Gene expression analysis of two extensively drug-resistant tuberculosis isolates show that two-component response systems enhance drug resistance

Summary

Global analysis of expression profiles using DNA microarrays was performed between a reference strain H37Rv and two clinical extensively drug-resistant isolates in response to three anti-tuberculosis drug exposures (isoniazid, capreomycin, and rifampicin). A deep analysis was then conducted using a combination of genome sequences of the resistant isolates, resistance information, and related public microarray data. Certain known resistance-associated gene sets were significantly overrepresented in upregulated genes in the resistant isolates relative to that observed in H37Rv, which suggested a link between resistance and expression levels of particular genes. In addition, isoniazid and capreomycin response genes, but not rifampicin, either obtained from published works or our data, were highly consistent with the differentially expressed genes of resistant isolates compared to those of H37Rv, indicating a strong association between drug resistance of the isolates and genes differentially regulated by isoniazid and capreomycin exposures. Based on these results, 92 genes of the studied isolates were identified as candidate resistance genes, 10 of which are known resistance-related genes. Regulatory network analysis of candidate resistance genes using published networks and literature mining showed that three two-component regulatory systems and regulator CRP play significant roles in the resistance of the isolates by mediating the production of essential envelope components. Finally, drug sensitivity testing indicated strong correlations between expression levels of these regulatory genes and sensitivity to multiple anti-tuberculosis drugs in Mycobacterium tuberculosis. These findings may provide novel insights into the mechanism underlying the emergence and development of drug resistance in resistant tuberculosis isolates and useful clues for further studies on this issue.

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.