Parool Gupta

Conference 2023 Live Talk

Talk title

L. donovani Cfd1 and Nbp35 proteins make stable cytosolic complex and contribute to the parasite’s survival and Amphotericin B resistance

Authors and Affiliations

Parool Gupta1,2, Sachidananda Behera1, Vahab Ali1

1. Department of Biochemistry, ICMR-Rajendra Memorial Research Institute of Medical Sciences, Patna, India 2. Department of Biotechnology, University of Calcutta, Kolkata, India

Abstract

Background

Leishmania donovani is a unicellular protozoan parasite that causes visceral leishmaniasis (VL), a neglected fatal tropical disease posing threat to public health. Iron-Sulfur (Fe-S) proteins are very essential for survival of Leishmania and involved in many biological functions; such as, electron transport, regulation of gene expression, enzymatic activities, etc. Biosynthesis and transfer of Fe-S clusters depend on ISC, CIA, SUF, NIF systems in various organisms. L. donovani possess ISC & CIA machineries for Fe-S cluster assembly. The ISC system acts as house-keeping machinery which generally involved in mitochondrial Fe-S cluster biogenesis and assist CIA machinery for maturation of both cytosolic and nuclear proteins, involved in DNA replication and repair, ribosome biogenesis and iron regulation, etc.

Methods

In this study, two main components of CIA machinery, Cfd1 and Nbp35 proteins, were purified using affinity column chromatography and their localization was demonstrated by using digitonin fractionation and confocal microscopy in the cytoplasm. Besides, we have used assays like co-purification, pull down, immunoprecipitation along with in-silico studies to confirm the interaction between these two proteins. Further we have applied qPCR and WB to check the expression of these proteins at transcriptional and translational level. We have performed transfection to generate over-expressior (OE) parasites and carried out drug sensitivity assay, enzymatic activities to compare Wild type (WT) vs. OE parasites.

Results

We have successfully purified these two proteins and confirmed interaction by in-silico analysis, co-purification, and pull down assays as well as immune-precipitation. Further, expression of Cfd1 and Nbp35 genes was analysed by semi-qPCR and Real Time-PCR, which showed higher expression of these genes in Amphotericin B resistant clinical isolates as compared to sensitive. We also confirmed the higher Cfd1 and Nbp35 proteins expression in the resistant isolates by western blotting suggesting their role in drug resistance. We have also generated Cfd1 and Nbp35 overexpressor strains of L. donovani; and IC50 value for Amp-B was found to be higher for overexpressor than WT sensitive strain. We also found that enzymatic activites are upregulated in the OE strains than the WT parasites.

Conclusions

We reported the CIA machinery component proteins (Cfd1 and Nbp35) in L. donovani parasites for the first time. We have demonstrated the existing interaction and formation of stable complex by these two proteins to facilitate Fe-S cluster biogenesis and helps in parasites growth. Our results cumulatively delineate the roles of Cfd1 and Nbp35 proteins in survival and Amp B resistance of L. donovani.