Drug discovery and resistanceGene expression analysis of two extensively drug-resistant tuberculosis isolates show that two-component response systems enhance drug resistance
Introduction
Since the introduction of several anti-tuberculosis drugs, the transmission of tuberculosis (TB) has been successfully controlled by advances in treatment regimens. However, the emergence of multidrug-resistant strains [MDR, resistance to at least rifampicin (RMP) and isoniazid (INH)] and extensively drug-resistant strains (XDR), with additional resistance to a fluoroquinolone, and any one of the injectable drugs kanamycin, amikacin, and capreomycin (CPM)] has rendered TB as one of the most prevalent and deadly infectious diseases worldwide.
A large number of mutations preferentially associated with various anti-TB drug resistance have been identified in resistant clinical isolates [49], [66]. Most of these mutations were located within the coding region, indicating that structural alterations of drug target protein play a major role in the emergence and development of resistance in clinical isolates. However, the detection of certain resistance-associated intergenic mutations is also suggestive of a close relationship between resistance and gene expression regulation [66]. It has been previously reported that the downregulated expression of katG results in INH resistance [3], and an increase in the transcription of embAB significantly alters resistance to ethambutol in Mycobacterium smegmatis (M. smegmatis) [52]; however, this occurrence has not been verified in Mycobacterium tuberculosis (Mtb) [2].
To explore the mechanism of drug resistance in Mtb., most research investigations have focused on resistance-associated genetic mutations, whereas those examining resistance-related gene expression are limited. Drug responsive genes, especially induced genes, might be a clue to understanding the underlying mechanisms behind drug action, and mutations within these genes could also induce resistance in clinical isolates, in addition to known resistance genes. To date, the genes with altered expression by INH exposure have been extensively studied in Mtb mainly by using microarray hybridization [1], [9], [23], [33], [60], [62]. In addition to known INH targets such as genes coding for type II fatty acid synthase enzymes, several genes that have not been previously characterized in terms of drug resistance were identified as INH-induced genes, several of which were subsequently found to be related to INH resistance in clinical isolates [42], indicating that the study of drug response genes (DRGs) is of clinical significance.
However, reports on the global expression analysis in relation to drug exposures in resistant clinical isolates are limited. In the current study, to further elucidate the correlation of drug response genes with resistance in clinical isolates, global expression analysis of two clinical XDR isolates, whose genomes have been previously [64]sequenced, and a reference strain H37Rv, was performed after exposure to three anti-TB drugs (INH, CPM, and RMP). The analysis showed that known drug resistance-associated genes were upregulated in the XDR isolates relative to that observed in H37Rv, indicating an association between gene expression and resistance. Moreover, the genes responded to INH and CPM, but not RMP, which was highly consistent with the differentially expressed genes in the XDR isolates compared to that observed in H37Rv. Based on this relationship, we identified 92 genes as candidate drug resistance genes (CRGs) in the XDRs, then pathway enrichment and transcriptional regulation analyses were conducted on the CRGs. Several regulatory genes were associated to resistance in Mtb, and then a drug sensitivity test was performed to investigate these relationships, exploring the roles of transcriptional regulation on drug resistance in Mtb.
Section snippets
Bacterial strains and drug treatment
Mtb strain TMC 102 [H37Rv] (ATCC Number 27294) was provided by the Chinese Institute for the Control of Pharmaceutical and Biologic Products. XDR1219 and XDR1221 were obtained from the Shanghai Pulmonary Hospital affiliated to Tongji University. The minimal inhibitory concentrations were determined using the BACTEC MGIT 960 system according to standard procedures [47], [48]. The genome sequences of the XDR isolates have been recently reported by our research group [64].
Strains were grown in
Identification of differentially expressed genes
Tukey's honest significance test combined with FDR and fold change were conducted to determine specifically expressed genes for strains or drug exposures. A large number of genes were displayed differentially expression in the two XDR strains compared to that observed in H37Rv, but dramatically deceased between XDRs (Table 1). In addition, <10 genes were detected as response genes for each drug (data not shown) by using this method. The observed expression variance across distinct arrays was
Discussion
It is seemingly obvious that genes with altered expression in response to anti-bacterial drugs may be correlated with resistance. However, as far we know, studies examining the relationship between drug response and resistance in Mtb clinical isolates are limited. In the present study, we chose two extensively drug-resistant strains (XDR1219 and XDR1221) and a reference strain, H37Rv, to explore the correlation of transcriptional response to three anti-TB compounds (INH, CPM, and RMP) with
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These authors contributed equally to this work.