SummaryLeishmania major aquaglyceroporin (LmjAQP1) adventitiously facilitates the uptake of antimonite [Sb(III)], an active form of Pentostam® or Glucantime®, which are the first line of defence against all forms of leishmaniasis. The present paper shows that LmjAQP1 activity is modulated by the mitogenactivated protein kinase, LmjMPK2. Leishmania parasites coexpressing LmjAQP1 and LmjMPK2 show increased Sb(III) uptake and increased Sb(III) sensitivity. When subjected to a hypo-osmotic stress, these cells show faster volume recovery than cells expressing LmjAQP1 alone. LmjAQP1 is phosphorylated in vivo at Thr-197 and this phosphorylation requires LmjMPK2 activity. Lys-42 of LmjMPK2 is critical for its kinase activity. Cells expressing altered T197A LmjAQP1 or K42A LmjMPK2 showed decreased Sb(III) influx and a slower volume recovery than cells expressing wild-type proteins. Phosphorylation of LmjAQP1 led to a decrease in its turnover rate affecting LmjAQP1 activity. Although LmjAQP1 is localized to the flagellum of promastigotes, upon phosphorylation, it is relocalized to the entire surface of the parasite. Leishmania mexicana promastigotes with an MPK2 deletion showed reduced Sb(III) uptake and slower volume recovery than wild-type cells. This is the first report where a parasite aquaglyceroporin activity is post-translationally modulated by a mitogen-activated protein kinase.
SIN3, a transcriptional corepressor has been implicated in varied functions both as transcription activator and repressor. Recent studies associated Sin3 with the macroautophagic/autophagic process as a negative regulator of Atg8 and Atg32. Though the role of SIN3 in autophagy is being explored, little is known about the overall effect of SIN3 deletion on the survival of an organism. In this study using a Caenorhabditis elegans sin-3(tm1279);him-5(e1490) strain, we demonstrate that under in vivo conditions SIN-3 differentially modulates autophagy and lifespan. We provide evidence that the enhanced autophagy and decreased lifespan observed in sin-3 deletion mutants is dependent on ROS and intracellular oxidative stress. Inability of the mutant worms to maintain redox balance along with dysregulation of enzymatic antioxidants, depletion of GSH and NADP reserves and elevation of ROS markers compromises the longevity of the worms. It is possible that the enhanced autophagic process observed in sin-3 (tm1279);him-5(e1490) worms is required to compensate for oxidative stress generated in these worms.
Increasing drug resistance towards first line antimony-derived compounds has forced the introduction of novel therapies in leishmaniasis endemic areas including amphotericin B and miltefosine. However, their use is threatened by the emergence and spread of drug-resistant strains. In order to discover stage-dependent resistance genes, we have adapted the Cos-Seq approach through the introduction of macrophage infections in the pipeline. A L. infantum intracellular amastigote population complemented with a L. infantum cosmid library was submitted to increasing concentrations of miltefosine, amphotericin B and pentavalent antimonials in experimental infections of THP-1 cells. For each step of selection, amastigotes were extracted and cosmids were isolated and submitted to next-generation sequencing, followed by subsequent gene-enrichment analyses. Cos-Seq screen in amastigotes revealed four highly enriched loci for antimony, five for miltefosine and one for amphotericin B. Of these, a total of seven cosmids were recovered and tested for resistance in both promastigotes and amastigotes. Candidate genes within the pinpointed genomic regions were validated using single gene overexpression in wild-type parasites and/or gene disruption by means of a CRISPR-Cas9-based approach. This led to the identification and validation of a stage-independent antimony-resistance gene (LinJ.06.1010) coding for a putative leucine rich repeat protein and a novel amastigote-specific miltefosine-resistance gene (LinJ.32.0050) coding for a member of the SEC13 family of WD-repeat proteins. This study further reinforces the power of Cos-Seq approach to discover novel drug-resistance genes, some of which are life-stages specific.
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