Identification and Analysis of Amino Acid Mutations in Porin IB That Mediate Intermediate-Level Resistance to Penicillin and Tetracycline in Neisseria gonorrhoeae

Abstract

PenB is the third resistance determinant in the stepwise acquisition of multiple resistance genes in chromosomally mediated resistant Neisseria gonorrhoeae (CMRNG). Alterations in por IB , one of two alleles at the por locus that encodes the outer membrane protein porin IB (PIB), were recently reported to be responsible for the increased resistance to penicillin and tetracycline conferred by penB, but the specific mutations conferring antibiotic resistance were not identified experimentally. To determine which amino acids in PIB confer increased resistance, we transformed a recipient strain with chimeras of the por IB genes from strains FA1090 and FA140 (penB2). These studies revealed that two amino acid changes, G120D and A121D, were both necessary and sufficient to confer increased resistance to penicillin and tetracycline. Site-saturation and site-directed mutagenesis of Gly-120 and Ala-121 revealed that both a single mutation, G120K, and the double mutations G120R A121H and G120P A121P also conferred antibiotic resistance to the recipient strain. The identical mutations in PIA increased penicillin and tetracycline resistance either moderately or not at all. Analysis of por IB genes present in the GenBank database from 51 clinical isolates demonstrated that lysine and aspartate mutations at positions 120 and/or 121 also occur in nature. These studies demonstrate that charged amino acids at positions 120 and 121 in PIB are highly preferential for conferring resistance to penicillin and tetracycline in N. gonorrhoeae.From 1945 to 1988, penicillin was the antibiotic of choice for treatment of gonococcal infections. During this time, however, the resistance of isolates gradually increased until treatment failure with penicillin became widespread and penicillin was discontinued as a first-line antibiotic. Resistance to other antibiotics, including erythromycin and tetracycline, also increased during this time. The antibiotics currently recommended for treatment of gonococcal infections are expandedspectrum cephalosporins (i.e., ceftriaxone and cefixime) or fluoroquinolones (7), but resistance to fluoroquinolones threatens to make these antibiotics obsolete as well (8).Resistance to penicillin and tetracycline in gonococci can be either plasmid mediated or chromosomally mediated (5). Plasmid-mediated resistance to penicillin is due to the production of a TEM-1-like ␤-lactamase, whereas plasmid-mediated resistance to tetracycline is due to expression of the TetM determinant acquired from Streptococcus pneumoniae (34). In contrast, chromosomally mediated resistance to penicillin and tetracycline in Neisseria gonorrhoeae results from the acquisition of multiple resistance genes, each of which confers an incremental increase in resistance until the cell becomes refractory to clinically achieved levels of the antibiotic. As demonstrated by the work of Sparling and others (15,18,39), these resistance genes can be transferred in the laboratory in a stepwise manner from a resistant strain to a susceptible strain by DNA upt...

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 . substitution of these amino-acid residues using targeted mutagenesis has clearly shown that they have a role in antibiotic diffusion 55,57 .…”

Adaptive and Mutational Resistance: Role of Porins and Efflux Pumps in Drug Resistance

“…For instance, a G-to-D mutation in loop 3 of the OmpF/OmpC-like protein of Enterobacter aerogenes leads to a loss of conductance of the pore and, as a result, to decreased susceptibility to ␤-lactams (48). Similarly, mutations in loop 3 of the porin PIB of Neisseria gonorrhoeae confer resistance to penicillin and tetracycline in strains that simultaneously overexpress an efflux pump due to an mtrR mutation (189,190). In Vibrio cholerae, a D116A mutation in OmpU, which was predicted to be an antibiotic binding site, leads to increased resistance to cephalosporins (193).…”