Wall teichoic acid substitution with glucose governs phage susceptibility of Staphylococcus epidermidis.

The species- and clone-specific susceptibility of Staphylococcus cells for bacteriophages is governed by the structures and glycosylation patterns of wall teichoic acid (WTA) glycopolymers. The glycosylation-dependent phage-WTA interactions in the opportunistic pathogen Staphylococcus epidermidis and in other coagulase-negative staphylococci (CoNS) have remained unknown. We report a new S. epidermidis WTA glycosyltransferase TagE whose deletion confers resistance to siphoviruses such as ΦE72 but enables binding of otherwise unbound podoviruses. S. epidermidis glycerolphosphate WTA was found to be modified with glucose in a tagE-dependent manner. TagE is encoded together with the enzymes PgcA and GtaB providing uridine diphosphate-activated glucose. ΦE72 transduced several other CoNS species encoding TagE homologs, suggesting that WTA glycosylation via TagE is a frequent trait among CoNS that permits interspecies horizontal gene transfer. Our study unravels a crucial mechanism of phage-Staphylococcus interaction and horizontal gene transfer, and it will help in the design of anti-staphylococcal phage therapies.IMPORTANCEPhages are highly specific for certain bacterial hosts, and some can transduce DNA even across species boundaries. How phages recognize cognate host cells remains incompletely understood. Phages infecting members of the genus Staphylococcus bind to wall teichoic acid (WTA) glycopolymers with highly variable structures and glycosylation patterns. How WTA is glycosylated in the opportunistic pathogen Staphylococcus epidermidis and in other coagulase-negative staphylococci (CoNS) species has remained unknown. We describe that S. epidermidis glycosylates its WTA backbone with glucose, and we identify a cluster of three genes responsible for glucose activation and transfer to WTA. Their inactivation strongly alters phage susceptibility patterns, yielding resistance to siphoviruses but susceptibility to podoviruses. Many different CoNS species with related glycosylation genes can exchange DNA via siphovirus ΦE72, suggesting that glucose-modified WTA is crucial for interspecies horizontal gene transfer. Our finding will help to develop antibacterial phage therapies and unravel routes of genetic exchange.

Cryo-EM analysis of S. aureus TarL, a polymerase in wall teichoic acid biogenesis central to virulence and antibiotic resistance.

Wall teichoic acid (WTA), a covalent adduct of Gram-positive bacterial cell wall peptidoglycan, contributes directly to virulence and antibiotic resistance in pathogenic species. Polymerization of the Staphylococcus aureus WTA ribitol-phosphate chain is catalyzed by TarL, a member of the largely uncharacterized TagF-like family of membrane-associated enzymes. We report the cryo-electron microscopy structure of TarL, showing a tetramer that forms an extensive membrane-binding platform of monotopic helices. TarL is composed of an amino-terminal immunoglobulin-like domain and a carboxyl-terminal glycosyltransferase-B domain for ribitol-phosphate polymerization. The active site of the latter is complexed to donor substrate cytidine diphosphate-ribitol, providing mechanistic insights into the catalyzed phosphotransfer reaction. Furthermore, the active site is surrounded by electropositive residues that serve to retain the lipid-linked acceptor for polymerization. Our data advance general insight into the architecture and membrane association of the still poorly characterized monotopic membrane protein class and present molecular details of ribitol-phosphate polymerization that may aid in the design of new antimicrobials.

Processing of LtaS restricts LTA assembly and YSIRK preprotein trafficking into Staphylococcus aureus cross-walls.

