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Downregulation of mitogen-activated protein kinase 1 of Leishmania donovani field isolates is associated with antimony resistance.

ABSTRACT: Emergence of resistance to pentavalent antimonials has become a severe obstacle in the treatment of visceral leishmaniasis (VL) on the Indian subcontinent. The mechanisms operating in laboratory-generated strains are somewhat known, but the determinants of clinical antimony resistance are not well understood. By utilizing a DNA microarray expression profiling approach, we identified a gene encoding mitogen-activated protein kinase 1 (MAPK1) for the kinetoplast protozoan Leishmania donovani (LdMAPK1) that was consistently downregulated in antimony-resistant field isolates. The expression level of the gene was validated by real-time PCR. Furthermore, decreased expression of LdMAPK1 was also confirmed at the protein level in resistant isolates. Primary structure analysis of LdMAPK1 revealed the presence of all of the characteristic features of MAPK1. When expressed in Escherichia coli, the recombinant enzyme showed kinase activity with myelin basic protein as the substrate and was inhibited by staurosporine. Interestingly, overexpression of this gene in a drug-sensitive laboratory strain and a resistant field isolate resulted in increased the sensitivity of the transfectants to potassium antimony tartrate, suggesting that it has a role in antimony resistance. Our results demonstrate that downregulation of LdMAPK1 may be in part correlated with antimony drug resistance in Indian VL isolates.

SUBMITTER: Ashutosh

PROVIDER: S-EPMC3256019 | biostudies-literature | 2012 Jan

REPOSITORIES: biostudies-literature

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Downregulation of mitogen-activated protein kinase 1 of Leishmania donovani field isolates is associated with antimony resistance.

Ashutosh Garg Mansi M Sundar Shyam S Duncan Robert R Nakhasi Hira L HL Goyal Neena N

Antimicrobial agents and chemotherapy 20111107 1

Emergence of resistance to pentavalent antimonials has become a severe obstacle in the treatment of visceral leishmaniasis (VL) on the Indian subcontinent. The mechanisms operating in laboratory-generated strains are somewhat known, but the determinants of clinical antimony resistance are not well understood. By utilizing a DNA microarray expression profiling approach, we identified a gene encoding mitogen-activated protein kinase 1 (MAPK1) for the kinetoplast protozoan Leishmania donovani (LdMA ...[more]

PMID: 22064540

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Project description:The inability of sodium antimony gluconate (SAG)-unresponsive kala-azar patients to clear Leishmania donovani (LD) infection despite SAG therapy is partly due to an ill-defined immune-dysfunction. Since dendritic cells (DCs) typically initiate anti-leishmanial immunity, a role for DCs in aberrant LD clearance was investigated. Accordingly, regulation of SAG-induced activation of murine DCs following infection with LD isolates exhibiting two distinct phenotypes such as antimony-resistant (Sb(R)LD) and antimony-sensitive (Sb(S)LD) was compared in vitro. Unlike Sb(S)LD, infection of DCs with Sb(R)LD induced more IL-10 production and inhibited SAG-induced secretion of proinflammatory cytokines, up-regulation of co-stimulatory molecules and leishmanicidal effects. Sb(R)LD inhibited these effects of SAG by blocking activation of PI3K/AKT and NF-kappaB pathways. In contrast, Sb(S)LD failed to block activation of SAG (20 microg/ml)-induced PI3K/AKT pathway; which continued to stimulate NF-kappaB signaling, induce leishmanicidal effects and promote DC activation. Notably, prolonged incubation of DCs with Sb(S)LD also inhibited SAG (20 microg/ml)-induced activation of PI3K/AKT and NF-kappaB pathways and leishmanicidal effects, which was restored by increasing the dose of SAG to 40 microg/ml. In contrast, Sb(R)LD inhibited these SAG-induced events regardless of duration of DC exposure to Sb(R)LD or dose of SAG. Interestingly, the inhibitory effects of isogenic Sb(S)LD expressing ATP-binding cassette (ABC) transporter MRPA on SAG-induced leishmanicidal effects mimicked that of Sb(R)LD to some extent, although antimony resistance in clinical LD isolates is known to be multifactorial. Furthermore, NF-kappaB was found to transcriptionally regulate expression of murine gammaglutamylcysteine synthetase heavy-chain (mgammaGCS(hc)) gene, presumably an important regulator of antimony resistance. Importantly, Sb(R)LD but not Sb(S)LD blocked SAG-induced mgammaGCS expression in DCs by preventing NF-kappaB binding to the mgammaGCS(hc) promoter. Our findings demonstrate that Sb(R)LD but not Sb(S)LD prevents SAG-induced DC activation by suppressing a PI3K-dependent NF-kappaB pathway and provide the evidence for differential host-pathogen interaction mediated by Sb(R)LD and Sb(S)LD.

Project description:Antimonials (Sb) were used for decades for chemotherapy of visceral leishmaniasis (VL). Now abandoned in the Indian subcontinent (ISC) because of Leishmania donovani resistance, this drug offers a unique model for understanding drug resistance dynamics. In a previous phylogenomic study, we found two distinct populations of L. donovani: the core group (CG) in the Gangetic plains and ISC1 in the Nepalese highlands. Sb resistance was only encountered within the CG, and a series of potential markers were identified. Here, we analyzed the development of resistance to trivalent antimonials (SbIII) upon experimental selection in ISC1 and CG strains. We observed that (i) baseline SbIII susceptibility of parasites was higher in ISC1 than in the CG, (ii) time to SbIII resistance was higher for ISC1 parasites than for CG strains, and (iii) untargeted genomic and metabolomic analyses revealed molecular changes along the selection process: these were more numerous in ISC1 than in the CG. Altogether these observations led to the hypothesis that CG parasites are preadapted to SbIII resistance. This hypothesis was experimentally confirmed by showing that only wild-type CG strains could survive a direct exposure to the maximal concentration of SbIII The main driver of this preadaptation was shown to be MRPA, a gene involved in SbIII sequestration and amplified in an intrachromosomal amplicon in all CG strains characterized so far. This amplicon emerged around 1850 in the CG, well before the implementation of antimonials for VL chemotherapy, and we discuss here several hypotheses of selective pressure that could have accompanied its emergence.IMPORTANCE The "antibiotic resistance crisis" is a major challenge for scientists and medical professionals. This steady rise in drug-resistant pathogens also extends to parasitic diseases, with antimony being the first anti-Leishmania drug that fell in the Indian subcontinent (ISC). Leishmaniasis is a major but neglected infectious disease with limited therapeutic options. Therefore, understanding how parasites became resistant to antimonials is of commanding importance. In this study, we experimentally characterized the dynamics of this resistance acquisition and show for the first time that some Leishmania populations of the ISC were preadapted to antimony resistance, likely driven by environmental factors or by drugs used in the 19th century.

Dataset Information

Downregulation of mitogen-activated protein kinase 1 of Leishmania donovani field isolates is associated with antimony resistance.

Publications

Downregulation of mitogen-activated protein kinase 1 of Leishmania donovani field isolates is associated with antimony resistance.

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