No abstract

Cj0859c, or FspA, is a small, acidic protein ofCampylobacter jejunithat is expressed by a σ28promoter. Analysis of thefspAgene in 41 isolates ofC. jejunirevealed two overall variants of the predicted protein, FspA1 and FspA2. Secretion of FspA occurs in broth-grown bacteria and requires a minimum flagellar structure. The addition of recombinant FspA2, but not FspA1, to INT407 cells in vitro resulted in a rapid induction of apoptosis. These data define a novelC. jejunivirulence factor, and the observed heterogeneity amongfspAalleles suggests alternate virulence potential among different strains.

Campylobacter jejuni 81-176 is capable of extensive replication within human monocytic cell vacuoles and induces apoptotic death via cytolethal distending toxin.Our understanding of the interaction of Campylobacter jejuni with immune effector cells is limited. C. jejuni can survive phagocytosis by human mononuclear cells and remain viable for extended periods (9), induce secretion of chemokines (8), and mediate apoptosis (20). C. jejuni elaborates a well-characterized toxin, cytolethal distending toxin (CjCDT), that induces cell cycle arrest and interleukin 8 (IL-8) secretion from intestinal epithelial cells in culture (2,7,13,22). CDTs from other bacteria are also known to target immune cells and ultimately induce apoptosis (3,19,21). There have been two reports of apoptotic responses to C. jejuni (20,24), and both suggested that mechanisms independent of CDT were responsible. Here, we confirm earlier observations of long-term campylobacter survival and replication within monocytes and the induction of apoptosis. Since CDTs of other pathogens induce apoptosis of lymphocytes (1,3,17,19,21), we reexamined the effect of CjCDT on human monocytes in culture.Growth of C. jejuni in human 28SC monocyte cultures. The human monocyte line 28SC was infected with 81-176 at a multiplicity of infection (MOI) of 100:1 for 2 h. Extracellular bacteria were killed with 100 g/ml gentamicin for an additional 2-h incubation. The cells were washed and reincubated, and bacterial counts were enumerated at various times. Peak levels of 81-176 were recovered from 48-hour cultures (Fig. 1), representing an approximate 3-log-unit increase in bacterial counts (from approximately 10 4 CFU/ml following gentamicin treatment to 10 7 CFU/ml after 48 h). Viable campylobacters were recovered up to day 7, consistent with the results of Kiehlbauch et al. (9). In contrast, in the absence of monocytes, 81-176 viability dropped rapidly, and no bacteria could be cultured after 72 h. Thus, replication of C. jejuni within monocytes was more extensive than the limited replication following internalization into epithelial cells (12).Green fluorescent protein (GFP)-tagged 81-176 was imaged within 28SC cells using fluorescence microscopy. Generalized fluorescence (Fig. 2B) could be seen intracellularly within enlarged and irregularly shaped areas of cells ( Fig. 2A). Using live/dead staining, the C. jejuni organisms appeared to be localized within vacuoles, either as single or multiple bacteria (Fig. 2C and D). The bacteria appeared motile within the vacuoles, consistent with expression of GFP under the control of the 28 promoter of the major flagellin (5). Induction of chemokines from 28SC cells is independent of CDT. Viable campylobacters mediated chemokine responses from 28SC cells in 24-h cultures (Fig. 3A). 81-176 induced 1,689 Ϯ 848 pg/ml IL-6 and 1,231 Ϯ 479 pg/ml IL-8 in nine independent experiments. Since CDT is localized to bacterial membranes and is known to induce IL-8 secretion from intestinal epithelial cells (7), we compared the abilities of pu...

A Campylobacter jejuni 81-176 mutant in Cj0977 was fully motile but reduced >3 logs compared to the parent in invasion of intestinal epithelial cells in vitro. The mutant was also attenuated in a ferret diarrheal disease model. Expression of Cj0977 protein was dependent on a minimal flagella structure.