Evaluation of Cysteine Protease C of Leishmania donovani in Comparison with Glycoprotein 63 and Elongation Factor 1α for Diagnosis of Human Visceral Leishmaniasis and for Posttreatment Follow-Up Response.

Visceral leishmaniasis (VL) is a threat in many developing countries. Much effort has been put to eliminating this disease, for which serodiagnosis remains the mainstay for VL control programs. New and improved antigens as diagnostic candidates are required, though, as the available antigens fail to demonstrate equal optimum performance in all areas of endemicity. Moreover, these diagnoses are dependent on invasive serum sampling. In the current study, we cloned and expressed Leishmania donovani cysteine protease C (CPC) and evaluated its diagnostic and test-of-cure possibilities by detecting the antibody levels in human serum and urine through ELISA and immunoblot assays. Two immunodominant antigens, recombinant glycoprotein 63 (GP63) and elongation factor 1α (EF1α), identified earlier by our group, were also assessed by employing human serum and urine samples. Of these three antigens in ELISAs, CPC demonstrated the highest sensitivities of 98.15% and 96% positive testing in serum and urine of VL patients, respectively. Moreover, CPC yielded 100% specificity with serum and urine of nonendemic healthy controls compared to GP63 and EF1α. Urine samples were found to be more specific than serum for distinguishing endemic healthy controls and other diseases by means of all three antigens. In all cases, CPC gave the most promising results. Unlike serum, urine tests demonstrated a significant decrease in antibody levels for CPC, GP63, and EF1α after 6 months of treatment. The diagnostic and test-of-cure performances of CPC in the immunoblot assay were found to be better than those of GP63 and EF1α. In conclusion, CPC, followed by GP63 and EF1α, may be utilized as candidates for diagnosis of VL and to assess treatment response.

Leishmania donovani Aurora kinase: A promising therapeutic target against visceral leishmaniasis.

BACKGROUND: Aurora kinases are key mitotic kinases executing multiple aspects of eukaryotic cell-division. The apicomplexan homologs being essential for survival, suggest that the Leishmania homolog, annotated LdAIRK, may be equally important. METHODS: Bioinformatics, stage-specific immunofluorescence microscopy, immunoblotting, RT-PCR, molecular docking, in-vitro kinase assay, anti-leishmanial activity assays, flow cytometry, fluorescence microscopy. RESULTS: Ldairk expression is seen to vary as the cell-cycle progresses from G1 through S and finally G2M and cytokinesis. Kinetic studies demonstrate their enzymatic activity exhibiting a Km and Vmax of 6.12µM and 82.9pmoles·min(-1)mg(-1) respectively against ATP using recombinant Leishmania donovani H3, its physiological substrate. Due to the failure of LdAIRK-/+ knock-out parasites to survive, we adopted a chemical knock-down approach. Based on the conservation of key active site residues, three mammalian Aurora kinase inhibitors were investigated to evaluate their potential as inhibitors of LdAIRK activity. Interestingly, the cell-cycle progressed unhindered, despite treatment with GSK-1070916 or Barasertib, inhibitors with greater potencies for the ATP-binding pocket compared to Hesperadin, which at nanomolar concentrations, severely compromised viability at IC50s 105.9 and 36.4nM for promastigotes and amastigotes, respectively. Cell-cycle and morphological studies implicated their role in both mitosis and cytokinesis. CONCLUSION: We identified an Aurora kinase homolog in L. donovani implicated in cell-cycle progression, whose inhibition led to aberrant changes in cell-cycle progression and reduced viability. GENERAL SIGNIFICANCE: Human homologs being actively pursued drug targets and the observations with LdAIRK in both promastigotes and amastigotes suggest their potential as therapeutic-targets. Importantly, our results encourage the exploration of other proteins identified herein as potential novel drug targets.

Cell Death in Leishmania donovani promastigotes in response to Mammalian Aurora Kinase B Inhibitor- Hesperadin.

