Intrinsic Resistance of Mycobacterium smegmatis to Fluoroquinolones May Be Influenced by New Pentapeptide Protein MfpA

Abstract

The fluoroquinolones (FQ) are used in the treatment of Mycobacterium tuberculosis, but the development of resistance could limit their effectiveness. FQ resistance (FQ R ) is a multistep process involving alterations in the type II topoisomerases and perhaps in the regulation of efflux pumps, but several of the steps remain unidentified. Recombinant plasmid pGADIV was selected from a genomic library of wild-type (WT), FQsensitive M. smegmatis by its ability to confer low-level resistance to sparfloxacin (SPX). In WT M. smegmatis, pGADIV increased the MICs of ciprofloxacin (CIP) by fourfold and of SPX by eightfold, and in M. bovis BCG it increased the MICs of both CIP and SPX by fourfold. It had no effect on the accumulation of 14 C-labeled CIP or SPX. The open reading frame responsible for the increase in FQ R , mfpA, encodes a putative protein belonging to the family of pentapeptides, in which almost every fifth amino acid is either leucine or phenylalanine. Very similar proteins are also present in M. tuberculosis and M. avium. The MICs of CIP and SPX were lower for an M. smegmatis mutant strain lacking an intact mfpA gene than for the WT strain, suggesting that, by some unknown mechanism, the gene product plays a role in determining the innate level of FQ R in M. smegmatis.Although the fluoroquinolones (FQ) are used against multidrug-resistant Mycobacterium tuberculosis (1), initial experience with ciprofloxacin (CIP) and ofloxacin (OFX) resulted in the rapid development of resistance (27,34) and the use of the more effective sparfloxacin (SPX) is limited by toxicity (24). Nevertheless, newer drugs, such as moxifloxacin and gatifloxacin, have higher therapeutic ratios (8) and appear promising. Therefore, continued study of the mechanisms by which FQ resistance (FQ R ) develops in mycobacteria seems warranted. Other bacteria, such as Escherichia coli, Streptococcus pneumoniae, and Staphylococcus aureus, develop FQ R in a stepwise process, principally involving alterations in the A and B subunits of both gyrase and topoisomerase IV (30). However, there appear to be other elements involved in FQ R , because a very high level of resistance may not be completely explained by mutations in topoisomerase genes and strains with a low level of resistance may lack mutations in these genes (39). The only other mechanism that has been implicated in FQ R is the increased expression of FQ-transporting efflux pumps (29), which could play a role in both low-level and very high-level resistance (25).Mycobacteria do not contain topoisomerase IV (7), and strains with moderate levels of resistance to CIP, OFX, or levofloxacin (LVX) (Ͼ3 g) or SPX (Ͼ1 g) all contain gyrA mutations (36 MATERIALS AND METHODSBacterial strains and growth conditions. Wild-type (WT), FQ-sensitive M. smegmatis strain mc 2 155 was used for genetic selections (14). Some plasmids were also tested for the ability to confer resistance in M. bovis BCG Pasteur. Mycobacteria were grown in 7H9 oleic acid-albumin-dextrose-0.05% Tween 80 (OAD-TW) liquid or on 7H10 ...

The Pentapeptide Repeat Proteins MfpA Mt and QnrB4 Exhibit Opposite Effects on DNA Gyrase Catalytic Reactions and on the Ternary Gyrase-DNA-Quinolone Complex

“…Genes encoding PRPs were found in the genomes of various bacteria, such as mfpA-like genes in M. tuberculosis and other mycobacteria (M. avium, M. bovis, and M. leprae) (29) or qnr-like genes in environmental bacteria such as Shewanella and Vibrionaceae (10,31,40). Plasmid-borne qnr genes detected in quinolone-resistant strains of Enterobacteriaceae are derived from these chromosome-borne genes.…”

“…The mechanisms of intrinsic quinolone resistance in M. tuberculosis are due to the low permeability of its thick, lipidcontaining cell wall (25) and to the low affinity of its gyrase for quinolones (10-fold lower than that of E. coli gyrase) (3). Since mfpA was described as a new fluoroquinolone resistance determinant in M. smegmatis (29), the corresponding protein in M. tuberculosis, MfpA Mt , was proposed to be another determinant of intrinsic quinolone resistance. Moreover, its similarity to Qnr proteins led to the hypothesis that it could mediate fluoroquinolone resistance by gyrase protection (43).…”

“…mfpA, a chromosomal gene that encodes a 192-amino-acid PRP, is an intrinsic quinolone resistance determinant of Mycobacterium smegmatis (29). A similar gene, mfpA Mt , was found in the M. tuberculosis genome, and MfpA Mt shows 67% identity with MfpA.…”

DNA Mimicry by Antirestriction and Pentapeptide Repeat (PPR) Proteins

“…However, the wide use of quinolones in medical practice resulted in the discovery of a new type of quinolone resistance. It was shown that the gene determining such type of resistance in M. smegmotis and M. tuberculosis encodes the MfpA protein, a specific inhibitor of DNA gyrase (Hegde et al, 2005;Montero et al, 2001). The MfpA proteins of M. tuberculosis and M. smegmotis consist of 183 and 192 residues correspondently; they share 67% identity.…”

“…MfpA and Qnr (A,B,C,D,S) are two newly characterized pentapeptide repeat proteins (PPRs) that interact with type II topoisomerase (DNA gyrase) and confer bacterial resistance to the drugs quinolone and fluoroquinolone [Hegde et al, 2005;Hedge et al, 2011). The mfpA gene is chromosome borne in Mycobacterium tuberculosis (Hegde et al, 2005;Montero et al, 2001), while qnr genes are plasmid borne in Gram-negative enterobacteria (Martinez-Martinez, L. et al,1998;Tran et al, 2005;Cattoin & Nordmann, 2009;Rodriguez-Martinez et al 2011). The size, shape, and surface potential of MfpA and Qnr proteins mimics duplex DNA (Hegde et al, 2005;Vetting et al, 2009;Hegde et al, 2011).…”