a National Center for Gonococcal Antimicrobial Susceptibility Surveillance, Department of Microbiology, School of Chemistry, Avda Gral Flores 2124, Montevideo 11800, Uruguay; b Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA 30332; c Laboratories of Microbial Pathogenesis, Medical Research Service, VA Medical Center (Atlanta), Decatur, GA 30333, USA
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
The mtr(CDE)-encoded efflux pump of N. gonorrhoeae mediates an energy-dependent efflux process of structurally diverse hydrophobic agents (HAs) in gonococci.26 The expression of this system is negatively regulated by the product of the adjacent but divergent mtr(R) gene.3,5 The molecular basis for azithromycin resistance in gonococci mediated by the mtr(CDE)-encoded efflux pump has been described recently.1 We reported previously that a single T:A bp deletion within the mtr(R) promoter region, which would decrease the level of expression of mtr(R), and a missense mutation at codon 45 in the mtr(R)-coding region, frequently occur in strains of gonococci isolated from our patient population. The presence of such mutations explains the decreased azithromycin susceptibility property in clinical isolates with a multiple resistance phenotype (e.g. expression of cross-resistance to erythromycin, Triton X-100 and crystal violet).1
During the course of our studies, we identified another group of gonococcal isolates that expressed decreased susceptibility to azithromycin and erythromycin because of the mtr(CDE)-encoded efflux pump. Interestingly, these strains remained susceptible to Triton X-100 even though mtr(R) mutations typically result in high levels of gonococcal resistance to Triton X-100.2,5 DNA sequence analysis of the mtr(R) promoter region of one such strain (9604) revealed that it contained a novel mutation, a double TT insertion within the 13 bp inverted repeated sequence. This dinucleotide insertion increased the spacing between the 10 and 35 hexamers of the mtr(R) promoter from an optimal 17 nucleotides to an unfavourable 19 nucleotides.1 We now report that this mutation can decrease mtr(R) gene expression in gonococci, resulting in enhanced mtr(CDE) gene expression and decreased bacterial susceptibility to azithromycin and erythromycin without changing susceptibility to other HAs.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Clinical isolate 9604 was recovered from a male patient with urethritis in 1996. Strain FA19 is wild-type with respect to the mtr(CDE)-encoded efflux pump system and is sensitive to HAs. Its isogenic transformant strain KH15 is hyper-resistant to HAs and contains a single base pair deletion in a 13 bp inverted repeat sequence within the promoter region of the mtr(R) gene (Table).2,3 All strains were grown on GC agar base containing glucose and iron supplements at 37°C in 3.8% (v/v) CO2.
|
Piliated colonies of strain FA19 were transformed to increased resistance to erythromycin with PCR products (see below) of the mtr(R) gene from strain 9604, as described previously.2 The susceptibilities of the transformants to azithromycin, erythromycin and Triton X-100, were determined along with the parental and the donor strains by the agar dilution method.1
PCR amplification and DNA sequencing studies
Chromosomal DNA from strain 9604 was used in PCRs with oligonucleotide primers RPMAL#2 and KH9#3 to amplify the complete mtr(R) gene, which included the promoter region.6 A PCR product that encompassed the mtr(R) promoter region and the first 200 bp of the mtr(R) coding region was obtained with oligonucleotide primers KH9#3 and KH9#1; the latter primer anneals in the mtr(R) coding region.6 Purified PCR products were used for the transformation experiments. Automated DNA sequencing on purified PCR products was performed on both DNA strands at the Emory University DNA Sequencing Core Facility.
