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Chapter 18 : Campylobacter jejuni Secretes Proteins via the Flagellar Type III Secretion System That Contribute to Host Cell Invasion and Gastroenteritis
Category: Bacterial Pathogenesis
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This chapter is divided into three major sections. In the first section, a model of Campylobacter jejuni-mediated enteritis is presented. The second section presents a general overview of the organism's pathogenic mechanisms and virulence determinants. Finally, in the third section, various aspects of C. jejuni-host cell invasion and protein secretion are discussed. Specifically, in the third section C. jejuni protein export via the flagellar type III secretion system (T3SS), the development of an assay to identify C. jejuni secreted proteins, the evolutionary relatedness of the flagellum and virulence T3SS, and the putative roles of C. jejuni secreted proteins in disease, are discussed. Although much remains unknown regarding the identity and functional characteristics of the proteins exported via the flagellar apparatus, the chapter highlights evidence supporting the proposal that these proteins contribute to C. jejuni-mediated enteritis. Motility, adherence, invasion, protein secretion, intracellular survival, and toxin production may contribute to the pathogenicity of a given C. jejuni strain. C. jejuni nonmotile strain can be either secretion positive (i.e., the flaA flaB+ mutant) or secretion negative (i.e., the flaA flaB mutant), and the ability of the bacterium to secrete proteins can result in an increase in its invasive potential. Although these data helped clarify the relationship between C. jejuni motility, secretion, and host cell invasion, the significance of protein secretion and host cell invasion in C. jejuni-mediated gastroenteritis was not known.
Model of C. jejuni pathogenesis. The clinical manifestation of C. jejuni infection is a result of bacterial activities (white box) and the host immune response (gray box). Dotted lines indicate processes that can potentiate infection.
Adherence, protein secretion, and invasion are a few of the C. jejuni virulence attributes that contribute to acute infection. As depicted, bacterial colonization of the intestinal tract can occur by different routes. Several virulence attributes may stimulate the host inflammatory response and in turn promote additional bacteria–host cell interactions. Other factors (not listed) are also capable of triggering the host inflammatory response. The dotted line represents the possibility that secreted proteins may enhance the cytokine response.
Secretion of the Cia proteins is dependent on an intact flagellar T3SS. Culture medium supplemented with fetal bovine serum (FBS) triggers secretion of the Cia proteins. C. jejuni were suspended in minimal essential medium, either with or without FBS, and radiolabeled with [35S]-methionine for 3 h. Supernatant fluids were harvested, concentrated fourfold, and solubilized in double-strength sample buffer. Equal volumes of samples were separated in a sodium dodecyl sulfate 12.5% polyacrylamide gel. The gel was dried and analyzed by phosphorimaging (Molecular Dynamics, Inc., ImageQuant, Sunnyvale, CA). Lanes: 1, C. jejuni 81-176 (+) FBS; 2, C. jejuni 81-176 (−) FBS.
C. jejuni type III secretion system (T3SS) is the flagellum that secretes the Campylobacter secreted proteins (Csp). A subset of the Csps, termed the Campylobacter invasion antigens (Cia), are required for maximal invasion. Both the Csp and Cia proteins harbor nonconsensus secretion signals, which are required for export.
Depiction of the pCSP50 shuttle vector and phospholipase indicator plate results for controls. The pCSP50 vector includes a tet cassette, a constitutive promoter (cat), a 5′ truncated yplA (lacking 150 nt encoding the native T3SS signal), and the yplB gene (cognate chaperone). The NdeI and BglII sites facilitate directional cloning of C. jejuni sequences as fusions with the truncated yplA. (A to E) Scans of phospholipase indicator plates under flagellar T3SS induction. (A) Y. enterocolitica JB580v (wild type); (B) Y. enterocolitica yplAB mutant; (C) Y. enterocolitica yplAB mutant with pCSP50; (D) Y. enterocolitica yplAB mutant with pCSP50: ciaB 1–108; (E) Y. enterocolitica yplAB mutant with pCSP50: cysM 1–108. All strains show strong growth; (A) and (D) show strong secretion of YplA and CiaB1–36:YplA fusion protein, respectively, resulting in a zone of precipitate due to hydrolysis of fatty acids by the phospholipase.
Functional categories of C. jejuni secreted proteins. Shown are the numbers of C. jejuni proteins, categorized according to function, encoding signal sequences that elicited strong secretion from both the Y. enterocolitica flagellar and Ysa T3SS.
Relative molecular mass of C. jejuni secreted proteins