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Background The effectiveness of systemic antimonial (sodium stibogluconate, Pentostam, SSG) treatment versus local heat therapy (Thermomed) for cutaneous leishmaniasis was studied previously and showed similar healing rates. We hypothesized that different curative immune responses might develop with systemic and local treatment modalities. Methods We studied the peripheral blood immune cells in a cohort of 54 cutaneous Leishmania major subjects treated with SSG or TM. Multiparameter flow cytometry, lymphoproliferative assays and cytokine production were analyzed in order to investigate the differences in the immune responses of subjects before, on and after treatment. Results Healing cutaneous leishmaniasis lead to a significant decline in circulating T cells and NKT-like cells, accompanied by an expansion in NK cells, regardless of treatment modality. Functional changes involved decreased antigen specific CD4+ T cell proliferation (hyporesponsiveness) seen with CD8+ T cell depletion. Moreover, the healing (or healed) state was characterized by fewer circulating regulatory T cells, reduced IFN-γ production and an overall contraction in polyfunctional CD4+ T cells. Conclusion Healing from cutaneous Leishmaniasis is a dynamic process that alters circulating lymphocyte populations and subsets of T, NK and NKT-like cells. Immunology of healing, through local or systemic treatments, culminated in similar changes in frequency, quality, and antigen specific responsiveness with immunomodulation possibly via a CD8+ T cell dependent mechanism. Understanding the evolving immunologic changes during healing of human leishmaniasis informs protective immune mechanisms., Author Summary Globally, leishmaniasis treatment relies on the use of antimonial drugs (i.e. SSG). In an earlier study we showed that skin lesions due to L. major treated by the ThermoMed (TM) device healed at a similar rate and with less associated systemic toxicity than lesions treated with intravenous SSG. The current study compared the immune responses of these two therapeutic groups before, during and after therapy which may be relevant to resistance to reinfection and also in consideration for the development of local (versus systemic) therapy. Antimonials have immune effects on both the host and parasite while heat treatment locally kills the parasite and induces inflammation from a secondary burn. We demonstrated that healing from cutaneous leishmaniasis is a dynamic process associated with a modulation of immune responses independent of treatment modalities.

Previously, we showed Leishmania donovani Ufm1 has a Gly residue conserved at the C-terminal region with a unique 17 amino acid residue extension that must be processed prior to conjugation to target proteins. In this report, we describe for the first time the isolation and characterization of the Leishmania Ufm1-specific protease Ufsp. Biochemical analysis of L. donovani Ufsp showed that this protein possesses the Ufm1 processing activity using sensitive FRET based activity probes. The Ufm1 cleavage activity was absent in a mutant Ufsp in which the active site cysteine is altered to a serine. To examine the effects of abolition of Ufm1 processing activity, we generated a L. donovani null mutant of Ufsp (LdUfsp−/−). Ufm1 processing activity was abolished in LdUfsp−/− mutant, and the processing defect was reversed by re-expression of wild type but not the cys>ser mutant in the LdUfsp−/− parasites. Further LdUfsp−/− mutants showed reduced survival as amastigotes in infected human macrophages but not as promastigotes. This growth defect in the amastigotes was reversed by re-expression of wild type but not the cys>ser mutant in the Ufsp−/− indicating the essential nature of this protease for Leishmania pathogenesis. Further, mouse infection experiments showed deletion of Ufsp results in reduced virulence of the parasites. Additionally, Ufsp activity was inhibited by an anti-leishmanial drug Amphotericin B. These studies provide an opportunity to test LdUfsp−/− parasites as drug and vaccine targets., Author Summary Ubiquitin and ubiquitin like proteins (Ubls) and the enzymes that mediate the conjugation/deconjugation reactions have not been well studied in protozoan parasites despite their widely recognized importance in a broad range of cellular functions in eukaryotes. We have previously reported that Ufm1 has distinct protein targets and cellular localization in the human parasite Leishmania donovani and deletion of Ufm1 in L. donovani adversely impacts the pathogenesis suggesting that Ufm1 associated enzymes could be exploited as drug targets. Using sensitive FRET based activity probes we identified the Ufm1 processing peptidase Ufsp in L. donovani. In addition, we show that deletion of Ufsp specifically reduces the survival of amastigotes, the parasite stage that is present in the humans thus altering the pathogenesis. Studies showing inhibition of Ufsp activity by anti-leishmanial drug further suggests that Leishmania Ufsp can serve as a novel target for pharmacological intervention for this parasite that causes deadly disease.

Background Gene expression analysis in Leishmania donovani (Ld) identified an orthologue of the urea cycle enzyme, argininosuccinate synthase (LdASS), that was more abundantly expressed in amastigotes than in promastigotes. In order to characterize in detail this newly identified protein in Leishmania, we determined its enzymatic activity, subcellular localization in the parasite and affect on virulence in vivo. Methodology/Principal Findings Two parasite cell lines either over expressing wild type LdASS or a mutant form (G128S) associated with severe cases of citrullinemia in humans were developed. In addition we also produced bacterially expressed recombinant forms of the same proteins. Our results demonstrated that LdASS has argininosuccinate synthase enzymatic activity that is abolished using an ASS specific inhibitor (MDLA: methyl-D-L-Aspartic acid). However, the mutant form of the protein is inactive. We demonstrate that though LdASS has a glycosomal targeting signal that binds the targeting apparatus in vitro, only a small proportion of the total cellular ASS is localized in a vesicle, as indicated by protection from protease digestion of the crude organelle fraction. The majority of LdASS was found to be in the cytosolic fraction that may include large cytosolic complexes as indicated by the punctate distribution in IFA. Surprisingly, comparison to known glycosomal proteins by IFA revealed that LdASS was located in a structure different from the known glycosomal vesicles. Significantly, parasites expressing a mutant form of LdASS associated with a loss of in vitro activity had reduced virulence in vivo in BALB/c mice as demonstrated by a significant reduction in the parasite load in spleen and liver. Conclusion/Significance Our study suggests that LdASS is an active enzyme, with unique localization and essential for parasite survival and growth in the mammalian host. Based on these observations LdASS could be further explored as a potential drug target., Author Summary Leishmaniasis is a neglected tropical disease that continues to pose a public health threat worldwide due to the absence of an effective vaccine, drug toxicity and parasite resistance. In an attempt to identify new potential drug targets, we focused our research on Leishmania donovani argininosuccinate synthase (LdASS), which is more highly expressed in the virulent form of the parasite. Using two cell lines that over expressed the wild type or a mutant form of LdASS, we demonstrated that LdASS has argininosuccinate synthase activity, which is absent in the mutant form containing the G128S point mutation. Infection of mice with the cell line over expressing a mutant LdASS had a negative dominant effect as indicated by the reduction in parasite load. LdASS is localized to large cytosolic complexes and a small portion is in a new vesicular subset different from the known glycosomes. Thus LdASS constitutes a new virulence factor that may be a potential drug target.