Sreenivas Gannavaram

Characterization of metacaspases with trypsin-like activity and their putative role in programmed cell death in the protozoan parasite Leishmania.

ABSTRACT In this report, we have characterized two metacaspases of Leishmania donovani, L. donovani metacaspase-1 (LdMC1) and LdMC2. These two proteins show 98% homology with each other, and both contain a characteristic C-terminal proline-rich domain. Both genes are transcribed in promastigotes and axenic amastigotes of L. donovani; however, LdMC1 shows increased mRNA levels in axenic amastigotes. An anti-LdMC antibody was obtained and showed reactivity with a single ∼42-kDa protein band in both promastigote and axenic amastigote parasite whole-cell lysates by Western blotting. Pulse-chase experiments suggest that LdMCs are not synthesized as proenzymes, and immunofluorescence studies show that LdMCs are associated with the acidocalcisome compartments of L. donovani. Enzymatic assays of immunoprecipitated LdMCs show that native LdMCs efficiently cleave trypsin substrates and are unable to cleave caspase-specific substrates. Consistently, LdMC activity is insensitive to caspase inhibitors and is efficiently inhibited by trypsin inhibitors, such as leupeptin, antipain, and Nα-tosyl-l-lysine-chloromethyl ketone (TLCK). In addition, our results show that LdMC activity was induced in parasites treated with hydrogen peroxide, a known trigger of programmed cell death (PCD) in Leishmania and that parasites overexpressing metacaspases are more sensitive to hydrogen peroxide-induced PCD. These findings suggest that Leishmania metacaspases are not responsible for the caspase-like activities reported in this organism and suggest a possible role for LdMCs as effector molecules in Leishmania PCD.

Conservation of the pro-apoptotic nuclease activity of endonuclease G in unicellular trypanosomatid parasites.

Endonuclease G is a mitochondrial protein implicated in DNA fragmentation during apoptosis in cell types ranging from fungi to mammals. Features of programmed cell death have been reported in a number of single-celled organisms, including the human trypanosomatid parasites Leishmania and Trypanosoma. However, the protozoan cell death pathways and the effector molecules involved in such processes remain to be identified. In this report, we describe the pro-apoptotic function of endonuclease G in trypanosomatid parasites. Similar to metazoans, trypanosome endoG showed intrinsic nuclease activity, is localized in mitochondria and is released from this organelle when cell death is triggered. Overexpression of endoG strongly promoted apoptotic cell death under oxidant or differentiation-related stress in Leishmania and, conversely, loss of endoG expression conferred robust resistance to oxidant-induced cell death in T. brucei. These data demonstrate the conservation of the pro-apoptotic endonuclease activity of endoG in these evolutionarily ancient eukaryotic organisms. Furthermore, nuclear DNA degradation by endoG upon release from mitochondria might represent a caspase-independent cell death mechanism in trypanosomatid parasites as genes encoding caspase-like proteins have not been identified in their genomes.

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.

Vaccination using live attenuated Leishmania donovani centrin deleted parasites induces protection in dogs against Leishmania infantum.

Live attenuated Leishmania donovani parasites such as LdCen(-/-) have been shown elicit protective immunity against leishmanial infection in mice and hamster models. Previously, we have reported on the induction of strong immunogenicity in dogs upon vaccination with LdCen(-/-) including an increase in immunoglobulin isotypes, higher lymphoproliferative response, higher frequencies of activated CD4(+) and CD8(+) T cells, IFN-γ production by CD8(+) T cells, increased secretion of TNF-α and IL-12/IL-23p40 and, finally, decreased secretion of IL-4. To further explore the potential of LdCen(-/-) parasites as vaccine candidates, we performed a 24-month follow up of LdCen(-/-) immunized dogs after challenge with virulent Leishmania infantum, aiming determination of parasite burden by qPCR, antibody production (ELISA) and cellular responses (T cell activation and cytokine production) by flow cytometry and sandwich ELISA. Our data demonstrated that vaccination with a single dose of LdCen(-/-) (without any adjuvant) resulted in the reduction of up to 87.3% of parasite burden after 18 months of virulent challenge. These results are comparable to those obtained with commercially available vaccine in Brazil (Leishmune(®)). The protection was associated with antibody production and CD4(+) and CD8(+) proliferative responses, as well as T cell activation and significantly higher production of IFN-γ, IL-12/IL-23p40 and TNF-α, which was comparable to responses induced by immunization with Leishmune(®), with significant differences when compared to control animals (Placebo). Moreover, only animals immunized with LdCen(-/-) expressed lower levels of IL-4 when compared to animals vaccinated either with Leishmune(®) or PBS. Our results support further studies aiming to demonstrate the potential of genetically modified live attenuated L. donovani vaccine to control L. infantum transmission in endemic areas for CVL.