Septal membranes of Staphylococcus aureus serve as the site of secretion for precursors endowed with the YSIRK motif. Depletion of ltaS, a gene required for lipoteichoic acid (LTA) synthesis, results in the loss of restricted trafficking of YSIRK precursors to septal membranes. Here, we seek to understand the mechanism that ties LTA assembly and trafficking of YSIRK precursors. We confirm that catalytically inactive lipoteichoic acid synthase (LtaS)T300A does not support YSIRK precursor trafficking to septa. We hypothesize that the enzyme's reactants [gentiobiosyldiacylglycerol (Glc2-DAG) and phosphatidylglycerol (PG)] or products [LTA and diacylglycerol (DAG)], not LtaS, must drive this process. Indeed, we observe that septal secretion of the staphylococcal protein A YSIRK precursor is lost in ypfP and ltaA mutants that produce glycerophosphate polymers [poly(Gro-P)] without the Glc2-DAG lipid anchor. These mutants display longer poly(Gro-P) chains, implying enhanced PG consumption and DAG production. Our experiments also reveal that in the absence of Glc2-DAG, the processing of LtaS to the extracellular catalytic domain, eLtaS, is impaired. Conversely, LTA polymerization is delayed in a strain producing LtaSS218P, a variant processed more slowly than LtaS. We conclude that Glc2-DAG binding to the enzyme couples catalysis by LtaS and the physical release of eLtaS. We propose a model for the temporal and localized assembly of LTA into cross-walls. When LtaS is not processed in a timely manner, eLtaS no longer diffuses upon daughter cell splitting, LTA assembly continues, and the unique septal-lipid pool, PG over DAG ratio, is not established. This results in profound physiological changes in S. aureus cells, including the inability to restrict the secretion of YSIRK precursors at septal membranes.IMPORTANCEIn Staphylococcus aureus, peptidoglycan is assembled at the septum. Dedicated cell division proteins coordinate septal formation and the fission of daughter cells. Lipoteichoic acid (LTA) assembly and trafficking of preproteins with a YSIRK motif also occur at the septum. This begs the question as to whether cell division components also recruit these two pathways. This study shows that the processing of lipoteichoic acid synthase (LtaS) to extracellular LtaS by signal peptidase is regulated by gentiobiosyldiacylglycerol (Glc2-DAG), the priming substrate for LTA assembly. A model is proposed whereby a key substrate controls the temporal and spatial activity of an enzyme. In turn, this mechanism enables the establishment of a unique and transient lipid pool that defines septal membranes as a targeting site for the secretion of YSIRK preproteins.

Wall Teichoic Acid Mediates Staphylococcus aureus Binding to Endothelial Cells via the Scavenger Receptor LOX-1.

The success of Staphylococcus aureus as a major cause for endovascular infections depends on effective interactions with blood-vessel walls. We have previously shown that S. aureus uses its wall teichoic acid (WTA), a surface glycopolymer, to attach to endothelial cells. However, the endothelial WTA receptor remained unknown. We show here that the endothelial oxidized low-density lipoprotein receptor 1 (LOX-1) interacts with S. aureus WTA and permits effective binding of S. aureus to human endothelial cells. Purified LOX-1 bound to isolated S. aureus WTA. Ectopic LOX-1 expression led to increased binding of S. aureus wild type but not of a WTA-deficient mutant to a cell line, and LOX-1 blockage prevented S. aureus binding to endothelial cells. Moreover, WTA and LOX-1 expression levels correlated with the efficacy of the S. aureus-endothelial interaction. Thus, LOX-1 is an endothelial ligand for S. aureus, whose blockage may help to prevent or treat severe endovascular infections.

Invasive Staphylococcus epidermidis uses a unique processive wall teichoic acid glycosyltransferase to evade immune recognition.

Staphylococcus epidermidis expresses glycerol phosphate wall teichoic acid (WTA), but some health care-associated methicillin-resistant S. epidermidis (HA-MRSE) clones produce a second, ribitol phosphate (RboP) WTA, resembling that of the aggressive pathogen Staphylococcus aureus. RboP-WTA promotes HA-MRSE persistence and virulence in bloodstream infections. We report here that the TarM enzyme of HA-MRSE [TarM(Se)] glycosylates RboP-WTA with glucose, instead of N-acetylglucosamine (GlcNAc) by TarM(Sa) in S. aureus. Replacement of GlcNAc with glucose in RboP-WTA impairs HA-MRSE detection by human immunoglobulin G, which may contribute to the immune-evasion capacities of many invasive S. epidermidis. Crystal structures of complexes with uridine diphosphate glucose (UDP-glucose), and with UDP and glycosylated poly(RboP), reveal the binding mode and glycosylation mechanism of this enzyme and explain why TarM(Se) and TarM(Sa) link different sugars to poly(RboP). These structural data provide evidence that TarM(Se) is a processive WTA glycosyltransferase. Our study will support the targeted inhibition of TarM enzymes, and the development of RboP-WTA targeting vaccines and phage therapies.

