Porin-Mediated Antibiotic Resistance in Neisseria gonorrhoeae : Ion, Solute, and Antibiotic Permeation through PIB Proteins with penB Mutations

Abstract

Neisseria gonorrhoeae has two porins, PIA and PIB, whose genes (porA and porB, respectively) are alleles of a single por locus. We recently demonstrated that penB mutations at positions 120 and 121 in PIB, which are presumed to reside in loop 3 that forms the pore constriction zone, confer intermediate-level resistance to penicillin and tetracycline (M. Olesky, M. Hobbs, and R. A. Nicholas, Antimicrob. Agents Chemother. 46: 2811-2820, 2002). In the present study, we investigated the electrophysiological properties as well as solute and antibiotic permeation rates of recombinant PIB proteins containing penB mutations (G120K, G120D/A121D, G120P/A121P, and G120R/A121H). In planar lipid bilayers, the predominant conducting state of each porin variant was 30 to 40% of the wild type, even though the anion selectivity and maximum channel conductance of each PIB variant was similar to that of the wild type. Liposome-swelling experiments revealed no significant differences in the permeation of sugars or ␤-lactam antibiotics through the wild type or PIB variants. Although these results are seemingly contradictory with the ability of these variants to increase antibiotic resistance, they are consistent with MIC data showing that these porin mutations confer resistance only in strains containing an mtrR mutation, which increases expression of the MtrC-MtrD-MtrE efflux pump. Moreover, both the mtrR and penB mutations were required to decrease in vivo permeation rates below those observed in the parental strain containing either mtrR or porin mutations alone. Thus, these data demonstrate a novel mechanism of porin-mediated resistance in which mutations in PIB have no affect on antibiotic permeation alone but instead act synergistically with the MtrC-MtrD-MtrE efflux pump in the development of antibiotic resistance in gonococci.In the 1970s and early 1980s, Neisseria gonorrhoeae, the etiological agent of the sexually transmitted infection gonorrhea, gradually became more resistant to penicillin and tetracycline until these antibiotics were discontinued as a first-line therapy. Resistance to these antibiotics can be either plasmid mediated or chromosomally mediated. Plasmid-mediated resistance to penicillin or tetracycline involves expression of a TEM-1-like ␤-lactamase (8, 30) or the TetM protein (25), respectively. In contrast, chromosomally mediated resistant N. gonorrhoeae (CMRNG) strains arise from cumulative mutations in endogenous genes or loci that gradually increase resistance until treatment failure occurs (3).Four genes or loci (penA, mtrR, penB, and ponA) are known to be involved in high-level penicillin resistance (MICs, Ն2 g/ml) in CMRNG strains, while the existence of a fifth resistance gene is inferred but not yet identified (3, 9, 32). penA (38, 39) and ponA (32, 33) encode alterations in the two essential penicillin-binding proteins (PBP 2 and PBP 1, respectively) that decrease their rates of acylation by ␤-lactam antibiotics. A single-base-pair deletion in the mtrR promoter abolishes transcription of the mtrR...

Structural basis for solute transport, nucleotide regulation, and immunological recognition of Neisseria meningitidis PorB

“…Nonselective substrate translocation pathways are challenging to identify by x-ray crystallography because the solutes are unlikely to make specific interactions with the channel. However, the location of bound heavy atoms in the derivative datasets and previous studies of antibiotic resistance mutations (15,16) hint at the location of a cation-selective pathway in PorB. The Lu 3þ and Er 3þ derivative datasets (SI Text and Table S2) identify an electronegative location approaching the pore funnel where cations may be attracted prior to translocation.…”

“…In N. gonorrhoeae, the PenB antibiotic resistance mutations map to the PorB (15,16). In conjunction with mutations in the PorA porin and the MtrR transcription factor, these PenB mutations confer intermediate resistance to penicillin and tetracycline.…”

The porin and the permeating antibiotic: a selective diffusion barrier in Gram-negative bacteria

“…sequence analyses of the por gene have identified aminoacid substitutions (G120D, a121D and G121K) in the putative internal loop 3 in various resistant strains 54,55 . Other mutations at these positions have been identified in resistant clinical isolates from a separate study, emphasizing the importance of these residues in the penicillinresistant phenotype 54,56 .…”

“…Other mutations at these positions have been identified in resistant clinical isolates from a separate study, emphasizing the importance of these residues in the penicillinresistant phenotype 54,56 . substitution of these amino-acid residues using targeted mutagenesis has clearly shown that they have a role in antibiotic diffusion 55,57 . both clinical and in vitro studies have reported that aberrant or modified P. aeruginosa OprD porin is linked to carbapenem resistance, with or without the production of a carbapenem hydrolyzing enzyme 58,59 .…”

Draft Genome Sequence of Neisseria gonorrhoeae Strain NG_869 with Penicillin, Tetracycline and Ciprofloxacin Resistance Determinants Isolated from Malaysia

“…C content), harboured a virulence-associated protein D (vapD). Analysis of other genes involved in high-level penicillin resistance revealed that isolate NG_869 possessed the penB resistance determinant with double mutations of Gly-120-to-Asp and Ala-121-to-Gly but the mtrR resistance determinant required by penB porin mutants to exhibit an increased resistance to penicillin and tetracycline was absent [14]. Furthermore, penicillin-binding protein 2 was not encoded by a mosaic allele and Leu-421-to-Pro substitution was not observed in penicillin-binding protein 1.…”