Identification and Characterization of Genes Involved in Leishmania Pathogenesis: The Potential for Drug Target Selection

Identifying and characterizing Leishmania donovani genes and the proteins they encode for their role in pathogenesis can reveal the value of this approach for finding new drug targets. Effective drug targets are likely to be proteins differentially expressed or required in the amastigote life cycle stage found in the patient. Several examples and their potential for chemotherapeutic disruption are presented. A pathway nearly ubiquitous in living cells targeted by anticancer drugs, the ubiquitin system, is examined. New findings in ubiquitin and ubiquitin-like modifiers in Leishmania show how disruption of those pathways could point to additional drug targets. The programmed cell death pathway, now recognized among protozoan parasites, is reviewed for some of its components and evidence that suggests they could be targeted for antiparasitic drug therapy. Finally, the endoplasmic reticulum quality control system is involved in secretion of many virulence factors. How disruptions in this pathway reduce virulence as evidence for potential drug targets is presented

Immunity to Visceral Leishmaniasis Using Genetically Defined Live-Attenuated Parasites

Leishmaniasis is a protozoan parasitic disease endemic to the tropical and subtropical regions of the world, with three major clinical forms, self-healing cutaneous leishmaniasis (CL), mucocutaneous leishmaniasis (MCL), and visceral leishmaniasis (VL). Drug treatments are expensive and often result in the development of drug resistance. No vaccine is available against leishmaniasis. Subunit Leishmania vaccine immunization in animal models has shown some efficacy but little or none in humans. However, individuals who recover from natural infection are protected from reinfection and develop life-long protection, suggesting that infection may be a prerequisite for immunological memory. Thus, genetically altered live-attenuated parasites with controlled infectivity could achieve such memory. In this paper, we discuss development and characteristics of genetically altered, live-attenuated Leishmania donovani parasites and their possible use as vaccine candidates against VL. In addition, we discuss the challenges and other considerations in the use of live-attenuated parasites

Deletion of ubiquitin fold modifier protein Ufm1 processing peptidase Ufsp in L. donovani abolishes Ufm1 processing and alters pathogenesis.

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

The Immunology of a Healing Response in Cutaneous Leishmaniasis Treated with Localized Heat or Systemic Antimonial Therapy.

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

Demographic characteristics and outcome presented by treatment arm.

<p>* Mann-Whitney</p><p>** Fisher exact</p><p>***Vassarstats</p><p>Demographic characteristics and outcome presented by treatment arm.</p

Characterization of lymphocyte populations by flow cytometry.

<p>Data is presented from 30 subjects (17 in the SSG arm and 13 in TM arm represented in circles and triangles respectively) for which cells from all three time points were available. <b>(A)</b> Percentage of lymphocytes positive for surface expression of CD3 (T cells), CD19 (B cells), CD16/CD56 (CD3^-: NK cells; CD3⁺: NKT-like cells). <b>(B)</b> Distribution of subpopulations of NK cells based on CD16 and CD56 expression. <b>(C)</b> T cell phenotype stratified by treatment arm. <b>(D)</b> Distribution of T cells into CD4⁺, CD8⁺ and CD4⁻CD8⁻ (double negative, DN) populations pre- and post-treatment. (<b>E-F</b>) Identification of TCR expression within the T cell populations. Aggregate data from 12 study subjects compared to data from 7 healthy controls for <b>(E)</b> αβ and (<b>F</b>) γδ respectively. Bars represent medians. <i>P</i> values were derived using the Wilcoxon matched pairs test.</p

Identification of activated and regulatory T cell populations by flow cytometry.

<p><b>(A)</b> Freshly isolated cells were stained for CD3, CD4, CD8 and CD25 for identification of activated T cells. Data obtained from 31 subjects (circles for SSG subjects and triangles for TM subjects) for which pre-treatment (black) and post-treatment (grey) cells were available. Bars represent medians. <b>(B)</b> Identification of Treg cells from thawed PBMC. Aggregate data from 20 donors for CD4⁺CD25^high Foxp3⁺. P values derived using the Wilcoxon matched pairs test.</p

Identification of responding populations by CFDA-SE labeling and flow cytometry analysis.

<p>Identification of proliferating lymphocytes based on expression of (<b>A</b>) CD4 and CD8 or (<b>B</b>) CD25. Circles represent SSG subjects and triangles represent TM subjects. Black and grey are for PRE and POST respectively. Bars represent medians.</p