Dynamic and functional analysis of enterococcus faecalis penicillin binding protein 4 pedestal domain

The National Institute of Allergy and Infectious Diseases awarded The Medical College of Wisconsin, Inc. $155,460 on December 1, 2025, under the Allergy and Infectious Diseases Research program (CFDA 93.855) to conduct dynamic and functional analysis of Enterococcus faecalis penicillin binding protein 4 pedestal domain.

The research aims to elucidate mechanisms of intrinsic cephalosporin resistance in enterococci, which account for 16 percent of central line-associated bloodstream infections and have emerged as a serious antibiotic resistance threat. By characterizing how the non-catalytic pedestal domain of penicillin binding protein 4 (PBP4) influences function, the work will lay the foundation for developing novel antimicrobials that disable PBP4 activity and restore cephalosporin sensitivity in enterococcal infections. Vancomycin-resistant enterococci, named a serious threat by the Centers for Disease Control as of 2021, caused an estimated 5,400 deaths and represent a critical target for therapeutic intervention.

Work is performed in Wauwatosa, Wisconsin, with a period of performance extending through June 30, 2030. This is a project grant, a form of assistance funding under which the recipient conducts the specified research activities.

Abstract

Central line-associated bloodstream infections (CLABSI) are associated with increased patient mortality, prolonged hospital stays, and increased cost burdens. Enterococci are intrinsically cephalosporin resistant opportunistic pathogens that account for 16% of CLABSI cases. When a patient is treated with cephalosporins, commensal enterococci in the gastrointestinal microbiome can flourish in the absence of cephalosporin sensitive microbes. After proliferation, enterococci can then disseminate throughout the body causing severe infections including CLABSI. Enterococci can acquire resistance to other antibiotics leading to infections which are increasingly difficult to treat. As of 2021, the center for disease control named vancomycin-resistant enterococci a serious threat which caused an estimated 5,400 deaths. Within this context, understanding the mechanism of intrinsic antibiotic resistance of enterococci has become paramount. Through elucidating these mechanisms, we will lay the foundation to enable development of novel therapeutics that disable intrinsic cephalosporin resistance and thus aid in the treatment of critically ILL patients. Enterococcus faecalis possesses three non-functionally redundant class B penicillin binding proteins (BPBPS) which crosslink peptidoglycan in the final steps of peptidoglycan wall synthesis. Previous work has shown that penicillin binding protein 4 (PBP4), a bpbp, demonstrates low-reactivity to cephalosporins and is required for cephalosporin resistance. While the catalytic activity of PBP4 has been studied, there is a lack of understanding of how non-catalytic domains of PBP4 impact function. Through understanding the role of the pedestal domain of PBP4, we may be able to exploit these mechanisms to develop antimicrobials which disable PBP4 activity, thus resulting in cephalosporin sensitivity. Overall, I hypothesize that the pedestal domain of PBP4 plays role in regulating protein function and therefore impacts enterococci cephalosporin resistance. To test this hypothesis, I propose two distinct but complementary aims. Aim 1: to define and characterize protein-protein interactions between PBP4 and proposed binding partners in vitro and in vivo. Aim 2: to characterize PBP4 pedestal domain mutations which result in a cephalosporin-sensitive phenotype. Through this proposed multidisciplinary project, I will gain mastery of biophysical, biochemical, and bacterial genetic techniques. This experience housed within the medical college of wisconsin medical scientist training program will provide opportunities for my growth into an independent physician scientist.