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.

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.

No abstract

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.

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.

In the present work we analyzed the complete set of GP63 sequences from the parasitic protozoa Trypa

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Hello Experts, I would like to know, how much Memory is support for Intel(R) Core(TM) i7-8750H CPU @ 2.20GHz and (Intel HM370 (Cannon Lake-H))? At Intel-Ark there is described, that 64 GB are supported. Intel Core i78750H Processor 9M Cache up to 4.10 GHz Product Specifications Mobile Intel HM370 Chipset Product Specifications Some Vendors (GP63 Leopard 8RF, XMG NEO 15 - XNE15M18) say that "only" 32 GB are supported. What is true? (I would like to use 64 GB, if supported) Thanks, for your feedback! Regards, vene