Enzymatic Activity of PBP1B Compensates for β-lactam Mediated Inhibition of PBP2 in Cells Overproducing ppGpp

The alarmone (p)ppGpp (ppGpp) accumulates in response to starvation and other stress, leading to inhibition of multiple biosynthetic pathways and, at high concentrations, suppression of bacterial growth. Growth suppression by ppGpp is implicated in the formation of persister cells, which survive antibiotic challenge only to regrow once the drug is removed. However, there is also evidence that low levels of ppGpp contribute to resistance to certain cell wall-active antibiotics in actively growing cells. To characterize ppGpp's contribution to antibiotic resistance, we measured MICs of a panel of β-lactams in actively growing Escherichia coli cells overexpressing a ppGpp synthase ( relA *). Cells engineered to modestly overproduce ppGpp exhibited up to 64-fold increases in resistance to PBP2-targeting β-lactams only, with mecillinam the most dramatically affected. Resistance required the transcription factor DksA and the class A penicillin binding protein (PBP) PBP1B. PBP1B variants defective for transpeptidase activity, glycosyltransferase activity, or both were incapable of mediating resistance, suggesting the full enzymatic activity of PBP1B is required for resistance. Transcriptomics revealed that ppGpp overproduction leads to increased expression of lpoB , which encodes an activator of PBP1B. LpoB was required for mecillinam resistance, with an lpoB deletion mutant exhibiting a loss of ppGpp-dependent resistance. Resistance was partially lost in an lpoB deletion strain expressing an LpoB-bypass variant of PBP1B ( mrcB *). Together these results support a model wherein overproduction of ppGpp favors cell wall synthesis by PBP1B, in part via increased expression of lpoB , allowing PBP1B to substitute for PBP2 when it is inhibited by β-lactams.

Antimicrobial resistance is an increasing global health threat, but the molecular mechanisms underlying resistance remain incompletely understood. This work clarifies the role of the alarmone ppGpp in mediating antibiotic resistance in the model bacterium Escherichia coli . Elevated levels of ppGpp were found to cause resistance to β-lactam antibiotics targeting the cell wall synthesis enzyme PBP2. Resistance required transcriptional regulation by ppGpp and enzymatic activity of the cell wall enzyme PBP1B. This work suggests that ppGpp causes resistance in part by increasing expression of lpoB , which encodes an activator of PBP1B. Because ppGpp levels are controlled by nutritional conditions, this work suggests that nutritional availability could impact the efficacy of antibiotics. Furthermore, this study adds to a number of studies demonstrating that cell wall synthesizing PBP enzymes can compensate for one another under certain conditions, leading to β-lactam resistance.