Differential gene expression analysis in antimony-unresponsive Indian kala azar (visceral leishmaniasis) clinical isolates by DNA microarray

Parasitology ◽  
2007 ◽  
Vol 134 (6) ◽  
pp. 777-787 ◽  
Author(s):  
N. SINGH ◽  
R. ALMEIDA ◽  
H. KOTHARI ◽  
P. KUMAR ◽  
G. MANDAL ◽  
...  
Keyword(s):  
Leishmania Donovani ◽  
Leishmania Major ◽  
Clinical Isolates ◽  
Resistant Isolate ◽  
Closely Related Species ◽  
Kala Azar ◽  
Antimony Resistance ◽  
Sensitive Isolate ◽  
Differential Gene

SUMMARYIn this study, cDNA microarray analysis of a closely related species,Leishmania major, was used as a screening tool to compare antimonial-resistant and susceptible clinical isolates ofLeishmania donovaniin order to to identify candidate genes on the basis of antimony resistance. Clinically confirmed resistant isolate 39 and sensitive isolate 2001 were used in this study. Many differentially regulated genes were identified whose expression levels differ in sodium antimony gluconate (SAG)-treated patients. Interestingly, genes on the array, showing changes in expression of over 2-fold revealed the identity of ABC transporters, which are known determinants of drug resistance in laboratory mutants. The functionality of the transporters was validated by flow cytometry which, being biologically informative, provides direct clues to gene function. The results suggest that isolate 39 could have developed resistance by an increased multidrug resistance protein (MRP)-like pump. This study provides preliminary clues to the role of a thiol-dependent efflux system in antimonial resistant clinical isolates ofLeishmania donovani.

scholarly journals Characterization of the Proliferating Cell Nuclear Antigen of Leishmania donovani Clinical Isolates and Its Association with Antimony Resistance

2014 ◽  
Vol 58 (6) ◽  
pp. 2997-3007 ◽  
Author(s):  
Rati Tandon ◽  
Sharat Chandra ◽  
Rajendra Kumar Baharia ◽  
Sanchita Das ◽  
Pragya Misra ◽  
...  
Keyword(s):  
Clinical Isolate ◽  
Proliferating Cell Nuclear Antigen ◽  
Leishmania Donovani ◽  
Clinical Isolates ◽  
Resistant Isolate ◽  
Nuclear Antigen ◽  
Wild Type ◽  
Content Type ◽  
Sensitive Isolate ◽  
Proliferating Cell

ABSTRACTPreviously, through a proteomic analysis, proliferating cell nuclear antigen (PCNA) was found to be overexpressed in the sodium antimony gluconate (SAG)-resistant clinical isolate compared to that in the SAG-sensitive clinical isolate ofLeishmania donovani. The present study was designed to explore the potential role of the PCNA protein in SAG resistance inL. donovani. For this purpose, the protein was cloned, overexpressed, purified, and modeled. Western blot (WB) and real-time PCR (RT-PCR) analyses confirmed that PCNA was overexpressed by ≥3-fold in the log phase, stationary phase, and peanut agglutinin isolated procyclic and metacyclic stages of the promastigote form and by ∼5-fold in the amastigote form of the SAG-resistant isolate compared to that in the SAG-sensitive isolate.L. donovaniPCNA (LdPCNA) was overexpressed as a green fluorescent protein (GFP) fusion protein in a SAG-sensitive clinical isolate ofL. donovani, and modulation of the sensitivities of the transfectants to pentavalent antimonial (SbV) and trivalent antimonial (SbIII) drugs was assessedin vitroagainst promastigotes and intracellular (J774A.1 cell line) amastigotes, respectively. Overexpression of LdPCNA in the SAG-sensitive isolate resulted in an increase in the 50% inhibitory concentrations (IC50) of SbV(from 41.2 ± 0.6 μg/ml to 66.5 ± 3.9 μg/ml) and SbIII(from 24.0 ± 0.3 μg/ml to 43.4 ± 1.8 μg/ml). Moreover, PCNA-overexpressing promastigote transfectants exhibited less DNA fragmentation compared to that of wild-type SAG-sensitive parasites upon SbIIItreatment. In addition, SAG-induced nitric oxide (NO) production was found to be significantly inhibited in the macrophages infected with the transfectants compared with that in wild-type SAG-sensitive parasites. Consequently, we infer that LdPCNA has a significant role in SAG resistance inL. donovaniclinical isolates, which warrants detailed investigations regarding its mechanism.

scholarly journals Application of kDNA Minicircle PCR-RFLP to Characterize Leishmania donovani Clinical Isolates Obtained from Post-Kala-Azar Dermal Leishmaniasis in Eastern Nepal

