Porin-mediated antibiotic resistance in Neisseria gonorrhoeae: ion, solute, and antibiotic permeation through PIB proteins with penB mutations.

Department of Pharmacology, University of North Carolina at Chapel Hill, CB#7365 Mary Ellen Jones Bldg., Chapel Hill, NC 27599-7365, USA.
Journal of Bacteriology (Impact Factor: 2.69). 05/2006; 188(7):2300-8. DOI: 10.1128/JB.188.7.2300-2308.2006
Source: PubMed

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 beta-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.

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    ABSTRACT: In this issue of the Journal of Bacteriology, Olesky et al. (13) report a novel observation regarding the mechanism by which Neisseria gonorrhoeae developed clinically significant levels of resistance to penicillin. Although yet to be fully defined, their results link changes in the structure of a gonococcal porin (PorB), which was presumed to modulate permeation of pen- icillin due to precedents set by studies with porins from Entero- bacteriaceae (1, 11), with overexpression of a multidrug efflux pump and the development of penicillin resistance in gono- cocci. The implications of their work for further advancing our knowledge regarding the structure-function relationships of the gram-negative cell envelope, differences between such bac- teria in this respect, and the connection of efflux pumps with other cell envelope proteins in the development of antibiotic resistance are substantial. Moreover, the findings justify con- tinued research on basic problems of antibiotic resistance even when the antibiotic in question is no longer used clinically to treat the disease in question. Historical review of chromosomally mediated penicillin re- sistance in gonococci. The introduction of antibiotics in gen- eral and penicillin specifically as a means to treat bacterial infections is arguably one of the greatest advances in modern medicine. Unfortunately, soon after its introduction, certain pathogens (e.g., Staphylococcus aureus) were noted to have quickly developed resistance to penicillin due to their produc- tion of penicillinase. Other infectious diseases, such as gonor- rhea, remained treatable with the relatively inexpensive peni- cillin G for several years. With respect to N. gonorrhoeae, strains expressing clinically significant levels of penicillin resis- tance emerged slowly. However, by the late 1960s and early 1970s, the peak of the gonorrhea epidemic in the United States, isolates were identified that displayed decreased sus- ceptibility to penicillin. Studies in the 1970s (6, 7, 16) and 1980s (2, 3, 17) showed that these strains contained chromosomally borne mutations that could additively increase resistance of gonococci to penicillin to a level approaching or at clinical significance (e.g., treatment failures). It is important to stress that these strains did not produce detectable penicillinase, although other (comparatively rare) strains bearing a plasmid encoding a TEM-1-type beta-lactamase were identified in the mid-1970s (15). With the report in 1985 (3) of a community-based outbreak of penicillin-resistant gonorrhea due to a strain not producing a beta-lactamase, the final blow to penicillin therapy for treat- ment of this sexually transmitted infection was, unfortunately, realized. The culprit strain (FA6140 (3)) from this outbreak contained (12) a number of chromosomal mutations (penA, penB, ponA, and mtr) that are known to alter cell envelope structure and/or function. In general terms, these mutations impact penicillin's accumulation in gonococci (penB and mtr) or affinity (penA and ponA) for penicillin-binding proteins; this commentary will be restricted to issues related to penB and mtr. The penB mutation was originally linked (7) to production of an altered major outer membrane protein (termed POMP or protein I) and was found to confer two- to fourfold increases in MIC levels of penicillin and tetracycline. Curiously, pheno- typic expression of penB required the presence of the mtr mutation, which was found to confer single-step resistance to structurally diverse hydrophobic antimicrobial agents (10) and was presumed to decrease cell envelope permeability to such agents (6).
    Journal of Bacteriology 05/2006; 188(7):2297-9. DOI:10.1128/JB.188.7.2297-2299.2006 · 2.69 Impact Factor
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    ABSTRACT: The molecular mechanisms of reduced susceptibility to cefixime in clinical isolates of Neisseria gonorrhoeae, particularly amino acid substitutions in mosaic penicillin-binding protein 2 (PBP2), were examined. The complete sequence of ponA, penA, and por genes, encoding, respectively, PBP1, PBP2, and porin, were determined for 58 strains isolated in 2002 from Japan. Replacement of leucine 421 by proline in PBP1 and the mosaic-like structure of PBP2 were detected in 48 strains (82.8%) and 28 strains (48.3%), respectively. The presence of mosaic PBP2 was the main cause of the elevated cefixime MIC (4- to 64-fold). In order to identify the mutations responsible for the reduced susceptibility to cefixime in isolates with mosaic PBP2, penA genes with various mutations were transferred to a susceptible strain by genetic transformation. The susceptibility of partial recombinants and site-directed mutants revealed that the replacement of glycine 545 by serine (G545S) was the primary mutation, which led to a two- to fourfold increase in resistance to cephems. Replacement of isoleucine 312 by methionine (I312M) and valine 316 by threonine (V316T), in the presence of the G545S mutation, reduced susceptibility to cefixime, ceftibuten, and cefpodoxime by an additional fourfold. Therefore, three mutations (G545S, I312M, and V316T) in mosaic PBP2 were identified as the amino acid substitutions responsible for reduced susceptibility to cefixime in N. gonorrhoeae.
    Antimicrobial Agents and Chemotherapy 12/2006; 50(11):3638-45. DOI:10.1128/AAC.00626-06 · 4.45 Impact Factor


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