Project description:In this study we set out to determine whole blood transcriptional profiles that might distinguish asymptomatic infection with Leishmania donovani from active disease, as well as to monitor the changes in transcriptional profiles that accompanied drug treatment. Amphotericin B provides improved therapy for visceral leishmaniasis (VL) caused by Leishmania donovani, with single dose liposomal-encapsulated Ambisome providing the best cure rates. The VL elimination program aims to reduce the incidence rate in the Indian subcontinent to <1/10,000 population/year. Ability to predict which asymptomatic individuals (e.g. anti-leishmanial IgG and/or Leishmania-specific modified Quantiferon positive) will progress to clinical VL would help in monitoring disease outbreaks. Here we examined whole blood transcriptional profiles associated with asymptomatic infection, active disease, and in treated cases. Two independent microarray experiments were performed, with analysis focussed primarily on differentially expressed genes (DEGs) concordant across both experiments. No DEGs were identified for IgG or Quantiferon positive asymptomatic groups compared to negative healthy endemic controls. We therefore concentrated on comparing concordant DEGs from active cases with all healthy controls, and in examining differences in the transcriptome following different regimens of drug treatment. In these comparisons 6 major themes emerged: (i) expression of genes and enrichment of gene sets associated with erythrocyte function in active cases; (ii) strong evidence for enrichment of gene sets involved in cell cycle in comparing active cases with healthy controls; (iii) identification of IFNG encoding interferon- as the major hub gene in concordant gene expression patterns across experiments comparing active cases with healthy controls or with treated cases; (iv) enrichment for interleukin signalling (IL-1/3/4/6/7/8) and a prominent role for CXCL10/9/11 and chemokine signalling pathways in comparing active cases with treated cases; (v) the novel identification of Aryl Hydrocarbon Receptor signalling as a significant canonical pathway when comparing active cases with healthy controls, and to a lesser extent with treated cases; and (vi) global expression profiling support for more effective cure at day 30 post-treatment with a single dose of liposomal encapsulated amphotericin B compared to multi-dose non-liposomal amphotericin B treatment over 30 days.

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.

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 it’s 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.

Project description:Amphotericin B provides improved therapy for visceral leishmaniasis (VL) caused by Leishmania donovani, with single dose liposomal-encapsulated Ambisome providing the best cure rates. The VL elimination program aims to reduce the incidence rate in the Indian subcontinent to <1/10,000 population/year. Ability to predict which asymptomatic individuals (e.g. anti-leishmanial IgG and/or Leishmania-specific modified Quantiferon positive) will progress to clinical VL would help in monitoring disease outbreaks. Here we examined whole blood transcriptional profiles associated with asymptomatic infection, active disease, and in treated cases. Two independent microarray experiments were performed, with analysis focussed primarily on differentially expressed genes (DEGs) concordant across both experiments. No DEGs were identified for IgG or Quantiferon positive asymptomatic groups compared to negative healthy endemic controls. We therefore concentrated on comparing concordant DEGs from active cases with all healthy controls, and in examining differences in the transcriptome following different regimens of drug treatment. In these comparisons 6 major themes emerged: (i) expression of genes and enrichment of gene sets associated with erythrocyte function in active cases; (ii) strong evidence for enrichment of gene sets involved in cell cycle in comparing active cases with healthy controls; (iii) identification of IFNG encoding interferon-γ as the major hub gene in concordant gene expression patterns across experiments comparing active cases with healthy controls or with treated cases; (iv) enrichment for interleukin signalling (IL-1/3/4/6/7/8) and a prominent role for CXCL10/9/11 and chemokine signalling pathways in comparing active cases with treated cases; (v) the novel identification of Aryl Hydrocarbon Receptor signalling as a significant canonical pathway when comparing active cases with healthy controls or with treated cases; and (vi) global expression profiling support for more effective cure at day 30 post-treatment with a single dose of liposomal encapsulated amphotericin B compared to multi-dose non-liposomal amphotericin B treatment over 30 days. (296 words; 300 words allowed).

Dataset Information

Transcriptional Blood Signatures for Active and Amphotericin B Treated Visceral Leishmaniasis in India [exp2]

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