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Integrative genomic and proteomic studies of Leishmania donovani strains revealed genetic features associated with drug resistance

ABSTRACT: Leishmaniasis is a neglected tropical disease affecting millions of people worldwide. Emerging drug resistance of Leishmania species poses threaten to the effective control and elimination of this neglected tropical disease. Here we conducted whole genome resequencing, proteome profiling, and comparative analyses of a drug-resistant clinical isolate and two drug-susceptible strains of Leishmania donovani to explore genetic features that might contribute to the establishment of drug resistance in this parasite. By comparative genomic analysis, exclusive variations were identified in the drug-resistant isolate of L. donovani, including 86 copy number variations, 271 frameshift mutations in protein-coding genes and two site mutations in non-coding genes. Comparative proteomic analysis indicated significant differences in protein expression between resistant and susceptible strains of L. donovani, including 69 exclusive detected molecules and 84 consistent down-/up-regulations in the former. Integrating the genomic mutations and proteomic specificities linked nine of the genomic mutations (gene duplication, insertion and deletion) to significantly altered protein expression changes in the drug-resistant clinical isolate. These genetic features were inferred to be associated with nucleotide-binding and fatty acid metabolism (biosynthesis and degradation), which might contribute to fitness-gains allowing for the drug-resistant phenotype of L. donovani. This comparative and integrative work provided deep insights into the molecular basis underlying resistance establishment, suggesting new aspects to be investigated for novel intervention strategies against L. donovani and related species.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Leishmania <subgenus>

SUBMITTER: Jiao Li

LAB HEAD: Jiao Li

PROVIDER: PXD017089 | Pride | 2021-09-09

REPOSITORIES: Pride

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Integrative genomic, proteomic and phenotypic studies of Leishmania donovani strains revealed genetic features associated with virulence and antimony-resistance.

Zheng Zhiwan Z Chen Jianping J Ma Guangxu G Satoskar Abhay R AR Li Jiao J

Parasites & vectors 20201012 1

<h4>Background</h4>Leishmaniasis is a neglected tropical disease affecting millions of people worldwide. Emerging drug resistance of Leishmania species poses threaten to the effective control and elimination programme of this neglected tropical disease.<h4>Methods</h4>In this work, we conducted drug-resistance testing, whole genome resequencing and proteome profiling for a recently reported clinical isolate with supposed drug resistance (HCZ), and two reference sensitive strains (DD8 and 9044) o ...[more]

PMID: 33046138

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Project description:Infection with antimony resistant (SbR) but not with sentitive (SbS) Leishmania donovani (LD) gives rise to aggressive pathology in mammalian hosts, the cause of which is far from clear. Some intracellular pathogens exploit autophagy for their own benefit. Here we show that induction of autophagy in normal macrophages (MF) by pharmacological mediators prior to infection with SbRLD (SbRLD-MF) enhanced their growth as compared to untreated MF, unlike SbSLD-MF. Autophagy was evident in SbRLD-MF from electron microscopical studies showing double membrane-bound compartment around amastigote. In SbRLD-MF there is induction of beclin 1, which forms the platform to recruit other interacting molecules to initiate autophagy. Knocking down the beclin 1 transcription factor Nrf2 and subsequent infection with SbRLD showed significantly lower organ parasites as compared to wild type BALB/c mice. Cessation of autophagy in SbRLD-MF at the later stage of infection is coupled with induction of miR-30a, whose binding to 3'UTR of beclin 1 leads to its post-transcriptional attenuation followed by rise in intracellular Ca++ and apoptosis. SbRLD mediated translocation of AP-1 transcription factor to the nucleus induce pri-miR-30a over-expression. Rise in Ca++ causes caspase 8 activtion leading to the cleavage of beclin 1 and initiation of apoptosis in SbRLD-MF. Apoptosis may favor parasite egress for cell to cell transmission. We also found that beclin 1 expression is present in splenocytes of kala-azar patients harbouring SbRLD but not SbSLD. Our results suggest that SbRLD has evolved a unique mechanism for its own benefit which explains, in part, the cause of aggressive pathology. Peritoneal exudate macrophages were isolated from mouse, grown in 60mm plates and infected with Leishmania donovani and total RNA was isolated from cells at 12, 18 and 24 hrs post infection. Leishmania infected macrophage miRNA expression signature was generated. Cells grown on 60mm plates and infected with Leishmania. The main objective of the microarray analysis of mmu-miRNA in antimony resistant and antimony sensitive Leishmania donovani infected macrophages are as follows: 1. To study how the expression of miRNA varies in either antimony resistant or antimony sensitive Leishmania infected macrophages as compared to the normal macrophages as a function of time. LPS was used as control. 2. To study the expression of those miRNAs which are differentially expressed in antimony resistant and antimony sensitive Leishmania infected macrophages at each time point post infection. 3. To identify those miRNAs which are responsible for degradation of autophagy initiating protein beclin 1 mRNA

Project description:Among the most central questions in Leishmania research is why some species remain in the skin dermis at the site of infection by the sand fly vector whereas other species migrate to visceral organs where they cause fatal visceral leishmaniasis. Although L. donovani is the species typically responsible for visceral leishmaniasis, an atypical L. donovani strain is the etiologic agent for cutaneous leishmaniasis in Sri Lanka. To identify molecular determinants for visceral disease, we have analysed the phenotype and genotype of two L. donovani clinical isolates from Sri Lanka where one isolate was derived from a cutaneous leishmaniasis patient (CL) and the other from a visceral leishmaniasis patient (VL). These isolates cause dramatically different pathology when introduced into mice; notably the CL isolate has lost the ability to survive in visceral organs while the VL isolate was highly virulent in visceral organs of BALB/c mice. Whole genome sequencing of the CL and VL isolates revealed that these genomes were very similar as there were no gene deletions and few individual gene amplifications. Indels resulting in frame shifts and loss/gain of stop codons resulted in 13 distinct pseudogenes present in each of the CL and VL isolates. There were 154 non-synonymous SNPs specific to the CL isolate and 193 non-synonymous SNPs specific to the VL isolate. Genome wide gene expression analysis revealed several transcript level differences, including the A2 virulence gene resulting in higher expression of A2 proteins in the VL isolate than in the CL isolate. Genotypic variations relevant to pathology and tropism in Leishmania can be interrogated by reverse genetics. Experimentally increasing A2 expression in the CL isolate through gene transfer significantly increased itM-bM-^@M-^Ys ability to survive in the spleen of BALB/c mice and conversely, down-regulating A2 expression in the VL isolate abrogated attenuated its survival in BALB/c mice. These observations reveal that there are relatively few genetic differences between the CL and VL isolates apart from the A2 genes, but collectively these have profound effects on human disease and experimentally infected mice. 6 Samples in total, 3 each from VL and CL causing isolates were analyzed by Splice Leader RNASeq. These three samples from each of the isolates were grown to form one of the following three lifestages, Promastigotes, Macrophage derived Amastigotes, Axenic Amastigotes.

Dataset Information

Integrative genomic and proteomic studies of Leishmania donovani strains revealed genetic features associated with drug resistance

Publications

Integrative genomic, proteomic and phenotypic studies of Leishmania donovani strains revealed genetic features associated with virulence and antimony-resistance.

Similar Datasets

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