The mecillinam resistome reveals a role for peptidoglycan endopeptidases in stimulating cell wall synthesis in Escherichia coli

Abstract

Bacterial cells are typically surrounded by an net-like macromolecule called the cell wall constructed from the heteropolymer peptidoglycan (PG). Biogenesis of this matrix is the target of penicillin and related beta-lactams. These drugs inhibit the transpeptidase activity of PG synthases called penicillin-binding proteins (PBPs), preventing the crosslinking of nascent wall material into the existing network. The beta-lactam mecillinam specifically targets the PBP2 enzyme in the cell elongation machinery of Escherichia coli. Low-throughput selections for mecillinam resistance have historically been useful in defining mechanisms involved in cell wall biogenesis and the killing activity of beta-lactam antibiotics. Here, we used transposon-sequencing (Tn-Seq) as a high-throughput method to identify nearly all mecillinam resistance loci in the E. coli genome, providing a comprehensive resource for uncovering new mechanisms underlying PG assembly and drug resistance. Induction of the stringent response or the Rcs envelope stress response has been previously implicated in mecillinam resistance. We therefore also performed the Tn-Seq analysis in mutants defective for these responses in addition to wild-type cells. Thus, the utility of the dataset was greatly enhanced by determining the stress response dependence of each resistance locus in the resistome. Reasoning that stress response-independent resistance loci are those most likely to identify direct modulators of cell wall biogenesis, we focused our downstream analysis on this subset of the resistome. Characterization of one of these alleles led to the surprising discovery that the overproduction of endopeptidase enzymes that cleave crosslinks in the cell wall promotes mecillinam resistance by stimulating PG synthesis by a subset of PBPs. Our analysis of this activation mechanism suggests that, contrary to the prevailing view in the field, PG synthases and PG cleaving enzymes need not function in multi-enzyme complexes to expand the cell wall matrix.

Structural basis of peptidoglycan endopeptidase regulation

“…To assess enhanced NGO_1686 toxicity more quantitatively, we also performed a mecillinam/moenomycin sensitivity assay. In E. coli , increased EP activity imparts mecillinam resistance, likely due to overactivation of aPBPs (52). We predicted that such aPBP overactivation should also render these cells more susceptible to moenomycin, an aPBP inhibitor (53).…”

“…While we have not identified the signal promoting this conformational switch, we hypothesize that this might be an interaction with cell wall synthases or the protein complexes they are embedded in, to enable tight coordination between synthesis and degradation. Importantly, however, the aPBPs themselves are not likely activators of conformational switching – data in E. coli suggests that EPs are limiting for aPBP activity (52); if aPBPs were necessary to activate EPs, this relationship would be reversed. In E. coli , the lipoprotein NlpI was recently identified as a multifunctional regulator of EP activity and coordinator of hydrolysis with PG synthesis functions (40).…”

Cell-wall synthases contribute to bacterial cell-envelope integrity by actively repairing defects

“…Since aPBPs and Lpo’s form envelope-spanning complexes ( Egan et al, 2014 ; Jean et al, 2014 ) they have been suggested to work as repair enzymes that activate at site of defects or large pores in the peptidoglycan ( Typas et al, 2012 ; Cho et al, 2016 ). In support of this idea, aPBP activity was increased ( Lai et al, 2017 ) upon over-expression of the DD-endopeptidase MepS, which cleaves peptide bonds ( Singh et al, 2012 ). Therefore, Rod complex and aPBPs might serve different functions despite catalyzing the same chemical reactions ( Zhao et al, 2017 ; Pazos et al, 2017 ).…”

“…Second, the flux of lipid-II precursors shared by both systems might not be fixed in E. coli . Instead, both systems might secure access somewhat independently, as also supported by the overall increase in peptidoglycan synthesis upon MepS over-expression ( Lai et al, 2017 ). Finally, LpoB might play an important limiting factor for PBP1b activity, which is absent in B. subtilis .…”

“…On the contrary, we confirmed that aPBPs are essential for mechanical cell-wall integrity. Second, we investigate how the major aPBP PBP1b contributes to mechanical integrity: simply through a higher overall rate of peptidoglycan insertion ( Caparrós et al, 1994 ), by constitutively stabilizing cell-wall, for example by inserting peptidoglycan in a more spatially homogeneous manner, or through active repair of local cell-wall damage, as previously suggested ( Typas et al, 2012 ; Lai et al, 2017 ; Cho et al, 2016 ). We first measured mechanical stability and rate of peptidoglycan insertion in cells with aPBP levels reduced three-fold.…”