Pyridoxal kinase gene deletion leads to impaired growth, deranged redox metabolism and cell cycle arrest in Leishmania donovani.

Pyridoxal kinase (PdxK) is a vitamin B6 salvage pathway enzyme which produces pyridoxal phosphate. We have investigated the impact of PdxK deletion in Leishmania donovani on parasite survivability, infectivity and cellular metabolism. LdPdxK mutants were generated by gene replacement strategy. All mutants showed significant reduction in growth in comparison to wild type. For PdxK mediated biochemical perturbations, only heterozygous mutants and complementation mutants were used as the growth of null mutants were compromised. Heterozygous mutant showed reduction invitro infectivity and higher cytosolic and mitochondrial ROS levels. Glutathione levels decreased significantly in heterozygous mutant indicating its involvement in cellular oxidative metabolism. Pyridoxal kinase gene deletion resulted in reduced ATP levels in parasites and arrest at G0/G1 phase of cell cycle. All these perturbations were rescued by PdxK gene complementation. This is the first report to confirm that LdPdxK plays an indispensable role in cell survival, pathogenicity, redox metabolism and cell cycle progression of L. donovani parasites. These results provide substantial evidence supporting PdxK as a therapeutic target for the development of specific antileishmanial drug candidates.

Clotrimazole causes membrane depolarization and induces sub G0 cell cycle arrest in Leishmania donovani.

Clotrimazole is an FDA approved drug and is widely used as an antifungal agent. An extensive body of research is available about its mechanism of action on various cell types but its mode of killing of Leishmania donovani parasites is unknown. L. donovani causes Visceral Leishmaniasis which is a public health problem with limited treatment options. Its present chemotherapy is expensive, has adverse effects and is plagued with drug resistance issues. In this study we have explored the possibility of repurposing clotrimazole as an antileishmanial drug. We have assessed its efficacy on the parasites and attempted to understand its mode of action. We found that it has a half-maximal inhibitory concentration (IC50) of 35.75 ± 1.06 µM, 12.75 ± 0.35 µM and 73 ± 1.41 µM in promastigotes, intracellular amastigotes and macrophages, respectively. Clotrimazole is 5.73 times more selective for the intracellular amastigotes as compared to the mammalian cell. Effect of clotrimazole was reduced by ergosterol supplementation. It leads to impaired parasite morphology. It alters plasma membrane permeability and disrupts plasma membrane potential. Mitochondrial function is compromised as is evident from increased ROS generation, depolarized mitochondrial membrane and decreased ATP levels. Cell cycle analysis of clotrimazole treated parasites shows arrest at sub-G0 phase suggesting apoptotic mode of cell death.

Leishmania donovani 6-phosphogluconolactonase: Crucial for growth and host infection?

The hexose monophosphate shunt is a crucial pathway in a variety of microorganisms owing to its vital metabolic products and intermediates such as NADPH, ribose 5-phosphate etc. The enzyme 6-phosphogluconolactonase catalyses the second step of this pathway, converting 6-phosphogluconolactone to 6-phosphogluconic acid. This enzyme has been known to have a significant involvement in growth, pathogenesis and sensitivity to oxidative stress in bacterial and protozoal pathogens. However, the functional role of kinetoplastid Leishmania donovani 6-phospohogluconolactonase (Ld6PGL) remains unexplored. L. donovani is the second largest parasitic killer and causative organism of life threatening visceral leishmaniasis. To understand its possible functional role in the parasite, the alleles of Ld6PGL were sequentially knocked-out followed by gene complementation. The Ld6PGL mutant cell lines showed decrease in transcriptional and translational expression as well as in the enzyme activity. In case of Ld6PGL null mutants, approximately 2-fold reduction was observed in growth. The null mutants also showed ∼38% decrease in infectivity, which recovered to ∼15% on complementation. Scanning electron microscopy showed a marked decrease in flagellar length in the knockout parasites. When treated with the standard drug miltefosine, the mutant strains had no significant change in the drug sensitivity. However, the Ld6PGL mutants were more susceptible to oxidative stress. Our findings suggest that 6PGL is required for parasite growth and infection, but it is not essential.

Molecular explorations of the Leishmania donovani 6-phosphogluconolactonase enzyme, a key player in the pentose phosphate pathway.

The enzymes of the pentose phosphate pathway are vital to survival in kinetoplastids. The second step of the pentose phosphate pathway involves hydrolytic cleavage of 6-phosphogluconolactone to 6-phosphogluconic acid by 6- phosphogluconolactonase (6PGL). In the present study, Leishmania donovani 6PGL (Ld6PGL) was cloned and overexpressed in bacterial expression system. Comparative sequence analysis revealed the conserved sequence motifs, functionally and structurally important residues in 6PGL family. In silico amino acid substitution study and interacting partners of 6PGL were predicted. The Ld6PGL enzyme was found to be active in the assay and in the parasites. Specificity was confirmed by Western blot analysis. The ∼30 kDa protein was found to be a dimer in MALDI, glutaraldehyde crosslinking and size exclusion chromatography studies. Kinetic analysis and structural stability studies of Ld6PGL were performed with denaturants and at varied temperature. Computational 3D Structural modelling of Ld6PGL elucidates that it has a similar α/ß hydrolase fold structural topology as in other members of 6PGL family. The three loops are found in extended form when the structure is compared with the human 6PGL (Hs6PGL). Further, enzyme substrate binding mode and its mechanism were investigated using the molecular docking and molecular simulation studies. Interesting dynamics action of substrate 6-phosphogluconolactone was observed into active site during MD simulation. Interesting differences were observed between host and parasite enzyme which pointed towards its potential to be explored as an antileishmanial drug target. This study forms the basis for further analysis of the role of Ld6PGL in combating oxidative stress in Leishmania.