Novel Electrophilic Scaffold for Imaging of Essential Penicillin-Binding Proteins in <i>Streptococcus pneumoniae</i>

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 effective 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 <i>Streptococcus pneumoniae</i>. 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

Selective Penicillin-Binding Protein Imaging Probes Reveal Substructure in Bacterial Cell Division

The peptidoglycan cell wall is a common target for antibiotic therapy, but its structure and assembly are only partially understood. Peptidoglycan synthesis requires a suite of penicillin-binding proteins (PBPs), the individual roles of which are difficult to determine because each enzyme is often dispensable for growth perhaps due to functional redundancy. To address this challenge, we sought to generate tools that would enable selective examination of a subset of PBPs. We designed and synthesized fluorescent and biotin derivatives of the β-lactam-containing antibiotic cephalosporin C. These probes facilitated specific <i>in vivo</i> labeling of active PBPs in both <i>Bacillus subtilis</i> PY79 and an unencapsulated derivative of D39 <i>Streptococcus pneumoniae</i>. Microscopy and gel-based analysis indicated that the cephalosporin C-based probes are more selective than BOCILLIN-FL, a commercially available penicillin V analogue, which labels all PBPs. Dual labeling of live cells performed by saturation of cephalosporin C-susceptible PBPs followed by tagging of the remaining PBP population with BOCILLIN-FL demonstrated that the two sets of PBPs are not co-localized. This suggests that even PBPs that are located at a particular site (<i>e.g.</i>, septum) are not all intermixed, but rather that PBP subpopulations are discretely localized. Accordingly, the Ceph C probes represent new tools to explore a subset of PBPs and have the potential to facilitate a deeper understand of the roles of this critical class of proteins

Selective Penicillin-Binding Protein Imaging Probes Reveal Substructure in Bacterial Cell Division

The peptidoglycan cell wall is a common target for antibiotic therapy, but its structure and assembly are only partially understood. Peptidoglycan synthesis requires a suite of penicillin-binding proteins (PBPs), the individual roles of which are difficult to determine because each enzyme is often dispensable for growth perhaps due to functional redundancy. To address this challenge, we sought to generate tools that would enable selective examination of a subset of PBPs. We designed and synthesized fluorescent and biotin derivatives of the β-lactam-containing antibiotic cephalosporin C. These probes facilitated specific <i>in vivo</i> labeling of active PBPs in both <i>Bacillus subtilis</i> PY79 and an unencapsulated derivative of D39 <i>Streptococcus pneumoniae</i>. Microscopy and gel-based analysis indicated that the cephalosporin C-based probes are more selective than BOCILLIN-FL, a commercially available penicillin V analogue, which labels all PBPs. Dual labeling of live cells performed by saturation of cephalosporin C-susceptible PBPs followed by tagging of the remaining PBP population with BOCILLIN-FL demonstrated that the two sets of PBPs are not co-localized. This suggests that even PBPs that are located at a particular site (<i>e.g.</i>, septum) are not all intermixed, but rather that PBP subpopulations are discretely localized. Accordingly, the Ceph C probes represent new tools to explore a subset of PBPs and have the potential to facilitate a deeper understand of the roles of this critical class of proteins

Selective Penicillin-Binding Protein Imaging Probes Reveal Substructure in Bacterial Cell Division

The peptidoglycan cell wall is a common target for antibiotic therapy, but its structure and assembly are only partially understood. Peptidoglycan synthesis requires a suite of penicillin-binding proteins (PBPs), the individual roles of which are difficult to determine because each enzyme is often dispensable for growth perhaps due to functional redundancy. To address this challenge, we sought to generate tools that would enable selective examination of a subset of PBPs. We designed and synthesized fluorescent and biotin derivatives of the β-lactam-containing antibiotic cephalosporin C. These probes facilitated specific <i>in vivo</i> labeling of active PBPs in both <i>Bacillus subtilis</i> PY79 and an unencapsulated derivative of D39 <i>Streptococcus pneumoniae</i>. Microscopy and gel-based analysis indicated that the cephalosporin C-based probes are more selective than BOCILLIN-FL, a commercially available penicillin V analogue, which labels all PBPs. Dual labeling of live cells performed by saturation of cephalosporin C-susceptible PBPs followed by tagging of the remaining PBP population with BOCILLIN-FL demonstrated that the two sets of PBPs are not co-localized. This suggests that even PBPs that are located at a particular site (<i>e.g.</i>, septum) are not all intermixed, but rather that PBP subpopulations are discretely localized. Accordingly, the Ceph C probes represent new tools to explore a subset of PBPs and have the potential to facilitate a deeper understand of the roles of this critical class of proteins

