A Proteomics Screen Implicates HSP83 and a Small Kinetoplastid Calpain-related Protein in Drug Resistance in Leishmania donovani Clinical Field Isolates by Modulating Drug-induced Programmed Cell Death

Abstract

The therapeutic mainstay against the protozoan parasite Leishmania is still based on the antiquated pentavalent antimonials (Sb(V)), but resistance is increasing in several parts of the world. Resistance is now partly understood in laboratory isolates, but our understanding of resistance in field isolates is lagging behind. We describe here a comparative analysis of a genetically related pair of Sb(V)-sensitive and -resistant Leishmania donovani strains isolated from kala-azar patients. The resistant isolate exhibited cross-resistance to other unrelated Leishmania drugs including miltefosine and amphotericin B. A comparative proteomics screen has highlighted a number of proteins differentially expressed suggesting that programmed cell death (PCD) is modified in the resistant parasite. Indeed drug-induced PCD progression was altered in the Sb(V)-resistant strain as determined using early and late markers of apoptosis. Two proteins, the heat shock protein HSP83 and the small kinetoplastid calpain-related protein (SKCRP14.1) were shown to be intimately implicated in the drug-induced PCD phenotype. HSP83 increased drug resistance and reduced drug-mediated PCD activation by interfering with the mitochondrial membrane potential, whereas SKCRP14

Expanding Members and Roles of the Calpain Superfamily and Their Genetically Modified Animals

“…In contrast to the autophagy system, which is found in almost all eukaryotes but not in bacteria and archaea, only 94 entries are found for bacteria, while no calpain species is found in any archaebacterium. It should be noted that Leishmania and Trypanosoma have around 20 calpain homologues, whose physiological roles probably involve cell morphogenesis, drug resistance, and stress response mechanisms [37,45,118,170]. Some trypanosome calpains have N-terminal domains weakly similar to calpastatin.…”

Contribution of proteomics of Leishmania spp. to the understanding of differentiation, drug resistance mechanisms, vaccine and drug development

“…Interestingly hsp83 over-expressing parasites were also cross-resistant to miltefosine. Over-expression of SKCRP14.1 increased the sensitivity to Sb(V) but decreased its susceptibility to miltefosine 52 .…”

“…Indian field isolates 52 to study resistance to this drug in L. donovani. In total 11 protein species were identified seven up-regulated (heat shock protein 83; hsp70-related protein 1, mitochondrial precursor, chaperonin containing tcp1, subunit 8; 14-3-3 protein; ATP-dependent RNA helicase; hSP70, dipeptidyl-peptidase III) and five down-regulated (hsp70-related protein 1, mitochondrial precursor; hypothetical protein, unknown function; enolase, 20 S proteasome α5 subunit, SKCRP14.1 (small kinetoplastid calpain related protein)).…”

“…Metabolomics on the other hand will support future proteomic studies and as an outcome novel drug targets are likely to be identified. Proteomic studies will also be useful to study further drug resistance in the field as it has already been shown to identify proteins 26,46,52,54 .…”

Biological Roles of Peptidases in Trypanosomatids~!2009-11-26~!2010-02-15~!2010-03-18~!

“…Our group demonstrated that MDL28170, a potent calpain inhibitor, is capable of reducing promastigote growth in culture and induces cell death [150], probable through apoptosis (unpublished data). A proteomics screen implicated a calpain-related protein in drug resistance in L. donovani clinical field isolates, probably by modulating drug-induced apoptosis [151]. Highly sensitive gene expression microarray analysis revealed that calpains, among other genes, are differentially expressed in Leishmania parasites isolated from post kala-azar dermal leishmaniasis (PKDL) patients in comparison with those from visceral leishmaniasis (VL) [152].…”