RNA preparation and RTPCR
Total RNA from strains FA19, KH15 and LZ6 [a transformant of strain FA19 obtained by transformation with mtr(R) gene from strain 9604] was prepared as described previously.7 cDNA was synthesized from 500 ng of total RNA. RTPCRs for the rmp (reduction modifiable protein) gene, which is not controlled by MtrR,3 were performed as an internal control to assure that equal amounts of RNA were used in the reaction. RNA was reverse transcribed with Superscript II RNase H reverse transcriptase (Gibco-BRL, Grand Island, NY, USA) using the following primers: rmp-2 (5'-GTG TTG GTG ATG ATT GCG TGC C-3'), RPMAL#2,6 mtrD-5 (5'-CAA GGA ACA CGG ACA AGA GCG-3'), DSD-5 (5'-CGG CCA TAA ACA ATG CCC GGC-3') for rmp, mtr(R), mtr(D) and mtr(F) genes, respectively.
![]() |
Results and discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
The dinucleotide insertion (TT) within the mtr(R) promoter region would increase the spacing between the 10 and 35 promoter hexamers. We hypothesized that this unfavourable distance would reduce the binding of RNA polymerase, and thereby repress and/or reduce transcription of the mtr(R) gene. Accordingly, we compared levels of the mtr(R) transcript by RTPCR in RNA preparations from isogenic transformants of strain FA19 that contained the double TT insertion (strain LZ6) or a single bp deletion in the mtr(R) promoter (strain KH15).2 Our results showed that both strains had a decreased level of the mtr(R) transcript compared with parental strain FA19 (Figure, a). We next examined whether the decreased transcription of mtr(R) observed with strain LZ6 would result in enhanced transcription of the mtr efflux pump protein encoding genes, as was observed previously for transcription of mtr(CDE) in strain KH15.3 For this purpose we examined the level of the mtr(D) transcript, which encodes the cytoplasmic membrane transporter protein (MtrD) of the mtr efflux pump.4 We found that in both strains KH15 and LZ6, the level of the mtr(D) transcript was elevated compared with that of strain FA19. Recently, we have identified a fourth protein that appears to be involved in expression of high-level HA resistance mediated by the mtr efflux pump, which is encoded by a gene [mtr(F)] downstream of mtr(R) (W. Veal & W. M. Shafer, manuscript in preparation). As with transcription of mtr(D), the level of mtr(F) transcription was increased in strains KH15 and LZ6 compared with parental strain FA19 (Figure
, b).
|
![]() |
Acknowledgments |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
2 . Hagman, K. E., Pan W., Spratt B. G., Balthazar J. T., Judd, R. C. & Shafer, W. M. (1995). Resistance of Neisseria gonorrhoeae to antimicrobial hydrophobic agents is modulated by the mtrRCDE efflux system. Microbiology 141, 61122.[Abstract]
3 . Hagman, K. E. & Shafer, W. M. (1995). Transcriptional control of the mtr efflux system of Neisseria gonorrhoeae. Journal of Bacteriolgy 177, 41625.
4 . Hagman, K. E., Lucas, C. E., Balthazar, J. T., Snyder, L., Nilles, M., Judd, R. C. & Shafer, W. M. (1997). The MtrD protein of Neis-seria gonorrhoeae is a member of the resistance/nodulation/ division protein family constituting part of an efflux system. Microbiology 143, 211725.[Abstract]
5 . Shafer, W. M., Balthazar, J. T., Hagman, K. E. & Morse, S. A. (1995). Missense mutations that alter the DNA-binding domain of the MtrR protein occur frequently in rectal isolates of Neisseria gonorrhoeae that are resistant to faecal lipids. Microbiology 141, 90711.[Abstract]
6 . Lucas, C. E., Balthazar, J. T., Hagman, K. E. & Shafer, W. M. (1997). The MtrR repressor binds the DNA sequence between the mtrR and mtrC genes of Neisseria gonorrhoeae. Journal of Bacteriology 179, 41238.[Abstract]
7 . Lee, E. H. & Shafer, W. M. (1999). The farAB-encoded efflux pump mediates resistance of gonococci to long-chained antibacterial fatty acids. Molecular Microbiology 33, 83945.[ISI][Medline]
Received 6 October 2000; returned 29 November 2000; revised 2 January 2001; accepted 19 January 2001