The Rhodococcus equi virulence protein VapA disrupts endolysosome function and stimulates lysosome biogenesis

Abstract

Rhodococcus equi (R. equi) is an important pulmonary pathogen in foals that often leads to the death of the horse. The bacterium harbors a virulence plasmid that encodes numerous virulence‐associated proteins (Vaps) including VapA that is essential for intracellular survival inside macrophages. However, little is known about the precise function of VapA. Here, we demonstrate that VapA causes perturbation to late endocytic organelles with swollen endolysosome organelles having reduced Cathepsin B activity and an accumulation of LBPA, LC3 and Rab7. The data are indicative of a loss of endolysosomal function, which leads cells to upregulate lysosome biogenesis to compensate for the loss of functional endolysosomes. Although there is a high degree of homology of the core region of VapA to other Vap proteins, only the highly conserved core region of VapA, and not VapD of VapG, gives the observed effects on endolysosomes. This is the first demonstration of how VapA works and implies that VapA aids R. equi survival by reducing the impact of lysosomes on phagocytosed bacteria.

Virulence‐associated protein A fromRhodococcus equiis an intercompartmental pH‐neutralising virulence factor

“…They were only in some cases strongly stained with LT ( Figure S2) whereas NRK lysosomes were always LT positive. In summary, VapA raises the pH in mouse macrophage lysosomes strongly whereas it does not in rat epithelial cell lysosomes, which is a possible reason for R. equi barely multiplying in NRK cells (Rofe et al, 2017).…”

“…(a) RAW264.7 macrophages were infected for 2 or 24 hr with the virulent, VapA-producing strain R. equi 103+ or with VapA-lacking strains 103− or ΔvapA. In summary, VapA raises the pH in mouse macrophage lysosomes strongly whereas it does not in rat epithelial cell lysosomes, which is a possible reason for R. equi barely multiplying in NRK cells (Rofe et al, 2017). Lysosomes were fluorescently labelled with 10 kDa dark red fluorescent dextran using a pulse-chase protocol.…”

“…Bars, 10 μm explained by our use of macrophages versus their kidney cells and that our study uses murine cells, and mice are used as models for R. equi infection (González-Iglesias et al, 2014) whereas rats are not. We therefore tested whether lysosomes in rVapA-fed NRK cells (following the protocol by Rofe et al, 2017) would be acidic. We confirmed the observation by Rofe et al that lysosomes in treated NRK cells are somewhat bigger than in untreated cells whereas the difference between lysosomes in RAW264.7 macrophage cells treated with rVapA was more pronounced ( Figure S2; see also Figures S3B,D and 3c).…”

Rescue of an intracellular avirulent <i>Rhodococcus equi</i> replication defect by the extracellular addition of virulence-associated protein A

“…The resulting RCVs possess some late endocytic markers, such as Rab7, BMP, LAMP 1 and 2, but acquire neither cathepsin D nor v-ATPase. Recently, it was reported that VapA was taken into endocytic compartments when it was added to the culture media, and that cells fed VapA formed swollen endolysosome organelles with reduced activity of cathepsin B and accumulation of LBPA, LC3 and Lab7 [ 10 ]. Here, we show that the extracellular addition of VapA can exert an influence on the growth of intraphagocytic R. equi .…”

“…Rofe et al . reported that the extracellular addition of VapA resulted in the formation of swollen endolysosome organelles with reduced cathepsin B activity and accumulation of late endocytic markers such as LBPA and Lab7, suggesting the loss of functional endolysosomes [ 10 ]. These features are similar to those found in RCVs [ 4 ].…”