Novel Electrophilic Scaffold for Imaging of Essential Penicillin-Binding Proteins in Streptococcus pneumoniae

Abstract

Peptidoglycan (PG) is a mesh-like heteropolymer made up of glycan chains cross-linked by short peptides and is the major scaffold of eubacterial cell walls, determining cell shape, size, and chaining. This structure, which is required for growth and survival, is located outside of the cytoplasmic membrane of bacterial cells, making it highly accessible to antibiotics. Penicillin-binding proteins (PBPs) are essential for construction of PG and perform transglycosylase activities to generate the glycan strands and transpeptidation to cross-link the appended peptides. The β-lactam antibiotics, which are among the most clinically effctive antibiotics for the treatment of bacterial infections, inhibit PBP transpeptidation, ultimately leading to cell lysis. Despite this importance, the discrete functions of individual PBP homologues have been difficult to determine. These major gaps in understanding of PBP activation and macromolecular interactions largely result from a lack of tools to assess the functional state of specific PBPs in bacterial cells. We have identified β-lactones as a privileged scaffold for the generation of PBP-selective probes and utilized these compounds for imaging of the essential proteins, PBP2x and PBP2b, in Streptococcus pneumoniae. We demonstrated that while PBP2b activity is restricted to a ring surrounding the division sites, PBP2x activity is present both at the septal center and at the surrounding ring. These spatially separate regions of PBP2x activity could not be detected by previous activity-based approaches, which highlights a critical strength of our PBP-selective imaging strategy.

Chemical Reporters for Exploring Microbiology and Microbiota Mechanisms

“…Alternatively, the Carlson laboratory has developed fluorescent and biotin derivatives of cephalosporin C (Figure B), which enables selective in vivo labeling of active PBPs . The Carlson laboratory has also developed β‐lactone based PBP‐selective reporters to distinguish the localization of PBP2x from PBP2b in S. pneumoniae (Figure C) . To explore other peptidoglycan biosynthetic enzymes, the Sieber laboratory has generated a chemical reporter of showdomycin (Figure D), a maleimide‐containing uridine‐based covalent inhibitor of MurA, a UDP‐ N ‐acetylglucosamine 1‐carboxyvinyltransferase that catalyzes the first step in cell wall biosynthesis and antibiotic drug target.…”

“…B) Cephalosporin‐based reporters for PBPs . C) β‐lactone‐based reporters for PBPs . D) Showdomycin‐based reporter for MurA1/2 .…”

“…[86] The Carlson laboratory has also developed b-lactoneb ased PBP-selective reporterst od istinguish the localization of PBP2x from PBP2b in S. pneumoniae (Figure 11 C). [87] To explore other peptidoglycan biosynthetice nzymes,t he Sieberl aboratory has generated ac hemical reportero fs howdomycin (Figure 11 D), [88] am aleimide-containing uridine-based covalent inhibitor of MurA, a UDP-N-acetylglucosamine 1-carboxyvinyltransferase that catalyzes the first step in cell wall biosynthesis and antibiotic drug target. In addition, chemical reporterso fa cylhomoserine lactones involved in bacterial quorum sensing have been developed by the Krom and Meijler laboratories to identify their potentialp rotein targets.…”

“…[86] C) b-lactone-based reporters for PBPs. [87] D) Showdomycin-based reporter for MurA1/2. [88] E) BSH activity-based reporter,Ch-AOMK.…”

Harnessing β-Lactam Antibiotics for Illumination of the Activity of Penicillin-Binding Proteins inBacillus subtilis

“…(1,(4)(5)(6) Although the importance of the PBPs as antibacterial targets has long been appreciated, the specific roles of individual PBPs, beyond their catalytic function, has remained elusive due to the lack of appropriate chemical and biological tools. (7,8) Functional redundancy within the PBPs has compounded the difficulties in establishing the role of individual PBPs in peptidoglycan biosynthesis. (9)(10)(11) Single PBPs have been studied by conjugation to fluorescently labeled protein tags.…”