OBJECTIVES:The ram locus, consisting of the romA-ramA genes, is repressed by the tetracycline-type regulator RamR, where regulation is abolished due to loss-of-function mutations within the protein or ligand interactions. The aim of this study was to determine whether the phenothiazines (chlorpromazine and thioridazine) directly interact with RamR to derepress ramA expression.METHODS:Quantitative real-time PCR analyses were performed to determine expression levels of the romA-ramA genes after exposure to the phenothiazines. Electrophoretic mobility shift assays (EMSAs) and in vitro transcription experiments were performed to show direct binding to and repression by RamR. Direct binding of the RamR protein to the phenothiazines was measured by fluorescence spectroscopy experiments and molecular docking models were generated using the RamR crystal structure.RESULTS:Exposure to either chlorpromazine or thioridazine resulted in the up-regulation of the romA-ramA genes. EMSAs and in vitro transcription experiments demonstrated that both agents reduce/abolish binding and enhance transcription of the target PI promoter upstream of the ramR-romA genes in Klebsiella pneumoniae compared with RamR alone. Fluorescence spectroscopy measurements demonstrated that RamR directly binds both chlorpromazine and thioridazine with micromolar affinity. Molecular docking analyses using the RamR crystal structure demonstrated that the phenothiazines interact with RamR protein through contacts described for other ligands, in addition to forming unique strong polar interactions at positions D152 and K63.CONCLUSIONS:These data demonstrate that phenothiazines can modulate loci linked to the microbe-drug response where RamR is an intracellular target for the phenothiazines, thus resulting in a transient non-mutational derepression of ramA concentrations.
Bibliographical noteReference text: 1. Ramos JL, Martinez-Bueno M, Molina-Henares AJ et al. The TetR family of transcriptional repressors. Microbiol Mol Biol Rev 2005; 69: 326–56.
2 Grkovic S, Brown MH, Skurray RA. Regulation of bacterial drug export systems. Microbiol Mol Biol Rev 2002; 66: 671–701.
3 Rosenblum R, Khan E, Gonzalez G et al. Genetic regulation of the ramA locus and its expression in clinical isolates of Klebsiella pneumoniae. Int J Antimicrob Agents 2011; 38: 39–45.
4 Schneiders T, Amyes SG, Levy SB. Role of AcrR and ramA in fluoroquinolone resistance in clinical Klebsiella pneumoniae isolates from Singapore. Antimicrob Agents Chemother 2003; 47: 2831–7.
5 George AM, Hall RM, Stokes HW. Multidrug resistance in Klebsiella pneumoniae: a novel gene, ramA, confers a multidrug resistance phenotype in Escherichia coli. Microbiology 1995; 141: 1909–20.
6 Bailey AM, Ivens A, Kingsley R et al. RamA, a member of the AraC/XylS family, influences both virulence and efflux in Salmonella enterica serovar Typhimurium. J Bacteriol 2010; 192: 1607–16.
7 Veleba M, De Majumdar S, Hornsey M et al. Genetic characterisation of tigecycline resistance in clinical isolates of Enterobacter cloacae and Enterobacter aerogenes. J Antimicrob Chemother 2013; 68: 1011–8.
8 Yamasaki S, Nikaido E, Nakashima R et al. The crystal structure of multidrug-resistance regulator RamR with multiple drugs. Nat Commun 2013; 4: 2078.
9 Bailey AM, Paulsen IT, Piddock LJ. RamA confers multidrug resistance in Salmonella enterica via increased expression of acrB, which is inhibited by chlorpromazine. Antimicrob Agents Chemother 2008; 52: 3604–11.
10 Kristiansen JE, Amaral L. The potential management of resistant infections with non-antibiotics. J Antimicrob Chemother 1997; 40: 319–27.
11 Martins M, Dastidar SG, Fanning S et al. Potential role of non-antibiotics (helper compounds) in the treatment of multidrug-resistant Gramnegative infections: mechanisms for their direct and indirect activities. Int J Antimicrob Agents 2008; 31: 198–208.
