Identification of Novel Efflux Proteins Rv0191, Rv3756c, Rv3008, and Rv1667c Involved in Pyrazinamide Resistance in Mycobacterium tuberculosis

Abstract

Pyrazinamide (PZA) is a critical drug used for the treatment of tuberculosis (TB). PZA is a prodrug that requires conversion to the active component pyrazinoic acid (POA) by pyrazinamidase (PZase) encoded by the pncA gene. Although resistance to PZA is mostly caused by pncA mutations and less commonly by rpsA, panD, and clpC1 mutations, clinical strains without these mutations are known to exist. While efflux of POA was demonstrated in Mycobacterium tuberculosis previously, the efflux proteins involved have not been identified. Here we performed POA binding studies with an M. tuberculosis proteome microarray and identified four efflux proteins (Rv0191, Rv3756c, Rv3008, and Rv1667c) that bind POA. Overexpression of the four efflux pump genes in M. tuberculosis caused low-level resistance to PZA and POA but not to other drugs. Furthermore, addition of efflux pump inhibitors such as reserpine, piperine, and verapamil caused increased susceptibility to PZA in M. tuberculosis strains overexpressing the efflux proteins Rv0191, Rv3756c, Rv3008, and Rv1667c. Our studies indicate that these four efflux proteins may be responsible for PZA/POA efflux and cause PZA resistance in M. tuberculosis. Future studies are needed to assess their roles in PZA resistance in clinical strains.

Phenotypic and genotypic characterization of pyrazinamide resistance among multidrug-resistant Mycobacterium tuberculosis clinical isolates in Hangzhou, China

“…Other genetic background mutations together with the pncA mutations may help determine the susceptibility of the isolates. Recently, new genes like clpC1 [36,37], rv2783 [38], four efflux genes [39] and mas/ppeA-E pathway genes [40] have been identified as contributors of PZA resistance, although their impact on MIC has been small. Second, the isolates were geographically limited to a single province, and the sequence analysis could therefore be limited to a single East Asian lineage of TB.…”

Function, essentiality, and expression of cytochrome P450 enzymes and their cognate redox partners in Mycobacterium tuberculosis: are they drug targets?

“…The expression of the three pks is downregulated except for pks17 and pks9 genes ( Rv1663 and Rv1664 , respectively) upon exposure to ciprofloxacin and rifampicin. In contrast, Rv1667c is also induced upon exposure to first-line antibiotics (Zhang et al 2017 ; Boot et al 2018 ). Furthermore, it can also be speculated that increased expression of cyp139a1 (or one of the other CYPs) contributes to the intrinsic resistance of Mtb to anti-TB agents.…”

Structure guided prediction of Pyrazinamide resistance mutations in pncA

“…postulated that the mechanism of action of PZA is through POA, which disrupts the bacterial membrane energetics and inhibits the membrane transport function which is necessary for the survival of the bacterium, at an acidic site of infection 16 . PZA resistance has been linked to mutations in a number of genes, including pncA, rpsA 17 , panD 18 , clpC1 19 , and the putative efflux pumps Rv0191, Rv3756c, Rv3008, and Rv1667c 20 , but mutations in pncA are the major mechanism for PZA resistance (70-97%) 21 . While sequencing the pncA gene can be a more reliable method to determine resistance than DST, which is prone to missing low-level pyrazinamide resistance caused by non-synonymous mutations in pncA 22 , the development of a genetics based resistance screen is complicated as resistant and non-resistant mutations are found across the entire protein.…”