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

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.

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

Parasitology ◽

10.1017/s0031182007002284 ◽

2007 ◽

Vol 134 (6) ◽

pp. 777-787 ◽

Cited By ~ 32

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.

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A Novel Bioimpedance-Based Detection of Miltefosine Susceptibility Among Clinical Leishmania donovani Isolates of the Indian Subcontinent Exhibiting Resistance to Multiple Drugs

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2021.768830 ◽

2021 ◽

Vol 11 ◽

Author(s):

Souradeepa Ghosh ◽

Souvik Biswas ◽

Sandip Mukherjee ◽

Arijit Pal ◽

Aaditya Saxena ◽

...

Keyword(s):

Leishmania Donovani ◽

Indian Subcontinent ◽

Accurate Determination ◽

Clinical Isolates ◽

Protein Thiol ◽

Hamster Model ◽

Spectra Analysis ◽

Thiol Content ◽

Multiple Drugs

The extent of susceptibility towards miltefosine (Mil), amphotericin B (AmpB), and paromomycin (Paro) was measured among 19 clinical isolates of Leishmania donovani (LD). Thirteen of these clinical isolates were reported to exhibit low susceptibility towards sodium stibogluconate (SSG-R), while six of them were highly susceptible (SSG-S). The degree of clearance of amastigotes (EC50) for these predefined SSG-R- and SSG-S-infected macrophages was determined against Mil, AmpB, and Paro. Two out of the 13 SSG-R isolates (BHU575 and BHU814) showed low susceptibility towards all three drugs studied, while the rest of the 11 SSG-R isolates showed varying degrees of susceptibility either towards none or only towards individual drugs. Interestingly, all the SSG-S isolates showed high susceptibility towards Mil/AmpB/Paro. The total intracellular non-protein thiol content of the LD promastigotes, which have been previously reported to be positively co-related with EC50 towards SSG, was found to be independent from the degree of susceptibility towards Mil/AmpB/Paro. Impedance spectra analysis, which quantifies membrane resistance, revealed lower impedimetric values for all those isolates exhibiting low efficacy to Mil (Mil-R). Our analysis points out that while non-protein thiol content can be an attribute of SSG-R, lower impedimetric values can be linked with lower Mil susceptibility, although neither of these parameters seems to get influenced by the degree of susceptibility towards AmpB/Paro. Finally, a correlation analysis with established biological methods suggests that impedance spectral analysis can be used for the accurate determination of lower Mil susceptibility among LD isolates, which is further validated in the LD-infected in vivo hamster model.

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

Canadian Journal of Infectious Diseases and Medical Microbiology ◽

10.1155/2019/9392414 ◽

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.

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Heat shock protein 70 (HSP70) expression in antimony susceptible/resistant clinical isolates of Leishmania donovani

Nepal Journal of Biotechnology ◽

10.3126/njb.v3i1.14225 ◽

2015 ◽

Vol 3 (1) ◽

pp. 22-28 ◽

Cited By ~ 4

Author(s):

Mahendra Maharjan ◽

Rentala Madhubala

Keyword(s):

Leishmania Donovani ◽

Gene Copy Number ◽

Clinical Isolates ◽

Gene Copy ◽

Hsp70 Expression ◽

First Line ◽

Over Expression ◽

Hsp70 Protein ◽

Pentavalent Antimony ◽

Different Parts

Pentavalent antimonials have long been the first line of defence against leishmaniasis, but resistance has been reported in different parts of the world. Pentavalent antimony is reduced into trivalent form in the cells and is a potential inducer of HSP70 in L. donovani. Expression profile of HSP70 in antimony susceptible and resistant L. donovani isolates were characterized by Southern blot, Northern blot and western blot analysis. HSP70 gene copy number, gene expression and HSP70 protein expression was found uniform in both antimony sensitive and resistant clinical isolates. In laboratory condition, Leishmania cells respond to antimonial drug stress by three fold over expression of the HSP70 protein. The observed results indicated that HSP70 play important role in stress tolerance against antimonial drug without differential expression in antimony sensitive and resistance clinical isolates of L. donovani.Nepal Journal of Biotechnology. Dec. 2015 Vol. 3, No. 1: 22-28

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

Nepal Journal of Biotechnology ◽

10.3126/njb.v4i1.16340 ◽

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.

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Increased levels of thiols protect antimony unresponsive Leishmania donovani field isolates against reactive oxygen species generated by trivalent antimony

Parasitology ◽

10.1017/s0031182007003150 ◽

2007 ◽

Vol 134 (12) ◽

pp. 1679-1687 ◽

Cited By ~ 71

Author(s):

G. MANDAL ◽

S. WYLLIE ◽

N. SINGH ◽

S. SUNDAR ◽

A. H. FAIRLAMB ◽

...

