Determining the value of PBP 7/8 as an antimicrobial target for XDR-A. baumannnii

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Anticipated Impacts on Veterans Health Care. The incidence of infections due to highly resistant Acinetobacter baumannii is increasing. True pan drug resistant (PDR) strains have been reported. Unfortunately, the newly approved antimicrobials ceftolozane-tazobactam, ceftazidime-avibactam, and meropenem/vaborbactam are poorly active against resistant A. baumannii. The need to identify new antimicrobials active against A. baumannii is pressing. Background. We have demonstrated that the A. baumannii low molecular mass (LMM) penicillin binding protein 7/8 (PBP 7/8) is essential in vivo (i.e., required for bacterial survival in a host) in rat pneumonia and subcutaneous abscess infection models, and is required for survival in human serum and ascites. Our initial studies on PBP 7/8 were performed in an antimicrobial sensitive strain AB307-0294. These findings have since been confirmed with the extensively drug resistant (XDR) strain HUMC1. Since our initial observation other investigators, using INseq and Tn-seq, have shown that PBP 7/8 is needed for A. baumannii survival in the Galleria mellonella insect infection model and serum respectively. PBP 7/8 has many characteristics that support its potential as a high value drug target. However, based on studies in rich laboratory medium, LMM PBPs including PBP 7/8 have been perceived to be non-essential. As a result, PBP 7/8 have received scant attention as an antibacterial target and has not been considered for A. baumannii. However, our data contradicts this paradigm and endorses the need for additional studies on PBP 7/8 in XDR A. baumannii. Therefore, our objective is to validate PBP 7/8 as a drug target, delineate mechanisms that mediate its ex vivo and in vivo essentiality, and to identify compounds that inactivate PBP 7/8. Objectives and Methods. To achieve these objectives, studies on PBP 7/8 will be extended to include additional XDR strains of A. baumannii to confirm our findings are generalizable (aim 1). Aim 1 also will also explore mechanisms by which PBP 7/8 enables growth/survival of A. baumannii ex vivo and in vivo and identify the innate host defense factors it protects against. Increasing data support the need to treat infections due to XDR isolates with multiple agents, thereby increasing efficacy and protecting against the development of resistance. Our demonstration that the loss of PBP 7/8 production increases susceptibility to complement and lysozyme mediated bactericidal activity and changes cell structure supports the hypothesis that the inability to produce PBP 7/8 affects permeability, a critical factor in the intrinsic antimicrobial resistance of A. baumannii. Therefore, an additional goal of this proposal will be to assess the whether the loss of PBP 7/8 production enhances the activity of adjunctive therapy with antimicrobials and/or monoclonal antibodies directed against the capsule (aim 2). Additional studies in aim 2 will assess whether the loss of PBP 7/8 affects the production of other PBPs and ampC expression. These data will generate additional mechanistic insights into the consequences of not being able to produce PBP 7/8. Lastly, aim 3 will begin the process of identifying a “tool” compound that is active against PBP 7/8. A live wild-type XDR strain will be screened against a chemical library to identify compounds directed against PBP 7/8 that possess bactericidal activity. Additional orthogonal assays will establish specificity, determine their quality, and prioritize identified inhibitors for downstream optimization and pre-clinical studies. Deliverables from this proposal will validate the paradigm shift on the value of PBP 7/8 as an antimicrobial target and open an untapped venue for the development of a new class of antimicrobial agents against this XDR pathogen.