The prevalence of the Burkholderia cepacia complex (Bcc), a group of multidrug-resistant (MDR) pathogens, is predicted to significantly increase in patients with pulmonary disorders (e.g., chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), and asthma) by 2024. Moreover, MDR Bcc isolates that are resistant to all currently recommended therapies are emerging. Unfortunately, the development of novel drugs against MDR Bcc is lacking as is our understanding of these unique pathogens. In a retrospective study, a 35% mortality rate in Veterans that acquired a Bcc infection was observed. Additionally, Veterans are shown to be disproportionately affected by COPD, which puts them at an increased risk of acquiring infections by Bcc. Indeed, the number of Bcc outbreaks around the world has doubled over the last decade. Identifying novel strategies to overcome antibiotic resistance in these highly complex organisms that possess multiple chromosomes is a significant unmet medical need and a substantial scientific challenge. ?-Lactams are one of the most prescribed and safest class of antibiotics and are often used to treat Bcc infections. However, the production of ?-lactamases is the most prevalent ?-lactam-resistance mechanism in members of the Bcc, which possess two chromosomally-encoded inducible ?-lactamases, blapenA and blaampC. As a result, the main objective of this application is to identify novel ways of overcoming ?-lactam resistance in Bcc. Building upon studies performed previously, mechanism-based approaches will be used to selectively inhibit the following proteins in Bcc: 1. PenA, a versatile carbapenemase; 2. AmpC, a unique cephalosporinase; 3. Penicillin binding proteins (PBPs), the biological target of ?-lactams and whose inhibition is linked to bla (?- lactamase gene) expression; and 4. PenRA, the transcription regulator of bla genes. To address these objectives, a mechanism-based approach will be used to restore susceptibility to MDR Bcc by testing selected ?-lactams alone and in combination with ?-lactamase inhibitors, performing biochemical and structural analysis of PenA and AmpC with the ?-lactams and ?-lactamase inhibitors, analyzing the genomes of MDR Bcc, and determining the in vivo efficacy of selected combinations. Moreover, the link between PBP inhibition and bla expression will be deciphered by identifying which ?-lactams effect bla expression, measuring the binding of ?-lactams to PBPs, visualizing cells exposed to ?-lactams via microscopy to reveal the impact of ?-lactams on cell morphology, and constructing pbp gene knockouts and assessing their phenotypes. In addition, PenRA will be targeted for inhibition in B. multivorans by using crystallography to define the binding pocket of the PenRA effector binding domain (EBD) and conducting a targeted small molecule inhibitor library screen using an in-house high-throughput fluorescence assay. The anticipated outcomes include identifying novel combinations to inhibit highly drug resistant Bcc by determining which compounds target PenA, AmpC, and/or PBPs. Moreover, a greater understanding of the link between PBP inhibition and bla expression will be gained, thus allowing clinicians to make better choices for therapy. The interactions between native ligand of PenRA as well as a selected panel of small molecules which resemble the native ligand will be determined, thus allowing for the identification of ?lead? compounds to target PenRA and inhibit bla expression. Based on the studies conducted herein, Veterans as well as other individuals that acquire a Bcc infection will have alternative therapeutic options compared to what is currently available, enabling clinicians to eradicate the organism and obtain clinical cure.

Public Health Relevance

The introduction of antibiotics >80 years ago was a revolutionary occurrence that saved the lives of millions of people. Yet, in 2014, a high-profile review estimated that the number of deaths due to untreatable infections could reach 10 million by 2050; thus, foreshadowing the evaporation of effective antibiotic therapies. ?- Lactamase-mediated resistance by Gram-negative pathogens is an existing threat to Veterans and the general population. Of these Gram-negatives, Burkholderia cepacia complex (Bcc) is emerging as important group of antibiotic-resistant pathogens and our understanding of these unique pathogens remains underdeveloped. Patients with lung diseases (i.e., chronic obstructive pulmonary disorder (COPD), asthma, and cystic fibrosis) are highly susceptible to acquire Bcc infections such as pneumonia and bacteremia. Moreover, in the last 17 years, Bcc was found throughout the US VA healthcare system and resulted in a 35% mortality rate for affected Veterans. The goal of this project is to discover novel therapies to treat highly drug-resistant Bcc infections.