2019 ◽  
Vol 2019 ◽  
pp. 1-8
Author(s):  
Ojesh Pokhrel ◽  
Keshav Rai ◽  
Narayan Raj Bhattarai ◽  
Suman Rijal ◽  
Arpana Rijal ◽  
...  
Keyword(s):  
Leishmania Donovani ◽  
Clinical Isolates ◽  
Rdna Genes ◽  
Kala Azar ◽  
Pcr Rflp ◽  
Potential Reservoir ◽  
Rflp Assay ◽  
Epidemiological Characterization

Post-kala-azar dermal leishmaniasis (PKDL) is a skin manifestation of visceral leishmaniasis (VL) which develops after apparent cure in some patients. PKDL is considered as the potential reservoir for the VL infection. Molecular epidemiological characterization of L. donovani isolates obtained from VL and PKDL isolates is essentially required in order to understand the transmission dynamics of the VL infection. To date, genetic variation among the VL and PKDL L. donovani isolates was not fully elucidated. Therefore, 14 clinical isolates from VL and 4 clinical isolates from PKDL were speciated by hsp70 and rDNA genes. Further characterization of L. donovani by haspB PCR demonstrates two different genotypes. All PKDL isolates have the same genetic structure. kDNA PCR-RFLP assay revealed 18 different genotypes; however, structural analysis showed the two distinct kDNA genotype population (k = 2). The kDNA fingerprint patterns of parasites from hilly districts were clustered separately from low-land districts. Therefore, further study with a large number of samples is urgently required for systematic characterization of the clinical isolates to track the molecular epidemiology of the Leishmania donovani causing VL and the role of PKDL as a reservoir.

scholarly journals Mutations in an Aquaglyceroporin as a Proven Marker of Antimony Clinical Resistance in the Parasite Leishmania donovani

2020 ◽  
Author(s):  
Jade-Eva Potvin ◽  
Philippe Leprohon ◽  
Marine Queffeulou ◽  
Shyam Sundar ◽  
Marc Ouellette
Keyword(s):  
Leishmania Donovani ◽  
Indian Subcontinent ◽  
Copy Number Variations ◽  
Clinical Isolates ◽  
Gene Copy ◽  
Specific Gene ◽  
Single Nucleotide Variants ◽  
Clinical Resistance ◽  
Antimony Resistance

Abstract Background Antimonial drugs have long been the mainstay to treat visceral leishmaniasis. Their use has been discontinued in the Indian subcontinent because of drug resistance, but they are still clinically useful elsewhere. The goal of this study was to find markers of antimony resistance in Leishmania donovani clinical isolates and validate experimentally their role in resistance. Methods The genomes of sensitive and antimony-resistant clinical isolates were sequenced. The role of a specific gene in contributing to resistance was studied by CRISPR-Cas9–mediated gene editing and intracellular drug sensitivity assays. Results Both gene copy number variations and single nucleotide variants were associated with antimony resistance. A homozygous insertion of 2 nucleotides was found in the gene coding for the aquaglyceroporin AQP1 in both resistant isolates. Restoring the wild-type AQP1 open reading frame re-sensitized the 2 independent resistant isolates to antimonials. Alternatively, editing the genome of a sensitive isolate by incorporating the 2-nucleotide insertion in its AQP1 gene led to antimony-resistant parasites. Conclusions Through genomic analysis and CRISPR-Cas9–mediated genome editing we have proven the role of the AQP1 mutations in antimony clinical resistance in L. donovani.

scholarly journals Assessing the Role of Potential Biomarkers in Antimony Susceptible and Resistant Clinical Isolates of L. donovani from India

2016 ◽  
Vol 4 (1) ◽  
pp. 1-13
Author(s):  
Mahendra Maharjan ◽  
Swati Mandal ◽  
Rentala Madhubala
Keyword(s):  
Protein A ◽  
Building Blocks ◽  
Underlying Mechanism ◽  
Multidrug Resistant ◽  
Clinical Isolates ◽  
Polyamine Biosynthesis ◽  
Glutathione Biosynthesis ◽  
Antimony Resistance ◽  
Rate Limiting

Failure of antimonial drugs, the mainstay therapy for leishmaniasis has become an escalating problem in the treatment of Indian leishmaniasis. Using 14 clinical isolates from both visceral (VL) and post-kala-azar dermal leismaniasis (PKDL) patients, we have examined the role of ATP-binding cassette transporter (ABC transporter) gene, multidrug resistant protein A (MRPA) and two building blocks of the major thiol, trypanothione namely, ornithine decarboxylase gene (ODC) (a rate limiting enzyme in the polyamine biosynthesis) and γ-glutamylcysteine synthetase (γ-GCS) (a rate limiting enzyme in glutathione biosynthesis) in antimony resistance. Amplification of these three genes was observed in some but not all clinical isolates. Increased expression of the three RNAs as determined by real-time PCR was observed in all SAG-R clinical isolates. Significant increase in cysteine and glutathione levels was observed in the resistant isolates. Our studies report the underlying mechanism of antimony resistance in the clinical isolates.