Selective Penicillin-Binding Protein Imaging Probes Reveal Substructure in Bacterial Cell Division

The peptidoglycan cell wall is a common target for antibiotic therapy, but its structure and assembly are only partially understood. Peptidoglycan synthesis requires a suite of penicillin-binding proteins (PBPs), the individual roles of which are difficult to determine because each enzyme is often dispensable for growth perhaps due to functional redundancy. To address this challenge, we sought to generate tools that would enable selective examination of a subset of PBPs. We designed and synthesized fluorescent and biotin derivatives of the β-lactam-containing antibiotic cephalosporin C. These probes facilitated specific <i>in vivo</i> labeling of active PBPs in both <i>Bacillus subtilis</i> PY79 and an unencapsulated derivative of D39 <i>Streptococcus pneumoniae</i>. Microscopy and gel-based analysis indicated that the cephalosporin C-based probes are more selective than BOCILLIN-FL, a commercially available penicillin V analogue, which labels all PBPs. Dual labeling of live cells performed by saturation of cephalosporin C-susceptible PBPs followed by tagging of the remaining PBP population with BOCILLIN-FL demonstrated that the two sets of PBPs are not co-localized. This suggests that even PBPs that are located at a particular site (<i>e.g.</i>, septum) are not all intermixed, but rather that PBP subpopulations are discretely localized. Accordingly, the Ceph C probes represent new tools to explore a subset of PBPs and have the potential to facilitate a deeper understand of the roles of this critical class of proteins

Selective Penicillin-Binding Protein Imaging Probes Reveal Substructure in Bacterial Cell Division

The peptidoglycan cell wall is a common target for antibiotic therapy, but its structure and assembly are only partially understood. Peptidoglycan synthesis requires a suite of penicillin-binding proteins (PBPs), the individual roles of which are difficult to determine because each enzyme is often dispensable for growth perhaps due to functional redundancy. To address this challenge, we sought to generate tools that would enable selective examination of a subset of PBPs. We designed and synthesized fluorescent and biotin derivatives of the β-lactam-containing antibiotic cephalosporin C. These probes facilitated specific <i>in vivo</i> labeling of active PBPs in both <i>Bacillus subtilis</i> PY79 and an unencapsulated derivative of D39 <i>Streptococcus pneumoniae</i>. Microscopy and gel-based analysis indicated that the cephalosporin C-based probes are more selective than BOCILLIN-FL, a commercially available penicillin V analogue, which labels all PBPs. Dual labeling of live cells performed by saturation of cephalosporin C-susceptible PBPs followed by tagging of the remaining PBP population with BOCILLIN-FL demonstrated that the two sets of PBPs are not co-localized. This suggests that even PBPs that are located at a particular site (<i>e.g.</i>, septum) are not all intermixed, but rather that PBP subpopulations are discretely localized. Accordingly, the Ceph C probes represent new tools to explore a subset of PBPs and have the potential to facilitate a deeper understand of the roles of this critical class of proteins

Selective Penicillin-Binding Protein Imaging Probes Reveal Substructure in Bacterial Cell Division

The peptidoglycan cell wall is a common target for antibiotic therapy, but its structure and assembly are only partially understood. Peptidoglycan synthesis requires a suite of penicillin-binding proteins (PBPs), the individual roles of which are difficult to determine because each enzyme is often dispensable for growth perhaps due to functional redundancy. To address this challenge, we sought to generate tools that would enable selective examination of a subset of PBPs. We designed and synthesized fluorescent and biotin derivatives of the β-lactam-containing antibiotic cephalosporin C. These probes facilitated specific <i>in vivo</i> labeling of active PBPs in both <i>Bacillus subtilis</i> PY79 and an unencapsulated derivative of D39 <i>Streptococcus pneumoniae</i>. Microscopy and gel-based analysis indicated that the cephalosporin C-based probes are more selective than BOCILLIN-FL, a commercially available penicillin V analogue, which labels all PBPs. Dual labeling of live cells performed by saturation of cephalosporin C-susceptible PBPs followed by tagging of the remaining PBP population with BOCILLIN-FL demonstrated that the two sets of PBPs are not co-localized. This suggests that even PBPs that are located at a particular site (<i>e.g.</i>, septum) are not all intermixed, but rather that PBP subpopulations are discretely localized. Accordingly, the Ceph C probes represent new tools to explore a subset of PBPs and have the potential to facilitate a deeper understand of the roles of this critical class of proteins