Deciphering how hesperadin, a repurposed mammalian aurora kinase B inhibitor, affects the cellular pathways in Leishmania donovani might be beneficial. This investigation sought to assess the physiological effects of hesperadin on promastigotes of L. donovani, by altering the duration of treatment following exposure to hesperadin. Groups pre-treated with inhibitors such as EGTA, NAC, and z-VAD-fmk before hesperadin exposure were also included. Morphological changes by microscopy, ATP and ROS changes by luminometry; DNA degradation using agarose gel electrophoresis and metacaspase levels through RT-PCR were assessed. Flow cytometry was used to study mitochondrial depolarization using JC-1 and MitoTracker Red; mitochondrial-superoxide accumulation using MitoSOX; plasma membrane modifications using Annexin-V and propidium iodide, and lastly, caspase activation using ApoStat. Significant alterations in promastigote morphology were noted. Caspase activity and mitochondrial-superoxide rose early after exposure whereas mitochondrial membrane potential demonstrated uncharacteristic variations, with significant functional disturbances such as leakage of superoxide radicals after prolonged treatments. ATP depletion and ROS accumulation demonstrated inverse patterns, genomic DNA showed fragmentation and plasma membrane showed Annexin-V binding, soon followed by propidium iodide uptake. Multilobed macronuclei and micronuclei accumulated in hesperadin exposed cells before they disintegrated into necrotic debris. The pathologic alterations were unlike the intrinsic or extrinsic pathways of classical apoptosis and suggest a caspase-mediated cell death most akin to mitotic-catastrophe. Most likely, a G2/M transition block caused accumulation of death signals, disorganized spindles and mechanical stresses, causing changes in morphology, organellar functions and ultimately promastigote death. Thus, death was a consequence of mitotic-arrest followed by ablation of kinetoplast functions, often implicated in L. donovani killing.

Liposomal Elongation Factor-1α Triggers Effector CD4 and CD8 T Cells for Induction of Long-Lasting Protective Immunity against Visceral Leishmaniasis.

Despite advances, identification and formulation of safe and effective vaccine for long-lasting protection against leishmaniasis is still inadequate. In this study, we have identified a novel antigen, leishmanial elongation factor-1α (EF1-α), as an immunodominant component of solubilized leishmanial membrane antigens that reacts with visceral leishmaniasis (VL) sera and induces cellular proliferative and cytokine response in PBMCs of cured VL subjects. Leishmanial EF1-α is a 50 kDa antigen that plays a crucial role in pathogen survival by regulating oxidative burst in the host phagocytes. Previously, immunodominant truncated forms of EF1-α from different species of Leishmania have been reported. Formulation of the L. donovani 36 kDa truncated as well as the cloned recombinant EF1-α in cationic liposomes induce strong resistance to parasitic burden in liver and spleen of BALB/c mice through induction of DTH and a IL-10 and TGF-ß suppressed mixed Th1/Th2 cytokine responses. Multiparametric analysis of splenocytes for generation of antigen-specific IFN-γ, IL2, and TNF-α producing lymphocytes indicate that cationic liposome facilitates expansion of both CD4+ as well as CD8+ memory and effector T cells. Liposomal EF1-α is a novel and potent vaccine formulation against VL that imparts long-term protective responses. Moreover, the flexibility of this formulation opens up the scope to combine additional adjuvants and epitope selected antigens for use in other disease forms also.

Genetically modified organisms and visceral leishmaniasis.

Vaccination is the most effective method of preventing infectious diseases. Since the eradication of small pox in 1976, many other potentially life compromising if not threatening diseases have been dealt with subsequently. This event was a major leap not only in the scientific world already burdened with many diseases but also in the mindset of the common man who became more receptive to novel treatment options. Among the many protozoan diseases, the leishmaniases have emerged as one of the largest parasite killers of the world, second only to malaria. There are three types of leishmaniasis namely cutaneous (CL), mucocutaneous (ML), and visceral (VL), caused by a group of more than 20 species of Leishmania parasites. Visceral leishmaniasis, also known as kala-azar is the most severe form and almost fatal if untreated. Since the first attempts at leishmanization, we have killed parasite vaccines, subunit protein, or DNA vaccines, and now we have live recombinant carrier vaccines and live attenuated parasite vaccines under various stages of development. Although some research has shown promising results, many more potential genes need to be evaluated as live attenuated vaccine candidates. This mini-review attempts to summarize the success and failures of genetically modified organisms used in vaccination against some of major parasitic diseases for their application in leishmaniasis.