Peptidoglycan remodeling under envelope stress: deciphering the interplay between DpaA and ActS

Emerging antimicrobial resistance in pathogenic bacteria has led to a strong interest in studying the underlying molecular mechanisms, such as the mode of action of L,D-transpeptidases (LDTs), which contribute to beta-lactam resistance in gram-negative bacteria by providing an alternative bypass to penicillin-binding proteins (PBPs). Compared to PBPs, LDTs catalyze 3 -> 3 cross-links in the peptidoglycan (PG), connecting two meso-diaminopimelic acids (DAP). The LDT family comprises six members, all of which contain a YkuD (LDT) domain, except the last one, DpaA (formerly LdtF), which lacks a conserved arginine residue. DpaA does not exhibit LD-TPase activity but instead detaches Braun’s lipoproteins (Lpp) from PG and becomes essential only when the activity of the Lpt machinery, which fuels the transport of lipopolysaccharides (LPS) to the outer membrane (OM), is impaired. Despite playing a role in PG remodeling during stress, DpaA also modulates the activity of the amidase activator ActS in an unknown manner. ActS is a predicted OM lipoprotein that activates PG amidases, among other proteins, during envelope stress conditions. Upon depletion of LptC, an essential player in the Lpt machinery and thus LPS transport, ActS becomes activated. Our previous data showed that the deletion of dpaA leads to cell lysis under LPS stress conditions, while the double deletion of dpaA and actS rescues the lysing phenotype, suggesting that DpaA negatively regulates ActS. Unpublished data indicate that actS expression is not activated under envelope stress conditions or by the deletion of dpaA. Furthermore, the amidase activation of ActS does not appear to result from DpaA’s catalytic activity, leading to the hypothesis of a post-translational regulation of ActS by additional interaction partner(s). This project aims to unravel the molecular mechanisms driving LDT activities and protein interactions under OM stress to help understand modes of antibacterial resistance.