Bacteria elicit a phage tolerance response subsequent to infection of their neighbors.

Appearance of plaques on a bacterial lawn is a sign of successive rounds of bacteriophage infection. Yet, mechanisms evolved by bacteria to limit plaque spread have been hardly explored. Here, we investigated the dynamics of plaque development by lytic phages infecting the bacterium Bacillus subtilis. We report that plaque expansion is followed by a constriction phase owing to bacterial growth into the plaque zone. This phenomenon exposed an adaptive process, herein termed "phage tolerance response", elicited by non-infected bacteria upon sensing infection of their neighbors. The temporary phage tolerance is executed by the stress-response RNA polymerase sigma factor σX (SigX). Artificial expression of SigX prior to phage attack largely eliminates infection. SigX tolerance is primarily conferred by activation of the dlt operon, encoding enzymes that catalyze D-alanylation of cell wall teichoic acid polymers, the major attachment sites for phages infecting Gram-positive bacteria. D-alanylation impedes phage binding and hence infection, thus enabling the uninfected bacteria to form a protective shield opposing phage spread.

Sensitizing methicillin-resistant Staphylococcus aureus (MRSA) to cefuroxime: the synergic effect of bicarbonate and the wall teichoic acid inhibitor ticlopidine.

Methicillin-resistant Staphylococcus aureus (MRSA) strains are a major challenge for clinicians due, in part, to their resistance to most ß-lactams, the first-line treatment for methicillin-susceptible S. aureus. A phenotype termed "NaHCO3-responsiveness" has been identified, wherein many clinical MRSA isolates are rendered susceptible to standard-of-care ß-lactams in the presence of physiologically relevant concentrations of NaHCO3, in vitro and ex vivo; moreover, such "NaHCO3-responsive" isolates can be effectively cleared by ß-lactams from target tissues in experimental infective endocarditis (IE). One mechanistic impact of NaHCO3 exposure on NaHCO3-responsive MRSA is to repress WTA synthesis. This NaHCO3 effect mimics the phenotype of tarO-deficient MRSA, including sensitization to the PBP2-targeting ß-lactam, cefuroxime (CFX). Herein, we further investigated the impacts of NaHCO3 exposure on CFX susceptibility in the presence and absence of a WTA synthesis inhibitor, ticlopidine (TCP), in a collection of clinical MRSA isolates from skin and soft tissue infections (SSTI) and bloodstream infections (BSI). NaHCO3 and/or TCP enhanced susceptibility to CFX in vitro, by both minimum inhibitor concentration (MIC) and time-kill assays, as well as in an ex vivo simulated endocarditis vegetations (SEV) model, in NaHCO3-responsive MRSA. Furthermore, in experimental IE (presumably in the presence of endogenous NaHCO3), pre-exposure to TCP prior to infection sensitized the NaHCO3-responsive MRSA strain (but not the non-responsive strain) to enhanced clearances by CFX in target tissues. These data support the notion that NaHCO3 is acting similarly to WTA synthesis inhibitors, and that such inhibitors have potential translational applications in the treatment of certain MRSA strains in conjunction with specific ß-lactam agents.

Isolation and Molecular Characterization of the LTA Precursor Molecule Glc2-DAG, a Potential Target for Antibiotics.

Bacterial infections present a major global health threat, often displaying resistance to various antibiotics. Lipoteichoic acid (LTA) is a vital component of bacterial cell envelopes of Gram-positive bacteria, crucial for cell integrity, cell division, and host inflammation. Due to its essential role for bacteria, LTA and its biosynthesis are also attractive drug targets, however, there is only scant molecular knowledge on LTA and its precursor molecules in membranes. Here, we report the isolation and molecular characterization of diglucosyldiacylglycerol (Glc2-DAG), the glycolipid precursor molecule that anchors LTA in the bacterial plasma-membrane. Using a tailored growth medium and purification protocols, we isolated 13C-isotope labelled Glc2-DAG from bacteria, which can then be used for high-resolution NMR studies. Using solution-state and solid-state NMR, we show an in-depth molecular characterization of Glc2-DAG, including in native-like membranes. Our approach may help to identify antibiotics that directly target LTA precursor molecules, and it offers a tool for future investigations into the role of Glc2-DAG in bacterial physiology.

