Structural basis of peptidoglycan endopeptidase regulation

Abstract

<jats:p>Most bacteria surround themselves with a cell wall, a strong meshwork consisting primarily of the polymerized aminosugar peptidoglycan (PG). PG is essential for structural maintenance of bacterial cells, and thus for viability. PG is also constantly synthesized and turned over; the latter process is mediated by PG cleavage enzymes, for example, the endopeptidases (EPs). EPs themselves are essential for growth but also promote lethal cell wall degradation after exposure to antibiotics that inhibit PG synthases (e.g., β-lactams). Thus, EPs are attractive targets for novel antibiotics and their adjuvants. However, we have a poor understanding of how these enzymes are regulated in vivo, depriving us of novel pathways for the development of such antibiotics. Here, we have solved crystal structures of the LysM/M23 family peptidase ShyA, the primary EP of the cholera pathogen<jats:italic>Vibrio cholerae</jats:italic>. Our data suggest that ShyA assumes two drastically different conformations: a more open form that allows for substrate binding and a closed form, which we predicted to be catalytically inactive. Mutations expected to promote the open conformation caused enhanced activity in vitro and in vivo, and these results were recapitulated in EPs from the divergent pathogens<jats:italic>Neisseria gonorrheae</jats:italic>and<jats:italic>Escherichia coli</jats:italic>. Our results suggest that LysM/M23 EPs are regulated via release of the inhibitory Domain 1 from the M23 active site, likely through conformational rearrangement in vivo.</jats:p>

Proximate and ultimate causes of the bactericidal action of antibiotics

“…In all these cases, as well as in E. coli, β-lactams trigger autolysin release by disturbing the balance between peptidoglycan synthesis and hydrolysis, which is necessary for growth of the cell wall 54 . Bacterial growth requires constant synthesis and turnover of the cell wall to insert new molecules, and the latter process relies on peptidoglycan cleavage enzymes, including glycosidases, amidases and endopeptidases 55 . The same enzymes can produce lethal damage -the physiology of growth can be converted into the physiology of death.…”

Class A Penicillin-Binding Protein-mediated cell wall synthesis promotes structural integrity during peptidoglycan endopeptidase insufficiency

“…We have previously shown that EP depletion in the ∆6 endo strain results in a dramatic increase in cell diameter and ultimately the generation of giant, bulky and contorted cells (34). Here, we asked to what extent these enlarged cells accumulated a PG cell wall.…”

“…4A, 5A). However, we recently demonstrated that EPs from diverse organisms (including E. coli and N. gonorrhoeae) are produced in an inactive form due to the inhibitory function of their domain 1 and likely activated in vivo by an unknown mechanism (34). Heterologously expressed enzymes are likely not subject to this activation pathway in V. cholerae (especially if the activator is a protein) and we thus instead expressed EP mutant versions with their inhibitory domain 1 deleted, which are expected to be constitutively active.…”

“…EPs in particular are a doubleedged sword as they can both promote cell wall synthesis (28) and play major roles in cell wall cleavage after beta lactam exposure (29,30). However, how EPs are regulated has only begun to be unravelled (31)(32)(33)(34), and at least in Gram-negative bacteria we lack a complete understanding of how EP cleavage activity relates to PG synthesis by the two distinct cell wall synthase complexes.…”

“…While the lac promoter is tightly repressed in the absence of inducer, a small number of molecules under its control might still be produced(53). ShyA is produced as an inactive precursor and the signal for activation is unkown(34). It is conceivable that complementation with a heterologously expressed EP might somehow enhance activation of this leaky background of ShyA molecules, e.g.…”