Beta-lactam antibiotics, which target the essential transpeptidases (penicillin-binding proteins or PBPs) that cross-link peptidoglycan strands, are important drugs in the treatment of bacterial diseases. Unfortunately, the emergence of antibiotic-resistant pathogenic bacteria is a growing problem and threatens to make these and other antibiotics obsolete. Penicillin and tetracycline are no longer used to treat gonococcal infections due to the emergence of resistant strains of N. gonorrhoeae. Moreover, resistance to fluoroquinolones and third-generation cephalosporins, the two classes of antibiotics current recommended in the treatment of gonorrhea, is increasing. Clearly there is an urgent need to develop new antimicrobials directed both against well-known molecular targets, such as PBPs, but also against novel targets such as transglycosylases (TGases), which catalyze the polymerization of glycan stands, and autolysins, which break down peptidoglycan during biosynthesis. Development of new antibiotics, however, has been hindered by a dearth of mechanistic information for these enzymes. In this proposal we describe genetic, biochemical and structural studies of three classes of enzyme involved in peptidoglycan metabolism in N. gonorrhoeae. Each has been selected to address one or more of the following aims: (a) to understand the biology of peptidoglycan synthesis, (b) to explore their interactions with antibiotics, (c) to elucidate the molecular basis for antibiotic resistance and (d) to examine their potential as targets for drug development. The molecular basis for antibiotic resistance will be investigated by structural and biochemical studies of a unique variant of PBP 2 from strains of N. gonorrhoeae with intermediate-level resistance to ceftriaxone. The role of the lytic TGase MltA as part of a multienzyme complex involved in cell division will be investigated by genetic studies. The suitability of the amidase AmiC as a novel target for antimicrobials will be examined both genetically and by solving its crystal structure. Finally, the crystal structure of a TGase domain will reveal the catalytic mechanism of these enzymes and pave the way for drug design. Together, these studies will provide insight into the functional roles of these proteins in peptidoglycan metabolism but also the essential molecular information needed to bolster the current repertoire of antimicrobials directed against pathogenic bacteria. The sexually transmitted disease gonorrhea is a growing public-health problem due to the emergence of strains harboring resistance to antibiotics such as penicillin. The development of new treatments for gonococcal disease requires detailed, three-dimensional pictures of essential proteins in these bacteria for use in drug discovery. This project will use X-ray crystallography to provide such information for a number of key proteins in N. gonorrhoeae that are involved in cell wall synthesis.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM066861-08
Application #
7769474
Study Section
Drug Discovery and Mechanisms of Antimicrobial Resistance Study Section (DDR)
Program Officer
Marino, Pamela
Project Start
2003-02-01
Project End
2012-02-29
Budget Start
2010-03-01
Budget End
2012-02-29
Support Year
8
Fiscal Year
2010
Total Cost
$289,080
Indirect Cost
Name
Medical University of South Carolina
Department
Biochemistry
Type
Schools of Medicine
DUNS #
183710748
City
Charleston
State
SC
Country
United States
Zip Code
29425
Wu, Qinglin; Fenton, Benjamin A; Wojtaszek, Jessica L et al. (2017) Probing the excited-state chemical shifts and exchange parameters by nitrogen-decoupled amide proton chemical exchange saturation transfer (HNdec-CEST). Chem Commun (Camb) 53:8541-8544
Tomberg, Joshua; Fedarovich, Alena; Vincent, Leah R et al. (2017) Alanine 501 Mutations in Penicillin-Binding Protein 2 from Neisseria gonorrhoeae: Structure, Mechanism, and Effects on Cephalosporin Resistance and Biological Fitness. Biochemistry 56:1140-1150
Nemmara, Venkatesh V; Nicholas, Robert A; Pratt, R F (2016) Synthesis and Kinetic Analysis of Two Conformationally Restricted Peptide Substrates of Escherichia coli Penicillin-Binding Protein 5. Biochemistry 55:4065-76
Lenz, Jonathan D; Stohl, Elizabeth A; Robertson, Rosanna M et al. (2016) Amidase Activity of AmiC Controls Cell Separation and Stem Peptide Release and Is Enhanced by NlpD in Neisseria gonorrhoeae. J Biol Chem 291:10916-33
Alawieh, Ali; Sabra, Zahraa; Bizri, Abdul Rahman et al. (2015) A computational model to monitor and predict trends in bacterial resistance. J Glob Antimicrob Resist 3:174-183
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
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
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

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