Keyword(s):

Reactive Oxygen Species ◽

Leishmania Donovani ◽

Indian Subcontinent ◽

Current Trend ◽

Reactive Oxygen ◽

Clinical Isolates ◽

Ros Generation ◽

Scavenging Activity ◽

Oxygen Species ◽

Field Isolates

SUMMARYThe current trend of antimony (Sb) unresponsiveness in the Indian subcontinent is a major impediment to effective chemotherapy of visceral leishmaniasis (VL). Although contributory mechanisms studied in laboratory-raised Sb-R parasites include an up-regulation of drug efflux pumps and increased thiols, their role in clinical isolates is not yet substantiated. Accordingly, our objectives were to study the contributory role of thiols in the generation of Sb unresponsiveness in clinical isolates. Promastigotes were isolated from VL patients who were either Sb responsive (n=2) or unresponsive (n=3). Levels of thiols as measured by HPLC and flow cytometry showed higher basal levels of thiols and a faster rate of thiol regeneration in Sb unresponsive strains as compared with sensitive strains. The effects of antimony on generation of reactive oxygen species (ROS) in normal and thiol-depleted conditions as also their H2O2 scavenging activity indicated that in unresponsive parasites, Sb-mediated ROS generation was curtailed, which could be reversed by depletion of thiols and was accompanied by a higher H2O2 scavenging activity. Higher levels of thiols in Sb-unresponsive field isolates from patients with VL protect parasites from Sb-mediated oxidative stress, thereby contributing to the antimony resistance phenotype.

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Casein kinase 1.2 over expression restores stress resistance to Leishmania donovani HSP23 null mutants

Scientific Reports ◽

10.1038/s41598-020-72724-x ◽

2020 ◽

Vol 10 (1) ◽

Cited By ~ 3

Author(s):

Constanze Kröber-Boncardo ◽

Stephan Lorenzen ◽

Christine Brinker ◽

Joachim Clos

Keyword(s):

Translational Control ◽

Leishmania Donovani ◽

Gene Copy Number ◽

Small Heat Shock Protein ◽

Copy Number Variations ◽

Casein Kinase ◽

Gene Copy ◽

Escape Mutant ◽

Over Expression

Abstract Leishmania donovani is a trypanosomatidic parasite and causes the lethal kala-azar fever, a neglected tropical disease. The Trypanosomatida are devoid of transcriptional gene regulation and rely on gene copy number variations and translational control for their adaption to changing conditions. To survive at mammalian tissue temperatures, L. donovani relies on the small heat shock protein HSP23, the loss of which renders the parasites stress sensitive and impairs their proliferation. Here, we analysed a spontaneous escape mutant with wild type-like in vitro growth. Further selection of this escape strains resulted in a complete reversion of the phenotype. Whole genome sequencing revealed a correlation between stress tolerance and the massive amplification of a six-gene cluster on chromosome 35, with further analysis showing over expression of the casein kinase 1.2 gene as responsible. In vitro phosphorylation experiments established both HSP23 and the related P23 co-chaperone as substrates and modulators of casein kinase 1.2, providing evidence for another crucial link between chaperones and signal transduction protein kinases in this early branching eukaryote.

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High-quality reference genome ofFasciola gigantica: Insights into the genomic signatures of transposon-mediated evolution and specific parasitic adaption in tropical regions

10.1101/2021.04.09.439143 ◽

2021 ◽

Author(s):

Xier Luo ◽

Kuiqing Cui ◽

Zhiqiang Wang ◽

Lijuan Yin ◽

Zhipeng Li ◽

...

Keyword(s):

Reference Genome ◽

Fasciola Hepatica ◽

Process Analysis ◽

Large Body ◽

Copy Number Variations ◽

Fasciola Gigantica ◽

Genomic Research ◽

Gene Copy ◽

Specific Gene ◽

High Quality

AbstractFasciola giganticaandFasciola hepaticaare causative pathogens offascioliasis, with the widest latitudinal, longitudinal, and altitudinal distribution; however, among parasites, they have the largest sequenced genomes, hindering genomic research. In the present study, we used various sequencing and assembly technologies to generate a new high-qualityFasciola giganticareference genome. We improved the integration of gene structure prediction, and identified two independent transposable element expansion events contributing to (1) the speciation between Fasciola and Fasciolopsis during the Cretaceous-Paleogene boundary mass extinction, and (2) the habitat switch to the liver during the Paleocene-Eocene Thermal Maximum, accompanied by gene length increment. Long interspersed element (LINE) duplication contributed to the second transposon-mediated alteration, showing an obvious trend of insertion into gene regions, regardless of strong purifying selection. Gene ontology analysis of genes with long LINE insertions identified membrane-associated and vesicle secretion process proteins, further implicating the functional alteration of the gene network. We identified 852 excretory/secretory proteins and 3300 protein-protein interactions betweenFasciola giganticaand its host. Among them, copper/zinc superoxide dismutase genes, with specific gene copy number variations, might play a central role in the phase I detoxification process. Analysis of 559 single-copy orthologs suggested thatFasciola giganticaandFasciola hepaticadiverged at 11.8 Ma near the Middle and Late Miocene Epoch boundary. We identified 98 rapidly evolving gene families, including actin and aquaporin, which might explain the large body size and the parasitic adaptive character resulting in these liver flukes becoming epidemic in tropical and subtropical regions.

