The γ‐glutamylcysteine synthetase gene of Leishmania is essential and involved in response to oxidants

Abstract

SummaryGamma-glutamylcysteine synthetase, encoded by the GSH1 gene, is the rate-limiting enzyme in the biosynthesis of glutathione and of trypanothione in Leishmania. The importance of GSH1 was assessed by generating GSH1 null mutants in Leishmania infantum. Removal of even a single wild-type allelic copy of GSH1 invariably led to the generation of an extra copy of GSH1, maintaining two intact wild-type alleles. However, by first supplementing the parasites with a rescue plasmid, we succeeded in obtaining both a single and null chromosomal GSH1 mutants. Parasites with one intact GSH1 chromosomal allele lost the rescuing plasmid but not the double knockout, when grown in the absence of antibiotic, indicating the essentiality of the GSH1 gene in Leishmania. Heterozygous mutants with one allele-inactivated transcribed less GSH1 mRNA and synthesized less glutathione and trypanothione. These mutants were more susceptible to oxidative stresses in vitro as promastigotes and showed decreased survival inside activated macrophages producing reactive oxygen or nitrogen species. These mutants showed a significant decreased survival in the presence of antimony (SbV) compared with control cells. All phenotypes were reverted in the add-back mutant, thus proving the importance of thiols in dealing with oxidants including the action of antimonials.

Glutathione biosynthesis in the yeast pathogens Candida glabrata and Candida albicans: essential in C. glabrata, and essential for virulence in C. albicans

“…The importance of oxidative stress responses in the virulence and survival of pathogens in their natural environment has been suggested by many studies, but the relative importance of the glutathione-dependent redox pathway, as opposed to other pathways for redox homeostasis and oxidative stress, has not been rigorously evaluated. In parasitic protozoans such as Leishmania infantum and Trypansoma brucei, however, glutathione biosynthesis (which leads to trypanothione synthesis), is essential (Huynh et al, 2003;Mukherjee et al, 2009), while in the malarial parasite Plasmodium berghei, glutathione biosynthesis has been shown to be essential for the survival of the protozoan during its passage through insects (VegaRodríguez et al, 2009). Further, in L. infantum, even the deletion of one of the two copies of the GSH1 gene is not tolerated by the organism.…”

Interplay between a cytosolic and a cell surface carbonic anhydrase in pH homeostasis and acid tolerance of Leishmania

“…Macrophage infection was performed as described previously, with minor modifications (Pal et al, 2015;Mukherjee et al, 2009). Briefly, J774A.1 macrophages were activated with 100 ng/ml lipopolysaccharide to enhance their chemotactic and phagocytic abilities (Wu et al, 2009).…”

Using Genomic Information to Understand Leishmania Biology

“…Targeted gene disruption is achievable in Leishmania by homologous recombination of linear DNA and it is a powerful and effective tool for genetic functional studies in the parasite [82]. Since gene replacement and double gene knockout strategies have been established in Leishmania [74,83], several essential and non-essential genes have been knocked-out and phenotypic changes have been analyzed [84][85][86][87][88]. Some of these studies confirmed the complexity of issues such as virulence, infectivity or pathogenesis [33][34][35][36].…”