Identifying the fitness cost of rifampicin-resistance in Mycobacterium tuberculosis through RNAseq profiling.
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Illumina HiSeq 2500
ORGANISM(S): Mycobacterium tuberculosis
SUBMITTER: Andrej Trauner
PROVIDER: E-MTAB-7359 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Antimicrobial resistance (AMR) poses a threat to global health and the economy. Rifampicin resistant Mycobacterium tuberculosis accounts for a third of the global AMR burden. Gaining the upper hand on AMR requires a deeper understanding of the physiology of resistance. AMR often results in the erosion of normal cell function: a fitness cost. Identifying intervention points in the mechanisms underpinning the cost of resistance in M. tuberculosis could play a pivotal role in strengthening future treatment regimens. We used a collection of M. tuberculosis strains providing an evolutionary and phylogenetic snapshot of rifampicin resistance and subjected them to genome-wide transcriptomic and proteomic profiling to identify key perturbations of normal physiology. We found that a rifampicin resistance-conferring mutation in RpoB imparts considerable gene expression changes, many of which are mitigated by a compensatory mutation in RpoC. However, our data also provide evidence for pervasive epistasis: the same resistance mutation imposed a different fitness cost and functionally unrelated changes to gene expression in clinical strains from unrelated genetic backgrounds. Rather than functional changes in specific pathways, our data suggest that the fitness cost of rifampicin resistance stems from a misallocation of resources: the greater the departure from the wild type baseline proteome investment, the greater the fitness cost of rifampicin resistance in a given strain. We summarize these observations in the “Burden of Expression” hypothesis of fitness cost and provide evidence that it can be used for suppressing the emergence of rifampicin resistance.
2021-03-12 | PXD011568 | Pride
Project description:Tuberculosis (TB) is still a major life-threatening infectious disease, within which especially the rise of multidrug resistant TB (MDR-TB) is currently worrying. This study focuses on mechanisms of development of rifampicin resistance, since rifampicin seems to play an important role in the development of MDR-TB. To provide further insight in rifampicin resistance, we performed a genome-wide transcriptional profile analysis for Mycobacterium tuberculosis (M. tuberculosis) using microarray technology and qRT-PCR analysis. We exposed a rifampicin-susceptible H37Rv wild type (H37Rv-WT) and a rifampicin-resistant progeny H37Rv strain with a H526Y mutation in the rpoB gene (H37Rv-H526Y) to several concentrations of rifampicin, to define the effect of rifampicin on the transcription profile. Our study showed that there are resistance-dependant differences in response between both M. tuberculosis strains. Gene clusters associated with efflux, transport and virulence were altered in the rifampicin-resistant H37Rv mutant compared to the rifampicin-susceptible H37Rv-WT strain after exposure to rifampicin. We conclude that the small gene cluster Rv0559c-Rv0560c in the H37Rv-H526Y strain was remarkably up-regulated in the microarray analysis and qRT-PCR results and appeared to be dependent on rifampicin concentration and time of exposure. Therefore this study suggests that Rv0559c and Rv0560c play a pivotal role in rifampicin resistance of M. tuberculosis. Further investigation of Rv0559c and Rv0560c is needed to reveal function and mechanism of both genes that were triggered upon rifampicin exposure. [Data is also available from http://bugs.sgul.ac.uk/E-BUGS-139]
2013-04-21 | E-BUGS-139 | biostudies-arrayexpress
Project description:The present study was aimed at analyzing (i) the biological cost of RNA polymerase (rpoB) mutations conferring rifampin resistance on H.pylori, (ii) the relationship between the cost of rpoB mutations and the chromosomal mutaion, (iii) the relationship between the cost of rpoB mutations and the transcription profile of sensitive and resistantrif strains of H.pylori (iv) and rpoB mutations in view of the possible fitness burden associated with resistance to another antibiotics. H.pylori reference strain 26695 was routinely maintained on Columbia agar plates and H. pylori-selective antibiotic mix Dent. Liquid culture was grown in BHI broth. Both plates and broth cultures were incubated at 37C under atmosphere enriched with 5% CO2 for 2-3 days . Mutant strains were selected by culturing H. pylori 26695 on selective plates containing rifampicin. In 5 days resistant colonies were picked up and passed under rifampicin pressure. RNA isolated was reverse transcribed and used to probe H. pylori home-made arrays
2010-11-30 | E-GEOD-25333 | biostudies-arrayexpress
Project description:The global diversity of Mycobacterium tuberculosis comprises at least seven lineages, each with its distinct geographic distribution. The aim of this experiment was to perform a comparative analysis of two of these lineages: Lineage 1 and Lineage 2. The former is found around the rim of the Indian ocean and in south-east Asia, while the latter is widely spread throughout Asia and shows an increasing global spread. We have chosen three fully drug susceptible clincal isolates to represent each of the two lineages. We performed RNAseq analysis on rRNA depleted samples isolated from cultures during mid-log phase. Each strain was measured in triplicate.