Insight into the molecular basis of substrate recognition by the wall teichoic acid glycosyltransferase TagA.

Wall teichoic acid (WTA) polymers are covalently affixed to the Gram-positive bacterial cell wall and have important functions in cell elongation, cell morphology, biofilm formation, and ß-lactam antibiotic resistance. The first committed step in WTA biosynthesis is catalyzed by the TagA glycosyltransferase (also called TarA), a peripheral membrane protein that produces the conserved linkage unit, which joins WTA to the cell wall peptidoglycan. TagA contains a conserved GT26 core domain followed by a C-terminal polypeptide tail that is important for catalysis and membrane binding. Here, we report the crystal structure of the Thermoanaerobacter italicus TagA enzyme bound to UDP-N-acetyl-d-mannosamine, revealing the molecular basis of substrate binding. Native MS experiments support the model that only monomeric TagA is enzymatically active and that it is stabilized by membrane binding. Molecular dynamics simulations and enzyme activity measurements indicate that the C-terminal polypeptide tail facilitates catalysis by encapsulating the UDP-N-acetyl-d-mannosamine substrate, presenting three highly conserved arginine residues to the active site that are important for catalysis (R214, R221, and R224). From these data, we present a mechanistic model of catalysis that ascribes functions for these residues. This work could facilitate the development of new antimicrobial compounds that disrupt WTA biosynthesis in pathogenic bacteria.

Structural variations and roles of rhamnose-rich cell wall polysaccharides in Gram-positive bacteria.

Rhamnose-rich cell wall polysaccharides (Rha-CWPSs) have emerged as crucial cell wall components of numerous Gram-positive, ovoid-shaped bacteria-including streptococci, enterococci, and lactococci-of which many are of clinical or biotechnological importance. Rha-CWPS are composed of a conserved polyrhamnose backbone with side-chain substituents of variable size and structure. Because these substituents contain phosphate groups, Rha-CWPS can also be classified as polyanionic glycopolymers, similar to wall teichoic acids, of which they appear to be functional homologs. Recent advances have highlighted the critical role of these side-chain substituents in bacterial cell growth and division, as well as in specific interactions between bacteria and infecting bacteriophages or eukaryotic hosts. Here, we review the current state of knowledge on the structure and biosynthesis of Rha-CWPS in several ovoid-shaped bacterial species. We emphasize the role played by multicomponent transmembrane glycosylation systems in the addition of side-chain substituents of various sizes as extracytoplasmic modifications of the polyrhamnose backbone. We provide an overview of the contribution of Rha-CWPS to cell wall architecture and biogenesis and discuss current hypotheses regarding their importance in the cell division process. Finally, we sum up the critical roles that Rha-CWPS can play as bacteriophage receptors or in escaping host defenses, roles that are mediated mainly through their side-chain substituents. From an applied perspective, increased knowledge of Rha-CWPS can lead to advancements in strategies for preventing phage infection of lactococci and streptococci in food fermentation and for combating pathogenic streptococci and enterococci.

Disruption of the tagF Orthologue in the epa Locus Variable Region of Enterococcus faecalis Causes Cell Surface Changes and Suppresses an eep-Dependent Lysozyme Resistance Phenotype.

