Growth Regulation

Analysis of the multi-protein systems that dictate bacterial growth, with focus on the penicillin-binding proteins

Most bacteria are surrounded by a cell wall composed of a complex polymeric structure called peptidoglycan (PG) that is essential for cell survival. The biosynthetic pathway for production of PG and the proteins required for its assembly have been the targets for many antibacterial agents. There are significant gaps in the fundamental knowledge regarding the various players within cell wall synthesis and their respective spatial and temporal regulation. One example is the class of proteins known as the penicillin-binding proteins (PBPs), which polymerize and crosslink strands of PG and are targets of the beta-lactam antibiotics such as penicillin (Figure 1). While these proteins have been effectively targeted by the beta-lactam antibiotics for the last century, most bacteria have multiple PBP isoforms which are highly structurally homologous and often functionally redundant, and to date the individual roles and regulations of each PBP isoform are not well understood. To address these issues, we are using a chemical biology approach to develop activity-based probes that target a conserved catalytic serine residue in the transpeptidase domain of all PBPs.

To this end, we have developed fluorescently labeled small molecule probes to visualize the activity of PBPs directly in live cells in a broad range of organisms, which affords both temporal control and dose-dependent selectivity (ACS Chem. Biol2012, 7, 1746-1753; Curr. Prot. Chem. Bio. 20135, 239-250Antimicrob. Agents Chemother201559, 2785-2790; Antimicrob. Agents Chemother201559, 3548-3555). To assess the PBP-selectivity of inhibitors of interest, we developed a competition-assay using Bocillin-FL, a fluorescent Penicillin V analogue that indiscriminately labels most PBPs in a given organism (Figure 2). From the molecules identified in our screens, we have developed a library of beta-lactam and beta-lactone probes that can be used for labeling PBPs in an activity-dependent manner, which is unique among current PBP labeling strategies (Curr. Top. Microbiol. and Immunol. 2019). From our beta-lactone library, we have identified two probes that are semi-selective for the two genetically essential PBPs in Streptococcus pneumonaie, PBP2x and PBP2b, and enabled the first visualization of the spatiotemporal localization of these two PBPs throughout cell division (ACS Chem. Biol. 2017, 12, 2849-2857) (Figure 3). Current efforts in the lab are focused on development of existing probes for new applications, as well as the design and discovery of new probes for selectively targeting other PBPs in S. pneumoniae and Bacillus subtilis (BioRxiv2019, doi.org/10.1101/2019.12.19.881714).

Despite the effectiveness of beta-lactam antibiotics, bacterial resistance has arisen very rapidly and is one of the most pressing issues facing global health today. A more detailed understanding of the mechanism of PG synthesis may be the key for design of new and more effective antibiotics. Additionally, further development of our chemical probes for proteomic studies will enable us to interrogate the protein-protein interactions of the PBPs and uncover new antibacterial targets.

What types of methods will you learn/use on this project?

Chemical biology, organic synthesis, chemical proteomics and mass spectrometry, fluorescence microscopy, bacterial physiology, molecular modeling, and biochemistry.

pbp1

Figure 1. Beta-lactam antibiotics target the penicillin-binding proteins, key regulators of bacterial cell wall synthesis and division.

inhibitor assay

Figure 2. We have screened for selective, activity-based PBP inhibition in multiple organisms using a fluorescent competition assay with Bocillin-FL, a known global PBP inhibitor based on the Penicillin V scaffold.

pbp3

Figure 3. Using selective lactone probes discovered in our library, we have directly visualized localization of different PBPs in an activity-dependent manner.