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Deciphering the genetic control of gene expression following Mycobacterium leprae antigens stimulation

ABSTRACT: Leprosy is a human infectious disease caused by Mycobacterium leprae. A strong host genetic contribution to leprosy susceptibility is well established. However, the modulation of the transcriptional response to infection and the mechanism(s) of disease control are poorly understood. To address this gap in knowledge of leprosy pathogenicity, we conducted a genome-wide search for expression quantitative trait loci (eQTL) that are associated with transcript variation –– before and after stimulation with M. leprae sonicate in whole blood cells. We show that M. leprae antigen stimulation mainly triggered the upregulation of immune related genes and that a substantial proportion of the differential gene expression is genetically controlled. Indeed, using stringent criteria, we identified 318 genes displaying cis-eQTL at an FDR of 0.01, including 66 genes displaying response-eQTL (reQTL), i.e. cis-eQTL that showed significant evidence for interaction with the M. leprae stimulus. Such reQTL correspond to regulatory variations that affect the interaction between human whole blood cells and M. leprae sonicate, and thus likely between the human host and M. leprae bacilli. We found that reQTL are significantly enriched among binding sites of transcription factors that are activated in response to infection, and that they were enriched among single nucleotide polymorphisms (SNPs) associated with susceptibility to leprosy per se and Type-I Reaction, as well as SNPs targeted by recent positive selection. Our study suggests that natural selection shaped our genomic diversity to face pathogen exposure including M. leprae infection.

ORGANISM(S): Homo sapiens

PROVIDER: GSE100853 | GEO | 2017/07/07

SECONDARY ACCESSION(S): PRJNA393277

REPOSITORIES: GEO

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Project description:Approximately half of M. lepraeâ??s transcriptome consists of inactive gene products. This has an impact on overall energy and resource consumption without potential benefit to this organism. However, multiple translational â??silencingâ?? mechanisms are present, reducing additional energy and resource expenditure required for protein production from these transcripts. The Mycobacterium leprae genome has less than 50% coding capacity and 1,133 pseudogenes. Preliminary evidence suggests that some pseudogenes are expressed. Therefore, defining pseudogene transcriptional and translational potentials should increase our understanding of their impact on M. leprae physiology. To address this, M. leprae was purified from the granulomatous hind footpad tissue of four individual nu/nu nude mice six months post-infection. M. leprae whole genome DNA microarrays representing the 1,614 annotated ORFs and 1,133 identified pseudogenes, were obtained from the Leprosy Research Support and Maintenance of an Armadillo Colony Post-Genome Era, Part I: Leprosy Research Support Contract (NO1 AI-25469) at Colorado State University. To validate 20% of genes positive by microarray analysis, RT-PCR was performed. Results of this study Gene expression analysis identified transcripts from 49% of all M. leprae genes including 57% of all ORFs and 43% of all pseudogenes in the genome. Pseudogenes were randomly distributed throughout the chromosome. Factors resulting in pseudogene transcription included: 1) co-orientation of transcribed pseudogenes with transcribed ORFs within or exclusive of operon-like structures; 2) the paucity of intrinsic stem-loop transcriptional terminators between transcribed ORFs and downstream pseudogenes; and 3) predicted pseudogene promoters. Mechanisms for translational silencing of pseudogene transcripts included the lack of both translational start codons and strong Shine-Dalgarno sequences. Transcribed pseudogenes also contained multiple in-frame stop codons and high Ka/Ks ratios, compared to that of homologs in M. tuberculosis and ORFs in M. leprae. A pseudogene transcript containing an active promoter, strong SD site, a start codon, but containing two in frame stop codons yielded a protein product when expressed in E. coli. Approximately half of M. leprae's transcriptome consists of inactive gene products consuming energy and resources without potential benefit to M. leprae. Presently it is unclear what additional detrimental affect(s) this large number of inactive mRNAs has on the functional capability of this organism. Translation of these pseudogenes may play an important role in overall energy consumption and resultant pathophysiological characteristics of M. leprae. However, this study also demonstrated that multiple translational silencing mechanisms are present, reducing additional energy and resource expenditure required for protein production from the vast majority of these transcripts. The overall design of this study was to identify the transcriptome of M. leprae in the granulomatous tissue of the mouse hind foot pad 6 months post infection.

Project description:Approximately half of M. leprae’s transcriptome consists of inactive gene products. This has an impact on overall energy and resource consumption without potential benefit to this organism. However, multiple translational ‘silencing’ mechanisms are present, reducing additional energy and resource expenditure required for protein production from these transcripts. The Mycobacterium leprae genome has less than 50% coding capacity and 1,133 pseudogenes. Preliminary evidence suggests that some pseudogenes are expressed. Therefore, defining pseudogene transcriptional and translational potentials should increase our understanding of their impact on M. leprae physiology. To address this, M. leprae was purified from the granulomatous hind footpad tissue of four individual nu/nu nude mice six months post-infection. M. leprae whole genome DNA microarrays representing the 1,614 annotated ORFs and 1,133 identified pseudogenes, were obtained from the Leprosy Research Support and Maintenance of an Armadillo Colony Post-Genome Era, Part I: Leprosy Research Support Contract (NO1 AI-25469) at Colorado State University. To validate 20% of genes positive by microarray analysis, RT-PCR was performed. Results of this study Gene expression analysis identified transcripts from 49% of all M. leprae genes including 57% of all ORFs and 43% of all pseudogenes in the genome. Pseudogenes were randomly distributed throughout the chromosome. Factors resulting in pseudogene transcription included: 1) co-orientation of transcribed pseudogenes with transcribed ORFs within or exclusive of operon-like structures; 2) the paucity of intrinsic stem-loop transcriptional terminators between transcribed ORFs and downstream pseudogenes; and 3) predicted pseudogene promoters. Mechanisms for translational silencing of pseudogene transcripts included the lack of both translational start codons and strong Shine-Dalgarno sequences. Transcribed pseudogenes also contained multiple in-frame stop codons and high Ka/Ks ratios, compared to that of homologs in M. tuberculosis and ORFs in M. leprae. A pseudogene transcript containing an active promoter, strong SD site, a start codon, but containing two in frame stop codons yielded a protein product when expressed in E. coli. Approximately half of M. leprae's transcriptome consists of inactive gene products consuming energy and resources without potential benefit to M. leprae. Presently it is unclear what additional detrimental affect(s) this large number of inactive mRNAs has on the functional capability of this organism. Translation of these pseudogenes may play an important role in overall energy consumption and resultant pathophysiological characteristics of M. leprae. However, this study also demonstrated that multiple translational silencing mechanisms are present, reducing additional energy and resource expenditure required for protein production from the vast majority of these transcripts.

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.

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Deciphering the genetic control of gene expression following Mycobacterium leprae antigens stimulation

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