Controlling biofilm and virulence properties of Gram-positive bacteria by targeting wall teichoic acid and lipoteichoic acid.

Wall teichoic acid (WTA) and lipoteichoic acid (LTA) are structural components of Gram-positive bacteria's peptidoglycan and cell membrane, which are mostly anionic glycopolymers. WTA confers numerous physiological, virulence, and pathogenic features to bacterial pathogens. It controls cell shape, cell division, and the localisation of autolytic enzymes and ion homeostasis. In the context of virulence and pathogenicity, it aids bacterial cell attachment and colonisation and protects against the host defence system and antibiotics. Having such a broad function in pathogenic bacteria's lifecycle, WTA/LTA become one of the potential targets for antibacterial agents to reduce bacterial infection in the host. The number of reports for targeting the WTA/LTA pathway has risen, mostly by focusing on three distinct targets: antivirulence targets, ß-lactam potentiator targets, and essential targets. The current review looked at the role of WTA/LTA in biofilm development and virulence in a range of Gram-positive pathogenic bacteria. Furthermore, alternate strategies, such as the application of natural and synthetic compounds that target the WTA/LTA pathway, have been thoroughly discussed. Moreover, the application of nanomaterials and a combination of drugs have also been discussed as a viable method for targeting the WTA/LTA in numerous Gram-positive bacteria. In addition, a future perspective for controlling bacterial infection by targeting the WTA/LTA is proposed.

Extensive remodelling of the cell wall during the development of Staphylococcus aureus bacteraemia.

The bloodstream represents a hostile environment that bacteria must overcome to cause bacteraemia. To understand how the major human pathogen Staphylococcus aureus manages this we have utilised a functional genomics approach to identify a number of new loci that affect the ability of the bacteria to survive exposure to serum, the critical first step in the development of bacteraemia. The expression of one of these genes, tcaA, was found to be induced upon exposure to serum, and we show that it is involved in the elaboration of a critical virulence factor, the wall teichoic acids (WTA), within the cell envelope. The activity of the TcaA protein alters the sensitivity of the bacteria to cell wall attacking agents, including antimicrobial peptides, human defence fatty acids, and several antibiotics. This protein also affects the autolytic activity and lysostaphin sensitivity of the bacteria, suggesting that in addition to changing WTA abundance in the cell envelope, it also plays a role in peptidoglycan crosslinking. With TcaA rendering the bacteria more susceptible to serum killing, while simultaneously increasing the abundance of WTA in the cell envelope, it was unclear what effect this protein may have during infection. To explore this, we examined human data and performed murine experimental infections. Collectively, our data suggests that whilst mutations in tcaA are selected for during bacteraemia, this protein positively contributes to the virulence of S. aureus through its involvement in altering the cell wall architecture of the bacteria, a process that appears to play a key role in the development of bacteraemia.

Molecular basis of the final step of cell division in Streptococcus pneumoniae.

Bacterial cell-wall hydrolases must be tightly regulated during bacterial cell division to prevent aberrant cell lysis and to allow final separation of viable daughter cells. In a multidisciplinary work, we disclose the molecular dialogue between the cell-wall hydrolase LytB, wall teichoic acids, and the eukaryotic-like protein kinase StkP in Streptococcus pneumoniae. After characterizing the peptidoglycan recognition mode by the catalytic domain of LytB, we further demonstrate that LytB possesses a modular organization allowing the specific binding to wall teichoic acids and to the protein kinase StkP. Structural and cellular studies notably reveal that the temporal and spatial localization of LytB is governed by the interaction between specific modules of LytB and the final PASTA domain of StkP. Our data collectively provide a comprehensive understanding of how LytB performs final separation of daughter cells and highlights the regulatory role of eukaryotic-like kinases on lytic machineries in the last step of cell division in streptococci.

