Overexpression of mfpA Gene Increases Ciprofloxacin Resistance in Mycobacterium smegmatis.

Carlos Aranaga,Aura Falco,Simona Nardoni,Howard Takiff,Ivan Ocampo

doi:10.1155/2021/6689186

Abstract

Fluoroquinolones (FQs) are antibiotics useful in the treatment of drug-resistant tuberculosis, but FQ-resistant mutants can be selected rapidly. Although mutations in the DNA gyrase are the principal cause of this resistance, pentapeptide proteins have been found to confer low-level FQ resistance in Gram-negative bacteria. MfpA is a pentapeptide repeat protein conserved in mycobacterial chromosomes, where it is adjacent to a group of four highly conserved genes termed a conservon. We wished to characterize the transcriptional regulation of the mfpA gene and relate its expression to ciprofloxacin resistance in M. smegmatis. Reverse transcription PCR showed that mfpA gene is part of an operon containing the conservon genes. Using a transcriptional fusion, we showed that a promoter was located 5′ to the mfpEA operon. We determined the promoter activity under different growth conditions and found that the expression of the operon increases slightly in late growth phases in basic pH and in subinhibitory concentrations of ciprofloxacin. Finally, by cloning the mfpA gene in an inducible vector, we showed that induced expression of mfpA increases the ciprofloxacin Minimal Inhibitory Concentration. These results confirm that increased expression of the mfpA gene, which is part of the mfpEA operon, increases ciprofloxacin resistance in M. smegmatis.

Highlights

Fluoroquinolones (FQs) are the most important antibiotics in drug regimens used for treating Multidrug-Resistant Tuberculosis (MDR-TB) [1]. ey exert a powerful bactericidal activity and can penetrate macrophages, where the tuberculosis-causing Mycobacterium tuberculosis bacilli reside [2]. ere are, reservations regarding FQ use because resistant mutants can be selected in a remarkably short time [1]. e targets of the FQs are the DNA gyrase and DNA Topoisomerase IV
Pentapeptides were initially associated with FQ resistance when it was found that a plasmid containing the mfpA (Mycobacterial Fluoroquinolone Resistance Protein) gene of International Journal of Microbiology
M. smegmatis mc2155 strains containing pJEM15 or pJEM-5-11 (Table 1) were grown in 7H9 broth with C2FDG substrate (0.33 μM), and promoter activity was measured as fluorescence resulting from the hydrolysis of C2FDG by beta-galactosidase. e fluorescence produced with plasmid pJEM-5-11 indicated that the cloned fragment contained a promoter whose activity correlated with bacterial growth (Figure 3)

Summary

Introduction

Fluoroquinolones (FQs) are the most important antibiotics in drug regimens used for treating Multidrug-Resistant Tuberculosis (MDR-TB) [1]. ey exert a powerful bactericidal activity and can penetrate macrophages, where the tuberculosis-causing Mycobacterium tuberculosis bacilli reside [2]. ere are, reservations regarding FQ use because resistant mutants can be selected in a remarkably short time [1]. e targets of the FQs are the DNA gyrase and DNA Topoisomerase IV. Resistance-Determining Regions (QRDR) of the GyrA and GyrB subunits, efflux pumps have been implicated in the development of resistance [3,4,5] Another novel mechanism of FQ resistance involves proteins belonging to the Pentapeptide Repeat Family (PRF). In Gram-negative bacteria, the genes of the qnr family encode pentapeptide proteins that are generally found on transmissible plasmids and confer low-level FQ resistance [6,7,8,9]. In M. tuberculosis, the gene Rv3361c encodes a pentapeptide protein (termed MtMfpA) of 183 amino acids that has 67% amino acid identity with the M. smegmatis MfpA. Because the FQs are an important drug for curing patients with MDR-TB [14, 15], we attempted to clarify the role of pentapeptide proteins in FQ resistance through genetic characterization of the mfpA gene in M. smegmatis. The mfpA gene was cloned into an inducible vector to increase its expression and thereby confirm that increased expression leads to increased FQ resistance. e results suggest a possible link between MfpA and FQ resistance in M. smegmatis, which could have important implications for FQ resistance in M. tuberculosis

Bacterial Strains and Plasmids

Results

Discussion