2020-12-31 | E-MTAB-8103 | biostudies-arrayexpress
Project description:The present study was aimed at analyzing (i) the biological cost of RNA polymerase (rpoB) mutations conferring rifampin resistance on H.pylori, (ii) the relationship between the cost of rpoB mutations and the chromosomal mutaion, (iii) the relationship between the cost of rpoB mutations and the transcription profile of sensitive and resistantrif strains of H.pylori (iv) and rpoB mutations in view of the possible fitness burden associated with resistance to another antibiotics.
2010-11-30 | GSE25333 | GEO
Project description:Mutations in the rifampicin (Rif)-binding site of RNA polymerase (RNAP) impart antibiotic resistance and inextricably affect transcription initiation, elongation, and termination properties as well. At each step of the transcription cycle, RNAP responds to non-essential transcription factors, signaling molecules, and substrate availability. As such, the non- essential genome and its impact on fitness cost potentially represent an untapped resource for new combination therapies. Using transposon sequencing (Tn-seq), we present a genome- wide analysis of resistance cost in a clinically common rpoB H526Y mutant. Our data show that cost-compounding genes include factors that promote high transcription elongation rate, whereas cost-mitigating genes function in cell wall synthesis and division. We demonstrate that cell wall synthesis and division defects in rpoB H526Y are a consequence of an abnormally high transcription elongation rate, which is further exacerbated by superfluous activity of the uracil salvage pathway and indifference of the mutant RNAP to alarmone ppGpp. Leveraging on this knowledge, we identified drugs that are highly potent against rpoB H526Y and other RifR alleles from the same phenotypic class. Thus, genome-wide analysis of fitness cost of antibiotic resistant mutants should expedite discovery of new combination therapies and delineate cellular pathways that underlie molecular mechanisms of cost.
2021-11-25 | PXD027810 | Pride
Project description:We measured steady-state E. coli transcript levels in 1) a pykF(C8Y) mutant, 2) a rpoB(T1037P) mutant, and 3) five rifampicin-resistant rpoB mutant strains selected from each of these single mutants, in the presence and absence of rifampicin.
2020-10-23 | GSE136977 | GEO
Project description:The alarming rise of antimicrobial resistance in Mycobacterium tuberculosis coupled with the shortage of new antibiotics has made tuberculosis (TB) control a global health priority. Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit the growth of multi-drug resistant isolates of M. tuberculosis. Repurposing NSAIDs, with known clinical properties and safety records, offers a direct route to clinical trials. Therefore we investigated the novel mechanisms of anti-mycobacterial action of the NSAID, carprofen. Integrative molecular and microbiological approaches revealed that carprofen, a bactericidal drug, inhibited bacterial drug efflux mechanisms. In addition, carprofen restricted mycobacterial biofilm-like growth, highlighting the requirement of efflux-mediated communicative systems for the formation of biofilms. Transcriptome profiling revealed that carprofen likely acts by inhibiting respiration through the disruption of membrane potential, which may explain why spontaneous drug-resistant mutants could not be raised due to the pleiotropic nature of carprofen’s anti-tubercular action. This immunomodulatory drug has the potential to reverse TB antimicrobial resistance by inhibiting drug efflux pumps and biofilm formation, and paves a new chemotherapeutic path for tackling tuberculosis.