The disease-producing capacity of the opportunistic pathogen Enterococcus faecalis is enhanced by the ability of the bacterium to evade killing by antimicrobial agents. Survival of E. faecalis in the presence of the human antimicrobial enzyme lysozyme is mediated in part by the site 2 metalloprotease Eep; however, a complete model of enterococcal lysozyme resistance has not been elucidated. To better understand the molecular basis for lysozyme resistance in E. faecalis, we analyzed Δeep suppressor mutants that acquire resistance to lysozyme through mutation of the gene OG1RF_11713, a predicted teichoic acid biosynthesis-encoding gene located within the variable region of the enterococcal polysaccharide antigen (epa) locus. Sequence comparisons revealed that OG1RF_11713 is most similar to the cytidine-5'-diphosphate (CDP)-glycerol:poly-(glycerolphosphate)glycerophosphotransferase TagF from Staphylococcus epidermidis. Inactivation of OG1RF_11713 in both the wild-type and Δeep genetic backgrounds was sufficient to increase the resistance of E. faecalis OG1RF to lysozyme. Minimal amounts of N-acetylgalactosamine were detectable in cell wall carbohydrate extracts of OG1RF_11713 deletion mutants, and this was associated with a reduction in negative cell surface charge. Targeted disruption of OG1RF_11713 was also associated with increased susceptibility to the antibiotic polymyxin B and membrane-targeting detergents and decreased susceptibility to the lantibiotic nisin. This work implicates OG1RF_11713 as a major determinant of cell envelope integrity and provides further validation that lysozyme resistance is intrinsically linked to the modification of enterococcal cell wall polysaccharides. IMPORTANCE Enterococcus faecalis is a leading cause of health-care-associated infections for which there are limited treatment options. E. faecalis is resistant to several antibiotics and to high concentrations of the human antimicrobial enzyme lysozyme. The molecular mechanisms that mediate lysozyme resistance in E. faecalis are complex and remain incompletely characterized. This work demonstrates that a gene located within the variable region of the enterococcal polysaccharide antigen locus of E. faecalis strain OG1RF (OG1RF_11713), which is predicted to encode a component of the teichoic acid biosynthesis machinery, is part of the lysozyme resistance circuitry and is important for enterococcal cell wall integrity. These findings suggest that OG1RF_11713 is a potential target for new therapeutic strategies to combat enterococcal infections.

Bacillus subtilis YngB contributes to wall teichoic acid glucosylation and glycolipid formation during anaerobic growth.

UTP-glucose-1-phosphate uridylyltransferases are enzymes that produce UDP-glucose from UTP and glucose-1-phosphate. In Bacillus subtilis 168, UDP-glucose is required for the decoration of wall teichoic acid (WTA) with glucose residues and the formation of glucolipids. The B. subtilis UGPase GtaB is essential for UDP-glucose production under standard aerobic growth conditions, and gtaB mutants display severe growth and morphological defects. However, bioinformatics predictions indicate that two other UTP-glucose-1-phosphate uridylyltransferases are present in B. subtilis. Here, we investigated the function of one of them named YngB. The crystal structure of YngB revealed that the protein has the typical fold and all necessary active site features of a functional UGPase. Furthermore, UGPase activity could be demonstrated in vitro using UTP and glucose-1-phosphate as substrates. Expression of YngB from a synthetic promoter in a B. subtilis gtaB mutant resulted in the reintroduction of glucose residues on WTA and production of glycolipids, demonstrating that the enzyme can function as UGPase in vivo. When WT and mutant B. subtilis strains were grown under anaerobic conditions, YngB-dependent glycolipid production and glucose decorations on WTA could be detected, revealing that YngB is expressed from its native promoter under anaerobic condition. Based on these findings, along with the structure of the operon containing yngB and the transcription factor thought to be required for its expression, we propose that besides WTA, potentially other cell wall components might be decorated with glucose residues during oxygen-limited growth condition.

Targeting the Achilles' Heel of Multidrug-Resistant Staphylococcus aureus by the Endocannabinoid Anandamide.