Biomarkers of Safety and Immune Protection for Genetically Modified Live Attenuated Leishmania Vaccines Against Visceral Leishmaniasis – Discovery and Implications

Despite intense efforts there is no safe and efficacious vaccine against visceral leishmaniasis, which is fatal and endemic in many tropical countries. A major shortcoming in the vaccine development against blood-borne parasitic agents such as Leishmania is the inadequate predictive power of the early immune responses mounted in the host against the experimental vaccines. Often immune correlates derived from in-bred animal models do not yield immune markers of protection that can be readily extrapolated to humans. The limited efficacy of vaccines based on DNA, subunit, heat killed parasites has led to the realization that acquisition of durable immunity against the protozoan parasites requires a controlled infection with a live attenuated organism. Recent success of irradiated malaria parasites as a vaccine candidate further strengthens this approach to vaccination. We developed several gene deletion mutants in Leishmania donovani as potential live attenuated vaccines and reported extensively on the immunogenicity of LdCentrin1 deleted mutant in mice, hamsters, and dogs. Additional limited studies using genetically modified live attenuated Leishmania parasites as vaccine candidates have been reported. However, for the live attenuated parasite vaccines, the primary barrier against widespread use remains the absence of clear biomarkers associated with protection and safety. Recent studies in evaluation of vaccines, e.g., influenza and yellow fever vaccines, using systems biology tools demonstrated the power of such strategies in understanding the immunological mechanisms that underpin a protective phenotype. Applying similar tools in isolated human tissues such as PBMCs from healthy individuals infected with live attenuated parasites such as LdCen−/− in vitro followed by human microarray hybridization experiments will enable us to understand how early vaccine-induced gene expression profiles and the associated immune responses are coordinately regulated in normal individuals. In addition, comparative analysis of biomarkers in PBMCs from asymptomatic or healed visceral leishmaniasis individuals in response to vaccine candidates including live attenuated parasites may provide clues about determinants of protective immunity and be helpful in shaping the final Leishmania vaccine formulation in the clinical trials.

Induction of immunogenicity by live attenuated Leishmania donovani centrin deleted parasites in dogs

Zoonotic visceral leishmaniasis, caused by the intracellular protozoan parasite Leishmania infantum, is a neglected tropical disease that is often fatal when untreated. Dogs are considered the main reservoir of L. infantum in zoonotic VL as the presence of infected dogs may increase the risk for human infection. Canine visceral leishmaniasis (CVL) is a major veterinary and public health problem in Southern Europe, Middle East and South America. Control of animal reservoirs relies on elimination of seropositive dogs in endemic areas. However, treatment of infected dogs is not considered a favorable approach as this can lead to emergence of drug resistance since the same drugs are used to treat human infections. Therefore, vaccination against CVL remains the best alternative in control of the animal reservoirs. In this study, we present data on the immunogenicity profile of a live attenuated parasite LdCen(-/-) in a canine infection model and compared it to that of Leishmune(®), a commercially available recombinant vaccine. The immunogenicity of the LdCen(-/-) parasites was evaluated by antibody secretion, production of intracytoplasmic and secreted cytokines, activation and proliferation of T cells. Vaccination with LdCen(-/-) resulted in high immunogenicity as revealed by the higher IgGTotal, IgG1, and IgG2 production and higher lymphoproliferative response. Further, LdCen(-/-) vaccinated dogs showed higher frequencies of activated CD4+ and CD8+ T cells, IFN-γ production by CD8+ T cells, increased secretion of TNF-α and IL-12/IL-23p40 and decreased secretion of IL-4. These results contribute to the understanding of immunogenicity elicited by live attenuated L. donovani parasites and, consequently, to the development of effective vaccines against visceral leishmaniasis.

Deletion of mitochondrial associated ubiquitin fold modifier protein Ufm1 in Leishmania donovani results in loss of β-oxidation of fatty acids and blocks cell division in the amastigote stage.

