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

Abstract

Professional phagocytic cells such as macrophages are a central part of innate immune defence. They ingest microorganisms into membrane-bound compartments (phagosomes), which acidify and eventually fuse with lysosomes, exposing their contents to a microbicidal environment. Gram-positive Rhodococcus equi can cause pneumonia in young foals and in immunocompromised humans. The possession of a virulence plasmid allows them to subvert host defence mechanisms and to multiply in macrophages. Here, we show that the plasmid-encoded and secreted virulenceassociated protein A (VapA) participates in exclusion of the proton-pumping vacuolar-ATPase complex from phagosomes and causes membrane permeabilisation, thus contributing to a pH-neutral phagosome lumen. Using fluorescence and electron microscopy, we show that VapA is also transferred from phagosomes to lysosomes where it permeabilises the limiting membranes for small ions such as protons. This permeabilisation process is different from that of known membrane pore formers as revealed by experiments with artificial lipid bilayers. We demonstrate that, at 24 hr of infection, virulent R. equi is contained in a vacuole, which is enriched in lysosome material, yet possesses a pH of 7.2 whereas phagosomes containing a vapA deletion mutant have a pH of 5.8 and those with virulence plasmid-less sister strains have a pH of 5.2. Experimentally neutralising the macrophage endocytic system allows avirulent R. equi to multiply. This observation is mirrored in the fact that virulent and avirulent R. equi multiply well in extracts of purified lysosomes at pH 7.2 but not at pH 5.1. Together these data indicate that the major function of VapA is to generate a pH-neutral and hence growth-promoting intracellular niche. VapA represents a new type of Gram-positive virulence factor by trafficking from one subcellular compartment to another, affecting membrane permeability, excluding proton-pumping ATPase, and consequently disarming host defences.

Mycoredoxins Are Required for Redox Homeostasis and Intracellular Survival in the Actinobacterial Pathogen Rhodococcus equi

“…When R. equi is inhaled, the pathogen is recognized by FcγRI and toll-like receptors on the surface of alveolar macrophages and phagocytized [24,56]. R. equi 103S + is then able to block the phagosome maturation with Vaps, known modulators of the intraphagolysosomal pH [14,57].…”

“…The pathogenicity islands of pVAP plasmids comprise several genes that encode virulence-associated proteins (Vaps), VapA being the most studied [13]. Particularly, it is known that VapA is required to preserve a neutral pH within the phagolysosome containing R. equi [14].…”

Effect of Macrolide and Rifampin Resistance on the Fitness of Rhodococcus equi

“…R. equi carries a virulence plasmid that is required for replication in macrophages and disease presentation in foals (20,(23)(24)(25). On this plasmid, the virulence-associated protein A (vapA) gene may interfere with normal phagosomal acidification, creating an environment conducive to intracellular survival and replication (26)(27)(28)(29). Two additional genes housed on the plasmid, virR and virS, regulate vapA expression under particular environmental cues, such as changes in temperature and pH (30,31).…”

The extracellular thioredoxin Etrx3 is required for macrophage infection in Rhodococcus equi

“…It is becoming clear that the virulence associated proteins (Vaps) of R. equi are major determinants of the control of intraphagolysosomal pH during cell infection [7]. Furthermore, different members of the pVAP megaplasmids family carry specific complements of vap genes, which are essential for the intracellular survival of R. equi and they are considered the main driving factor of the host tropism of this pathogen [8].…”