LygA retention on the surface of Listeria monocytogenes via its interaction with wall teichoic acid modulates bacterial homeostasis and virulence.

Wall teichoic acid (WTA) is the abundant cell wall-associated glycopolymer in Gram-positive bacteria, playing crucial roles in surface proteins retention, bacterial homeostasis, and virulence. The WTA glycosylation of Listeria monocytogenes is essential for surface anchoring of virulence factors, whereas the nature and function of the noncovalent interactions between cell wall-associated proteins and WTA are less unknown. In this study, we found that galactosylated WTA (Gal-WTA) of serovar (SV) 4h L. monocytogenes plays a key role in modulating the novel glycine-tryptophan (GW) domain-containing autolysin protein LygA through direct interactions. Gal-deficient WTA of Lm XYSN (ΔgalT) showed a dramatic reduction of LygA on the cell surface. We demonstrated that LygA binds to Gal-WTA through the GW domains, and the binding affinity is associated with the number of GW motifs. Moreover, we confirmed the direct Gal-dependent binding of the GW protein Auto from the type I WTA strain, which has no interaction with rhamnosylated WTA, indicating that the complexity of both WTA and GW proteins affect the coordination patterns. Importantly, we revealed the crucial roles of LygA in facilitating bacterial homeostasis as well as crossing the intestinal and blood-brain barriers. Altogether, our findings suggest that both the glycosylation patterns of WTA and a fixed numbers of GW domains are closely associated with the retention of LygA on the cell surface, which promotes the pathogenesis of L. monocytogenes within the host.

Synthesis of structure-defined ß-1,4-GlcNAc-modified wall teichoic acids as potential vaccine against methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is a high priority pathogen due to its life-threating infections to human health. Development of prophylactic or therapeutic anti-MRSA vaccine is a potential approach to treat S. aureus infections and overcome the resistance crisis. ß-1,4-GlcNAc glycosylated wall teichoic acids (WTAs) derived from S. aureus are a new type of antigen that is closely associated with ß-lactam resistance. In this study, structure-defined ß-1,4-GlcNAc-modified WTAs varied in chain length and numbers of GlcNAc modification were synthesized by an ionic liquid-supported oligosaccharide synthesis (ILSOS) strategy in high efficiency and chromatography-free approach. Then the obtained WTAs were conjugated with tetanus toxin (TT) as vaccine candidates and were further evaluated in a mouse model to determine the structure-immunogenicity relationship. In vivo immunological studies revealed that the WTAs-TT conjugates provoked robust T cell-dependent responses and elicited high levels of specific anti-WTAs IgG antibodies production associated with the WTAs structure including chain length as well as the ß-1,4-GlcNAc modification pattern. Heptamer WTAs conjugate T6, carrying three copy of ß-1,4-GlcNAc modified RboP, was identified to elicit the highest titers of specific antibody production. The T6 antisera exhibited the highest recognition and binding affinity and the most potent OP-killing activities to MSSA and MRSA cells. This study demonstrated that ß-1,4-GlcNAc glycosylated WTAs are promising antigens for further development against MRSA.

Mechanism of D-alanine transfer to teichoic acids shows how bacteria acylate cell envelope polymers.

Bacterial cell envelope polymers are often modified with acyl esters that modulate physiology, enhance pathogenesis and provide antibiotic resistance. Here, using the D-alanylation of lipoteichoic acid (Dlt) pathway as a paradigm, we have identified a widespread strategy for how acylation of cell envelope polymers occurs. In this strategy, a membrane-bound O-acyltransferase (MBOAT) protein transfers an acyl group from an intracellular thioester onto the tyrosine of an extracytoplasmic C-terminal hexapeptide motif. This motif shuttles the acyl group to a serine on a separate transferase that moves the cargo to its destination. In the Dlt pathway, here studied in Staphylococcus aureus and Streptococcus thermophilus, the C-terminal 'acyl shuttle' motif that forms the crucial pathway intermediate is found on a transmembrane microprotein that holds the MBOAT protein and the other transferase together in a complex. In other systems, found in both Gram-negative and Gram-positive bacteria as well as some archaea, the motif is fused to the MBOAT protein, which interacts directly with the other transferase. The conserved chemistry uncovered here is widely used for acylation throughout the prokaryotic world.