Antibiotic-resistant Staphylococcus aureus is a major health issue that requires new therapeutic approaches. Accumulating data suggest that it is possible to sensitize these bacteria to antibiotics by combining them with inhibitors targeting efflux pumps, the low-affinity penicillin-binding protein PBP2a, cell wall teichoic acid, or the cell division protein FtsZ. We have previously shown that the endocannabinoid Anandamide (N-arachidonoylethanolamine; AEA) could sensitize drug-resistant S. aureus to a variety of antibiotics, among others, through growth arrest and inhibition of drug efflux. Here, we looked at biochemical alterations caused by AEA. We observed that AEA increased the intracellular drug concentration of a fluorescent penicillin and augmented its binding to membrane proteins with concomitant altered membrane distribution of these proteins. AEA also prevented the secretion of exopolysaccharides (EPS) and reduced the cell wall teichoic acid content, both processes known to require transporter proteins. Notably, AEA was found to inhibit membrane ATPase activity that is necessary for transmembrane transport. AEA did not affect the membrane GTPase activity, and the GTPase cell division protein FtsZ formed the Z-ring of the divisome normally in the presence of AEA. Rather, AEA caused a reduction in murein hydrolase activities involved in daughter cell separation. Altogether, this study shows that AEA affects several biochemical processes that culminate in the sensitization of the drug-resistant bacteria to antibiotics.

Crystallographic analysis of TarI and TarJ, a cytidylyltransferase and reductase pair for CDP-ribitol synthesis in Staphylococcus aureus wall teichoic acid biogenesis.

The cell wall of many pathogenic Gram-positive bacteria contains ribitol-phosphate wall teichoic acid (WTA), a polymer that is linked to virulence and regulation of essential physiological processes including cell division. CDP-ribitol, the activated precursor for ribitol-phosphate polymerization, is synthesized by a cytidylyltransferase and reductase pair known as TarI and TarJ, respectively. In this study, we present crystal structures of Staphylococcus aureus TarI and TarJ in their apo forms and in complex with substrates and products. The TarI structures illustrate the mechanism of CDP-ribitol synthesis from CTP and ribitol-phosphate and reveal structural changes required for substrate binding and catalysis. Insights into the upstream step of ribulose-phosphate reduction to ribitol-phosphate is provided by the structures of TarJ. Furthermore, we propose a general topology of the enzymes in a heterotetrameric form built using restraints from crosslinking mass spectrometry analysis. Together, our data present molecular details of CDP-ribitol production that may aid in the design of inhibitors against WTA biosynthesis.

Phosphoglycerol-type wall and lipoteichoic acids are enantiomeric polymers differentiated by the stereospecific glycerophosphodiesterase GlpQ.

The cell envelope of Gram-positive bacteria generally comprises two types of polyanionic polymers linked to either peptidoglycan (wall teichoic acids; WTA) or to membrane glycolipids (lipoteichoic acids; LTA). In some bacteria, including Bacillus subtilis strain 168, both WTA and LTA are glycerolphosphate polymers yet are synthesized through different pathways and have distinct but incompletely understood morphogenetic functions during cell elongation and division. We show here that the exolytic sn-glycerol-3-phosphodiesterase GlpQ can discriminate between B. subtilis WTA and LTA. GlpQ completely degraded unsubstituted WTA, which lacks substituents at the glycerol residues, by sequentially removing glycerolphosphates from the free end of the polymer up to the peptidoglycan linker. In contrast, GlpQ could not degrade unsubstituted LTA unless it was partially precleaved, allowing access of GlpQ to the other end of the polymer, which, in the intact molecule, is protected by a connection to the lipid anchor. Differences in stereochemistry between WTA and LTA have been suggested previously on the basis of differences in their biosynthetic precursors and chemical degradation products. The differential cleavage of WTA and LTA by GlpQ reported here represents the first direct evidence that they are enantiomeric polymers: WTA is made of sn-glycerol-3-phosphate, and LTA is made of sn-glycerol-1-phosphate. Their distinct stereochemistries reflect the dissimilar physiological and immunogenic properties of WTA and LTA. It also enables differential degradation of the two polymers within the same envelope compartment in vivo, particularly under phosphate-limiting conditions, when B. subtilis specifically degrades WTA and replaces it with phosphate-free teichuronic acids.

