The MtrD protein of Neisseria gonorrhoeae is a member of the resistance/nodulation/division protein family constituting part of an efflux system

Abstract

The mfr (multiple transferable resistance) system of Neisseria gonorrhoeae mediates resistance of gonococci to structurally diverse hydrophobic agents (HAS) through an energy-dependent efflux process. Recently, complete or partial ORFs that encode membrane proteins (MtrG MtrD, MtrE) forming an efflux pump responsible for removal of HAS from gonococci were identified and appeared to constitute a single transcriptional unit. In this study, the complete nucleotide sequence of the mtrD gene was determined, permitting the characterization of the MtrD protein. The full-length MtrD protein has a predicted molecular mass of nearly 114 kDa, putatively containing a 56 amino acid signal peptide. MtrD displays significant amino acid sequence similarity to a f am i I y of cytoplasmic membrane proteins, termed resistancehodu lat ion/ division (RND) proteins, which function as energy-dependent transporters of antibacterial agents and secrete bacterial products to the extracellular fluid. The predicted topology of the MtrD transporter protein revealed 12 potential membrane-spanning domains, which were clustered within the central and Cterminal regions of the primary sequence. Loss of MtrD due to insertional inactivation of the mtrD gene rendered gonococci hypersusceptible to several structurally diverse HAS, including two fatty acids (capric acid and palmitic acid) and a bile salt (cholic acid), but not hydrophilic antibiotics such as ciprof loxacin and streptomycin. Since gonococci often infect mucosal sites rich in toxic fatty acids and bile salts, the expression of the mtr efflux system may promote growth of gonococci under hostile conditions encountered in vivo.

Modulation of Neisseria gonorrhoeae susceptibility to vertebrate antibacterial peptides due to a member of the resistance/nodulation/division efflux pump family

“…Using the microtiter well growth inhibition assay described above, we found ( Table 1) that isogenic transformant strains bearing insertionally inactivated mtrC (strain KH12), mtrD (strain KH14), or mtrE (strain RD1) genes were significantly (P ϭ 0.001) more susceptible to PG-1 (MGICs range of 0.2-0.75 g͞ml) than parental strain FA19 (MGIC of 4.37 g͞ml). The extent of enhanced PG-1 susceptibility of these strains was similar to that observed previously for other antimicrobial compounds that are removed from gonococci by the mtr efflux system (14)(15)(16)(17)(18).…”

“…2B). Because the kanamycin-resistance cassette within the mtrD gene, which encodes the membrane transporter As BR54 but mtrD54 † 7.14 (Ϯ2.67) *The construction of transformant strains KH12, KH14, and RD1 has been described (14)(15)(16)(17)(18). Strain BR54 was kindly provided by P. F. Sparling (21).…”

“…Table 1 is a description of the genotype and source of the isogenic gonococcal strains used in this investigation. Strain FA19 is the wild-type parental strain that was used in the genetic construction of strains KH12, KH14, and RD1 (14,15,18). Strain BR54 is a transformant of the FA19 genetic lineage constructed previously (21) and is hypersusceptible to HAs such as erythromycin, Triton X-100, and crystal violet.…”

“…It was verified that the MtrC-MtrD-MtrE efflux pump was indeed responsible for PG-1 export by comparing the accumulation of radiolabeled PG-1 (0.25 g͞ml) by isogenic strains FA19 and KH14, which revealed that strain KH14 accumulated at least 50% more PG-1 than parental strain FA19 over a 35-min incubation period (Fig. 4B) The MtrC-MtrD-MtrE efflux pump can recognize a broad range of substrates that includes structurally diverse antibiotics, dyes, and detergents (14)(15)(16)(17)(18). Based on genetic and biochemical results obtained in this study, this list now includes certain antibacterial peptides that can assume ␤-sheet (PG-1) or ␣-helical (PC-8 and LL-37) structures.…”

Resistance ofNeisseria meningitidisto the Toxic Effects of Heme Iron and Other Hydrophobic Agents Requires Expression ofght

“…Toxicity of HAs and mechanisms of HA resistance (HA r ) have been extensively studied in the gonococci (2,10,11,26,36). The gonococcal Mtr efflux system is a member of the resistance-nodulation-division family of energy-dependent efflux pumps.…”