Rhodococcus equi Escapes Macrophage Autophagy: VapA Inhibits ATP6V0d1-Mediated Lysosomal Degradation

Rhodococcus equi is a gram-positive actinomycetales bacterial, which cause a pneumonia in foals and in immunocompromised humans. Virulence associated protein A (VapA) encoded from virulence plasmid is essential for intracellular proliferation in macrophage. It has reported that VapA participates in exclusion of proton-pumping vacuolar-ATPase complex from phagosome and causes membrane permeabilization, thus contributing to a pH-neutral phagosome lumen. However, there have been no reports about VapA-induced autophagy or its mechanism. In this study, we sought to determine the role of VapA in macrophage autophagy using western blotting, immunofluorescence, pull-down, Co-IP, LS/MS, pH detection LysoTracker and CFU analysis. We show that VapA inhibiting the macrophage autophagy by detecting the expression of autophagy related protein LC3II and P62 and autophagy flux in VapA treated macrophage. Furthermore, we using pull-down and MS analysis to selected ATP6V0D1 could interact with VapA, Co-IP was used to confirm its interaction. Furthermore, VapA treatment in ATP6V0D1 overexpressed cells weaken the lyso Tracker red stain comparing with ATP6V0D1 overexpressed J774A.1 cells. In addition, the expression of ATP6V0D1 significantly increased in J774A.1 post infected with vapA deletion strain, R. equi 103+/{Delta}VapA by comparing with wild type R. equi 103+ post-infection. And raise of raise of ATP6V0D1 actually reduce R. equi 103+ intracellular multiplication. These results further suggesting a decrease of ATP6V0D1 were caused the exit of VapA in R. equi. Taken those date together, we reported that VapA mediated lysosome acidification by inhibiting the expression of ATP6V0D1, which contributing the R. equi escape macrophage autophagy.