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Proteomic Analysis of Leishmania donovani Membrane Components Reveals the Role of Activated Protein C Kinase in Host-Parasite Interaction.

ABSTRACT: Visceral leishmaniasis (VL), mainly caused by the Leishmania donovani parasitic infection, constitutes a potentially fatal disease, for which treatment is primarily dependent on chemotherapy. The emergence of a resistant parasite towards current antileishmanial agents and increasing reports of relapses are the major concerns. Detailed research on the molecular interaction at the host-parasite interface may provide the identification of the parasite and the host-related factors operating during disease development. Genomic and proteomic studies highlighted several essential secretory and cytosolic proteins that play vital roles during Leishmania pathogenesis. The aim of this study was to identify membrane proteins from the Leishmania donovani parasite and the host macrophage that interact with each other using 2-DE/MALDI-TOF/MS. We identified membrane proteins including activated protein C kinase, peroxidoxin, small myristoylated protein 1 (SMP-1), and cytochrome C oxidase from the parasite, while identifying filamin A interacting protein 1(FILIP1) and β-actin from macrophages. We further investigated parasite replication and persistence within macrophages following the macrophage-amastigote model in the presence or absence of withaferin (WA), an inhibitor of activated C kinase. WA significantly reduced Leishmania donovani replication within host macrophages. This study sheds light on the important interacting proteins for parasite proliferation and virulence, and the establishment of infection within host cells, which can be targeted further to develop a strategy for chemotherapeutic intervention.

SUBMITTER: Verma S

PROVIDER: S-EPMC8465321 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Visceral leishmaniasis is associated with hepato-splenomegaly and altered immune and hematological parameters in both preclinical animal models and humans. We studied mouse experimental visceral leishmaniasis caused by Leishmania infantum and Leishmania donovani in BALB/c mice using dual RNA-seq to investigate the transcriptional response of host and parasite in liver and spleen. We identified only 4 species-specific parasite expressed genes (SSPEGs; log2FC >1, FDR <0.05) in the infected spleen, and none in the infected liver. For the host transcriptome, we found 789 differentially expressed genes (DEGs; log2FC >1, FDR <0.05) in the spleen that were common to both infections, with IFNγ signaling and complement and coagulation cascade pathways highly enriched, and an additional 286 and 186 DEGs that were selective to L. donovani and L. infantum infection, respectively. Among those, there were network interactions between genes of amino acid metabolism and PPAR signaling in L. donovani infection and increased IL1β and positive regulation of fatty acid transport in L. infantum infection, although no pathway enrichment was observed. In the liver, there were 1,939 DEGs in mice infected with either L. infantum or L. donovani in comparison to uninfected mice, and the most enriched pathways were IFNγ signaling, neutrophil mediated immunity, complement and coagulation, cytokine-chemokine responses, and hemostasis. Additionally, 221 DEGs were selective in L. donovani and 429 DEGs in L. infantum infections. These data show that the host response for these two visceral leishmaniasis infection models is broadly similar, and ∼10% of host DEGs vary in infections with either parasite species. IMPORTANCE Visceral leishmaniasis (VL) is caused by two species of Leishmania parasites, L. donovani in the Old World and L. infantum in the New World and countries bordering the Mediterranean. Although cardinal features such as hepato-splenomegaly and alterations in blood and immune function are evident, clinical presentation may vary by geography, with for example severe bleeding often associated with VL in Brazil. Although animal models of both L. donovani and L. infantum have been widely used to study disease pathogenesis, a direct side-by-side comparison of how these parasites species impact the infected host and/or how they might respond to the stresses of mammalian infection has not been previously reported. Identifying common and distinct pathways to pathogenesis will be important to ensure that new therapeutic or prophylactic approaches will be applicable across all forms of VL.

Project description:Leishmania secretes over 151 proteins during in vitro cultivation. Cellular functions of one such novel protein: mevalonate kinase is discussed here; signifying its importance in Leishmania infection. Visceral Leishmaniasis is a persistent infection, caused by Leishmania donovani in Indian subcontinent. This persistence is partly due to phagocytosis and evasion of host immune response. The underlying mechanism involves secretory proteins of Leishmania parasite; however, related studies are meagre. We have identified a novel secretory Leishmania donovani glycoprotein, Mevalonate kinase (MVK), and shown its importance in parasite internalization and immuno-modulation. In our studies, MVK was found to be secreted maximum after 1 h temperature stress at 37°C. Its secretion was increased by 6.5-fold in phagolysosome-like condition (pH ~5.5, 37°C) than at pH ~7.4 and 25°C. Treatment with MVK modulated host immune system by inducing interleukin-10 and interleukin-4 secretion, suppressing host's ability to kill the parasite. Peripheral blood mononuclear cell (PBMC)-derived macrophages infected with mevalonate kinase-overexpressing parasites showed an increase in intracellular parasite burden in comparison to infection with vector control parasites. Mechanism behind the increase in phagocytosis and immunosuppression was found to be phosphorylation of mitogen-activated protein (MAP) kinase pathway protein, Extracellular signal-regulated kinases-1/2, and actin scaffold protein, cortactin. Thus, we conclude that Leishmania donovani Mevalonate kinase aids in parasite engulfment and subvert the immune system by interfering with signal transduction pathways in host cells, which causes suppression of the protective response and facilitates their persistence in the host. Our work elucidates the involvement of Leishmania in the process of phagocytosis which is thought to be dependent largely on macrophages and contributes towards better understanding of host pathogen interactions.

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Proteomic Analysis of Leishmania donovani Membrane Components Reveals the Role of Activated Protein C Kinase in Host-Parasite Interaction.

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