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:We used next generation sequencing to examine global changes in gene expression in the spleen and splenic macrophages in a hamster model of progressive visceral leishmaniasis. We used RNA sequencing coupled with de novo transcriptome assembly, because the Syrian hamster does not have a fully sequenced and annotated reference genome. Libraries for deep sequencing were constructed from poly-A RNA isolated from the spleens or splenic macrophages of uninfected and 28-day L. donovani-infected hamsters (n=4 per group) using standard Illumina protocols. The library quality was confirmed by Agilent Bioanalyzer. Paired-end 50-base sequencing was performed using TruSeq SBS kit v3 (Illumina) on an Illumina HiSeq 1000.The quality of raw sequencing reads was checked using FastQC (v0.10.1). To avoid the contamination of pathogen sequences, we filtered out reads (using the FASTX Toolkit v0.0.13) that aligned to the Leishmania donovani BPK282A1 genome (NCBI BioProject PRJEA61817) using Bowtie 2 (v2.0.0-beta5) under default options. To reduce the effect of low quality reads, we further filtered out artifacts and reads having a phred score <28 in more than 10% of nucleotides using FASTX Toolkit (v0.0.13). Both forward and reverse reads were removed if any of them failed to pass the filters. To obtain a complete transcriptome, we used two steps. First, the cleaned sequencing reads from different spleen samples were pooled together and de novo assembled using Trinity software. Second, the resulting transcriptome was combined with all cleaned reads from hamster spleen and splenic macrophages and the CHO-K1 RefSeq genome to perform a second de novo assembly using BRANCH. We first created a customized reference library using Rattus norvegicus (Rnor_5.0.73) and Mus musculus (GRCm38.73) genomes. We then used BLAST (v 2.2.28+) to align each transcript generated from BRANCH against the customized library to assign it a gene name based upon sequence similarity. An E-value cutoff of <1e-3 was used. Additionally, we compared the Trinity transcripts with the CHO-K1 Ref Seq genome. All the non-Leishmania-like raw sequencing reads were first mapped to our de novo reference genome using Bowtie2 (v2.1.0) with default options, but allowing one read to map to as many as 500 different transcripts. We then measured the expression abundance, count of reads mapped to each transcript, using the software eXpress. To identify differentially expressed transcripts in each experiment, 3 different approaches were applied: exact tests of Robinson and Smyth and generalized linear models with the likelihood ratio test using the BioConductor R package EdgeR, and the Wald test using DESeq2. Only transcripts with at least 1 count per million in at least 3 out of 4 samples in the control or experimental group were included in the analysis. A transcript was considered differentially expressed only when it was identified by all three different approaches. Analysis of differentially expressed transcripts identified a highly inflammatory spleen environment with abundant expression of type I and type II interferon response genes. However, high IFN-γ expression was ineffective in directing exclusive M1 macrophage polarization, suppressing M2-associated gene expression, and restraining parasite replication and disease. While many IFN-inducible transcripts were upregulated in the infected spleen, fewer were induced in splenic macrophages in VL suggesting suppression of macrophage responsiveness to activating cytokines. The transcription factor STAT3 was predicted and confirmed to be activated, and found to orchestrate an exuberant counter-regulatory response that favors parasite replication.

Dataset Information

Tissue-specific transcriptomic changes associated with AmBisome treatment of mice with experimental visceral leishmaniasis [LV9]

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