Synergy unleashed: Leishmania donovani GP63 paralogues cooperatively function to drive parasite infectivity at various pathogenic stages and regulate host survival mechanisms

Leishmania donovani (Ld), the etiological agent of visceral leishmaniasis, poses a significant global health burden due to its complex dixenous lifecycle involving both insect vectors and mammalian hosts. Successful infection in mammals requires the coordinated activity of stage-specific virulence factors. The zinc metalloprotease glycoprotease 63 (GP63) is a well-established virulence determinant critical for host cell attachment and invasion by insect-stage promastigotes. For humans, subsequent parasite propagation depends exclusively on intracellular amastigotes arising from lysed macrophages. Classical GP63 expression and function in Ld amastigotes remain poorly understood, and GP63 null mutants reportedly retain infectivity in mice, raising fundamental questions about virulence factor complementation during mammalian infection. By employing comparative transcriptomics, CRISPR-based mutagenesis, complemented with cell biology, and biochemical assays, this work identifies and characterizes multiple Ld GP63 paralogues with distinct roles in mammalian infection. While both copies of GP63 encoded on chromosome 10 (LdGP63_10.51 and 10.52) were functionally redundant, LdGP63_28 encoded on chromosome 28 proved essential for intracellular amastigote survival by suppressing host cell pyroptosis. Moreover, LdGP63_31 (chromosome 31) was found to primarily mediate promastigote attachment to the host macrophages with minimal contribution from LdGP63_28, facilitating initial infection establishment and amastigote genesis. Importantly, the absence of LdGP63_28 impacted amastigote infection more severely as compared to LdGP63_31. Structural and enzymatic analyses revealed divergent localization and substrate specificities to fulfil functional requirement of these divergent proteases, which have evolved independently to carry out diverse function in establishing infection. Collectively, this study indicates evolutionary divergence and functional specialization among GP63 isoforms in Ld by demonstrating that amastigote-specific and promastigote-specific GP63 isoforms synergistically mediate infection establishment and persistence.