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.

Peptidoglycan NlpC/P60 peptidases in bacterial physiology and host interactions.

The bacterial cell wall is composed of a highly crosslinked matrix of glycopeptide polymers known as peptidoglycan that dictates bacterial cell morphology and protects against environmental stresses. Regulation of peptidoglycan turnover is therefore crucial for bacterial survival and growth and is mediated by key protein complexes and enzyme families. Here, we review the prevalence, structure, and activity of NlpC/P60 peptidases, a family of peptidoglycan hydrolases that are crucial for cell wall turnover and division as well as interactions with antibiotics and different hosts. Understanding the molecular functions of NlpC/P60 peptidases should provide important insight into bacterial physiology, their interactions with different kingdoms of life, and the development of new therapeutic approaches.

The Histone H1-Like Protein AlgP Facilitates Even Spacing of Polyphosphate Granules in Pseudomonas aeruginosa.

Synthesis of polyphosphate (polyP) is an ancient and universal stress and starvation response in bacteria. In many bacteria, polyP chains come together to form granular superstructures within cells. Some species appear to regulate polyP granule subcellular organization. Despite the critical role of polyP in starvation fitness, the composition of these structures, mechanism(s) underpinning their organization, and functional significance of such organization are poorly understood. We previously determined that granules become transiently evenly spaced on the cell's long axis during nitrogen starvation in the opportunistic human pathogen Pseudomonas aeruginosa. Here, we developed a granule-enrichment protocol to screen for polyP granule-localizing proteins. We identified AlgP as a protein that associates with polyP granules. We further discovered that AlgP is required for the even spacing of polyP granules. AlgP is a DNA-binding protein with a 154 amino acid C-terminal domain enriched in "KPAA" repeats and variants of this repeat, with an overall sequence composition similar to the C-terminal tail of eukaryotic histone H1. Granule size, number, and spacing are significantly perturbed in the absence of AlgP, or when AlgP is truncated to remove the C-terminus. The ΔalgP and algPΔCTD mutants have fewer, larger granules. We speculate that AlgP may contribute to spacing by tethering polyP granules to the chromosome, thereby inhibiting fusion with neighboring granules. Our discovery that AlgP facilitates granule spacing allows us for the first time to directly uncouple granule biogenesis from even spacing, and will inform future efforts to explore the functional significance of granule organization on fitness during starvation. IMPORTANCE The mechanisms underpinning polyP's pleiotropic effects on bacterial starvation physiology remain elusive. This simple polyanion's lack of protein binding specificity has impeded validation of bona fide polyP-binding proteins. However, polyP forms granule superstructures with spatial specificity. Our granule enrichment protocol identified a polyP granule-associated protein in Pseudomonas aeruginosa, AlgP. AlgP was originally reported as a regulator of alginate, an extracellular polysaccharide important in biofilm formation, including in cystic fibrosis (CF) chronic infections. AlgP's putative role in alginate biosynthesis has recently been called into question. We establish a distinct, previously unknown function for AlgP in modulating the subcellular organization of polyP, another polymer important for pathogenesis. In CF clinical isolates, the C-terminal repeat domain of AlgP is a hot spot for genetic rearrangements. Our finding that the C-terminus of AlgP is required for granule organization lays the groundwork for exploring the functional significance of these mutations in the evolutionary trajectory of chronic infections.

A NusG Specialized Paralog That Exhibits Specific, High-Affinity RNA-Binding Activity.

Bacterial NusG associates with RNA polymerase (RNAP) through its N-terminal domain, while the C-terminal domain (CTD) forms dynamic interactions with Rho, S10, NusB and NusA to affect transcription elongation. While virtually all bacteria encode for a core NusG, many also synthesize paralogs that transiently bind RNAP to alter expression of targeted genes. Yet, despite the importance of the genes they regulate, most of the subfamilies of NusG paralogs (e.g., UpxY, TaA, ActX and LoaP) have not been investigated in depth. Herein, we discover that LoaP requires a small RNA hairpin located within the 5' leader region of its targeted operons. LoaP binds the RNA element with nanomolar affinity and high specificity, in contrast to other NusG proteins, which have not been shown to exhibit RNA-binding activity. These data reveal a sequence feature that can be used to identify LoaP-regulated operons. This discovery also expands the repertoire of macromolecular interactions exhibited by the NusG CTD during transcription elongation to include an RNA ligand.

LoaP is a broadly conserved antiterminator protein that regulates antibiotic gene clusters in Bacillus amyloliquefaciens.

A valuable resource available in the search for new natural products is the diverse microbial life that spans the planet. A large subset of these microorganisms synthesize complex specialized metabolites exhibiting biomedically important activities. A limiting step to the characterization of these compounds is an elucidation of the genetic regulatory mechanisms that oversee their production. Although proteins that control transcription initiation of specialized metabolite gene clusters have been identified, those affecting transcription elongation have not been broadly investigated. In this study, we analysed the phylogenetic distribution of the large, widespread NusG family of transcription elongation proteins and found that it includes a cohesive outgroup of paralogues (herein coined LoaP), which are often positioned adjacent or within gene clusters for specialized metabolites. We established Bacillus amyloliquefaciens LoaP as a paradigm for this protein subgroup and showed that it regulated the transcriptional readthrough of termination sites located within two different antibiotic biosynthesis operons. Both of these antibiotics have been implicated in plant-protective activities, demonstrating that LoaP controls an important regulon of specialized metabolite genes for this microorganism. These data therefore reveal transcription elongation as a point of regulatory control for specialized metabolite pathways and introduce a subgroup of NusG proteins for this purpose.