Neisseria gonorrhoeae is the causative agent for the sexually transmitted disease gonorrhea and was responsible for over 350,000 infections in the U.S. in 2007. The steady and inexorable increase of resistance in this organism toward multiple classes of antibiotics has severely limited treatment options for gonococcal infections and, after the recent withdrawal of fluoroquinolones, the expanded-spectrum cephalosporin ceftriaxone is now the only single-dose treatment recommended by the CDC in the U.S. Unfortunately, new strains of N. gonorrhoeae have emerged that exhibit resistance to cefixime and ceftriaxone, and treatment failures are now being reported. This precarious position endangers public health and demands a better understanding of antibiotic resistance at the molecular level, as well as strategies to develop new antimicrobials directed against N. gonorrhoeae. This renewal application will address this need by investigating two enzymes of N. gonorrhoeae involved in peptidoglycan synthesis. One is penicillin-binding protein 2 (PBP 2), a transpeptidase that forms peptide cross-links during the latter stages of cell wall synthesis, and the clinical target for ?-lactam antibiotics directed against this organism. Cephalosporin-resistant strains of N. gonorrhoeae harbor mutations in PBP 2 and a key goal is to determine the structural mechanisms that lower reactivity of PBP 2 with these antibiotics. We will also apply NMR relaxation methods to test the hypothesis that the molecular mechanism governing penicillin and cephalosporin resistance mediated by PBP 2 involves dynamic states of the protein. In recognition that other enzymes involved in peptidoglycan metabolism are potential targets for antimicrobials, we will also investigate N-acetylmuramyl-L-alanine amidase (AmiC), an autolysin that is required for proper cell division of N. gonorrhoeae. We have discovered that this enzyme exhibits autolytic activity in its N-terminal domain in addition to its known amidase activity in the C-terminal domain and therefore is a bifunctional autolysin. To understand the functional role of AmiC in peptidoglycan breakdown, but also to pave the way for drug discovery against its two active sites, we will obtain essential structural and biochemical information for AmiC.

Public Health Relevance

The steady and inexorable increase of antibiotic resistance in N. gonorrhoeae, the causative agent for the sexually transmitted disease gonorrhea, has severely limited treatment options for gonococcal infections such that the expanded-spectrum cephalosporin ceftriaxone is now the only single-dose treatment recommended by the CDC in the U.S. Unfortunately, new strains of N. gonorrhoeae have emerged that exhibit marked increases in resistance to the expandedspectrum cephalosporins, cefixime and ceftriaxone, and are now overcoming established breakpoints for these antibiotics. This application will address this impending crisis in public health by elucidating the molecular mechanism of antibiotic resistance caused by mutations in a penicillin-binding protein and by investigation of an autolysin required for cell division as a potential new target for antimicrobial development.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM066861-11
Application #
8601094
Study Section
Special Emphasis Panel (ZRG1-DDR-T (09))
Program Officer
Marino, Pamela
Project Start
2003-02-01
Project End
2015-12-31
Budget Start
2014-01-01
Budget End
2014-12-31
Support Year
11
Fiscal Year
2014
Total Cost
$302,386
Indirect Cost
$72,886
Name
Medical University of South Carolina
Department
Biochemistry
Type
Schools of Medicine
DUNS #
183710748
City
Charleston
State
SC
Country
United States
Zip Code
29425
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
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
Tomberg, Joshua; Unemo, Magnus; Davies, Christopher et al. (2010) Molecular and structural analysis of mosaic variants of penicillin-binding protein 2 conferring decreased susceptibility to expanded-spectrum cephalosporins in Neisseria gonorrhoeae: role of epistatic mutations. Biochemistry 49:8062-70
Fedarovich, Alena; Nicholas, Robert A; Davies, Christopher (2010) Unusual conformation of the SxN motif in the crystal structure of penicillin-binding protein A from Mycobacterium tuberculosis. J Mol Biol 398:54-65
Nicola, George; Tomberg, Joshua; Pratt, R F et al. (2010) Crystal structures of covalent complexes of ýý-lactam antibiotics with Escherichia coli penicillin-binding protein 5: toward an understanding of antibiotic specificity. Biochemistry 49:8094-104
Zhao, Shuqing; Duncan, Margaret; Tomberg, Joshua et al. (2009) Genetics of chromosomally mediated intermediate resistance to ceftriaxone and cefixime in Neisseria gonorrhoeae. Antimicrob Agents Chemother 53:3744-51
Powell, Ailsa J; Tomberg, Joshua; Deacon, Ashley M et al. (2009) Crystal structures of penicillin-binding protein 2 from penicillin-susceptible and -resistant strains of Neisseria gonorrhoeae reveal an unexpectedly subtle mechanism for antibiotic resistance. J Biol Chem 284:1202-12
Sauvage, Eric; Powell, Ailsa J; Heilemann, Jason et al. (2008) Crystal structures of complexes of bacterial DD-peptidases with peptidoglycan-mimetic ligands: the substrate specificity puzzle. J Mol Biol 381:383-93
Powell, Ailsa J; Liu, Zhi-Jie; Nicholas, Robert A et al. (2006) Crystal structures of the lytic transglycosylase MltA from N.gonorrhoeae and E.coli: insights into interdomain movements and substrate binding. J Mol Biol 359:122-36

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