Characterization of efflux pumps genes involved in second-line drug resistance of tuberculosis

Abstract

In this study, we used molecular and proteomic methods to detect novel changes within drug-resistant tuberculosis (TB) strains. Our goal was to detect changes within efflux pump (EP) genes of extensively drug resistant (XDR-TB) strains using genomic and transcriptomic methods. We firstly sequenced multiple genes in discordant samples that lacked molecular markers present on the GenoType® MTBDRsl assay. Further analysis by whole genome sequencing was done on XDR-TB strains. Transcriptomic changes of EP genes were detected by RNA sequencing strains. The minimum inhibitory concentration (MIC) of second-line drugs in the presence and absence of efflux pump inhibitors was also measured. Proteomic expression by cloning and expression of three efflux pump genes (Rv1258c, Rv1634 and Rv0194) was done and effect on MICs of second-line drugs measured in the presence and absence of efflux pump inhibitors. We analysed molecular markers responsible for resistance to second-line drugs of ofloxacin (OFX), amikacin (AMK), kanamycin (KAN) and capreomycin (CAP) in 636 drug-resistant strains using GenoType® MTBDRsl. We compared GenoType® MTBDRsl with phenotypic second-line drug susceptibility testing. After comparison, 102 (14.8%) strains were discordant between the two methods. In the discordant population, genetic regions of gyrA, gyrB, rrs, eis, tlyA and EP genes (Rv1634, Rv1258c and Rv0194) were sequenced and analysed in search of mutations. Combining sequencing and GenoType® MTBDRsl significantly improved the diagnosis of XDR-TB and second-line drugs. The Rv0194 belongs to the ATP (adenosine triphosphate) binding cassette (ABC) family while Rv1258c and Rv1634 belong to the major facilitator superfamily (MFS) transporters. Since these genes are implicated in multiple drug resistance, our hypothesis was that possible mutations in these genes could confer cross-resistance. Our analysis revealed the appearance of Rv1258c and Rv0194 mutations in strains with cross-resistance to second-line injectable drugs. Further analysis revealed rrs G878A mutation that was specific to EuroAmerican X3 lineage (P<0.001) and linked to CAP resistance. The inclusion of G878A in new rapid assays might be beneficial for rapid CAP resistance detection. Whole genome sequencing with increased resolution and depth was used to study two XDR-TB strains. Drug resistant mutations were detected for all other drugs except for OFX in one strain. We further analysed EP genes for mutations. Bioinformatic prediction tools detected protein changes related to EP gene mutations belonging to Rv0987, Rv2039 and Rv0402. Two of the efflux pump genes (Rv0987, Rv2039) belong to the ABC family, while Rv0402 is of resistance nodulation-cell division (RND) family. Mutations within lipid metabolism and secretion pathways were also detected. To fully understand the role of EP gene mechanisms at a transcriptional level, we sequenced RNA molecules of 11 XDR-TB, five MDR and two susceptible strains. The RNA signatures of EP and lipid metabolism genes detected in XDR-TB strains were characterized. Further analysis of four XDR strains with MIC data with or without efflux pump inhibitors (EPIs) was performed in relation to RNA sequenced data. The ABC Rv2686/87/88c operon was significantly over-expressed in the background on strains with gyrA mutations causing OFX resistance. The Rv1258c, Rv0194 and Rv1634 EP genes were consistently over-expressed in XDR TB strains. Efflux pump inhibitor of piperine was effective in reducing MICs to hydrophilic drugs of AMK and OFX whereas verapamil reduced MIC of hydrophobic CAP drug. Protein-protein interaction pathways revealed novel associations between ABC, RND and type VII secretion (T7S) proteins. Finally, we cloned and expressed Rv1258c, Rv0194 and Rv1634 EP genes in Mycobacterium vectors. These genes have the potential to cause multidrug resistance. We did not detect increased MIC levels of second-line drugs in the presence of the clones. However, a reduction of MICs in the presence of the EPI, piperine, was observed. Bioinformatic approaches also revealed transmembrane motifs, domains and loops. Since EP genes are implicated in transport of substrates across cell membrane, we used biofilm formation assays to determine the role of each clone. Both Rv1258c and Rv0194 clones showed biofilm formation. Such discovery highlights the secretion of lipid bodies on the cell wall of the bacteria through EP genes/proteins. This information will allow us to develop novel strategies to treat drug-resistant TB. Our study emphasises the importance of EP gene mechanisms in causing drug resistance. The combination of EP and target genes is important in the detection of second-line drugs. Furthermore, it is suggested that EPIs combined with second-line drugs might be effective in the treatment of XDR-TB.