Structure-function analysis of Lactiplantibacillus plantarum DltE reveals D-alanylated lipoteichoic acids as direct cues supporting Drosophila juvenile growth.

Metazoans establish mutually beneficial interactions with their resident microorganisms. However, our understanding of the microbial cues contributing to host physiology remains elusive. Previously, we identified a bacterial machinery encoded by the dlt operon involved in Drosophila melanogaster's juvenile growth promotion by Lactiplantibacillus plantarum. Here, using crystallography combined with biochemical and cellular approaches, we investigate the physiological role of an uncharacterized protein (DltE) encoded by this operon. We show that lipoteichoic acids (LTAs) but not wall teichoic acids are D-alanylated in Lactiplantibacillus plantarumNC8 cell envelope and demonstrate that DltE is a D-Ala carboxyesterase removing D-Ala from LTA. Using the mutualistic association of L. plantarumNC8 and Drosophila melanogaster as a symbiosis model, we establish that D-alanylated LTAs (D-Ala-LTAs) are direct cues supporting intestinal peptidase expression and juvenile growth in Drosophila. Our results pave the way to probing the contribution of D-Ala-LTAs to host physiology in other symbiotic models.

Visualization of Wall Teichoic Acid Decoration in Bacillus subtilis.

Teichoic acids are important for the maintenance of cell shape and growth in Gram-positive bacteria. Bacillus subtilis produces major and minor forms of wall teichoic acid (WTA) and lipoteichoic acid during vegetative growth. We found that newly synthesized WTA attachment to peptidoglycan occurs in a patch-like manner on the sidewall with the fluorescent labeling compound of the concanavalin A lectin. Similarly, WTA biosynthesis enzymes fused to the epitope tags were localized in similar patch-like patterns on the cylindrical part of the cell, and WTA transporter TagH was frequently colocalized with WTA polymerase TagF, WTA ligase TagT, and actin homolog MreB, respectively. Moreover, we found that the nascent cell wall patches, decorated with the newly glucosylated WTA, were colocalized with TagH and WTA ligase TagV. In the cylindrical part, the newly glucosylated WTA patchily inserted into the bottom of the cell wall layer and finally reached the outermost layer of the cell wall after approximately half an hour. Incorporation of newly glucosylated WTA was arrested with the addition of vancomycin but restored with the removal of the antibiotic. These results are consistent with the prevailing model that WTA precursors are attached to newly synthesized peptidoglycan. IMPORTANCE In Gram-positive bacteria, the cell wall is composed of mesh-like peptidoglycan and covalently linked wall teichoic acid (WTA). It is unclear where WTA decorates peptidoglycan to create a cell wall architecture. Here, we demonstrate that nascent WTA decoration occurred in a patch-like manner at the peptidoglycan synthesis sites on the cytoplasmic membrane. The incorporated cell wall with newly glucosylated WTA in the cell wall layer then reached the outermost layer of the cell wall after approximately half an hour. Incorporation of newly glucosylated WTA was arrested with the addition of vancomycin but restored with the removal of the antibiotic. These results are consistent with the prevailing model that WTA precursors are attached to newly synthesized peptidoglycan.

Assembly of Bacterial Surface Glycopolymers as an Antibiotic Target.

Bacterial cells are covered with various glycopolymers such as peptidoglycan (PG), lipopolysaccharides (LPS), teichoic acids, and capsules. Among these glycopolymers, PG assembly is the target of some of our most effective antibiotics, consistent with its essentiality and uniqueness to bacterial cells. Biosynthesis of other surface glycopolymers have also been acknowledged as potential targets for developing therapies to control bacterial infections, because of their importance for bacterial survival in the host environment. Moreover, biosynthesis of most surface glycopolymers are closely related to PG assembly because the same lipid carrier is shared for glycopolymer syntheses. In this review, I provide an overview of PG assembly and antibiotics that target this pathway. Then, I discuss the implications of a common lipid carrier being used for assembly of PG and other surface glycopolymers in antibiotic development.