scholarly journals Inhibition of ABC Transporters Abolishes Antimony Resistance in Leishmania Infection

2007 ◽  
Vol 52 (3) ◽  
pp. 1080-1093 ◽  
Author(s):  
Jayati Mookerjee Basu ◽  
Ananda Mookerjee ◽  
Rajdeep Banerjee ◽  
Manik Saha ◽  
Subhankar Singh ◽  
...  
Keyword(s):  
Abc Transporters ◽  
Leishmania Donovani ◽  
Host Cells ◽  
Leishmania Infection ◽  
Blood Monocytes ◽  
Glycoprotein P ◽  
Kala Azar ◽  
Antimony Resistance ◽  
Parasite Replication

ABSTRACT The emergence of antimony (Sb) resistance has jeopardized the treatment of visceral leishmaniasis in various countries. Previous studies have considered the part played by leishmanial parasites in antimony resistance, but the involvement of host factors in the clinical scenario remained to be investigated. Here we show that unlike infection with Sb-sensitive (Sbs) Leishmania donovani, infection with Sb-resistant (Sbr) L. donovani induces the upregulation of multidrug resistance-associated protein 1 (MRP1) and permeability glycoprotein (P-gp) in host cells, resulting in a nonaccumulation of intracellular Sb following treatment with sodium antimony gluconate (SAG) favoring parasite replication. The inhibition of MRP1 and P-gp with resistance-modifying agents such as lovastatin allows Sb accumulation and parasite killing within macrophages and offers protection in an animal model in which infection with Sbr L. donovani is otherwise lethal. The occurrence of a similar scenario in clinical cases is supported by the findings that unlike monocytes from SAG-sensitive kala-azar (KA) patients, monocytes from SAG-unresponsive KA patients overexpress P-gp and MRP1 and fail to accumulate Sb following in vitro SAG treatment unless pretreated with inhibitors of ABC transporters. Thus, the expression status of MRP1 and P-gp in blood monocytes may be used as a diagnostic marker for Sb resistance and the treatment strategy can be designed accordingly. Our results also indicate that lovastatin, which can inhibit both P-gp and MRP1, might be beneficial for reverting Sb resistance in leishmaniasis as well as drug resistance in other clinical situations, including cancer.

scholarly journals Exploring the Possible Role of Nature in Curing Leishmaniasis

2019 ◽  
pp. 1-21
Author(s):  
Ravinder Sharma ◽  
Vikas Gupta ◽  
Viney Chawla ◽  
Pooja Chawla
Keyword(s):  
Natural Products ◽  
Leishmania Donovani ◽  
Leishmania Major ◽  
Chemotherapeutic Agents ◽  
Leishmania Braziliensis ◽  
Leishmania Chagasi ◽  
Major Health Problem ◽  
Major Health ◽  
Number Of Plant Species

Background: Communicable diseases have always been a threat to mankind since times immemorial. Leishmaniasis, an infectious disease caused by protozoan of various species of leishmania, is a major health problem spreading across 98 countries and about 350 million people stand the risk of this infection worldwide. Medical research has struggled a lot to combat this disease. Objective: Among the various approaches available for treatment of Leishmaniasis, many are costly so there is a need to develop effective but economical and easily available antileishmanial agents. Methods: Natural products are important source of various new medicaments and their derivatives can be used for synthetic modification and bioactivity optimization. Therefore, in order to fulfil the need for novel, economical, more effective and safer chemotherapeutic agents, scientists have explored Mother Nature in detail. Results: A number of plant species possess inhibitory activity against certain types of parasites such as Leishmania major, Leishmania amazonensis, Leishmania aethiopica, Leishmania braziliensis, Leishmania mexicana, Leishmania infantum, Leishmania chagasi and Leishmania donovani. Moreover natural products are economical, safer, more effective and without considerable side effects. Conclusion: The present review highlights the leishmanicidal activity of various natural products with an insight in to their possible mechanism.

scholarly journals Accelerated Control of Visceral Leishmania donovani Infection in Interleukin-6-Deficient Mice