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High-quality reference genome of Fasciola gigantica: Insights into the genomic signatures of transposon-mediated evolution and specific parasitic adaption in tropical regions

PLoS Neglected Tropical Diseases ◽

10.1371/journal.pntd.0009750 ◽

2021 ◽

Vol 15 (10) ◽

pp. e0009750

Author(s):

Xier Luo ◽

Kuiqing Cui ◽

Zhiqiang Wang ◽

Zhipeng Li ◽

Zhengjiao Wu ◽

...

Keyword(s):

Reference Genome ◽

Fasciola Hepatica ◽

Process Analysis ◽

Large Body ◽

Copy Number Variations ◽

Fasciola Gigantica ◽

Genomic Research ◽

Gene Copy ◽

Specific Gene ◽

High Quality

Fasciola gigantica and Fasciola hepatica are causative pathogens of fascioliasis, with the widest latitudinal, longitudinal, and altitudinal distribution; however, among parasites, they have the largest sequenced genomes, hindering genomic research. In the present study, we used various sequencing and assembly technologies to generate a new high-quality Fasciola gigantica reference genome. We improved the integration of gene structure prediction, and identified two independent transposable element expansion events contributing to (1) the speciation between Fasciola and Fasciolopsis during the Cretaceous-Paleogene boundary mass extinction, and (2) the habitat switch to the liver during the Paleocene-Eocene Thermal Maximum, accompanied by gene length increment. Long interspersed element (LINE) duplication contributed to the second transposon-mediated alteration, showing an obvious trend of insertion into gene regions, regardless of strong purifying effect. Gene ontology analysis of genes with long LINE insertions identified membrane-associated and vesicle secretion process proteins, further implicating the functional alteration of the gene network. We identified 852 predicted excretory/secretory proteins and 3300 protein-protein interactions between Fasciola gigantica and its host. Among them, copper/zinc superoxide dismutase genes, with specific gene copy number variations, might play a central role in the phase I detoxification process. Analysis of 559 single-copy orthologs suggested that Fasciola gigantica and Fasciola hepatica diverged at 11.8 Ma near the Middle and Late Miocene Epoch boundary. We identified 98 rapidly evolving gene families, including actin and aquaporin, which might explain the large body size and the parasitic adaptive character resulting in these liver flukes becoming epidemic in tropical and subtropical regions.

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Lipase Precursor-Like Protein Promotes Miltefosine Tolerance in Leishmania donovani by Enhancing Parasite Infectivity and Eliciting Anti-inflammatory Responses in Host Macrophages

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00666-18 ◽

2018 ◽

Vol 62 (12) ◽

Cited By ~ 3

Author(s):

Deepak Kumar Deep ◽

Ruchi Singh ◽

Arpita Kulshrestha ◽

Saima Wajid ◽

Poonam Salotra

Keyword(s):

Leishmania Donovani ◽

Inflammatory Responses ◽

Indian Subcontinent ◽

Interleukin 10 ◽

Oral Drug ◽

Necrosis Factor Alpha ◽

Content Type ◽

Tnf Α ◽

Antileishmanial Drug

ABSTRACT The oral drug miltefosine (MIL) was introduced in the Indian subcontinent in the year 2002 for the treatment of visceral leishmaniasis (VL). However, recent reports on its declining efficacy and increasing relapse rates pose a serious concern. An understanding of the factors contributing to MIL tolerance in Leishmania parasites is critical. In the present study, we assessed the role of the lipase precursor-like protein (Lip) in conferring tolerance to miltefosine by episomally overexpressing Lip in Leishmania donovani (LdLip++). We observed a significant increase (∼3-fold) in the MIL 50% inhibitory concentration (IC50) at both the promastigote (3.90 ± 0.68 µM; P < 0.05) and intracellular amastigote (9.10 ± 0.60 µM; P < 0.05) stages compared to the wild-type counterpart (LdNeo) (MIL IC50s of 1.49 ± 0.20 µM at the promastigote stage and 3.95 ± 0.45 µM at the amastigote stage). LdLip++ parasites exhibited significantly (P < 0.05) increased infectivity to host macrophages and increased metacyclogenesis and tolerance to MIL-induced oxidative stress. The susceptibility of LdLip++ to other antileishmanial drugs (sodium antimony gluconate and amphotericin B) remained unchanged. In comparison to LdNeo, the LdLip++ parasites elicited high host interleukin-10 (IL-10) cytokine expression levels (1.6-fold; P < 0.05) with reduced expression of the cytokine tumor necrosis factor alpha (TNF-α) (1.5-fold; P < 0.05), leading to a significantly (P < 0.01) increased ratio of IL-10/TNF-α. The above-described findings suggest a role of lipase precursor-like protein in conferring tolerance to the oral antileishmanial drug MIL in L. donovani parasites.

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