The Campylobacter jejuni CiaC virulence protein is secreted from the flagellum and delivered to the cytosol of host cells

Jason M Neal-Mckinney,Michael E Konkel

doi:10.3389/fcimb.2012.00031

Abstract

Campylobacter jejuni is a leading cause of bacterial gastroenteritis worldwide. Acute C. jejuni-mediated disease (campylobacteriosis) involves C. jejuni invasion of host epithelial cells using adhesins (e.g., CadF and FlpA) and secreted proteins [e.g., the Campylobacter invasion antigens (Cia)]. The genes encoding the Cia proteins are up-regulated upon co-culture of C. jejuni with epithelial cells. One of the Cia proteins, CiaC, is required for maximal invasion of host cells by C. jejuni. Previous work has also revealed that CiaC is, in part, responsible for host cell cytoskeletal rearrangements that result in membrane ruffling. This study was performed to test the hypothesis that CiaC is delivered to the cytosol of host cells. To detect the delivery of CiaC into cultured epithelial cells, we used the adenylate cyclase domain (ACD) of Bordetella pertussis CyaA as a reporter. In this study, we found that export and delivery of the C. jejuni Cia proteins into human INT 407 epithelial cells required a functional flagellar hook complex composed of FlgE, FlgK, and FlgL. Assays performed with bacterial culture supernatants supported the hypothesis that CiaC delivery requires bacteria-host cell contact. We also found that CiaC was delivered to host cells by cell-associated (bound) bacteria, as judged by experiments performed with inhibitors that specifically target the cell signaling pathways utilized by C. jejuni for cell invasion. Interestingly, the C. jejuni flgL mutant, which is incapable of exporting and delivering the Cia proteins, did not induce INT 407 cell membrane ruffles. Complementation of the flgL mutant with plasmid-encoded flgL restored the motility and membrane ruffling. These data support the hypothesis that the C. jejuni Cia proteins, which are exported from the flagellum, are delivered to the cytosol of host cells.

Highlights

Campylobacter jejuni remains a leading bacterial cause of gastroenteritis, with an estimated annual incidence of 2.4 million cases per year in the US (Mead et al, 1999), and 400–500 million cases worldwide (Konkel et al, 2001; Ruiz-Palacios, 2007)
We found that CiaC was delivered to host cells by cell-associated bacteria, as judged by experiments performed with inhibitors that target the cell signaling pathways utilized by C. jejuni for cell invasion
The number of internalized ciaC mutant bacteria did not change in response to additional wild-type bacteria. These results suggest that the Campylobacter invasion antigens (Cia) proteins act in a localized manner within the cell, and that the membrane ruffling induced by wild-type C. jejuni is not sufficient to facilitate the uptake of a bacterium attached to another location on the host cell

Summary

Introduction

Campylobacter jejuni remains a leading bacterial cause of gastroenteritis, with an estimated annual incidence of 2.4 million cases per year in the US (Mead et al, 1999), and 400–500 million cases worldwide (Konkel et al, 2001; Ruiz-Palacios, 2007). Most infections with C. jejuni are linked to the consumption of undercooked poultry or foods that have been contaminated with raw poultry (Konkel et al, 2001). This is due to the fact that C. jejuni is a commensal organism in chickens and is able to colonize the ceca of birds at levels greater than 108 CFU/gram of cecal contents (Sahin et al, 2002). Infection with C. jejuni can lead to Guillain–Barré syndrome, an acute demyelinating neuropathy characterized by flaccid paralysis (Konkel et al, 2001)

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