2008 ◽  
Vol 76 (9) ◽  
pp. 4088-4091 ◽  
Author(s):  
Henry W. Murray
Keyword(s):  
Visceral Leishmaniasis ◽  
Interleukin 6 ◽  
Leishmania Donovani ◽  
Wild Type ◽  
Therapeutic Blockade ◽  
Th1 Cell ◽  
Kala Azar ◽  
Type Response ◽  
Deficient Mice

ABSTRACT In patients with visceral leishmaniasis, increased levels of circulating interleukin-6 (IL-6) regularly accompany fully expressed, progressive infections (kala-azar). To experimentally test the role of IL-6, responses to an intracellular Leishmania donovani infection in the livers of IL-6−/− and wild-type mice were compared. IL-6−/− mice showed an enhanced control of the infection and earlier, rapid parasite killing along with additional evidence of a stimulated antileishmanial Th1-cell-type response: increased levels of circulating gamma interferon, accelerated granuloma assembly, and heightened responsiveness to chemotherapy. In this model of visceral leishmaniasis, IL-6 appears to act in a suppressive, macrophage-deactivating fashion, which identifies it as a potential target for therapeutic blockade.

scholarly journals Spatial analysis ofLeishmania donovaniexposure in humans and domestic animals in a recent kala azar focus in Nepal

Parasitology ◽  
2010 ◽  
Vol 137 (11) ◽  
pp. 1597-1603 ◽  
Author(s):  
BASUDHA KHANAL ◽  
ALBERT PICADO ◽  
NARAYAN RAJ BHATTARAI ◽  
GERT VAN DER AUWERA ◽  
MURARI LAL DAS ◽  
...  
Keyword(s):  
Leishmania Donovani ◽  
Indian Subcontinent ◽  
Public Health Problem ◽  
Domestic Animals ◽  
Major Public Health Problem ◽  
Scan Statistic ◽  
Transmission Cycle ◽  
Kala Azar ◽  
Information System Technology

SUMMARYVisceral leishmaniasis (VL) is a major public health problem in the Indian subcontinent where theLeishmania donovanitransmission cycle is described as anthroponotic. However, the role of animals (in particular domestic animals) in the persistence and expansion of VL is still a matter of debate. We combined Direct Agglutination Test (DAT) results in humans and domestic animals with Geographic Information System technology (i.e. extraction maps and scan statistic) to evaluate the exposure toL. donovanion these 2 populations in a recent VL focus in Nepal. A Poisson regression model was used to assess the risk of infection in humans associated with, among other factors, the proportion of DAT-positive animals in the proximities of the household. The serological results showed that both humans and domestic animals were exposed toL. donovani. DAT-positive animals and humans were spatially clustered. The presence of serologically positive goats (IRR=9·71), past VL cases (IRR=2·62) and the proximity to a forest island dividing the study area (IRR=3·67) increased the risk of being DAT-positive in humans. Even if they are not a reservoir, domestic animals, and specially goats, may play a role in the distribution ofL. donovani, in particular in this new VL focus.

Proteophosphoglycan is differentially expressed in sodium stibogluconate-sensitive and resistant Indian clinical isolates of Leishmania donovani

Parasitology ◽  
2007 ◽  
Vol 134 (9) ◽  
pp. 1175-1184 ◽  
Author(s):  
M. SAMANT ◽  
A. A. SAHASRABUDDHE ◽  
N. SINGH ◽  
S. K. GUPTA ◽  
S. SUNDAR ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Western Blotting ◽  
Leishmania Donovani ◽  
Clinical Isolates ◽  
Drug Resistant ◽  
Sodium Stibogluconate ◽  
Kala Azar ◽  
Microscopy Analysis ◽  
And Function ◽  
First Time

SUMMARYLeishmania produce several types of mucin-like glycoproteins called proteophosphoglycans (PPGs) some of which are secreted while others are found on the surface of promastigotes and amastigotes. These proteins are thought to be important in the transmission, invasion and subsequent intracellular survival of parasites. The structure and function of PPGs are species and stage-specific in the case of L. major and L. mexicana, but no such information has hitherto been available for L. donovani. This study presents, for the first time, an initial characterization (localization) of PPG in sodium stibogluconate (SSG)-resistant and sensitive clinical isolates of L. donovani from Bihar (India) by confocal microscopy, flow cytometry and Western blotting using antibodies to L. major PPG. Confocal microscopy analysis revealed that both promastigotes and amastigotes possess PPG on their cell membrane and flagellar pocket membrane but its expression was variable in different isolates. The quantitative analysis by FACS and Western blotting showed that the expression and intensity of PPG bands was higher in SSG-resistant isolates. This study suggests the possibilities of involvement of PPG in drug-resistant mechanisms and of using PPG abundance as a marker for identifying drug-resistant clinical isolates in Indian kala azar.

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