12 Amaral L, Viveiros M, Kristiansen JE. “Non-Antibiotics”: alternative therapy for the management of MDRTB and MRSA in economically disadvantaged countries. Curr Drug Targets 2006; 7: 887–91.
13 Trautwein C, Ku¨mmerer K. Ready biodegradability of trifluoromethylated phenothiazine drugs, structural elucidation of their aquatic transformation products, and identification of environmental risks studied by LC-MSn and QSAR. Environ Sci Pollut Res Int 2012; 19:3162–77.
14 Forage RG, Lin EC. DHA system mediating aerobic and anaerobic dissimilation of glycerol in Klebsiella pneumoniae NCIB 418. J Bacteriol 1982; 151: 591–9.
15 Fookes M, Yu J, De Majumdar S et al. Genome sequence of Klebsiella pneumoniae Ecl8, a reference strain for targeted genetic manipulation. Genome Announc 2013; 1: e00027–12.
16 Andrews JM. BSAC standardized disc susceptibility testing method (version 8). J Antimicrob Chemother 2009; 64: 454–89.
17 Schneiders T, Barbosa TM, McMurry LM et al. The Escherichia coli transcriptional regulator MarA directly represses transcription of purA and hdeA. J Biol Chem 2004; 279: 9037–42.
18 Gill SC, von Hippel PH. Calculation of protein extinction coefficients from amino acid sequence data. Anal Biochem 1989; 182: 319–26.
19 Rohl CA, Baldwin RL. Comparison of NH exchange and circular dichroism as techniques for measuring the parameters of the helix-coil transition in peptides. Biochemistry 1997; 36: 8435–42.
20 Muller B, Restle T, Reinstein J et al. Interaction of fluorescently labelled dideoxynucleotides with HIV-1 reverse transcriptase. Biochemistry 1991; 30:3709–15.
21 Glide. New York: Schro¨dinger LLC, 2009.
22 Maestro 8.5. New York: Schro¨dinger LLC, 2009.
23 Tahlan K, Yu Z, Xu Y et al. Ligand recognition by ActR, a TetR-like regulator of actinorhodin export. J Mol Biol 2008; 383: 753–61.
24 Abouzeed YM, Baucheron S, Cloeckaert A. ramR mutations involved in efflux-mediated multidrug resistance in Salmonella enterica serovar Typhimurium. Antimicrob Agents Chemother 2008; 52: 2428–34.
25 Kristiansen JE, Thomsen VF, Martins A et al. Non-antibiotics reverse resistance of bacteria to antibiotics. In Vivo 2010; 24: 751–4.
26 Lawler AJ, Ricci V, Busby SJ et al. Genetic inactivation of acrAB or inhibition of efflux induces expression of ramA. J Antimicrob Chemother 2013; 68:
27 Bolla JR, Do SV, Long F et al. Structural and functional analysis of the transcriptional regulator Rv3066 of Mycobacterium tuberculosis. Nucleic Acids Res 2012; 40: 9340–55.
28 Lei HT, Shen Z, Surana P et al. Crystal structures of CmeR-bile acid complexes
from Campylobacter jejuni. Protein Sci 2011; 20: 712–23.
29 Agari Y, Agari K, Sakamoto K et al. TetR-family transcriptional repressor Thermus thermophilus FadR controls fatty acid degradation. Microbiology 2011; 157: 1589–601.
30 Bialek-Davenet S, Marcon E, Leflon-Guibout V et al. In vitro selection of ramR and soxR mutants overexpressing efflux systems by fluoroquinolones as well as cefoxitin in Klebsiella pneumoniae. Antimicrob Agents Chemother 2011; 55: 2795–802.
31 Hentschke M, Wolters M, Sobottka I et al. ramR mutations in clinical isolates of Klebsiella pneumoniae with reduced susceptibility to tigecycline. Antimicrob Agents Chemother 2010; 54: 2720–3.
32 Barbosa TM, Levy SB. Activation of the Escherichia coli nfnB gene by MarA through a highly divergent marbox in a class II promoter. Mol Microbiol 2002; 45: 191–202.
- ligand interactions