Uncovering the activities, biological roles, and regulation of bacterial cell wall hydrolases and tailoring enzymes.

Bacteria account for 1000-fold more biomass than humans. They vary widely in shape and size. The morphological diversity of bacteria is due largely to the different peptidoglycan-based cell wall structures that encase bacterial cells. Although the basic structure of peptidoglycan is highly conserved, consisting of long glycan strands that are cross-linked by short peptide chains, the mature cell wall is chemically diverse. Peptidoglycan hydrolases and cell wall-tailoring enzymes that regulate glycan strand length, the degree of cross-linking, and the addition of other modifications to peptidoglycan are central in determining the final architecture of the bacterial cell wall. Historically, it has been difficult to biochemically characterize these enzymes that act on peptidoglycan because suitable peptidoglycan substrates were inaccessible. In this review, we discuss fundamental aspects of bacterial cell wall synthesis, describe the regulation and diverse biochemical and functional activities of peptidoglycan hydrolases, and highlight recently developed methods to make and label defined peptidoglycan substrates. We also review how access to these substrates has now enabled biochemical studies that deepen our understanding of how bacterial cell wall enzymes cooperate to build a mature cell wall. Such improved understanding is critical to the development of new antibiotics that disrupt cell wall biogenesis, a process essential to the survival of bacteria.

Crystallographic analysis of Staphylococcus aureus LcpA, the primary wall teichoic acid ligase.

Gram-positive bacteria, including major clinical pathogens such as Staphylococcus aureus, are becoming increasingly drug-resistant. Their cell walls are composed of a thick layer of peptidoglycan (PG) modified by the attachment of wall teichoic acid (WTA), an anionic glycopolymer that is linked to pathogenicity and regulation of cell division and PG synthesis. The transfer of WTA from lipid carriers to PG, catalyzed by the LytR-CpsA-Psr (LCP) enzyme family, offers a unique extracellular target for the development of new anti-infective agents. Inhibitors of LCP enzymes have the potential to manage a wide range of bacterial infections because the target enzymes are implicated in the assembly of many other bacterial cell wall polymers, including capsular polysaccharide of streptococcal species and arabinogalactan of mycobacterial species. In this study, we present the first crystal structure of S. aureus LcpA with bound substrate at 1.9 Å resolution and those of Bacillus subtilis LCP enzymes, TagT, TagU, and TagV, in the apo form at 1.6-2.8 Å resolution. The structures of these WTA transferases provide new insight into the binding of lipid-linked WTA and enable assignment of the catalytic roles of conserved active-site residues. Furthermore, we identified potential subsites for binding the saccharide core of PG using computational docking experiments, and multiangle light-scattering experiments disclosed novel oligomeric states of the LCP enzymes. The crystal structures and modeled substrate-bound complexes of the LCP enzymes reported here provide insights into key features linked to substrate binding and catalysis and may aid the structure-guided design of specific LCP inhibitors.

Lipoteichoic acid, a cell wall component of Gram-positive bacteria, induces sleep and fever and suppresses feeding.

Fragments of the bacterial cell wall are bioactive microbial molecules that have profound effects on the function of the brain. Some of the cell wall constituents are common to both Gram-positive and Gram-negative bacteria, e.g., peptidoglycans, while other cell wall components are specific to either Gram-positive or Gram-negative microbes. Lipopolysaccharide (LPS), also called endotoxin, is found exclusively in Gram-negative bacteria, while lipoteichoic acid (LTA) is specific to Gram-positive bacteria. The effects of peptidoglycans, their fragments, and LPS are well characterized, they induce sleep, fever and anorexia. In the present study, we investigated the sleep, body temperature and food intake modulating effects of LTA. We found that intraperitoneal injection of 100 and 250 µg LTA from B. subtilis and S. aureus increases non-rapid-eye movement sleep (NREMS) in mice. The effects were dose-dependent, and the changes were accompanied by decreased motor activity and feeding as well as febrile responses. Intraperitoneal injection of 10 µg LTA induced monophasic increases in body temperature, while 100 and 250 µg LTA from B. subtilis induced initial hypothermia followed by fever. Treatment with 250 µg LTA from S. aureus elicited monophasic hypothermia. Administration of 300 µg/kg LTA from S. aureus directly into the portal vein elicited similar sleep responses in rats but did not affect body temperature. The sleep-modulating effects of LTA were similar to that of LPS in mice, although LTA appears to be less potent. These findings suggest that the role of LTA in signaling by Gram-positive bacteria in the host body is analogous to the role of LPS/endotoxin in signaling by Gram-negative microbes. LTA may play a role in the development of sickness response in clinically manifest Gram-positive bacterial infections and may contribute to sleep signaling by the commensal intestinal microbiota.

