Wall teichoic acids are anionic phosphate-rich polymers that are part of the complex meshwork of carbohydrates that make up the gram-positive cell wall. These polymers are essential to the proper rod-shaped morphology of Bacillus subtilis and have been shown to be an important virulence determinant in the nosocomial opportunistic pathogen Staphylococcus aureus. Together, sequence-based studies, in vitro experiments with biosynthetic proteins, and analyses of the chemical structure of wall teichoic acid have begun to shed considerable light on our understanding of the biogenesis of this polymer. Nevertheless, some paradoxes remain unresolved. One of these involves a putative duplication of genes linked to CDP-ribitol synthesis (tarI′J′ and tarIJ) as well as poly(ribitol phosphate) polymerization (tarK and tarL) in S. aureus. In the work reported here, we performed careful studies of the dispensability of each gene and discovered a functional redundancy in the duplicated gene clusters. We were able to create mutants in either of the putative ribitol phosphate polymerases (encoded by tarK and tarL) without affecting teichoic acid levels in the S. aureus cell wall. Although genes linked to CDP-ribitol synthesis are also duplicated, a null mutant in only one of these (tarI′J′) could be obtained, while tarIJ remained essential. Suppression analysis of the tarIJ null mutant indicated that the mechanism of dysfunction in tarI′J′ is due to poor translation of the TarJ′ enzyme, which catalyzes the rate-limiting step in CDP-ribitol formation. This work provides new insights into understanding the complex synthetic steps of the ribitol phosphate polymer in S. aureus and has implications on specifically targeting enzymes involved in polymer biosynthesis for antimicrobial design.
There have been considerable strides made in the characterization of the dispensability of teichoic acid biosynthesis genes in recent years. A notable omission thus far has been an early gene in teichoic acid synthesis encoding the N-acetylmannosamine transferase (tagA in Bacillus subtilis; tarA in Staphylococcus aureus), which adds N-acetylmannosamine to complete the synthesis of undecaprenol pyrophosphate-linked disaccharide. Here, we show that the N-acetylmannosamine transferases are dispensable for growth in vitro, making this biosynthetic enzyme the last dispensable gene in the pathway, suggesting that tagA (or tarA) encodes the first committed step in wall teichoic acid synthesis.
An extensive study of teichoic acid biosynthesis in the model organism Bacillus subtilis has established teichoic acid polymers as essential components of the gram-positive cell wall. However, similar studies pertaining to therapeutically relevant organisms, such as Staphylococcus aureus, are scarce. In this study we have carried out a meticulous examination of the dispensability of teichoic acid biosynthetic enzymes in S. aureus. By use of an allelic replacement methodology, we examined all facets of teichoic acid assembly, including intracellular polymer production and export. Using this approach we confirmed that the first-acting enzyme (TarO) was dispensable for growth, in contrast to dispensability studies in B. subtilis. Upon further characterization, we demonstrated that later-acting gene products (TarB, TarD, TarF, TarIJ, and TarH) responsible for polymer formation and export were essential for viability. We resolved this paradox by demonstrating that all of the apparently indispensable genes became dispensable in a tarO null genetic background. This work suggests a lethal gain-of-function mechanism where lesions beyond the initial step in wall teichoic acid biosynthesis render S. aureus nonviable. This discovery poses questions regarding the conventional understanding of essential gene sets, garnered through single-gene knockout experiments in bacteria and higher organisms, and points to a novel drug development strategy targeting late steps in teichoic acid synthesis for the infectious pathogen S. aureus.
Wall teichoic acids are anionic, phosphate-rich polymers linked to the peptidoglycan of gram-positive bacteria. In Bacillus subtilis, the predominant wall teichoic acid types are poly(glycerol phosphate) in strain 168 and poly(ribitol phosphate) in strain W23, and they are synthesized by the tag and tar gene products, respectively. Growing evidence suggests that wall teichoic acids are essential in B. subtilis; however, it is widely believed that teichoic acids are dispensable under phosphate-limiting conditions. In the work reported here, we carefully studied the dispensability of teichoic acid under phosphate-limiting conditions by constructing three new mutants. These strains, having precise deletions in tagB, tagF, and tarD, were dependent on xylose-inducible complementation from a distal locus (amyE) for growth. The tarD deletion interrupted poly(ribitol phosphate) synthesis in B. subtilis and represents a unique deletion of a tar gene. When teichoic acid biosynthetic proteins were depleted, the mutants showed a coccoid morphology and cell wall thickening. The new wall teichoic acid biogenesis mutants generated in this work and a previously reported tagD mutant were not viable under phosphate-limiting conditions in the absence of complementation. Cell wall analysis of B. subtilis grown under phosphate-limited conditions showed that teichoic acid contributed approximately one-third of the wall anionic content. These data suggest that wall teichoic acid has an essential function in B. subtilis that cannot be replaced by teichuronic acid.
