Campylobacter jejuni 81-176 Associates with Microtubules and Dynein during Invasion of Human Intestinal Cells

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American Society for Microbiology

RESUMO

Campylobacter jejuni uptake into cultured INT407 cells was analyzed kinetically over a wide range of starting multiplicities of infection (MOI; from 0.02 to 20,000 bacteria/epithelial cell). The efficiency of internalization was the highest at MOI of 0.02 and decreased steadily at higher MOIs, presumably due to reported C. jejuni autoagglutination at higher densities. Total internalized Campylobacter CFU increased gradually from an MOI of 0.02 to a peak at an MOI of 200 (reaching an average of two bacteria internalized per epithelial cell) and decreased at higher MOIs. The invasion process was apparently saturated within 2 h at an MOI of 200, indicating stringent host cell limitations on this entry process. Furthermore, whereas control Salmonella typhi invaded all monolayer cells within 1 h, only two-thirds of monolayer cells were infected after 2 h with C. jejuni at MOIs of 200 to 2,000. The percentage of Campylobacter-infected host cells gradually increased to 85% after 7 h of infection, suggesting that C. jejuni entry may be host cell cycle dependent. Direct evidence of the involvement of microtubules in C. jejuni internalization, suggested previously by biochemical inhibitor studies, was obtained by time course immunofluorescence microscopic analyses. Bacteria initially bound to the tips of host cell membrane extensions containing microtubules, then aligned in parallel with microtubules during entry, colocalized specifically with microtubules and dynein but not with microfilaments, and moved over 4 h, presumably via microtubules to the perinuclear region of host cells. Orthovanadate, which inhibits dynein activity, specifically reduced C. jejuni 81-176 entry, suggesting that this molecular motor is involved in entry and endosome trafficking during this novel bacterial internalization process. Collectively, these data suggest that C. jejuni enters host cells in a targeted and tightly controlled process leading to uptake into an endosomal vacuole which apparently moves intracellularly along microtubules via the molecular motor, dynein, to the perinuclear region.

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