Genomic Differences between Listeria monocytogenes EGDe Isolates Reveal Crucial Roles for SigB and Wall Rhamnosylation in Biofilm Formation.

Listeria monocytogenes is a Gram-positive firmicute that causes foodborne infections, in part due to its ability to use multiple strategies, including biofilm formation, to survive adverse growth conditions. As a potential way to screen for genes required for biofilm formation, we harnessed the ability of bacteria to accumulate mutations in the genome over time, diverging the properties of seemingly identical strains. By sequencing the genomes of four laboratory reference strains of the commonly used L. monocytogenes EGDe, we showed that each isolate contains single nucleotide polymorphisms (SNPs) compared with the reference genome. We discovered that two SNPs, contained in two independent genes within one of the isolates, impacted biofilm formation. Using bacterial genetics and phenotypic assays, we confirmed that rsbU and rmlA influence biofilm formation. RsbU is the upstream regulator of the alternative sigma factor SigB, and mutation of either rsbU or sigB increased biofilm formation. In contrast, deletion of rmlA, which encodes the first enzyme for TDP-l-rhamnose biosynthesis, resulted in a reduction in the amount of biofilm formed. Further analysis of biofilm formation in a strain that still produces TDP-l-rhamnose but which cannot decorate the wall teichoic acid with rhamnose (rmlT mutant) showed that it is the decorated wall teichoic acid that is required for adhesion of the cells to surfaces. Together, these data uncover novel routes by which biofilm formation by L. monocytogenes can be impacted.IMPORTANCE Biofilms are an important mode of growth in many settings. Here, we looked at small differences in the genomes of the bacterium Listeria monocytogenes isolate EGDe and used them to find out how biofilms form. This important fundamental information may help new treatments to be developed and also highlights the fact that isolates of the same identity often diverge.

Protective activity of anti-lipoteichoic acid monoclonal antibody in single or combination therapies in methicillin-resistant Staphylococcus aureus-induced murine sepsis models.

Previously, we generated and screened a panel of monoclonal antibodies (mAbs) against methicillin-resistant Staphylococcus aureus (MRSA) to identify protective mAbs in mouse infection models. One of these mAbs, ZBIA3H, bound to lipoteichoic acid (LTA) and exerted protective effects in a mouse sepsis model. To reinforce the ability of the mAb to protect against infection, combination therapies with the mAb and antibiotics need to be examined. Therefore, herein, we studied the efficacy of ZBIA3H (in combination or alone) in a mouse sepsis model. ZBIA3H improved the survival rate in the mouse models of sepsis induced by highly virulent or refractory S. aureus (community-acquired MRSA strain MW2, vancomycin-intermediate S. aureus strain Mu3, or vancomycin-resistant S. aureus strain VRS1). Furthermore, ZBIA3H remarkably improved the survival rate in combination with antimicrobial agents (vancomycin, daptomycin, or linezolid) in mouse sepsis models. From these results we conclude that anti-LTA mAb ZBIA3H or its humanized form is a promising mAb individually, or in combination with antibiotics, against clinical refractory infection of S. aureus.