Recently, we described the existence of the ubiquitin fold modifier 1 (Ufm1) and its conjugation pathway in Leishmania donovani. We demonstrated the conjugation of Ufm1 to proteins such as mitochondrial trifunctional protein (MTP) that catalyses β‐oxidation of fatty acids in L. donovani. To elucidate the biological roles of the Ufm1‐mediated modifications, we made an L. donovani Ufm1 null mutant (Ufm1−/−). Loss of Ufm1 and consequently absence of Ufm1 conjugation with MTP resulted in diminished acetyl‐CoA, the end‐product of the β‐oxidation in the Ufm1−/− amastigote stage. The Ufm1−/− mutants showed reduced survival in the amastigote stage in vitro and ex vivo in human macrophages. This survival was restored by re‐expression of wild‐type Ufm1 with concomitant induction of acetyl‐CoA but not by re‐expressing the non‐conjugatable Ufm1, indicating the essential nature of Ufm1 conjugation and β‐oxidation. Both cell cycle analysis and ultrastructural studies of Ufm1−/− parasites confirmed the role of Ufm1 in amastigote growth. The defect in vitro growth of amastigotes in human macrophages was further substantiated by reduced survival. Therefore, these studies suggest the importance of Ufm1 in Leishmania pathogenesis with larger impact on other organisms and further provide an opportunity to test Ufm1−/− parasites as drug and vaccine targets.

Intradermal Immunization of Leishmania donovani Centrin Knock-Out Parasites in Combination with Salivary Protein LJM19 from Sand Fly Vector Induces a Durable Protective Immune Response in Hamsters

Background Visceral leishmaniasis (VL) is a neglected tropical disease and is fatal if untreated. There is no vaccine available against leishmaniasis. The majority of patients with cutaneous leishmaniasis (CL) or VL develop a long-term protective immunity after cure from infection, which indicates that development of an effective vaccine against leishmaniasis is possible. Such protection may also be achieved by immunization with live attenuated parasites that do not cause disease. We have previously reported a protective response in mice, hamsters and dogs with Leishmania donovani centrin gene knock-out parasites (LdCen-/-), a live attenuated parasite with a cell division specific centrin1 gene deletion. In this study we have explored the effects of salivary protein LJM19 as an adjuvant and intradermal (ID) route of immunization on the efficacy of LdCen-/- parasites as a vaccine against virulent L. donovani. Methodology/Principal Findings To explore the potential of a combination of LdCen-/- parasites and salivary protein LJM19 as vaccine antigens, LdCen-/- ID immunization followed by ID challenge with virulent L. donovani were performed in hamsters in a 9-month follow up study. We determined parasite burden (serial dilution), antibody production (ELISA) and cytokine expression (qPCR) in these animals. Compared to controls, animals immunized with LdCen-/- + LJM19 induced a strong antibody response, a reduction in spleen and liver parasite burden and a higher expression of pro-inflammatory cytokines after immunization and one month post-challenge. Additionally, a low parasite load in lymph nodes, spleen and liver, and a non-inflamed spleen was observed in immunized animals 9 months after the challenge infection. Conclusions Our results demonstrate that an ID vaccination using LdCen-/-parasites in combination with sand fly salivary protein LJM19 has the capability to confer long lasting protection against visceral leishmaniasis that is comparable to intravenous or intracardial immunization.

Mitochondrial Associated Ubiquitin Fold Modifier-1 Mediated Protein Conjugation in Leishmania donovani

In this report, we demonstrate the existence of the ubiquitin fold modifier-1 (Ufm1) and its conjugation pathway in trypanosomatid parasite Leishmania donovani. LdUfm1 is activated by E1-like enzyme LdUba5. LdUfc1 (E2) specifically interacted with LdUfm1 and LdUba5 to conjugate LdUfm1 to proteinaceous targets. Mass spectrometry analysis revealed that LdUfm1 is conjugated to Leishmania protein targets that are associated with mitochondria. Immunofluorescence experiments showed that Leishmania Ufm1, Uba5 and Ufc1 are associated with the mitochondria. The demonstration that all the components of this system as well as the substrates are associated with mitochondrion suggests it may have physiological roles not yet described in any other organism. Overexpression of a non-conjugatable form of LdUfm1 and an active site mutant of LdUba5 resulted in reduced survival of Leishmania in the macrophage. Since mitochondrial activities are developmentally regulated in the life cycle of trypanosomatids, Ufm1 mediated modifications of mitochondrial proteins may be important in such regulation. Thus, Ufm1 conjugation pathway in Leishmania could be explored as a potential drug target in the control of Leishmaniasis.

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.