Substrate Preferences Establish the Order of Cell Wall Assembly in Staphylococcus aureus

Abstract

The Gram-positive bacterial cell wall is a large supramolecular structure and its assembly requires coordination of complex biosynthetic pathways. In the step that merges the two major biosynthetic pathways in Staphylococcus aureus cell wall assembly, conserved protein ligases attach wall teichoic acids to peptidoglycan, but the order of biosynthetic events is a longstanding question. Here, we use a chemical approach to define which of the possible peptidoglycan intermediates are substrates for wall-teichoic acid ligases, thereby establishing the order of cell wall assembly. We have developed a strategy to make defined glycan chain-length polymers of either un-cross-linked or cross-linked peptidoglycan, and we find that wall teichoic acid ligases cannot transfer wall teichoic acid precursors to the cross-linked substrates. A 1.9 Å crystal structure of a LytR-CpsA-Psr (LCP) family ligase in complex with a wall teichoic acid precursor defines the location of the peptidoglycan binding site as a long, narrow groove, and suggests that the basis for selectivity is steric exclusion of cross-linked peptidoglycan. Consistent with this hypothesis, we have found that chitin oligomers are good substrates for transfer, showing that LCPs do not discriminate cross-linked from un-cross-linked peptidoglycan substrates by recognizing features of the un-cross-linked stem peptide. We conclude that wall teichoic acids are coupled to un-cross-linked peptidoglycan chains at an early stage of peptidoglycan synthesis and may create marks that define the proper spacing of subsequent cross-links.

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

“…S. aureus LcpB, a prototypical LCP enzyme, only attaches WTA glycopolymers to uncross-linked peptidoglycan oligomers that are at least 4 sugars long (122). Neither Lipid II nor cross-linked peptidoglycan were substrates (123). Similarly, evidence suggests that S. aureus LcpC transfers CPS at an early stage of peptidoglycan synthesis as well (124).…”

“…In this case, LcpC appears to couple CPS to Lipid II, although the products were not characterized. Structural studies have revealed a possible mechanism for substrate selection by the LCP proteins based on steric exclusion (123). The LCP protein has an extended narrow groove that likely excludes cross-linked substrates.…”

“…This type of logic has been commonly used in the field of natural product biosynthesis, where elucidating the order of assembly often relies on testing the ability of a biosynthetic enzyme to convert a pathway intermediate to a product that can be used in the subsequent step. In S. aureus, WTA glycopolymers can only be transferred to nascent (uncross-linked) peptidoglycan strands, and it has been shown that these WTA-modified polymers can then be crosslinked (123). Indeed, it has even been proposed that WTAs regulate the localization of the PBP4 transpeptidase to control peptidoglycan cross-linking (128).…”

The cell cycle in Staphylococcus aureus is regulated by an amidase that controls peptidoglycan synthesis

“…To directly assess substrate preferences, we needed matched substrates that only differed in whether they were crosslinked. We made uncrosslinked glycan strands from native S. aureus Lipid II, introduced a radiolabel on the glycan backbone, and subjected a portion of the labeled material to crosslinking 22 (Supplementary Fig. 11b).…”

Rapid Intrabacterial Hydrolysis for Activating Antibiotics against Gram-negative Bacteria

“…4 Thus, there is an urgent need for developing new antimicrobial approaches against MDR bacterial pathogens. Besides investigating novel agents against new targets in bacteria [6][7][8][9] via previously unexplored mechanisms, another strategy is to enhance the efficacy and safety of the existing antibiotics via drug combinations or increasing specificity. For example, Kahne et al demonstrated that the use of novobiocin analogs greatly lower the dose of polymyxins against Gram-negative bacteria via inhibiting DNA gyrase, binding LptB, and disrupting outer membrane.…”