A Leishmania-specific gene upregulated at the amastigote stage is crucial for parasite survival.

The morphological and biochemical alterations between the two life stages of Leishmania are governed by stage-regulated expression of several genes. Amastigote-specific genes are believed to have a role in the survival and replication of the parasite in the hostile environment of the mammalian host. Previously, we have reported the upregulated expression of A1 gene (LdA1) at the amastigote stage at RNA level. In the present study, we have further characterized LdA1, in order to understand its role in Leishmania. Sequence homology search revealed that LdA1 is unique to the Leishmania genus. Sequence- and structure-level functional annotations predicted the involvement of LdA1 in a range of biological processes critical for survival of the parasites. Western blot confirmed the upregulated expression of LdA1 at the protein level at the amastigote stage. Overexpression of LdA1 in Leishmania did not affect its growth, phenotype, or infectivity. Attempts to generate null mutants of LdA1 by homologous recombination were not successful. Repeated inability to create null mutants of LdA1 was suggestive of gene essentiality. Mutant parasites with a single allele deletion of LdA1 (LdA1+/-) showed reduction in motility, size, and growth rate at both the life stages in vitro, which was restored following gene add-back by episomal expression of LdA1 in mutant parasites. Although LdA1+/- parasites were able to infect macrophages ex vivo, their capacity to survive inside macrophages was reduced significantly (P < 0.01) beyond 72 h of infection. In conclusion, LdA1 is a Leishmania-specific gene having upregulated expression at the amastigote stage and is important for survival of Leishmania parasite.

Genetic diversity of Leishmania donovani that causes cutaneous leishmaniasis in Sri Lanka: a cross sectional study with regional comparisons.

BACKGROUND: Leishmania donovani is the etiological agent of visceral leishmaniasis (VL) in the Indian subcontinent. However, it is also known to cause cutaneous leishmaniasis (CL) in Sri Lanka. Sri Lankan L. donovani differs from other L. donovani strains, both at the molecular and biochemical level. To investigate the different species or strain-specific differences of L. donovani in Sri Lanka we evaluated sequence variation of the kinetoplastid DNA (kDNA). METHODS: Parasites isolated from skin lesions of 34 CL patients and bone marrow aspirates from 4 VL patients were genotyped using the kDNA minicircle PCR analysis. A total of 301 minicircle sequences that included sequences from Sri Lanka, India, Nepal and six reference species of Leishmania were analyzed. RESULTS: Haplotype diversity of Sri Lankan isolates were high (H d = 0.757) with strong inter-geographical genetic differentiation (F ST > 0.25). In this study, L. donovani isolates clustered according to their geographic origin, while Sri Lankan isolates formed a separate cluster and were clearly distinct from other Leishmania species. Within the Sri Lankan group, there were three distinct sub-clusters formed, from CL patients who responded to standard antimony therapy, CL patients who responded poorly to antimony therapy and from VL patients. There was no specific clustering of sequences based on geographical origin within Sri Lanka. CONCLUSION: This study reveals high levels of haplotype diversity of L. donovani in Sri Lanka with a distinct genetic association with clinically relevant phenotypic characteristics. The use of genetic tools to identify clinically relevant features of Leishmania parasites has important therapeutic implications for leishmaniasis.

Role of Mast Cells in clearance of Leishmania through extracellular trap formation.

Mast Cells (MCs) are one of the first immune cells encountered by invading pathogens. Their presence in large numbers in the superficial dermis, where Leishmania is encountered, suggests that they may play a critical role in immune responses to Leishmania. In this study the interactions of Leishmania donovani, the causative agent of visceral Leishmaniasis, and Leishmania tropica, the causative agent of cutaneous Leishmaniasis with MCs were studied. Co-culture of Leishmania with Peritoneal Mast Cells (PMCs) from BALB/c mice and Rat Basophilic Leukaemia (RBL-2H3) MCs led to significant killing of L. tropica and to a lesser extent of L. donovani. Also, while there was significant uptake of L. tropica by MCs, L. donovani was not phagocytosed. There was significant generation of Reactive Oxygen Species (ROS) by MCs on co-culture with these species of Leishmania which may contribute to their clearance. Interactions of MCs with Leishmania led to generation of MC extracellular traps comprising of DNA, histones and tryptase probably to ensnare these pathogens. These results clearly establish that MCs may contribute to host defences to Leishmania in a differential manner, by actively taking up these pathogens, and also by mounting effector responses for their clearance by extracellular means.