Biophysical Society Thematic Meeting - November 16-20, 2015

93 / 101

Biophysics in the Understanding, Diagnosis, and Treatment of Infectious Diseases Poster Abstracts
88
12-POS Board 12
Whole Transcriptome Analysis of Clinical Isolates of Mycobacterium Tuberculosis Using
RNA-Seq
Lynne De Welzen1, Vegard Eldholm2, Keira A. Cohen3,1, Kashmeel Maharaj 1, Kaitlin Stouffer 1,
Alexander S. Pym1 .
1KwaZulu-Natal Research Institute for Tuberculosis and HIV (K-RITH), Durban, KwaZulu-
Natal, South Africa, 2 Norwegian Institute of Public Health, Oslo, Norway, 3Pulmonary and
Critical Care Division, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA,
USA.
Background: Drug resistance of Mycobacterium tuberculosis ( 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 may identify additional mechanisms of drug resistance.
Aims: To identify transcriptional changes in that lead to drug resistance or compensate for
loss of fitness due to a drug resistance conferring mutation.
Methods: Twelve clinical isolates of 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 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.