Secreted antigen A peptidoglycan hydrolase is essential for Enterococcus faecium cell separation and priming of immune checkpoint inhibitor therapy.

Enterococcus faecium is a microbiota species in humans that can modulate host immunity (Griffin and Hang, 2022), but has also acquired antibiotic resistance and is a major cause of hospital-associated infections (Van Tyne and Gilmore, 2014). Notably, diverse strains of E. faecium produce SagA, a highly conserved peptidoglycan hydrolase that is sufficient to promote intestinal immunity (Rangan et al., 2016; Pedicord et al., 2016; Kim et al., 2019) and immune checkpoint inhibitor antitumor activity (Griffin et al., 2021). However, the functions of SagA in E. faecium were unknown. Here, we report that deletion of sagA impaired E. faecium growth and resulted in bulged and clustered enterococci due to defective peptidoglycan cleavage and cell separation. Moreover, ΔsagA showed increased antibiotic sensitivity, yielded lower levels of active muropeptides, displayed reduced activation of the peptidoglycan pattern-recognition receptor NOD2, and failed to promote cancer immunotherapy. Importantly, the plasmid-based expression of SagA, but not its catalytically inactive mutant, restored ΔsagA growth, production of active muropeptides, and NOD2 activation. SagA is, therefore, essential for E. faecium growth, stress resistance, and activation of host immunity.

Secreted antigen A peptidoglycan hydrolase is essential for Enterococcus faecium cell separation and priming of immune checkpoint inhibitor cancer therapy.

Enterococcus faecium is a microbiota species in humans that can modulate host immunity, but has also acquired antibiotic resistance and is a major cause of hospital-associated infections. Notably, diverse strains of E. faecium produce SagA, a highly conserved peptidoglycan hydrolase that is sufficient to promote intestinal immunity and immune checkpoint inhibitor antitumor activity. However, the functions of SagA in E. faecium were unknown. Here we report that deletion of sagA impaired E. faecium growth and resulted in bulged and clustered enterococci due to defective peptidoglycan cleavage and cell separation. Moreover, Δ sagA showed increased antibiotic sensitivity, yielded lower levels of active muropeptides, displayed reduced activation of the peptidoglycan pattern-recognition receptor NOD2, and failed to promote cancer immunotherapy. Importantly, plasmid-based expression of SagA, but not its catalytically-inactive mutant, restored Δ sagA growth, production of active muropeptides and NOD2 activation. SagA is therefore essential for E. faecium growth, stress resistance and activation of host immunity.

Reentrant DNA shells tune polyphosphate condensate size.

The ancient, inorganic biopolymer polyphosphate (polyP) occurs in all three domains of life and affects myriad cellular processes. An intriguing feature of polyP is its frequent proximity to chromatin, and in the case of many bacteria, its occurrence in the form of magnesium-enriched condensates embedded in the nucleoid, particularly in response to stress. The physical basis of the interaction between polyP and DNA, two fundamental anionic biopolymers, and the resulting effects on the organization of both the nucleoid and polyP condensates remain poorly understood. Given the essential role of magnesium ions in the coordination of polymeric phosphate species, we hypothesized that a minimal system of polyP, magnesium ions, and DNA (polyP-Mg2+-DNA) would capture key features of the interplay between the condensates and bacterial chromatin. We find that DNA can profoundly affect polyP-Mg2+ coacervation even at concentrations several orders of magnitude lower than found in the cell. The DNA forms shells around polyP-Mg2+ condensates and these shells show reentrant behavior, primarily forming in the concentration range close to polyP-Mg2+ charge neutralization. This surface association tunes both condensate size and DNA morphology in a manner dependent on DNA properties, including length and concentration. Our work identifies three components that could form the basis of a central and tunable interaction hub that interfaces with cellular interactors. These studies will inform future efforts to understand the basis of polyP granule composition and consolidation, as well as the potential capacity of these mesoscale assemblies to remodel chromatin in response to diverse stressors at different length and time scales.