The cytosol of eukaryotic host cells is an intrinsically hostile environment for bacteria. Understanding how cytosolic pathogens adapt to and survive in the cytosol is critical to developing novel therapeutic interventions for these pathogens. The cytosolic pathogen Listeria monocytogenes requires glmR (previously known as yvcK), a gene of unknown function, for resistance to cell wall stress, cytosolic survival, inflammasome avoidance and ultimately virulence in vivo. A genetic suppressor screen revealed that blocking utilization of UDP-GlcNAc by a non-essential wall teichoic acid decoration pathway restored resistance to cell wall stress and partially restored virulence of ΔglmR mutants. In parallel, metabolomics revealed that ΔglmR mutants are impaired in the production of UDP-GlcNAc, an essential peptidoglycan and wall teichoic acid (WTA) precursor. We next demonstrated that purified GlmR can directly catalyze the synthesis of UDP-GlcNAc from GlcNAc-1P and UTP, suggesting that it is an accessory uridyltransferase. Biochemical analysis of GlmR orthologues suggest that uridyltransferase activity is conserved. Finally, mutational analysis resulting in a GlmR mutant with impaired catalytic activity demonstrated that uridyltransferase activity was essential to facilitate cell wall stress responses and virulence in vivo. Taken together these studies indicate that GlmR is an evolutionary conserved accessory uridyltransferase required for cytosolic survival and virulence of L. monocytogenes.ImportanceBacterial pathogens must adapt to their host environment in order to cause disease. The cytosolic bacterial pathogen Listeria monocytogenes requires a highly conserved protein of unknown function, GlmR (previously known as YvcK) to survive in the host cytosol. GlmR is important for resistance to some cell wall stresses and is essential for virulence. The ΔglmR mutant is deficient in production of an essential cell wall metabolite, UDP-GlcNAc, and suppressors which increase metabolite levels also restore virulence. Purified GlmR can directly catalyze the synthesis of UDP-GlcNAc and this enzymatic activity is conserved in pathogens from Firmicutes and Actinobacteria phyla. These results highlight the importance accessory cell wall metabolism enzymes in responding to cell wall stress in a variety of bacterial pathogens.

Neisseria gonorrhoeae releases peptidoglycan fragments during growth, and these molecules induce an inflammatory response in the human host. The proinflammatory molecules include peptidoglycan monomers, peptidoglycan dimers, and free peptides. These molecules can be released by the actions of lytic transglycosylases or an amidase. However, Ͼ40% of the gonococcal cell wall is cross-linked, where the peptide stem on one peptidoglycan strand is linked to the peptide stem on a neighboring strand, suggesting that endopeptidases may be required for the release of many peptidoglycan fragments. Therefore, we characterized mutants with individual or combined mutations in genes for the low-molecular-mass penicillin-binding proteins PBP3 and PBP4. Mutations in either dacB, encoding PBP3, or pbpG, encoding PBP4, did not significantly reduce the release of peptidoglycan monomers or free peptides. A mutation in dacB caused the appearance of a larger-sized peptidoglycan monomer, the pentapeptide monomer, and an increased release of peptidoglycan dimers, suggesting the involvement of this enzyme in both the removal of C-terminal D-Ala residues from stem peptides and the cleavage of cross-linked peptidoglycan. Mutation of both dacB and pbpG eliminated the release of tripeptide-containing peptidoglycan fragments concomitantly with the appearance of pentapeptide and dipeptide peptidoglycan fragments and higher-molecular-weight peptidoglycan dimers. In accord with the loss of tripeptide peptidoglycan fragments, the level of human NOD1 activation by the dacB pbpG mutants was significantly lower than that by the wild type. We conclude that PBP3 and PBP4 overlap in function for cross-link cleavage and that these endopeptidases act in the normal release of peptidoglycan fragments during growth. KEYWORDS NOD1, NOD1 agonist, carboxypeptidase, endopeptidase, penicillinbinding proteins, peptidoglycan Citation Schaub RE, Perez-Medina KM, Hackett KT, Garcia DL, Dillard JP. 2019. Neisseria gonorrhoeae PBP3 and PBP4 facilitate NOD1 agonist peptidoglycan fragment release and survival in stationary phase. Infect Immun

Bacterial pathogens must adapt to their host environment in order to cause disease. The cytosolic bacterial pathogen Listeria monocytogenes requires a highly conserved protein of unknown function, GlmR (previously known as YvcK), to survive in the host cytosol.

Lytic transglycosylases function to degrade peptidoglycan strands that comprise the bacterial cell wall. Degradation of peptidoglycan at the septum following cell division is necessary for cell separation, and a deletion ofltgCin Neisseria gonorrhoeae results in growth in clusters of around 6-20 cells rather than as normal diplococci or monococci.N. gonorrhoeaeLtgC is a homolog ofEscherichia coliMltA, and comparison of the two proteins shows that LtgC has an extra domain not found in MltA, referred to as domain 3. To better understand the function of LtgC, we characterizedN. gonorrhoeaemutants with substitutions in amino acids predicted to be necessary for enzymatic activity or amino acids predicted to be on the surface of domain 3, and we characterized a mutant lacking domain 3. All the mutants showed defects in cell separation, and the bacteria failed to release peptidoglycan-derived disaccharides into the medium. Purified LtgC proteins with the amino acid substitutions had reduced peptidoglycan degradation activity. LtgC was found to bind AmiC in bacterial 2-hybrid assays, and domain 3 mutations reduced binding. In human blood, anltgCmutant showed decreased survival, suggesting the cell wall defects in the mutant make the bacteria more sensitive to innate immune system components.