Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Comparative analysis of gene expression in HAT4-nulls and wild type cells in Leishmania donovani

ABSTRACT: Histone acetylations are known to impact gene transcription. Here, we have attempted to examine the role of Leishmania donovani histone acetyltransferase HAT4 in regulating global gene expression. The transcriptome of HAT4-null promastigotes (Samples 2A, 2B) have been compared with the transcriptome of wild-type Leishmania promastigotes (Samples 1A, 1B) and fold change in gene expression with respect to the control wild-type has been determined. DNA microarray analysis was carried out with biological replicates, using RNA isolated from logarithmically growing promastigotes in two separate experiments. Bioanalyzer profiles of the isolated RNA samples were used to assess purity and integrity of the isolated RNA. Sample 1A and sample 1B are analyses carried out using RNA isolated from wild-type Leishmania donovani promastigotes. Sample 2A and sample 2B are analyses carried out using RNA isolated from HAT4-null promastigotes.

ORGANISM(S): Leishmania donovani

SUBMITTER: Swati Saha

PROVIDER: E-GEOD-76574 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Protozoa of the genus Leishmania are the causative agents of leishmaniasis in humans. These parasites cycle between promastigotes in the sand fly mid-gut and amastigotes in phagolysosome of mammalian macrophages. During infection, host up-regulate nitric oxide synthase and parasite induce host arginase expression, both of which use arginine as a substrate. These elevated activities deplete macrophage arginine pools, a situation that invading Leishmania must overcome since it is an essential amino acid. Leishmania donovani imports exogenous arginine via a mono-specific amino acid transporter (AAP3) and utilizes it primarily through the polyamine pathway to provide precursors for trypanothione biosynthesis. Here we report the discovery of a pathway whereby promastigote and amastigote forms of the Leishmania sense the lack of environmental arginine and respond with rapid up-regulation in AAP3 expression and activity, as well as several other transporters. Significantly, this arginine deprivation response is also activated in parasites during macrophage infection. Phosphoproteomic analyses of L. donovani promastigotes have implicated a Mitogen-Activated Protein Kinase 2 (MPK2)-mediated signaling cascade in this response and L. mexicana mutants lacking MPK2 are unable to respond to arginine deprivation. In this study, we established that Leishmania cells sense the absence of arginine in their environment; both in culture (axenic promastigotes and amastigotes) and in macrophages during infection (amastigotes). This study describes the first amino acid deprivation sensing mechanism and the pathway that transduce this response, and reveals a novel host-pathogen metabolic interplay. Total RNA from Ten Leishmania donovani samples were analyzed using RNA-Seq. Cells from two life stages (promastigotes and amastigotes) were grown in axenic culture in the presence and absense of arginine. For each condition two biological replicates were grown and analyzed. In addition two macrophage grown amastigotes were analyzed.

Project description:Transcriptome analysis via high-throughput sequencing (RNA-Seq) captures significant changes in gene expression. The purpose of this study is to elucidate the transcriptomic changes in profilin deficient Leishmania promastigotes (LdPfn+/-) as compared to wild type control (LdPfn+/+). This research will enable us to gain insight into the role of profilin in Leishmania cells. The data has been validated by qRT-PCR. Data analysis results are further supported by flow cytometry, immunofluorescence experiments and by western blot demonstrating a depletion in the protein expression of a eukaryotic translation initiation factor in the cells with depleted levels of profilin protein. Methods: Total RNA from three independent biological replicates from each of wild-type (LdPfn+/+) and profilin knockout (LdPfn+/-) Leishmania promastigotes was extracted and after library preparation, RNA sequencing was carried out on Illumina HiSeq 2500. The sequence reads that passed quality filters were aligned to the Leishmania donovani (BPK282A1) genomic data obtained from TriTrypDB version 51 using Bowtie2 (-x option). All analyses were carried out using the Tophat pipeline with the following versions: Tophat v2.1.1, Bowtie2 v2.3.5.1. The HTSeq version 0.12.4 (htseq-count -f option) was used to count the number of reads aligned to protein-coding genes. DeSeq tool was used for differential gene expression analysis between samples in protein-coding genes. qRT-PCR validation was performed using SYBR Green assay. Results: RNA-Seq datasets generated in this study with each sample control (LdPfn+/+) and profilin knockout (LdPfn+/-) have at least 30 million read pairs. RNA-seq data were aligned to the L.donovani genome (Leishmania donovani BPK282A1, NCBI taxon ID: 981087) and 8135 transcripts were identified in our data set. Data analysis revealed that a total of 254 genes were differentially expressed (DE) having a p-value <0.05. The data obtained from RNA-Seq have been validated by real-time PCR of 18 genes, 8 UP regulated and 10 down-regulated. Results of qRT-PCR analysis showed a strong correlation with the RNA-seq data. Inferences drawn from transcriptomic analysis were further validated by flow cytometry, immunofluorescence experiments and western blotting, revealing a critical role during the early phases of Leishmania cell division. Conclusions: Present study represents a comprehensive report elucidating the transcriptomic changes in profilin deficient Leishmania promastigotes (LdPfn+/-) as compared to wild type (LdPfn+/+). This RNA-Seq data along with validation by qPCR, flow cytometry experiments, and immunofluorescent microscopy experiments indicate that profilin might play a critical role during the early phases of Leishmania cell division.

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Comparative analysis of gene expression in HAT4-nulls and wild type cells in Leishmania donovani

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