Campylobacter jejuni 81-176 associates with microtubules and dynein duringinvasion of human intestinal cells

Campylobacter jejuni uptake into cultured INT407 cells was analyzed kinetic ally 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, presu mably due to reported C. jejuni autoagglutination at higher densities. Tota l 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 hos t cell limitations on this entry process. Furthermore, whereas control Salm onella typhi invaded all monolayer cells within 1 h, only two-thirds of mon olayer 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 t o 85% after 7 h of infection, suggesting that C. jejuni entry may be host c ell cycle dependent. Direct evidence of the involvement of microtubules in C. jejuni internalization, suggested previously by biochemical inhibitor st udies, was obtained by time course immunofluorescence microscopic analyses. Bacteria initially bound to the tips of host cell membrane extensions cont aining microtubules, then aligned in parallel with microtubules during entr y, colocalized specifically with microtubules and dynein but not with micro filaments, and moved over 4 h, presumably via min rotubules to the perinucl ear region of host cells. Orthovanadate, which inhibits dynein activity, sp ecifically reduced C. jejuni 81-176 entry, suggesting that this molecular m otor is involved in entry and endosome trafficking during this novel bacter ial internalization process. Collectively, these data suggest that C. jejun i enters host cells in a targeted and tightly controlled process leading to uptake into an endosomal vacuole which apparently moves intracellularly al ong microtubules via the molecular motor, dynein, to the perinuclear region .