Insights into the Roles of Lipoteichoic Acids and MprF in Bacillus subtilis.

Gram-positive bacterial cells are protected from the environment by a cell envelope that is comprised of a thick layer of peptidoglycan that maintains cell shape and teichoic acid polymers whose biological function remains unclear. In Bacillus subtilis, the loss of all class A penicillin-binding proteins (aPBPs), which function in peptidoglycan synthesis, is conditionally lethal. Here, we show that this lethality is associated with an alteration of lipoteichoic acids (LTAs) and the accumulation of the major autolysin LytE in the cell wall. Our analysis provides further evidence that the length and abundance of LTAs act to regulate the cellular level and activity of autolytic enzymes, specifically LytE. Importantly, we identify a novel function for the aminoacyl-phosphatidylglycerol synthase MprF in the modulation of LTA biosynthesis in both B. subtilis and Staphylococcus aureus. This finding has implications for our understanding of antimicrobial resistance (particularly to daptomycin) in clinically relevant bacteria and the involvement of MprF in the virulence of pathogens such as methicillin-resistant S. aureus (MRSA). IMPORTANCE In Gram-positive bacteria such as Bacillus subtilis and Staphylococcus aureus, the cell envelope is a structure that protects the cells from the environment but is also dynamic in that it must be modified in a controlled way to allow cell growth. In this study, we show that lipoteichoic acids (LTAs), which are anionic polymers attached to the membrane, have a direct role in modulating the cellular abundance of cell wall-degrading enzymes. We also find that the apparent length of the LTA is modulated by the activity of the enzyme MprF, previously implicated in modifications of the cell membrane leading to resistance to antimicrobial peptides. These findings are important contributions to our understanding of how bacteria balance cell wall synthesis and degradation to permit controlled growth and division. These results also have implications for the interpretation of antibiotic resistance, particularly for the clinical treatment of MRSA infections.

Wall Teichoic Acids Facilitate the Release of Toxins from the Surface of Staphylococcus aureus.

A major feature of the pathogenicity of Staphylococcus aureus is its ability to secrete cytolytic toxins. This process involves the translocation of the toxins from the cytoplasm through the bacterial membrane and the cell wall to the external environment. The process of their movement through the membrane is relatively well defined, involving both general and toxin-specific secretory systems. Movement of the toxins through the cell wall was considered to involve the passive diffusion of the proteins through the porous cell wall structures; however, recent work suggests that this is more complex, and here we demonstrate a role for the wall teichoic acids (WTA) in this process. Utilizing a genome-wide association approach, we identified a polymorphism in the locus encoding the WTA biosynthetic machinery as associated with the cytolytic activity of the bacteria. We verified this association using an isogenic mutant set and found that WTA are required for the release of several cytolytic toxins from the bacterial cells. We show that this effect is mediated by a change in the electrostatic charge across the cell envelope that results from the loss of WTA. As a major target for the development of novel therapeutics, it is important that we fully understand the entire process of cytolytic toxin production and release. These findings open up a new aspect to the process of toxin release by a major human pathogen while also demonstrating that clinical isolates can utilize WTA production to vary their cytotoxicity, thereby altering their pathogenic capabilities. IMPORTANCE The production and release of cytolytic toxins is a critical aspect for the pathogenicity of many bacterial pathogens. In this study, we demonstrate a role for wall teichoic acids, molecules that are anchored to the peptidoglycan of the bacterial cell wall, in the release of toxins from S. aureus cells into the extracellular environment. Our findings suggest that this effect is mediated by a gradient of electrostatic charge which the presence of the negatively charged WTA molecules create across the cell envelope. This work brings an entirely new aspect to our understanding of the cytotoxicity of S. aureus and demonstrates a further means by which this major human pathogen can adapt its pathogenic capabilities.

