RNA-Seq Revealed Expression of Many Novel Genes Associated With Leishmania donovani Persistence and Clearance in the Host Macrophage.

Host- as well as parasite-specific factors are equally crucial in allowing either the Leishmania parasites to dominate, or host macrophages to resist infection. To identify such factors, we infected murine peritoneal macrophages with either the virulent (vAG83) or the non-virulent (nvAG83) parasites of L. donovani. Then, through dual RNA-seq, we simultaneously elucidated the transcriptomic changes occurring both in the host and the parasites. Through Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the differentially expressed (DE) genes, we showed that the vAG83-infected macrophages exhibit biased anti-inflammatory responses compared to the macrophages infected with the nvAG83. Moreover, the vAG83-infected macrophages displayed suppression of many important cellular processes, including protein synthesis. Further, through protein-protein interaction study, we showed significant downregulation in the expression of many hubs and hub-bottleneck genes in macrophages infected with vAG83 as compared to nvAG83. Cell signaling study showed that these two parasites activated the MAPK and PI3K-AKT signaling pathways differentially in the host cells. Through gene ontology analyses of the parasite-specific genes, we discovered that the genes for virulent factors and parasite survival were significantly upregulated in the intracellular amastigotes of vAG83. In contrast, genes involved in the immune stimulations, and those involved in negative regulation of the cell cycle and transcriptional regulation, were upregulated in the nvAG83. Collectively, these results depicted a differential regulation in the host and the parasite-specific molecules during in vitro persistence and clearance of the parasites.

Apoptosis-like cell death in Leishmania donovani treated with KalsomeTM10, a new liposomal amphotericin B.

OBJECTIVE: The present study aimed to elucidate the cell death mechanism in Leishmania donovani upon treatment with KalsomeTM10, a new liposomal amphotericin B. METHODOLOGY/PRINCIPAL FINDINGS: We studied morphological alterations in promastigotes through phase contrast and scanning electron microscopy. Phosphatidylserine (PS) exposure, loss of mitochondrial membrane potential and disruption of mitochondrial integrity was determined by flow cytometry using annexinV-FITC, JC-1 and mitotraker, respectively. For analysing oxidative stress, generation of H2O2 (bioluminescence kit) and mitochondrial superoxide O2- (mitosox) were measured. DNA fragmentation was evaluated using terminal deoxyribonucleotidyl transferase mediated dUTP nick-end labelling (TUNEL) and DNA laddering assay. We found that KalsomeTM10 is more effective then Ambisome against the promastigote as well as intracellular amastigote forms. The mechanistic study showed that KalsomeTM10 induced several morphological alterations in promastigotes typical of apoptosis. KalsomeTM10 treatment showed a dose- and time-dependent exposure of PS in promastigotes. Further, study on mitochondrial pathway revealed loss of mitochondrial membrane potential as well as disruption in mitochondrial integrity with depletion of intracellular pool of ATP. KalsomeTM10 treated promastigotes showed increased ROS production, diminished GSH levels and increased caspase-like activity. DNA fragmentation and cell cycle arrest was observed in KalsomeTM10 treated promastigotes. Apoptotic DNA fragmentation was also observed in KalsomeTM10 treated intracellular amastigotes. KalsomeTM10 induced generation of ROS and nitric oxide leads to the killing of the intracellular parasites. Moreover, endocytosis is indispensable for KalsomeTM10 mediated anti-leishmanial effect in host macrophage. CONCLUSIONS: KalsomeTM10 induces apoptotic-like cell death in L. donovani parasites to exhibit its anti-leishmanial function.