Leishmanial GP63 acts as a protease for the small pore forming toxin aerolysin.

The eukaryotic pathogen Leishmania major causes disfiguring cutaneous lesions, whose resolution can be complicated by secondary bacterial infections. Bacteria, including Aeromonas spp., also interact with L. major promastigotes in the sandfly midgut. The mechanisms by which L. major competes with bacteria and resists their toxins are poorly defined. Prior work proposed that L. major resists the Aeromonas -produced pore-forming toxin aerolysin using an altered GPI-anchor. However, we found that L. major is sensitive to aerolysin. Here, we determined the mechanism by which L. major promastigotes are sensitive to aerolysin, using flow cytometry and biochemical approaches to analyze promastigotes genetically deficient in enzymes that produce key membrane components. The virulence factor lipophosphoglycan protected L. major from aerolysin cytotoxicity. The metalloproteinase GP63 exerted the necessary furin-like protease activity to activate aerolysin. Leishmanial GPI-anchored proteins were necessary for aerolysin heptamerization and killing of L. major promastigotes. Finally, mutation of the GPI-anchor binding domain of aerolysin crippled its cytotoxicity, consistent with its reliance on the GPI-anchor binding site to engage GPI anchors on the surface of L. major promastigotes. Taken together, we propose the L. major virulence factor lipophosphoglycan defends against pore-forming toxins made by bacterial competitors, while the GP63 metalloproteinase activates pro-aerolysin like furin. Overall, this study highlights approaches microbes use to compete with each other.