Phosphorylcholine esterase is critical for Dolichos biflorus and Helix pomatia agglutinin binding to pneumococcal teichoic acid.

Streptococcus pneumoniae (the pneumococcus) has wall teichoic acid (WTA) and lipoteichoic acid (LTA) expressing the Forssman antigen (FA). Two lectins, Dolichos biflorus agglutinin (DBA) and Helix pomatia agglutinin (HPA), are known to bind FA. To determine the molecular structure targeted by these two lectins, different pneumococcal strains were studied for DBA/HPA binding with flow cytometry and fluorescence microscopy. Genetic experiments were used to further examine the lectins' molecular target. Twelve strains were positive for DBA binding, whereas three were negative. Super-resolution microscopy showed that DBA stained only the subcapsular area of pneumococci. The three DBA nonbinders showed no phosphorylcholine esterase (Pce) activity in vitro, whereas 10 DBA binders displayed Pce activity (the remaining two strains were DBA binders with no Pce activity in vitro). The pcegene sequence for 10 representative strains revealed two functional pce alleles, the previously recognized "allele A" and a newly discovered "allele B" (with 12 additional nucleotides). Isolates with allele B showed no Pce activity in vitro but did bind to DBA, indicating allele B Pce is functional in vivo. Genetic transfer experiments confirmed that either allele is sufficient (and necessary) for DBA binding. The three DBA nonbinders had various mutations that affected Pce function. Observations with HPA were identical to those with DBA. We show that DBA and HPA bind only to the WTA/LTA of pneumococcal isolates with a functional Pce enzyme. A newly discovered Pce variant (allele B) is functional in vivo but nonfunctional when assayed in vitro.

Lipoteichoic Acid Biosynthesis Inhibitors as Potent Inhibitors of S. aureus and E. faecalis Growth and Biofilm Formation.

Methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecalis (VRE) have been deemed as serious threats by the CDC. Many chronic MRSA and VRE infections are due to biofilm formation. Biofilm are considered to be between 10-10,000 times more resistant to antibiotics, and therefore new chemical entities that inhibit and/or eradicate biofilm formation are needed. Teichoic acids, such as lipoteichoic acids (LTAs) and wall teichoic acids (WTAs), play pivotal roles in Gram-positive bacteria's ability to grow, replicate, and form biofilms, making the inhibition of these teichoic acids a promising approach to fight infections by biofilm forming bacteria. Here, we describe the potent biofilm inhibition activity against MRSA and VRE biofilms by two LTA biosynthesis inhibitors HSGN-94 and HSGN-189 with MBICs as low as 0.0625 µg/mL against MRSA biofilms and 0.5 µg/mL against VRE biofilms. Additionally, both HSGN-94 and HSGN-189 were shown to potently synergize with the WTA inhibitor Tunicamycin in inhibiting MRSA and VRE biofilm formation.

Discovery of genes required for lipoteichoic acid glycosylation predicts two distinct mechanisms for wall teichoic acid glycosylation.

The bacterial cell wall is an important and highly complex structure that is essential for bacterial growth because it protects bacteria from cell lysis and environmental insults. A typical Gram-positive bacterial cell wall is composed of peptidoglycan and the secondary cell wall polymers, wall teichoic acid (WTA) and lipoteichoic acid (LTA). In many Gram-positive bacteria, LTA is a polyglycerol-phosphate chain that is decorated with d-alanine and sugar residues. However, the function of and proteins responsible for the glycosylation of LTA are either unknown or not well-characterized. Here, using bioinformatics, genetic, and NMR spectroscopy approaches, we found that the Bacillus subtilis csbB and yfhO genes are essential for LTA glycosylation. Interestingly, the Listeria monocytogenes gene lmo1079, which encodes a YfhO homolog, was not required for LTA glycosylation, but instead was essential for WTA glycosylation. LTA is polymerized on the outside of the cell and hence can only be glycosylated extracellularly. Based on the similarity of the genes coding for YfhO homologs that are required in B. subtilis for LTA glycosylation or in L. monocytogenes for WTA glycosylation, we hypothesize that WTA glycosylation might also occur extracellularly in Listeria species. Finally, we discovered that in L. monocytogenes, lmo0626 (gtlB) was required for LTA glycosylation, indicating that the encoded protein has a function similar to that of YfhO, although the proteins are not homologous. Together, our results enable us to propose an updated model for LTA glycosylation and also indicate that glycosylation of WTA might occur through two different mechanisms in Gram-positive bacteria.

