Peptidoglycan Cross-Linking Preferences of Staphylococcus aureus Penicillin-Binding Proteins Have Implications for Treating MRSA Infections

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA) infections are a global public health problem. MRSA strains have acquired a non-native penicillin-binding protein called PBP2a that crosslinks peptidoglycan when the native S. aureus PBPs are inhibited by β-lactams. If assembly of the pentaglycine branch on the cell wall precursor Lipid II is genetically blocked, MRSA strains become susceptible to β-lactams. Therefore, it has been proposed that PBP2a can only crosslink peptidoglycan strands bearing a complete pentaglycine branch. This hypothesis has never been tested because the necessary substrates have not been available. Here, we obtained S. aureus Lipid II variants having shorter glycine branches and have tested whether PBP2a and two other S. aureus transpeptidases, PBP2 and PBP4, can crosslink peptidoglycan strands made from the variants. There are striking differences in enzymatic activity among these enzymes depending on the length of the glycine branch, but we find that PBP2a can, in fact, crosslink glycan strands bearing triglycine. We report experiments in cells that are consistent with our in vitro findings about the crosslinking preferences of these PBPs. In addition to providing insights into the cell wall physiology of a major pathogen, our studies identify the best target for β-lactam potentiators to treat MRSA.

Uncovering the activities, biological roles, and regulation of bacterial cell wall hydrolases and tailoring enzymes

“…In S. aureus, the Fem proteins sequentially JBC REVIEWS: Strategies to study cell wall-modifying enzymes install five glycines to the side chain of L-Lys in the stem peptide: FemX adds the first glycine, FemA adds the second and third glycines, and FemB adds the final two glycines. Lipid II-monoglycine and Lipid II-triglycine have been prepared from S. aureus ⌬femAB and ⌬femB mutants, respectively (99). Similarly, an S. pneumoniae ⌬murMN mutant provides access to Lipid II with unmodified lysine at the third position (100).…”

Remodeling of Crossbridges Controls Peptidoglycan Cross-linking Levels in Bacterial Cell Walls

“…Yet, for the most part, their physiological impact remains elusive. [11][12][13][14] The 3 rd position with the PG stem peptide has defining structural characteristics. As an example, the difference of a carboxylic acid group on L-Lys verus meso-2,6-diaminopimelic acid (m-DAP) has important implications in the activation of the human innate immune system.…”

“…[16][17][18][19][20][21] Furthermore, elegant prior in vitro studies using synthetic PG analogs have provided insight into the substrate structural requirements of TPs. 12,[22][23][24][25] However, fewer reports have described probes that decipher TP substrate requirements in live bacterial cells. A recent example of a synthetic L-Lys-D-Ala-D-Ala stem peptide mimic led to the demonstration of extraseptal TP activity in Staphylococcus aureus (S. aureus).…”

“…[33][34][35] Recently, Walker and co-workers isolated PG precursors corresponding to one, three, and five glycine units and showed in vitro that PBP transpeptidases have varying levels of preference for the length of the glycine chain. 12 We built a library of tripeptide S. aureus probes that complement their approach by assessing the acyl-acceptor preference in live bacterial cells. We synthesized TriQG1-6 (Figure 4b), corresponding to glycine chains from 1 to 6 units, and measured their incorporation into the PG scaffold of S. aureus cells.…”

Synthesis of Lipid‐Carbohydrate‐Peptidyl‐RNA Conjugates to Explore the Limits Imposed by the Substrate Specificity of Cell Wall Enzymes on the Acquisition of Drug Resistance

“…[9] We focus on FmhB since this enzymei se ssential presumably because the side-chain of peptidoglycan precursors directly participates in the peptidoglycanc ross-linking reaction ( Figure 1e). [9][10] To investigate the specificityo fF mhB, we have synthesized d-Alaand d-Lac-containings ubstrates analogues( Figure 2) and lipidcarbohydrate-peptidyl-RNA conjugates acting as inhibitors (Figure 1c). This strategy was designed to assess the impact of the d-Ala to d-Lac substitution both on the catalytic efficacy of the transfer of Gly from Gly-tRNA Gly to the peptidoglycanp recursors and on the affinity of FmhB fori nhibitors that mimic both substrates of the enzyme( referred to as "bi-substrates").…”