Antibiotic resistance in Neisseria gonorrhoeae remains a very important problem. Penicillin and tetracycline, which were once the antibiotics of choice for treatment of gonococcal infections, are no longer used due to the preponderance of strains resistant to these agents. Resistance to currently recommended antibiotics is also increasing. My laboratory is interested in the mechanisms of chromosomally-mediated antibiotic resistance in the gonococcus, especially those that promote high- level resistance and subsequent treatment failure. Intermediate- level chromosomally-mediated resistance to penicillin and tetracycline is due to three resistance loci. These include the penA gene encoding altered forms of penicillin-binding protein 2 (PBP 2), the mtr loci conferring resistance to hydrophobic agents, and the penB gene, which decreases outer membrane permeability. The genes involved in mediating high-level penicillin resistance, however, have been difficult to identify. Our work during the last funding period has identified two resistance genes, ponA and penC, which together mediate high- level penicillin resistance, and a third gene, tetGC, which confers high-level tetracycline resistance. This proposal outlines experiments to clone and characterize the penC and tetGC genes and to elucidate the mechanisms by which they increase resistance. In addition, we propose experiments that follow up on our structure/function studies of the penB gene product, porin IB, to understand how mutations in this protein increase both penicillin and tetracycline resistance. We also propose studies to complete our work on the crystal structure of penicillin- binding protein 2 (PBP 2), an essential penicillin target, and several mutant forms that display a lower affinity for beta- lactam antibiotics. In addition, we will engage in new structural studies of wild-type and mutant forms of porin IB to explicate in molecular detail how mutations in this protein decrease antibiotic permeability. The combination of genetic, biochemical, biophysical, and structural approaches outlined in this proposal will provide important insight into the mechanisms by which this important human pathogen becomes resistant to antibiotics.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
2R01AI036901-06
Application #
6399099
Study Section
Bacteriology and Mycology Subcommittee 2 (BM)
Program Officer
Quackenbush, Robert L
Project Start
1996-04-01
Project End
2006-05-31
Budget Start
2001-07-01
Budget End
2002-05-31
Support Year
6
Fiscal Year
2001
Total Cost
$313,465
Indirect Cost
Name
University of North Carolina Chapel Hill
Department
Pharmacology
Type
Schools of Medicine
DUNS #
078861598
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
Nandi, Sobhan; Swanson, Shauna; Tomberg, Joshua et al. (2015) Diffusion of antibiotics through the PilQ secretin in Neisseria gonorrhoeae occurs through the immature, sodium dodecyl sulfate-labile form. J Bacteriol 197:1308-21
Fedarovich, Alena; Cook, Edward; Tomberg, Joshua et al. (2014) Structural effect of the Asp345a insertion in penicillin-binding protein 2 from penicillin-resistant strains of Neisseria gonorrhoeae. Biochemistry 53:7596-603
Tomberg, Joshua; Unemo, Magnus; Ohnishi, Makoto et al. (2013) Identification of amino acids conferring high-level resistance to expanded-spectrum cephalosporins in the penA gene from Neisseria gonorrhoeae strain H041. Antimicrob Agents Chemother 57:3029-36
Fedarovich, Alena; Nicholas, Robert A; Davies, Christopher (2012) The role of the ýý5-ýý11 loop in the active-site dynamics of acylated penicillin-binding protein A from Mycobacterium tuberculosis. J Mol Biol 418:316-30
Unemo, Magnus; Golparian, Daniel; Nicholas, Robert et al. (2012) High-level cefixime- and ceftriaxone-resistant Neisseria gonorrhoeae in France: novel penA mosaic allele in a successful international clone causes treatment failure. Antimicrob Agents Chemother 56:1273-80
Fedarovich, Alena; Djordjevic, Kevin A; Swanson, Shauna M et al. (2012) High-throughput screening for novel inhibitors of Neisseria gonorrhoeae penicillin-binding protein 2. PLoS One 7:e44918
Unemo, Magnus; Nicholas, Robert A (2012) Emergence of multidrug-resistant, extensively drug-resistant and untreatable gonorrhea. Future Microbiol 7:1401-22
Tomberg, Joshua; Temple, Brenda; Fedarovich, Alena et al. (2012) A highly conserved interaction involving the middle residue of the SXN active-site motif is crucial for function of class B penicillin-binding proteins: mutational and computational analysis of PBP 2 from N. gonorrhoeae. Biochemistry 51:2775-84
Nemmara, Venkatesh V; Dzhekieva, Liudmila; Sarkar, Kumar Subarno et al. (2011) Substrate specificity of low-molecular mass bacterial DD-peptidases. Biochemistry 50:10091-101
Lee, Chul-Jin; Liang, Xiaofei; Chen, Xin et al. (2011) Species-specific and inhibitor-dependent conformations of LpxC: implications for antibiotic design. Chem Biol 18:38-47

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