DltC acts as an interaction hub for AcpS, DltA and DltB in the teichoic acid D-alanylation pathway of Lactiplantibacillus plantarum.

Teichoic acids (TA) are crucial for the homeostasis of the bacterial cell wall as well as their developmental behavior and interplay with the environment. TA can be decorated by different modifications, modulating thus their biochemical properties. One major modification consists in the esterification of TA by D-alanine, a process known as D-alanylation. TA D-alanylation is performed by the Dlt pathway, which starts in the cytoplasm and continues extracellularly after D-Ala transportation through the membrane. In this study, we combined structural biology and in vivo approaches to dissect the cytoplasmic steps of this pathway in Lactiplantibacillus plantarum, a bacterial species conferring health benefits to its animal host. After establishing that AcpS, DltB, DltC1 and DltA are required for the promotion of Drosophila juvenile growth under chronic undernutrition, we solved their crystal structure and/or used NMR and molecular modeling to study their interactions. Our work demonstrates that the suite of interactions between these proteins is ordered with a conserved surface of DltC1 docking sequentially AcpS, DltA and eventually DltB. Altogether, we conclude that DltC1 acts as an interaction hub for all the successive cytoplasmic steps of the TA D-alanylation pathway.

D-alanylation of lipoteichoic acid contributes to biofilm formation and acidogenesis capacity of Streptococcusmutans.

BACKGROUND: D-alanylation of Lipoteichoic acid (LTA) is considered to be essential for virulence factors expression in Gram-positive microorganism. The effects of the D-alanylation of LTA on biofilm formation and acidogenesis of Streptococcus mutans (S. mutans) are still not clearly understood. AIM: This study was designed to investigate the impact of D-alanylation of LTA on biofilm formation and acidogenesis of S. mutans and explore the related mechanisms. METHODS AND MATERIAL: We compared the biofilm formation process by fluorescence microscope observation of LTA D-alanylation blocking strain with that of the wildtype strain. Auto-aggregation, cell surface charge, and polysaccharide production assays were performed to investigate the related mechanisms. pH drop assay and glycolysis pH drop-down analysis were carried out to evaluate the acidogenesis capacity of S. mutans after LTA D-alanylation blocking. To identify the biofilm formation and adhesive-related genes expressions of S. mutans mutant, qRT-PCR was performed. RESULTS: After blocking off the D-alanylation of LTA, S. mutans could not form the three-dimensional structural biofilm, in which cells were scattered on the substratum as small clusters. The auto-aggregation was prompted due to the mutant strain cell morphology change (*p < 0.05). Furthermore, more negative charges were found on the mutant strain cells surfaces and fewer water-insoluble glucans were produced in mutant biofilm (*p < 0.05). The adhesion capacity of the S. mutans biofilm was impaired after LTA D-alanylation blocking (*p < 0.05). Biofilm formation and adhesive-related genes expressions decreased (*p < 0.05), especially at the early stages of biofilm formation. S. mutans mutant strains exhibited suppressed acidogenesis because its glycolytic activity was impaired. CONCLUSION: The results of this study suggest that blocking of LTA D-alanylation disrupts normal biofilm formation in S. mutans predominantly if not entirely by altering intercellular auto-aggregation, cell adhesion, and extracellular matrix formation. Moreover, our study results suggest that the LTA D-alanylation plays an important role in S. mutans acidogenesis by altering glycolytic activity. These findings add to the knowledge about mechanisms underlying biofilm formation and acid tolerance in S. mutans.

GpsB Coordinates Cell Division and Cell Surface Decoration by Wall Teichoic Acids in Staphylococcus aureus.

