Selective penicillin-binding protein imaging probes reveal substructure in bacterial cell division

Abstract

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.

Figure 1

Comparison of the transpeptidation reaction performed by penicillin-binding proteins (PBPs) and their inactivation by the β-lactam antibiotic, penicillin. (a) PBPs catalyze transpeptidation by cleavage of the terminal D-Ala residue of the stem peptide resulting in formation of an acyl-enzyme intermediate. In most Gram-positive bacteria, this ester is subsequently attacked by the lysine residue of a neighboring stem peptide to crosslink the chains. M = N-acetylmuramic acid, G = N-acetylglucosamine. In B. subtilis, L-Lys is replaced with meso-diaminopimelic acid. (b) Penicillin is a structural mimetic of the natural substrate for PBPs. (c) The active site serine in PBPs attacks the highly strained β-lactam ring resulting in opening of the lactam. This reaction leads to irreversible covalent modification of the PBP.

Figure 2

Structures of the cephalosporin C-based probes and utilization in gel-based analysis in comparison to BOCILLIN-FL. (a) Ceph C probes were designed with three reporter tags. Ceph C core (R), TAMRA (Ceph C-T), fluorescein (Ceph C-F) and biotin (Ceph C-B). (b) Gel-based analysis of the PBP profiles of B. subtilis PY79 cells that are labeled with Ceph C-T (in vivo or in vitro labeling of membrane proteome) or Boc-FL (in vivo labeling shown, in vitro profile was similar). Several bands show differential labeling by Ceph C-T when this compound is applied to live cells versus membrane proteome. Whereas Boc-FL labels all active PBPs in the sample, Ceph C labels only four PBPs when utilized in in vivo experiments. These proteins were subsequently identified as PBPs 1a/1b, 2b, 2c and 4.

Figure 3

Localization of Ceph C-F and Boc-FL labeled PBPs in B. subtilis PY79. Cells were stained with either Ceph C-F or Boc-FL and then subjected to a membrane dye (FM-4-64). The two probes yielded similar staining patterns with the PBPs labeled by Ceph C-F and Boc-FL both being localized on every other division septa. Scale bar, 2 μm.

Figure 4

Imaging of B. subtilis PBPs after dual labeling with Ceph C-T (red) and Boc-FL (green) in vivo as visualized with gel-based and wide-field fluorescence microscopy methods. (a) Labeling was performed first with Ceph C-T (30 min) followed by treatment with by Boc-FL (10 min). The selectivity profile observed when labeling the cells with only Ceph C-T was preserved (PBPs 1a/b, 2b, 2c and 4). (b) Wide-field microscopy images of dual labeled cells show almost complete co-staining with Ceph C-T and Boc-FL. 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate (TMA-DPH) was used to dye the entire membrane (blue). Scale bars, 2 μm.

Figure 5

Imaging of B. subtilis PBPs after dual labeling with Ceph C-T (red) and Boc-FL (green) in vivo as visualized with 3D-SIM super-resolution microscope. Labeling was performed first with Ceph C-T (30 min) followed by treatment with by Boc-FL (10 min). TMA-DPH was used to stain the entire membrane (blue). Images demonstrate that Ceph C-T and Boc-FL labeling at the division septa are not co-localized indicating that the PBPs targeted by the probes are found in different regions. Top images are of the whole cell while bottom panels show an end on view of the divisional septa. (a) Cells with the earliest divisional septa showed crescent-shaped fluorescent labeling with asymmetric intensity. (b) Cells marked by invaginations of the membrane show PBP localization as a disk. (c) Following division, crescent shapes were retained at the pole in each daughter cell (c). Scale bars, 1 μm.

Figure 6

PBP labeling of S. pneumoniae with Ceph C-T (red) or Boc-FL (green). (a) Gel-based analysis of S. pneumoniae PBPs labeled with Ceph C-T or Boc-FL. Mutant strains were used to assign the proteins labeled with Ceph C-T. This probe labels PBP1a (faint), 1b and 3. Boc-FL labels all active PBPs including PBPs 2a, 2b and 2x, which are not labeled by Ceph C-T. (b) Staining patterns of Ceph-T and Boc-FL labeled PBPs in live cells as detected by wide-field fluorescence microscopy. Scale bar, 2 μm. (c) Gel-based analysis of S. pneumoniae PBPs labeled first with Ceph C-T (30 min) followed by treatment with by Boc-FL (10 min). The weak labeling of PBP1a by Ceph C-T is outcompeted with Boc-FL. In addition, PBP3 is labeled by both probes (two bands are the result of slight variation in migration of the two labeled proteins).

Figure 7

Super-resolution (3D-SIM) images of S. pneumoniae IU1945 PBPs after dual labeling with Ceph C-T (red) and Boc-FL (green) in vivo. Both dyes were seen in the septal and equatorial regions but little co-localization of the two PBP subsets was observed indicating differential distribution. Scale bar, 1 μm.