Neisseria gonorrhoeae, the causative agent for the sexually transmitted infection gonorrhea, was responsible for an estimated 850,000 infections in the U.S. in 2012 and 106 million cases worldwide. Untreated or untreatable infections can lead to infertility, pelvic inflammatory disease (PID) in females, gonococcal arthritis in both sexes, and an increased risk of both contracting and transmitting HIV. Over the past few years, the steady and inexorable increase of resistance in this organism toward multiple classes of antibiotics has severely limited treatment options for gonorrhea infections. Most alarmingly, verified treatment failures against the extended-spectrum cephalosporins (ESCs), cefixime and ceftriaxone, have now been reported, prompting the Centers for Disease Control to recommend dual antibiotic therapy to replace the monotherapy that has been effective for ~80 years. Further increases in antibiotic resistance appear inevitable. This precarious position endangers public health and demands a better understanding of antibiotic resistance at the molecular level to enable countermeasures. A sentinel event in the development of cephalosporin resistance (CephR) in N. gonorrhoeae was the emergence of the first pan-resistant strain, called H041. Comparison of the known resistance mechanisms in H041 with those of cephalosporin-intermediate resistant strains (CephI) shows that alterations in the penA gene are responsible for the elevation to CephR. penA encodes penicillin-binding protein (PBP) 2, an essential transpeptidase (TPase) that functions in peptidoglycan synthesis. This renewal application proposes an investigation of the structural mechanisms underpinning cephalosporin resistance in N. gonorrhoeae due to mutations in PBP2. Crystal structures of PBP2, including complexes with ?-lactams, will be solved to elucidate how key mutations present in penA from H041 lower reactivity of the enzyme with ESCs. We will also conduct biochemical and structural investigations of the interactions between PBP2 variants and PG substrates, including boronic acid probes of the transition state, to determine how mutations selectively discriminate against ?-lactams without abrogating TPase activity. Finally, to examine our hypothesis that protein dynamics plays a critical role in antibiotic resistance, we will use NMR approaches to investigate whether mutations in PBP2 conferring cephalosporin resistance enhance or reduce the population of an alternative conformational state, and whether this state represents a productive or inhibitory conformation for the acylation reaction with both ?-lactams and PG substrate. By revealing the molecular mechanisms of how PBPs overcome the lethal action of ?-lactams, these investigations will enable new strategies for the development of replacement anti-gonococcal agents.

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 and dual therapy is now recommended by the CDC in the U.S. A major development over the past four years has been the emergence of strains that exhibit resistance to the expanded-spectrum cephalosporins, cefixime and ceftriaxone. This application will address the looming crisis in public health by elucidating the molecular mechanisms of cephalosporin resistance caused by mutations in N. gonorrhoeae penicillin-binding protein 2, with the ultimate goal of developing antimicrobials that circumvent such mechanisms.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM066861-16
Application #
9626407
Study Section
Drug Discovery and Mechanisms of Antimicrobial Resistance Study Section (DDR)
Program Officer
Marino, Pamela
Project Start
2003-02-01
Project End
2020-01-31
Budget Start
2019-02-01
Budget End
2020-01-31
Support Year
16
Fiscal Year
2019
Total Cost
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
29403
Zhu, Weiyan; Tomberg, Joshua; Knilans, Kayla J et al. (2018) Properly folded and functional PorB from Neisseria gonorrhoeae inhibits dendritic cell stimulation of CD4+ T cell proliferation. J Biol Chem 293:11218-11229
Young, Brandon F; Roth, Braden M; Davies, Christopher (2018) 1H, 13C, and 15N resonance assignments of N-acetylmuramyl-L-alanine amidase (AmiC) N-terminal domain (NTD) from Neisseria gonorrhoeae. Biomol NMR Assign :
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; 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

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