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Quantitative proteomics of infected macrophages reveals novel Leishmania virulence factors

ABSTRACT: Leishmaniasis is a major public health problem, causing diseases ranging from self-healing skin lesions to life-threatening chronic infections. Understanding how Leishmania parasites evade the host defense system is crucial for understanding the different manifestations of the disease and for improving diagnostic tools and drug development. We performed high-resolution proteome profiling of Leishmania spp. across three species during macrophage infection and identified distinct temporal expression patterns. Clustering analysis revealed unique protein expression profiles for each Leishmania species, whereas pairwise enrichment analysis revealed specific up- and downregulation patterns at different infection stages. Our results confirmed known virulence factors and highlighted new ones, demonstrating how our dataset could be used. We validated the dataset by showing that deletion of putative L. mexicana virulence factors resulted in reduced stage differentiation capacity and infectivity. This novel Leishmania infectome database (LInfDB) is available online for further exploration (https://butterlab.imb-mainz.de/LInfDB), providing a valuable resource for future research.

INSTRUMENT(S):

ORGANISM(S): Leishmania Major Leishmania Mexicana Mexicana Leishmania Infantum Mus Musculus (mouse)

SUBMITTER: Falk Butter

LAB HEAD: Falk Butter

PROVIDER: PXD058427 | Pride | 2026-01-28

REPOSITORIES: Pride

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			Action	DRS
	20201221_SFB1_FBU_FB_macro_LeishInfant_t000_1.raw	Raw
	20201221_SFB1_FBU_FB_macro_LeishInfant_t000_2.raw	Raw
	20201221_SFB1_FBU_FB_macro_LeishInfant_t000_3.raw	Raw
	20201221_SFB1_FBU_FB_macro_LeishInfant_t000_4.raw	Raw
	20201221_SFB1_FBU_FB_macro_LeishInfant_t005_1.raw	Raw

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Project description:Protozoa of the genus Leishmania are the causative agents of leishmaniasis in humans. These parasites cycle between promastigotes in the sand fly mid-gut and amastigotes in phagolysosome of mammalian macrophages. During infection, host up-regulate nitric oxide synthase and parasite induce host arginase expression, both of which use arginine as a substrate. These elevated activities deplete macrophage arginine pools, a situation that invading Leishmania must overcome since it is an essential amino acid. Leishmania donovani imports exogenous arginine via a mono-specific amino acid transporter (AAP3) and utilizes it primarily through the polyamine pathway to provide precursors for trypanothione biosynthesis. Here we report the discovery of a pathway whereby promastigote and amastigote forms of the Leishmania sense the lack of environmental arginine and respond with rapid up-regulation in AAP3 expression and activity, as well as several other transporters. Significantly, this arginine deprivation response is also activated in parasites during macrophage infection. Phosphoproteomic analyses of L. donovani promastigotes have implicated a Mitogen-Activated Protein Kinase 2 (MPK2)-mediated signaling cascade in this response and L. mexicana mutants lacking MPK2 are unable to respond to arginine deprivation. In this study, we established that Leishmania cells sense the absence of arginine in their environment; both in culture (axenic promastigotes and amastigotes) and in macrophages during infection (amastigotes). This study describes the first amino acid deprivation sensing mechanism and the pathway that transduce this response, and reveals a novel host-pathogen metabolic interplay. Total RNA from Ten Leishmania donovani samples were analyzed using RNA-Seq. Cells from two life stages (promastigotes and amastigotes) were grown in axenic culture in the presence and absense of arginine. For each condition two biological replicates were grown and analyzed. In addition two macrophage grown amastigotes were analyzed.

Project description:The ability of Leishmania to survive in their insect or mammalian host is dependent upon an ability to sense and adapt to changes in the microenvironment. However, little is known about the molecular mechanisms underlying the parasite response to environmental changes, such as nutrient availability. To elucidate nutrient stress response pathways in Leishmania donovani, we have used purine starvation as the paradigm. The salvage of purines from the host milieu is obligatory for parasite replication; nevertheless, purine-starved parasites can persist in culture without supplementary purine for over 3 months, indicating that the response to purine starvation is robust and engenders parasite survival under conditions of extreme scarcity. To understand metabolic reprogramming during purine starvation we have employed global approaches. Whole proteome comparisons between purine-starved and purine-replete parasites over a 6-48 h span have revealed a temporal and coordinated response to purine starvation. Purine transporters and enzymes involved in acquisition at the cell surface are upregulated within a few hours of purine removal from the media, while other key purine salvage components are upregulated later in the time-course and more modestly.Â After 48 h, the proteome of purine-starved parasites is extensively remodeled and adaptations to purine stress appear tailored to deal with both purine deprivation and general stress. To probe the molecular mechanisms affecting proteome remodeling in response to purine starvation, comparative RNA-seq analyses, qRT-PCR, and luciferase reporter assays were performed on purine-starved versus purine-replete parasites.Â While the regulation of a minority of proteins tracked with changes at the mRNA level, for many regulated proteins it appears that proteome remodeling during purine stress occurs primarily via translational and post-translational mechanisms. One mRNA sample from each of Purine-Starved and Purine-Replete cells were analyzed using SL RNA-Seq methodology

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Quantitative proteomics of infected macrophages reveals novel Leishmania virulence factors

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