Staphylococcus aureus counters phosphate limitation by scavenging wall teichoic acids from other staphylococci via the teichoicase GlpQ.

Staphylococcus aureus is part of the human nasal and skin microbiomes along with other bacterial commensals and opportunistic pathogens. Nutrients are scarce in these habitats, demanding effective nutrient acquisition and competition strategies. How S. aureus copes with phosphate limitation is still unknown. Wall teichoic acid (WTA), a polyol-phosphate polymer, could serve as a phosphate source, but whether S. aureus can utilize it during phosphate starvation remains unknown. S. aureus secretes a glycerophosphodiesterase, GlpQ, that cleaves a broad variety of glycerol-3-phosphate (GroP) headgroups of deacylated phospholipids, providing this bacterium with GroP as a carbon and phosphate source. Here we demonstrate that GlpQ can also use glycerophosphoglycerol derived from GroP WTA from coagulase-negative Staphylococcus lugdunensis, Staphylococcus capitis, and Staphylococcus epidermidis, which share the nasal and skin habitats with S. aureus Therefore, S. aureus GlpQ is the first reported WTA-hydrolyzing enzyme, or teichoicase, from Staphylococcus Activity assays revealed that unmodified WTA is the preferred GlpQ substrate, and the results from MS analysis suggested that GlpQ uses an exolytic cleavage mechanism. Importantly, GlpQ did not hydrolyze the ribitol-5-phosphate WTA polymers of S. aureus, underscoring its role in interspecies competition rather than in S. aureus cell wall homeostasis or WTA recycling. glpQ expression was strongly up-regulated under phosphate limitation, and GlpQ allowed S. aureus to grow in the presence of GroP WTA as the sole phosphate source. Our study reveals a novel and unprecedented strategy of S. aureus for acquiring phosphate from bacterial competitors under the phosphate-limiting conditions in the nasal or skin environments.

A partial reconstitution implicates DltD in catalyzing lipoteichoic acid d-alanylation.

Modifications to the Gram-positive bacterial cell wall play important roles in antibiotic resistance and pathogenesis, but the pathway for the d-alanylation of teichoic acids (DLT pathway), a ubiquitous modification, is poorly understood. The d-alanylation machinery includes two membrane proteins of unclear function, DltB and DltD, which are somehow involved in transfer of d-alanine from a carrier protein inside the cell to teichoic acids on the cell surface. Here, we probed the role of DltD in the human pathogen Staphylococcus aureus using both cell-based and biochemical assays. We first exploited a known synthetic lethal interaction to establish the essentiality of each gene in the DLT pathway for d-alanylation of lipoteichoic acid (LTA) and confirmed this by directly detecting radiolabeled d-Ala-LTA both in cells and in vesicles prepared from mutant strains of S. aureus We developed a partial reconstitution of the pathway by using cell-derived vesicles containing DltB, but no other components of the d-alanylation pathway, and showed that d-alanylation of previously formed lipoteichoic acid in the DltB vesicles requires the presence of purified and reconstituted DltA, DltC, and DltD, but not of the LTA synthase LtaS. Finally, based on the activity of DltD mutants in cells and in our reconstituted system, we determined that Ser-70 and His-361 are essential for d-alanylation activity, and we propose that DltD uses a catalytic dyad to transfer d-alanine to LTA. In summary, we have developed a suite of assays for investigating the bacterial DLT pathway and uncovered a role for DltD in LTA d-alanylation.