes (P=0.0064)

The Pathogenic Potential of Campylobacter concisus Strains Associated with Chronic Intestinal Diseases

Campylobacter concisus has garnered increasing attention due to its association with intestinal disease, thus, the pathogenic potential of strains isolated from different intestinal diseases was investigated. A method to isolate C. concisus was developed and the ability of eight strains from chronic and acute intestinal diseases to adhere to and invade intestinal epithelial cells was determined. Features associated with bacterial invasion were investigated using comparative genomic analyses and the effect of C. concisus on host protein expression was examined using proteomics. Our isolation method from intestinal biopsies resulted in the isolation of three C. concisus strains from children with Crohn's disease or chronic gastroenteritis. Four C. concisus strains from patients with chronic intestinal diseases can attach to and invade host cells using mechanisms such as chemoattraction to mucin, aggregation, flagellum-mediated attachment, “membrane ruffling”, cell penetration and damage. C. concisus strains isolated from patients with chronic intestinal diseases have significantly higher invasive potential than those from acute intestinal diseases. Investigation of the cause of this increased pathogenic potential revealed a plasmid to be responsible. 78 and 47 proteins were upregulated and downregulated in cells infected with C. concisus, respectively. Functional analysis of these proteins showed that C. concisus infection regulated processes related to interleukin-12 production, proteasome activation and NF-κB activation. Infection with all eight C. concisus strains resulted in host cells producing high levels of interleukin-12, however, only strains capable of invading host cells resulted in interferon-γ production as confirmed by ELISA. These findings considerably support the emergence of C. concisus as an intestinal pathogen, but more significantly, provide novel insights into the host immune response and an explanation for the heterogeneity observed in the outcome of C. concisus infection. Moreover, response to infection with invasive strains has substantial similarities to that observed in the inflamed mucosa of Crohn's disease patients

Expression patterns and role of the CadF protein in Campylobacter jejuni and Campylobacter coli

Binding of Campylobacter jejuni and Campylobacter coli to host fibronectin is mediated by the 37 kDa outer membrane protein CadF. Immunoblot analysis of 58 C. jejuni and C. coli isolates of human and animal origin showed that CadF is expressed in every strain. In most C. jejuni isolates, a 37 kDa band (p37) and a less-prominent 32 kDa band (p32) reacted with the antibodies. In C. coli isolates, CadF was consistently larger with sizes of 39 kDa (p39) and 34 kDa (p34), respectively. PCR analysis and sequencing revealed the presence of a 39-bp insertion sequence in the cadF gene of C. coli strains, explaining the increased molecular size. Infection assays revealed that C. jejuni bound and invaded INT-407 epithelial cells much more efficiently than C. coli and that this difference was considerably reduced in isogenic cadF mutants. These results demonstrate that CadF is an important pathogenicity factor. The difference between CadF of C. jejuni and C. coli may potentially be exploited to discriminate these species in food and clinical specimens