Biomarkers of antimony resistance: need for expression analysis of multiple genes to distinguish resistance phenotype in clinical isolates of Leishmania donovani

Abstract

Resistance to antimony is a major cause of failure to therapy in a large proportion of visceral leishmaniasis cases. Methods to distinguish resistant and sensitive parasite are urgently needed as the standard in vitro intracellular drug susceptibility assays are cumbersome and time consuming. Differential expression profiling studies have led to the identification of several antimony resistance-associated genes; however, their efficacy as a potential biomarker for monitoring antimony resistance remains imprecise. We analysed the expression of eight genes [antimony metabolism-associated genes - multidrug resistance protein A (MRPA), γ-glutamylcysteine synthetase (γ-GCS) and aquaporin-1 (AQP1) - and genes identified by proteome/transcriptome profiling—heat shock protein 83, mitogen-activated protein kinase 1 and histones H1, H2A and H4) in antimony-resistant (n=10) and antimony-sensitive (n=4) clinical isolates of Leishmania donovani by quantitative real-time PCR, in comparison with a lab-generated resistant and a standard sensitive isolate. We observed a significant differential expression of MRPA, histone H1 (p<0.01), γ-GCS, HSP83 (p<0.005) and histone H2A and H4 (p<0.0001) in a group of sodium antimony gluconate-resistant isolates compared to sensitive isolates. Preferential AQP1 expression was observed in all the sensitive isolates (p<0.0001). Overall, expression profile in field isolates for all the genes studied showed altered expression in majority of isolates, while in some, the expression was static. All the isolates showed a mosaic of expression pattern of the genes analysed indicating constellation of genes contributes towards the drug susceptibility of parasite. As none of the genes exhibit an absolute correlation with phenotype, targeted expression analysis of a set of genes should be considered as biomarker for distinguishing the antimony-resistant and antimony-sensitive parasite.

Heterogeneity of Molecular Resistance Patterns in Antimony-Resistant Field Isolates of Leishmania Species from the Western Mediterranean Area

“…GSH1, which is generally overexpressed in either in vitro-selected mutants or clinical isolates, was overexpressed in 3 resistant isolates (10,11,45). Our survey is also in accordance with an earlier study that demonstrated MAPK and H4 overexpression (45). Furthermore, the overexpression of TRPER, ARGG, 14-3-3, and P299 observed in resistant isolates is in accordance with previous proteomic findings (12,31,48,49).…”

“…The overexpression of MRPA observed in 2 resistant isolates in the current study was also described previously (11,45). GSH1, which is generally overexpressed in either in vitro-selected mutants or clinical isolates, was overexpressed in 3 resistant isolates (10,11,45). Our survey is also in accordance with an earlier study that demonstrated MAPK and H4 overexpression (45).…”

“…Overall, our results indicate that molecular resistance in Leishmania is multifactorial. These findings are generally in accordance with several multigene studies focused on other areas where leishmaniasis is endemic (45,46). …”

“…MRPA was the first molecular target that displayed gene amplification in Leishmania in vitro mutants selected with heavy metals, and this amplification was subsequently demonstrated in resistant field isolates (8)(9)(10). The overexpression of MRPA observed in 2 resistant isolates in the current study was also described previously (11,45). GSH1, which is generally overexpressed in either in vitro-selected mutants or clinical isolates, was overexpressed in 3 resistant isolates (10,11,45).…”

“…In fact, AQP1 is a membranous channel through which the trivalent antimony enters the parasite, and AQP1 overexpression is typically observed in susceptible isolates (11,45,47). However, a few cases of AQP1 overexpression in L. donovaniresistant clinical isolates have been reported (18,19).…”