Biophysical Society Thematic Meeting - November 16-20, 2015

Biophysics in the Understanding, Diagnosis, and Treatment of Infectious Diseases Poster Abstracts

12-POS Board 12 Whole Transcriptome Analysis of Clinical Isolates of Mycobacterium Tuberculosis Using RNA-Seq Lynne De Welzen 1 , Vegard Eldholm 2 , Keira A. Cohen 3,1 , Kashmeel Maharaj 1 , Kaitlin Stouffer 1 , Alexander S. Pym 1 . 1 KwaZulu-Natal Research Institute for Tuberculosis and HIV (K-RITH), Durban, KwaZulu- Natal, South Africa, 2 Norwegian Institute of Public Health, Oslo, Norway, 3 Pulmonary and Critical Care Division, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA. Background: Drug resistance of Mycobacterium tuberculosis ( M.tb ) has become an epidemic of global proportions. While much drug resistance can be attributed to known mechanisms, some resistance remains unexplained. Investigation of transcriptional adaptations among clinical isolates of drug resistant M.tb may identify additional mechanisms of drug resistance. Aims: To identify transcriptional changes in M.tb that lead to drug resistance or compensate for loss of fitness due to a drug resistance conferring mutation. Methods: Twelve clinical isolates of M.tb were selected for RNA-seq. These included Beijing and KZN spoligotypes, with varying drug susceptibilities. RNA sequencing and full genome sequencing was conducted on the Illumina HiSeq 2000 platform to identify mutations that could account for transcriptional changes. Sequenced reads were aligned to the M.tb reference genome H37Rv, and fold change differences were calculated using DNASTAR. A dual colour fluorescent protein promoter assay, in conjunction with flow cytometry, was used to assess the promoter activity of intergenic mutations. Results: Within the Beijing spoligotype the number of genes that were up or down regulated, relative to drug susceptible strains, was greater in MDR and PXDR strains compared to mono resistant strains. A genomic-transcriptomic analysis identified 3 out of 81 and 2 out of 37 intergenic mutations for the Beijing and KZN spoligotypes respectively that were associated with a 4-fold or greater differential regulation in the adjacent gene. The fluorescent reporter assay identified a significant decrease in promoter activity relative to wild-type in a mutation associated with Rv3854c. Discussion: The evolution of drug resistance can result in a large variation in the global transcriptional profile of XDR strains relative to drug susceptible strains. The minority of these are due to promoter mutations, suggesting the involvement of other global regulatory pathways.

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