A mitochondrial HSP70 (HSPA9B) is linked to miltefosine resistance and stress response in Leishmania donovani

Abstract

BackgroundProtozoan parasites of the genus Leishmania are responsible for leishmaniasis, a neglected tropical disease affecting millions worldwide. Visceral leishmaniasis (VL), caused by Leishmania donovani, is the most severe form of leishmaniasis with high rates of mortality if left untreated. Current treatments include pentavalent antimonials and amphotericin B. However, high toxicity and emergence of resistance hinder the success of these options. Miltefosine (HePC) is the first oral treatment available for leishmaniasis. While treatment with HePC has proven effective, higher tolerance to the drug has been observed, and experimental resistance is easily developed in an in vitro environment. Several studies, including ours, have revealed that HePC resistance has a multi-factorial origin and this work aims to shed light on this complex mechanism.Methods2D-DIGE quantitative proteomics comparing the soluble proteomes of sensitive and HePC resistant L. donovani lines identified a protein of interest tentatively involved in drug resistance. To test this link, we employed a gain-of-function approach followed by mutagenesis analysis. Functional studies were complemented with flow cytometry to measure HePC incorporation and cell death.ResultsWe identified a mitochondrial HSP70 (HSPA9B) downregulated in HePC-resistant L. donovani promastigotes. The overexpression of HSPA9B in WT lines confers an increased sensitivity to HePC, regardless of whether the expression is ectopic or integrative. Moreover, the increased sensitivity to HePC is specific to the HSPA9B overexpression since dominant negative mutant lines were able to restore HePC susceptibility to WT values. Interestingly, the augmented susceptibility to HePC did not correlate with an increased HePC uptake. Leishmania donovani promastigotes overexpressing HSPA9B were subjected to different environmental stimuli. Our data suggest that HSPA9B is capable of protecting cells from stressful conditions such as low pH and high temperature. This phenotype was further corroborated in axenic amastigotes overexpressing HSPA9B.ConclusionsThe results from this study provide evidence to support the involvement of a mitochondrial HSP70 (HSPA9B) in experimental HePC resistance, a mechanism that is not yet fully understood, and reveal potential fundamental roles of HSPA9B in the biology of Leishmania. Overall, our findings are relevant for current and future antileishmanial chemotherapy strategies.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1904-8) contains supplementary material, which is available to authorized users.

Unravelling the proteomic signature of extracellular vesicles released by drug-resistant Leishmania infantum parasites

“…For example, the HS70-related protein-1 mitochondrial precursor (HSPA9B) has been recently linked to miltefosine resistance and response to oxidative stress in L . donovani [ 89 ]. In addition to GP63, several other virulence factors, such as disulfide-isomerase and cysteine peptidase B (CPB) proteins, were found in MF200.5 EVs, further confirming that Leishmania EVs play a major role as virulence factors [ 21 , 65 ].…”

Differentially modulated proteins associated with Leishmaniasis—a systematic review of in-vivo and in-vitro studies

“…Carnielli et al [ 67 ] concluded that the up-regulation of proteins NADP-dependent alcohol dehydrogenase and translation elongation factor 1-beta play a key role in redox homeostasis and thereby could aid in miltefosine resistance. Vacchina et al [ 69 ] reported that the mitochondrial HSP70 is involved in miltefosine resistance as well as in the stress response of the parasite. In addition, Veronica et al [ 68 ] reported that the protein iron superoxide dismutase contributes to the miltefosine resistance.…”

Experimental Strategies to Explore Drug Action and Resistance in Kinetoplastid Parasites

“…2D-DIGE is the mostly used technique in the study of MoR/MoA in kinetoplastids but is known to predominantly detect soluble and abundant proteins [ 139 , 146 , 147 ]. A way to evaluate a broader spectrum of the proteome is to additionally extract proteins from various specific cellular compartments or the use of differential extraction [ 138 , 139 , 148 , 149 ], such as the separate isolation of membrane-enriched and cytosolic proteins or the isolation of phosphoproteins, mitochondrial components, or the analysis of the secretome [ 68 , 150 , 151 , 152 , 153 , 154 , 155 , 156 ]. Most studies employing proteomics, especially in Leishmania , compared sensitive to resistant strains to elucidate the MoR of drugs.…”