2020-08-15 | E-MTAB-6191 | biostudies-arrayexpress
Project description:Three types of phenotypic expression of M-CM-^_-lactam resistance has been reported in MRSA: heterogeneous-, homogeneous-, and Eagle-type resistance. Heterogeneous to homogeneous (hetero-to-homo) conversion of M-CM-^_-lactam resistance is postulated to be caused by a chromosomal mutation (chr*) together with mecA-gene expression. The Eagle-type resistance is a special pattern of chr* expression in the pre-MRSA strain N315 under the strong mecI-gene mediated repression of mecA gene transcription. Here, for the identification of chr*, experiments were conducted using an in-vitro derived homogeneously imipenem-resistant MRSA strain N315M-bM-^HM-^FIPH5 (M-bM-^HM-^FIPH5). The strain was selected with imipenem 8 mg/L from the heterogeneously imipenem-resistant MRSA strain N315M-bM-^HM-^FIP (M-bM-^HM-^FIP). The whole genome sequencing of M-bM-^HM-^FIPH5 revealed the presence of a unique mutation in the rpoB gene, rpoB(N967I), causing the amino-acid (AA) substitution of Asp by Ile at the 967th AA position of the RNA polymerase M-CM-^_ subunit. The effect of the mutation on the phenotypic change was confirmed by constructing and studying the phenotype of the strains H5rpoB(I967N), a M-bM-^HM-^FIPH5-derived strain cured of the rpoB mutation, and N315rpoB(N967I), a N315-derived strain introduced with the mutated rpoB gene. H5rpoB(I967N) regained the hetero-MRSA phenotype, and the mutant strain N315rpoB(N967I) showed an Eagle-type phenotype similar to that of N315h4. Furthermore, subsequent whole genome sequencing revealed that N315h4 also had a missense mutation in the rpoB gene rpoB(R644H). The rpoB mutations caused decreased autolysis, prolonged doubling-time, and tolerance to bactericidal concentrations of methicillin. We concluded that the certain rpoB mutations are chr* responsible for the hetero-to-homo phenotypic conversion of MRSA. We compared the gene expression profiles of the wild-type strain and rpoB mutant (N967I) using a 60mer oligo array.
2013-08-27 | E-GEOD-42218 | biostudies-arrayexpress
Project description:This series of microarry is the study of the transcriptional profile asm gene clusster of high Ansamitocin P3 producing mutants derived from isolating rifampicin resistant mutants. Background The Actinomycete Actinosynnema pretiosum ssp. auranticum has commercial importance due to its production of ansamitocin P-3 (AP-3), a potent antitumor agent. One method to improve the productivity of A. pretiosum is to genetically alter the regulation of ansamitocin biosynthesis by manipulating selected genes. To identify potential targets for genetic engineering, the transcriptional profile of an A. pretiosum mutant with enhanced AP-3 yield was compared to the parental strain. High-producing mutants were isolated from rifampicin-resistance screens using a plate-based bioassay. Transcriptional profiling of genes in the ansamitocin biosynthetic cluster was carried out using a custom-designed A. pretiosum microarray and verified with quantitative RT-PCR. Results Genes involved in the synthesis of the AP-3 precursor, 3-amino-5-hydroxybenzoic acid (AHBA), including asm43, asm45 and asm47, were significantly up-regulated (P-value<0.05) by more than two-fold in the high-producing mutant relative to the parental strain. In addition, genes involved in polyketide synthesis (asmB and asmD), bicyclomycin resistance (asm35) and transcriptional regulation (asm29) were up-regulated, while the transcriptional repressor, asm2, was down-regulated. Interestingly, the two AHBA synthase gene homologues, asm24 and asm43, had divergent expression profiles despite their strong sequence similarity and functional complementarities. The rifampicin-resistance mutation associated with increased AP-3 production was mapped to the gene encoding RNA polymerase beta subunit, rpoB. While distinct multiple mutations in RpoB were noted for each of the mutants sequenced, a common H436R mutation was identified in cluster I of rpoB in all four mutants sequenced. Conclusions The transcriptional analysis has provided useful insights into the mechanism underlying the increased AP-3 production in the high-producer mutant and expanded our understanding of the function and regulation of genes in the ansamitocin biosynthetic cluster. In addition, the differentially expressed genes identified are potential targets for genetic manipulation; alternatively, they can be used in reporter-based selections to isolate mutants with greater AP-3 productivity. Keywords: genetic modification, time course, boutique microarray, quantile normalization Microarray chip was hybdridized with cDNA obtained from samples vs sheared A. pretiosum gDNA. cDNA obtained from cultures of A. pretiosum (ATCC:31565) and r50D1 mutant were used. Cell pellets were harvested on days 2,4,6 and 8 of each culture.Three biological replicates were performed for each cell line with two technical replicates for each biological replicate:
2008-12-01 | E-GEOD-12739 | biostudies-arrayexpress