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 in vivo labeling of active PBPs in both Bacillus subtilis PY79 and an unencapsulated derivative of D39 Streptococcus pneumoniae. 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 (e.g., 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.
Department of Chemistry, Indiana University, Bloomington, Indiana 47405, United States.
Ozden
O
Kocaoglu
Rebecca A
RA
Calvo
Lok-To
LT
Sham
Loralyn M
LM
Cozy
Bryan R
BR
Lanning
Samson
S
Francis
Malcolm E
ME
Winkler
Daniel B
DB
Kearns
Erin E
EE
Carlson
D000900:Anti-Bacterial Agents / Q000138:chemical synthesis / Q000737:chemistry; D001412:Bacillus subtilis / Q000737:chemistry; D002455:Cell Division / Q000502:physiology*; D002511:Cephalosporins / Q000138:chemical synthesis / Q000737:chemistry; D005456:Fluorescent Dyes; D008856:Microscopy, Fluorescence; D046915:Penicillin-Binding Proteins / Q000737:chemistry* / Q000648:ultrastructure*; D010455:Peptides / Q000737:chemistry; D013296:Streptococcus pneumoniae / Q000737:chemistry
D000900:Anti-Bacterial Agents; D002511:Cephalosporins; D005456:Fluorescent Dyes; D046915:Penicillin-Binding Proteins; D010455:Peptides; C025163:cephalosporin C