Activation Of Host Phospholipase D Is Coupled To The Invasion Of Listeria Monocytogenes Into Vero Cells

ObjectiveIn our study, we stimulated Vero cells with L.monocytogenes, which act as a model of entry into nonphagocytic cells by bacteria, to study whether PLD is activated during the invasion of L.monocytogenes into Vero cells and analyze the effect of PLD activity on the extent of L.monocytogenes invasion.Methods1,The Vero cells were incubated with L.monocytogenes at different multiplicity of infection(MOI, bacteria:cell) for different time to allow the L.monocytogenes enter, then the invasion of L.monocytogenes into Vero cells and the activation of PLD stimulated by L.monocytogenes in Vero cells were detected. The quantitative assay of invasion was determined by the number of viable bacteria released from the cells. Cellular phospholipids were radiolabeled with [9,10-3H] oleic acid, the activity of PLD was determined by formation of the PLD-specific product [3H]phosphatydylethanol in the presence of ethanol and expressed as the percentage of [3H]phosphatydylethanol relative to total [3H] phospholipids to correct for potential differences in labeling .2,Before stimulated by L.monocytogenes, Vero cells were pretreated with two inhibitors of PLD, 1-butanol and 2,3-diposphoglycerate, respectively, then the L.monocytogenes-stimulated PLD activity in Vero cells and the invasion of L.monocytogenes into Vero cells were detected to evaluate the role of PLD in L.monocytogenes entry into Vero cells.3,Before stimulated by L.monocytogenes, Vero cells were preinfected with recombinant adenoviruses expressing hPLD1wt, hPLD1-K898R, mPLD2wt, mPLD2-K758R, respectively, then the L.monocytogenes-stimulated PLD activity and the invasion of L.monocytogenes into Vero cells were determined to identify the PLD isoform(s) involved in the L.monocytogenes entry into Vero cells.Results1,The invasion of L.monocytogenes into Vero cells was significantly enhauced with the increase of MOI(100:1,200:1,400:1) and the duration of infection(30min,60min,120min). The internalization of L.monocytogenes by Vero cells was associated with activation of host PLD,the PLD activity stimulated by L.monocytogenes increased 4-fold compared to basal ones(no stimuli). The PLD activation in Vero cells infected by L.monocytogenes shows time-dependent kinetics during the 15-90min assay: an increase in PLD activity was noted 15min after infection and the levels of PLD activity reached a maximum between 40-90min.2,The pretreatment of Vero cells with 1-butanol resulted in the decrease of PLD activity stimulated by L.monocytogenes(-60%), which was associated with the reductions in the invasion of L.monocytogenes into Vero cells( - 68%). The inhibition of L.monocytogenes-stimulated PLD avtivity by 2,3-diposphoglycerate was also associated with the reductions in the entry of L.monocytogenes into Vero cells.3,All of the recombinant adenoviruses can effectively infect Vero cells, and have no effect on basal PLD activity. While the overexpression of hPLD1-K898R or mPLD2-K758R protein can result in an 40% decreasing in the L.monocytogenes-stimulated PLD activity, the overexpression of hPLD1wt, but not mPLD2wt protein in Vero cells can lead to an 50% increase in the PLD activity, which responsed to L.monocytogenes. In addition, the inhibition of PLD activity by the overexpression of hPLD1-K898R or mPLD2-K758R protein can also inhibit the invasion of L.monocytogene into Vero cells with a reduction of 51% and 58% respectively.ConclusionThe L.monocytogenes-induced phagocytosis by Vero cells is associated with activation of host PLD. The reduction of L.monocytogenes-stimulated PLD activity can inhibit the invasion of L.monocytogenes into Vero cells to some extent. Both PLD1 and PLD2 may be activated during L.monocytogenes invading Vero cells and coordinately regulate the invasion of L.monocytogenes into Vero cells. The activate mechanism and important physiological role of PLD in this phathogen-host interaction process need to be further studied.