Agency
National Institute of Health (NIH)
Institute
Veterans Affairs (VA)
Type
Non-HHS Research Projects (I01)
Project #
2I01BX002872-05
Application #
9763694
Study Section
Special Emphasis Panel (ZRD1)
Project Start
2015-10-01
Project End
2023-09-30
Budget Start
2019-10-01
Budget End
2020-09-30
Support Year
5
Fiscal Year
2020
Total Cost
Indirect Cost
Name
Louis Stokes Cleveland VA Medical Center
Department
Type
DUNS #
093016124
City
Cleveland
State
OH
Country
United States
Zip Code
44141
Becka, Scott A; Zeiser, Elise T; Barnes, Melissa D et al. (2018) Characterization of the AmpC ?-Lactamase from Burkholderia multivorans. Antimicrob Agents Chemother 62:
Barnes, Melissa D; Bethel, Christopher R; Alsop, Jim et al. (2018) Inactivation of the Pseudomonas-Derived Cephalosporinase-3 (PDC-3) by Relebactam. Antimicrob Agents Chemother 62:
Nukaga, Michiyoshi; Papp-Wallace, Krisztina M; Hoshino, Tyuji et al. (2018) Probing the Mechanism of Inactivation of the FOX-4 Cephamycinase by Avibactam. Antimicrob Agents Chemother 62:
Becka, Scott A; Zeiser, Elise T; Marshall, Steven H et al. (2018) Sequence heterogeneity of the PenA carbapenemase in clinical isolates of Burkholderia multivorans. Diagn Microbiol Infect Dis 92:253-258
Papp-Wallace, Krisztina M; Barnes, Melissa D; Alsop, Jim et al. (2018) Relebactam Is a Potent Inhibitor of the KPC-2 ?-Lactamase and Restores Imipenem Susceptibility in KPC-Producing Enterobacteriaceae. Antimicrob Agents Chemother 62:
Papp-Wallace, Krisztina M; Nguyen, Nhu Q; Jacobs, Michael R et al. (2018) Strategic Approaches to Overcome Resistance against Gram-Negative Pathogens Using ?-Lactamase Inhibitors and ?-Lactam Enhancers: Activity of Three Novel Diazabicyclooctanes WCK 5153, Zidebactam (WCK 5107), and WCK 4234. J Med Chem 61:4067-4086
Barnes, Melissa D; Taracila, Magdalena A; Rutter, Joseph D et al. (2018) Deciphering the Evolution of Cephalosporin Resistance to Ceftolozane-Tazobactam in Pseudomonas aeruginosa. MBio 9:
Mojica, Maria F; Papp-Wallace, Krisztina M; Taracila, Magdalena A et al. (2017) Avibactam Restores the Susceptibility of Clinical Isolates of Stenotrophomonas maltophilia to Aztreonam. Antimicrob Agents Chemother 61:
Papp-Wallace, Krisztina M; Becka, Scott A; Taracila, Magdalena A et al. (2017) Exploring the Role of the ?-Loop in the Evolution of Ceftazidime Resistance in the PenA ?-Lactamase from Burkholderia multivorans, an Important Cystic Fibrosis Pathogen. Antimicrob Agents Chemother 61:
Barnes, Melissa D; Winkler, Marisa L; Taracila, Magdalena A et al. (2017) Klebsiella pneumoniae Carbapenemase-2 (KPC-2), Substitutions at Ambler Position Asp179, and Resistance to Ceftazidime-Avibactam: Unique Antibiotic-Resistant Phenotypes Emerge from ?-Lactamase Protein Engineering. MBio 8:

Showing the most recent 10 out of 21 publications