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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.
Journal of Bacteriology 05/2006; 188(7):2300-8. · 3.83 Impact Factor
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ABSTRACT: Herpes simplex virus 1 (HSV-1) ICP27 and ICP8 proteins have both been implicated in the transcription of late genes and regulation of viral gene expression. We showed previously that ICP27 and ICP8 associate with the RNAP II holoenzyme (Zhou and Knipe, J. Virol. 76, 5893-5904). Here, we demonstrate that ICP27 and ICP8 coprecipitate from lysates of HSV-1-infected HEp2 cells and from lysates of insect cells expressing ICP27 and ICP8, the latter being in the absence of other HSV-1 proteins. By expressing and purifying hexahistidine-tagged ICP8 (His-ICP8) and maltose binding protein (MBP)-tagged ICP27 (MBP-27) proteins and performing in vitro immunoprecipitation and pull-down assays, we also demonstrate that ICP27 and ICP8 coprecipitate in the absence of other viral or cellular proteins. Taken together, these data provide evidence that ICP27 and ICP8 interact directly in vitro and in infected cells. We hypothesize that the ICP27-ICP8 interaction plays a role in the stimulation of late gene transcription.
Virology 02/2005; 331(1):94-105. · 3.35 Impact Factor
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ABSTRACT: The herpes simplex virus (HSV) immediate early ICP27 protein plays an essential role in stimulating viral early and late gene expression. ICP27 appears to be multifunctional in that it has been reported to stimulate viral late gene transcription, polyadenylation site usage, and RNA export. We report here on proteomic studies involving immunoprecipitation of ICP27 and mass spectrometric identification of co-precipitated proteins. These studies show an association of ICP27 with the cellular translation initiation factors poly A binding protein (PABP), eukaryotic initiation factor 3 (eIF3), and eukaryotic initiation factor 4G (eIF4G) in infected cells. Immunoprecipitation-western blot studies confirmed these associations. Finally, purified MBP-tagged ICP27 (MBP-27) can interact with eIF3 subunits p47 and p116 in vitro. These results suggest that ICP27 may also play a role in stimulating translation of certain viral and host mRNAs and/or in inhibiting host mRNA translation.
Virology 01/2005; 330(2):487-92. · 3.35 Impact Factor
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ABSTRACT: A soluble form of penicillin-binding protein 3 (PBP 3) from Neisseria gonorrhoeae was expressed and purified from Escherichia coli and characterized for its interaction with beta-lactam antibiotics, its catalytic properties with peptide and peptidoglycan substrates, and its role in cell viability and morphology. PBP 3 had an unusually high k(2)/K' value relative to other PBPs for acylation with penicillin (7.7 x 10(5) M(-1) s(-1)) at pH 8.5 at 25 degrees C and hydrolyzed bound antibiotic very slowly (k(3) < 4.6 x 10(-5) s(-1), t(1/2) > 230 min). PBP 3 also demonstrated exceptionally high carboxypeptidase activity with a k(cat) of 580 s(-1) and a k(cat)/K(m) of 1.8 x 10(5) M(-1) s(-1) with the substrate N(alpha)-Boc-N(epsilon)-Cbz-L-Lys-D-Ala-D-Ala. This is the highest k(cat) value yet reported for a PBP or other serine peptidases. Activity against a approximately D-Ala-D-Lac peptide substrate was approximately 2-fold lower than against the analogous approximately D-Ala-D-Ala peptide substrate, indicating that deacylation is rate determining for both amide and ester hydrolysis. The pH dependence profiles of both carboxypeptidase activity and beta-lactam acylation were bell-shaped with maximal activity at pH 8.0-8.5. PBP 3 displayed weak transpeptidase activity in a model transpeptidase reaction but was active as an endopeptidase, cleaving dimeric peptide cross-links. Deletion of PBP 3 alone had little effect on viability, growth rate, and morphology of N. gonorrhoeae, although deletion of both PBP 3 and PBP 4, the other low-molecular-mass PBP in N. gonorrhoeae, resulted in a decreased growth rate and marked morphological abnormalities.
Biochemistry 01/2004; 42(49):14614-25. · 3.42 Impact Factor
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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.
Antimicrobial Agents and Chemotherapy 10/2002; 46(9):2811-20. · 4.84 Impact Factor
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ABSTRACT: Chromosomally mediated penicillin resistance in Neisseria gonorrhoeae occurs in part through alterations in penicillin-binding proteins (PBPs) and a decrease in outer membrane permeability. However, the genetic and molecular mechanisms of transformation of a penicillin-susceptible strain of N. gonorrhoeae to high-level penicillin resistance have not been clearly elucidated. Previous studies suggested that alterations in PBP 1 were involved in high-level penicillin resistance. In this study, we identified a single amino acid mutation in PBP 1 located 40 amino acids N terminal to the active-site serine residue that was present in all chromosomally mediated resistant N. gonorrhoeae (CMRNG) strains for which MICs of penicillin were > or = 1 microg/ml. PBP 1 harboring this point mutation (PBP 1*) had a three- to fourfold lower rate of acylation (k2/K') than wild-type PBP 1 with a variety of beta-lactam antibiotics. Consistent with its involvement in high-level penicillin resistance, replacement of the altered ponA gene (ponA1) in several CMRNG strains with the wild-type ponA gene resulted in a twofold decrease in the MICs of penicillin. Surprisingly, transformation of an intermediate-level penicillin-resistant strain (PR100; FA19 penA4 mtr penB5) with the ponA1 gene did not increase the MIC of penicillin for this strain. However, we identified an additional resistance locus, termed penC, which was required along with ponA1 to increase penicillin resistance of PR100 to a high level (MIC = 4 microg/ml). The penC locus by itself, when present in PR100, increases the MICs of penicillin and tetracycline twofold each. These data indicate that an additional locus, penC, is required along with ponA1 to achieve high-level penicillin resistance.
Antimicrobial Agents and Chemotherapy 03/2002; 46(3):769-77. · 4.84 Impact Factor
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Melanie. Olesky
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ABSTRACT: Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2002. " ... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Pharmacology." Includes bibliographical references (leaves : 158-191). Microfiche. s
