Virulence-associated protein A from Rhodococcus equi is an intercompartmental pH-neutralising virulence factor

Area of Science:

• Microbiology
• Immunology
• Cell Biology

Background:

• Macrophages are key to innate immunity, engulfing pathogens in phagosomes that normally acidify.
• Gram-positive Rhodococcus equi causes disease in foals and immunocompromised humans by subverting macrophage defenses.
• Virulence plasmid-encoded factors enable R. equi to survive and multiply within host cells.

Purpose of the Study:

• To investigate the role of virulence-associated protein A (VapA) in R. equi pathogenesis.
• To elucidate the mechanism by which VapA allows R. equi to multiply within macrophages.
• To characterize VapA's effect on phagosome and lysosome properties.

Main Methods:

• Fluorescence and electron microscopy to track VapA localization.
• Experiments with artificial lipid bilayers to study membrane permeabilization.
• pH measurements of phagosomes containing wild-type, mutant, and plasmid-less R. equi strains.
• Macrophage culture and infection assays.

Main Results:

• VapA excludes the proton-pumping vacuolar-ATPase from phagosomes, leading to a neutral pH (7.2) compared to acidic phagosomes (pH 5.8-5.2) with mutants.
• VapA is transferred to lysosomes, permeabilizing their membranes to protons.
• Neutralizing macrophage endocytic system pH allows even avirulent R. equi to multiply.
• Virulent and avirulent R. equi strains multiply in purified lysosomes at neutral pH but not acidic pH.

Conclusions:

• VapA's primary function is to create a neutral, growth-promoting intracellular niche for R. equi.
• VapA acts as a novel Gram-positive virulence factor by manipulating vacuolar compartments and host defenses.
• Understanding VapA's mechanism offers insights into bacterial pathogenesis and potential therapeutic targets.