Over-Expression of Cysteine Leucine Rich Protein Is Related to SAG Resistance in Clinical Isolates of Leishmania donovani

Abstract

BackgroundResistance emergence against antileishmanial drugs, particularly Sodium Antimony Gluconate (SAG) has severely hampered the therapeutic strategy against visceral leishmaniasis, the mechanism of resistance being indistinguishable. Cysteine leucine rich protein (CLrP), was recognized as one of the overexpressed proteins in resistant isolates, as observed in differential proteomics between sensitive and resistant isolates of L. donovani. The present study deals with the characterization of CLrP and for its possible connection with SAG resistance.Methodology and Principal FindingsIn pursuance of deciphering the role of CLrP in SAG resistance, gene was cloned, over-expressed in E. coli system and thereafter antibody was raised. The expression profile of CLrP and was found to be over-expressed in SAG resistant clinical isolates of L. donovani as compared to SAG sensitive ones when investigated by real-time PCR and western blotting. CLrP has been characterized through bioinformatics, immunoblotting and immunolocalization analysis, which reveals its post-translational modification along with its dual existence in the nucleus as well as in the membrane of the parasite. Further investigation using a ChIP assay confirmed its DNA binding potential. Over-expression of CLrP in sensitive isolate of L. donovani significantly decreased its responsiveness to SAG (SbV and SbIII) and a shift towards the resistant mode was observed. Further, a significant increase in its infectivity in murine macrophages has been observed.Conclusion/SignificanceThe study reports the differential expression of CLrP in SAG sensitive and resistant isolates of L. donovani. Functional intricacy of CLrP increases with dual localization, glycosylation and DNA binding potential of the protein. Further over-expressing CLrP in sensitive isolate of L. donovani shows significantly decreased sensitivity towards SAG and increased infectivity as well, thus assisting the parasite in securing a safe niche. Results indicates the possible contribution of CLrP to antimonial resistance in L. donovani by assisting the parasite growth in the macrophages.

High-throughput Cos-Seq screen with intracellular Leishmania infantum for the discovery of novel drug-resistance mechanisms

“…Moreover, overexpression of this gene also induced resistance to paromomycin in intracellular amastigotes ( Gazanion et al, 2016 ). There is growing evidence that LRR proteins interact with macrophages ( Kedzierski et al, 2004 ) and contribute to drug resistance, especially against antimony, by assisting the parasite growth in the host cell ( Das et al, 2015 ). The LinJ.06.1010 gene product may provide Leishmania with a dual advantage that would likely contribute to drug resistance (in both promastigotes and amastigotes) as well as a better adaptation to macrophage infections, as previously seen for other LRR-proteins such as PPG and PSA-2 ( Jimenez-Ruiz et al, 1998 ; Ilg et al, 1999 ; Kedzierski et al, 2004 ; Mukherjee et al, 2016 ).…”

Functional Involvement of Leishmania donovani Tryparedoxin Peroxidases during Infection and Drug Treatment

“…Promastigotes were lysed in Laemmli buffer (20 mM Tris-HCl at pH 7.4, 5 mM EDTA, 150 mM NaCl, 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 1 mM aprotinin, and 1 M leupeptin), and the protein content was quantified using a CBX protein assay kit (G-Biosciences, St. Louis, MO). SDS-PAGE and Western blot assays were carried out as described previously (10,39). Transblotted proteins were probed with mouse anti-GFP antibody at 1:7,500, rabbit anti-␤-tubulin at 1:10,000, rabbit anti-mTXNPx at 1:500, and rabbit anti-cTXNPx at 1:10,000 dilutions in 1ϫ PBS containing 0.1% Tween 20.…”

“…The mTXNPx gene (cloned in pXG-GFPϩ=) and the cTXNPx gene (cloned in pXG-GFPϩ2), along with genes coding for inactive enzymes, mTXNPx (Δ81) and cTXNPx (Δ52), were overexpressed in parasites. For the control groups, pXG-GFPϩ2, pXG-GFPϩ=, and mitochondrial targeting signal (MTS) were cloned in pXG-GFPϩ= (data not shown) (10,12,39,40). The overexpression of the proteins was confirmed by Western blotting with the respective antibody, as well as anti-GFP antibody.…”

“…Successful transfection was further confirmed by measuring GFP expression in a BD Calibur flow cytometer (Becton Dickinson, San Jose, CA) equipped with a 15-mV, 488-nm air-cooled argon ion laser. The localization of the overexpressed proteins was further checked by imaging of transfectants using a confocal microscope (10,39).…”

Reverse Epidemiology: An Experimental Framework to Drive Leishmania Biomarker Discovery in situ by Functional Genetic Screening Using Relevant Animal Models

“…However, even though these loci may represent potential biomarkers with important prognostic value, there are no dedicated, concerted efforts for their validation in clinically relevant settings. One exception includes CLrP, whose increased abundance on RNA and protein levels were correlated with increased Sb resistance in field isolates, albeit only a small number of isolates were used in these studies (Kumar et al, 2010b ; Das et al, 2015 ). For other loci, clinical validation of the functional screening results can be ambiguous, with for example the MRPA and PTR1 genes of the H-locus having been either strictly, partially, or not correlated to Sb resistance in different epidemiological studies (Decuypere et al, 2005 , 2012 ; Mittal et al, 2007 ; Mukherjee et al, 2007 ; Mukhopadhyay et al, 2011 ).…”