Bacterial cell division is a complex and highly regulated process requiring the coordination of many different proteins. Despite substantial work in model organisms, our understanding of the systems regulating cell division in noncanonical organisms, including critical human pathogens, is far from complete. One such organism is Staphylococcus aureus, a spherical bacterium that lacks known cell division regulatory proteins. Recent studies on GpsB, a protein conserved within the Firmicutes phylum, have provided insight into cell division regulation in S. aureus and other related organisms. It has been revealed that GpsB coordinates cell division and cell wall synthesis in multiple species. In S. aureus, we have previously shown that GpsB directly regulates FtsZ polymerization. In this study, using Bacillus subtilis as a tool, we isolated spontaneous suppressors that abrogate the lethality of S. aureus GpsB overproduction in B. subtilis. Through characterization, we identified several residues important for the function of GpsB. Furthermore, we discovered an additional role for GpsB in wall teichoic acid (WTA) biosynthesis in S. aureus. Specifically, we show that GpsB directly interacts with the WTA export protein TarG. We also identified a region in GpsB that is crucial for this interaction. Analysis of TarG localization in S. aureus suggests that WTA machinery is part of the divisome complex. Taken together, this research illustrates how GpsB performs an essential function in S. aureus by directly linking the tightly regulated cell cycle processes of cell division and WTA-mediated cell surface decoration. IMPORTANCE Cytokinesis in bacteria involves an intricate orchestration of several key cell division proteins and other factors involved in building a robust cell envelope. Presence of teichoic acids is a signature characteristic of the Gram-positive cell wall. By characterizing the role of Staphylococcus aureus GpsB, an essential cell division protein in this organism, we have uncovered an additional role for GpsB in wall teichoic acid (WTA) biosynthesis. We show that GpsB directly interacts with TarG of the WTA export complex. We also show that this function of GpsB may be conserved in other GpsB homologs as GpsB and the WTA exporter complex follow similar localization patterns. It has been suggested that WTA acts as a molecular signal to control the activity of autolytic enzymes, especially during the separation of conjoined daughter cells. Thus, our results reveal that GpsB, in addition to playing a role in cell division, may also help coordinate WTA biogenesis.

Streptococcus gordonii Poised for Glycan Feeding through a MUC5B-Discriminating, Lipoteichoic Acid-Mediated Outside-In Signaling Circuit.

Many oral bacteria employ cell wall-anchored adhesins to bind to the salivary films coating the teeth and mucosal surfaces. Surface binding prevents clearance and facilitates catabolism of salivary film glycoproteins. We asked whether Streptococcus gordonii adhesin expression changes in response to surface salivary cues using a eukaryote-like, outside-in recognition and signaling circuit. To determine whether the cues were discriminated, S. gordonii was tested during cell adhesion and biofilm formation on a MUC5B-rich or lower-molecular-mass salivary fraction or an uncoated abiotic surface. Cells were recovered and analyzed for differences in gene expression and proteins in cell wall fractions. In salivary-free conditions, planktonic S. gordonii presented three prominent cell wall LPXTG-motif proteins, SGO_1487, SGO_0890, and MbpA (mucin-binding protein A; SGO_0707). During biofilm formation on MUC5B-coated surfaces, MbpA, a MUC5B-binding protein, and key genes in the tagatose and quorum-sensing pathways were strongly promoted. The response to MUC5B required the two-component system (TCS), streptococcal regulator of adhesins sensor and regulator (SraSR, SGO_1180/81), lipoteichoic acid (LTA), and the homologous paired adhesins, SspA and SspB (SspAB). LTA appears to link the outside signal (MUC5B) to intramembrane SraSR. Tagatose pathway gene expression may poise cells to metabolize MUC5B glycans and, with a quorum-sensing gene (luxS), may direct formation of a consortium to facilitate glycan cross-feeding by S. gordonii. We now show that a Gram-positive bacterium discriminates specific surface environmental cues using an outside-in signaling mechanism to apparently optimize colonization of saliva-coated surfaces. IMPORTANCE All organisms throughout the tree of life sense and respond to their surface environments. To discriminate among mucosal surface environmental cues, we report that Streptococcus gordonii recognizes a high-molecular-weight mucin glycoprotein, MUC5B, using the paired adhesins SspAB and lipoteichoic acid; the latter bridges the outside signal to an intramembrane two-component system to transcriptionally regulate a MUC5B-specific adhesin and genes that may facilitate glycan catabolism.