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Analysis of RNA Based Transcriptome of Mycobacterium leprae genome from Environmental and Clinical Samples

ABSTRACT: BACKGROUND: This study was conducted to analyse M. leprae gene expression profiles in biopsy of leprosy patient and in environmental soil samples. Investigation was carried out to find out the expression profile for the minimal gene set necessary for growth of environmental M. leprae and its genes potentially require for infection and pathogenesis of leprosy. METHOD: RNA was extracted from human biopsy and environmental soil samples and total RNA obtained by subtractive hybridization of rRNA, was reverse transcribed and amplified using random primers. The whole genome was tiled at 10bp to obtain probes having 60 mer oligonucleotides in sense orientation. 179963 probes were designed in both sense and antisense orientation. Blast was performed against the mRNA sequence databases to check the specificity of the probes. Finally, 359926 probes were designed and 56579 specific probes were replicated to fill the remaining spots. RESULTS: Up regulation of several functional genes of M. leprae genome were observed from soil samples responsible for metabolism of Carbon compounds [9], Amino acids and amines [4] , Fatty acids synthesis [21], Lipid Biosynthesis [33], Phosphorous compounds [2]. Up regulation of genes were observed from soil samples in energy metabolism function such as Glycolysis [6], Pyruvate dehydrogenase [1], TCA cycle [12], Glyoxylate bypass [12]. In up regulation of respiration function [11] genes were observed and some of genes responsible for miscellaneous oxidoreductases and oxygenases [22] function, ATP-proton motive force [4] were also observed. Up regulation of genes for cell envelope function [65], conserved membrane proteins [107], cell processes [62], central intermediary metabolism [79], polyamine synthesis[38], biosynthesis of cofactors, prosthetic groups and carriers [36], broad regulatory functions [35], macromolecule metabolism[103], degradation of macromolecules [25], miscellaneous transferases [36], conserved hypothetical [204] and unknowns [101] were observed. Significant up regulation of Virulence genes [9], PE and PPE families [3] were also observed from soil samples. CONCLUSION: Transcriptome analysis indicated that up regulation of essential genes of M. leprae transcripts in environmental samples as compared to M. leprae from human biopsy samples which strongly suggested that several M. leprae genes are expressed more in environment for its survival and may act as source of infection. RNA was extracted from human biopsy and environmental soil samples and total RNA obtained by subtractive hybridization of rRNA, was reverse transcribed and amplified using random primers. The whole genome was tiled at 10bp to obtain probes having 60 mer oligonucleotides in sense orientation. 179963 probes were designed in both sense and antisense orientation. Blast was performed against the mRNA sequence databases to check the specificity of the probes. Finally, 359926 probes were designed and 56579 specific probes were replicated to fill the remaining spots.

ORGANISM(S): Mycobacterium leprae

SUBMITTER: Genotypic technology

PROVIDER: E-GEOD-68192 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:BACKGROUND: This study was conducted to analyse M. leprae gene expression profiles in biopsy of leprosy patient and in environmental soil samples. Investigation was carried out to find out the expression profile for the minimal gene set necessary for growth of environmental M. leprae and its genes potentially require for infection and pathogenesis of leprosy. METHOD: RNA was extracted from human biopsy and environmental soil samples and total RNA obtained by subtractive hybridization of rRNA, was reverse transcribed and amplified using random primers. The whole genome was tiled at 10bp to obtain probes having 60 mer oligonucleotides in sense orientation. 179963 probes were designed in both sense and antisense orientation. Blast was performed against the mRNA sequence databases to check the specificity of the probes. Finally, 359926 probes were designed and 56579 specific probes were replicated to fill the remaining spots. RESULTS: Up regulation of several functional genes of M. leprae genome were observed from soil samples responsible for metabolism of Carbon compounds [9], Amino acids and amines [4] , Fatty acids synthesis [21], Lipid Biosynthesis [33], Phosphorous compounds [2]. Up regulation of genes were observed from soil samples in energy metabolism function such as Glycolysis [6], Pyruvate dehydrogenase [1], TCA cycle [12], Glyoxylate bypass [12]. In up regulation of respiration function [11] genes were observed and some of genes responsible for miscellaneous oxidoreductases and oxygenases [22] function, ATP-proton motive force [4] were also observed. Up regulation of genes for cell envelope function [65], conserved membrane proteins [107], cell processes [62], central intermediary metabolism [79], polyamine synthesis[38], biosynthesis of cofactors, prosthetic groups and carriers [36], broad regulatory functions [35], macromolecule metabolism[103], degradation of macromolecules [25], miscellaneous transferases [36], conserved hypothetical [204] and unknowns [101] were observed. Significant up regulation of Virulence genes [9], PE and PPE families [3] were also observed from soil samples. CONCLUSION: Transcriptome analysis indicated that up regulation of essential genes of M. leprae transcripts in environmental samples as compared to M. leprae from human biopsy samples which strongly suggested that several M. leprae genes are expressed more in environment for its survival and may act as source of infection.

Project description:Background: Reactions in Leprosy are immune exacerbations that cause debilitating consequences like nerve damage and permanent deformities. Prediction of these reactional states using appropriate biomarkers would enable early treatment interventions to prevent nerve function impairment. The current study investigated whole transcriptomic expression profiles of Mycobacterium leprae (M. leprae) that differentiate leprosy cases in type 2 (Erythema Nodosum Leprosum) reactions with those without reactions in host skin tissue derived RNA. Methods: Post clinical examination, excisional skin biopsy specimens were collected from skin lesions of subjects with and without type 2 reaction. Total RNA was extracted following the Trizol protocol and bacterial RNA was enriched in the samples. A 2 x 400K gene expression array (whole genome tiling array) was designed with the probes having 60-mer oligonucleotides tiling every 10bp of the genome sequence of M. leprae (NC_011896.1). The array comprised 420288 features which include probes and Agilent controls. The quality of RNA was estimated using BioAnalyzer (Agilent Technologies) followed by labelling, reverse transcription, amplification and hybridization to the arrays. The hybridized slides were scanned on a G2600D scanner (Agilent Technologies). The data thus acquired is analysed using GeneSpring GX Version 12.1 software. Data was normalized and fold difference in expression was noted from 359,922 probes which include sense and antisense orientations of 179,961 probes. The differentially expressing M. leprae genomic regions between type 2 reactions and non reactional cases were noted. Results: Considering a statistical cut-off value of 0.6 for fold difference in expression between the test and the control samples, a set of 107 genes indicated statistically significant up-regulation with volcano plot p-values less than 0.05. Functional characterization revealed higher-expression of genes encoding transmembrane proteins (12), regulatory proteins (9), fatty acid biosynthesis (6), amino acid metabolism (13), nucleic acid metabolism (7), DNA replication and repair (7), Secretory proteins (2) Krebs Cycle (1), Glycolysis (1), Drug Efflux Protein (1),Stress Response Protein (1), Energy Metabolism (2), Pantothenate biosynthesis (1), Metalloproteins (3), Hydrolases (1) and Hypothetical Proteins (40). Additionally there are 157 genes that are down regulated in cases with reaction. Conclusion: Differential expression of genes in the human skin biopsy specimens among leprosy cases with type 2 reaction in contrast to those without reaction suggests the role of pathogen associated gene expression triggers with the aetiology of these reactions. As most of the transmembrane and cell wall proteins possess epitope and surface exposed domains, higher expression levels of genes encoding these proteins may have a possible role in enhancing host immune responses characteristic of type 2 reactions in leprosy.

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Analysis of RNA Based Transcriptome of Mycobacterium leprae genome from Environmental and Clinical Samples

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