Wall teichoic acids are cell wall polymers that maintain the integrity of the cellular envelope and contribute to the virulence of Staphylococcus aureus. Despite the central role of wall teichoic acid in S. aureus virulence, details concerning the biosynthetic pathway of the predominant wall teichoic acid polymer are lacking, and workers have relied on a presumed similarity to the putative polyribitol phosphate wall teichoic acid pathway in Bacillus subtilis. Using high-resolution polyacrylamide gel electrophoresis for analysis of wall teichoic acid extracted from gene deletion mutants, a revised assembly pathway for the late-stage ribitol phosphate-utilizing enzymes is proposed. Complementation studies show that a putative ribitol phosphate polymerase, TarL, catalyzes both the addition of the priming ribitol phosphate onto the linkage unit and the subsequent polymerization of the polyribitol chain. It is known that the putative ribitol primase, TarK, is also a bifunctional enzyme that catalyzes both ribitol phosphate priming and polymerization. TarK directs the synthesis of a second, electrophoretically distinct polyribitol-containing teichoic acid that we designate K-WTA. The biosynthesis of K-WTA in S. aureus strain NCTC8325 is repressed by the accessory gene regulator (agr) system. The demonstration of regulated wall teichoic acid biosynthesis has implications for cell envelope remodeling in relation to S. aureus adhesion and pathogenesis.
Streptococcus pneumoniae has unusually complex cell wall teichoic acid and lipoteichoic acid, both of which contain a ribitol phosphate moiety. The lic region of the pneumococcal genome contains genes for the uptake and activation of choline, the attachment of phosphorylcholine to teichoic acid precursors, and the transport of these precursors across the cytoplasmic membrane. The role of two other, so far uncharacterized, genes, spr1148 and spr1149, in the lic region was determined. TarJ (spr1148) encodes an NADPH-dependent alcohol dehydrogenase for the synthesis of ribitol 5-phosphate from ribulose 5-phosphate. TarI (spr1149) encodes a cytidylyl transferase for the synthesis of cytidine 5′-diphosphate (CDP)-ribitol from ribitol 5-phosphate and cytidine 5′-triphosphate. We also present the crystal structure of TarI with and without bound CDP, and the structures present a rationale for the substrate specificity of this key enzyme. No transformants were obtained with insertion plasmids designed to interrupt the tarIJ genes, indicating that their function could be essential for cell growth. CDP-activated ribitol is a precursor for the synthesis of pneumococcal teichoic acids and some of the capsular polysaccharides. Thus, all eight genes in the lic region have a role in teichoic acid synthesis.
Wall teichoic acids (WTAs) are anionic polymers that play key roles in bacterial cell shape, cell division, envelope integrity, biofilm formation, and pathogenesis. B. subtilis W23 and S. aureus both make polyribitol-phosphate (RboP) WTAs and contain similar sets of biosynthetic genes. We use in vitro reconstitution combined with genetics to show the pathways for WTA biosynthesis in B. subtilis W23 and S. aureus are different. S. aureus requires a glycerol-phosphate primase called TarF in order make RboP-WTAs; B. subtilis W23 contains a TarF homolog, but this enzyme makes glycerol-phosphate polymers and is not involved RboP-WTA synthesis. Instead, B. subtilis TarK functions in place of TarF to prime the WTA intermediate for chain extension by TarL. This work highlights the enzymatic diversity of the poorly characterized family of phosphotransferases involved in WTA biosynthesis in Gram-positive organisms.
The function(s) of gram-positive wall teichoic acid is emerging with recent findings that it is an important virulence factor in the pathogen Staphylococcus aureus and that it is crucial to proper rod-shaped cell morphology of Bacillus subtilis. Despite its importance, our understanding of teichoic acid biosynthesis remains incomplete. The TagB protein has been implicated in the priming step of poly(glycerol phosphate) wall teichoic acid synthesis in B. subtilis. Work to date indicates that the TagB protein is localized to the membrane, where it adds a single glycerol phosphate residue to the nonreducing end of the undecaprenol-phosphate-linked N-acetylmannosamine-β(1,4)-N-acetylglucosamine-1-phosphate. Thus, membrane association is critical to TagB function. In this work we elucidate the mechanism of TagB membrane localization. We report the identification of a membrane targeting determinant at the amino terminus of TagB that is necessary and sufficient for membrane localization. The putative amphipathicity of this membrane targeting determinant was characterized and shown to be required for TagB function but not localization. This work shows for the first time that the amino terminus of TagB mediates membrane targeting and protein function.
Using a previously reported conditional expression system for use in Bacillus subtilis (A. P. Bhavsar, X. Zhao, and E. D. Brown, Appl. Environ. Microbiol. 67:403–410, 2001), we report the first precise deletion of a teichoic acid biosynthesis (tag) gene, tagD, in B. subtilis. This teichoic acid mutant showed a lethal phenotype when characterized at a physiological temperature and in a defined genetic background. This tagD mutant was subject to full phenotypic rescue upon expression of the complementing copy of tagD. Depletion of the tagD gene product (glycerol 3-phosphate cytidylyltransferase) via modulated expression of tagD from the amyE locus revealed structural defects centered on shape, septation, and division. Thickening of the wall and ultimately lysis followed these events.
Polyanionic polymers, including lipoteichoic acid and wall teichoic acid, are important determinants of the charged character of the staphylococcal cell wall. This study was designed to investigate the extent to which teichoic acid contributes to protection from anionic azo dyes and to identify barriers to drug penetration for development of new antibiotics for multidrug-resistant Staphylococcus aureus infection.
We studied antimicrobial activity of azo dyes against S. aureus strains with or without inhibition of teichoic acid in vitro and in vivo.
We observed that inhibition of wall teichoic acid expression resulted in an ∼1000-fold increase in susceptibility to azo dyes such as Congo red, reducing its MIC from >1024 to <4 mg/L. Sensitization occurred when the first step in the wall teichoic acid pathway, catalysed by TarO, was inhibited either by mutation or by chemical inhibition. In contrast, genetic blockade of lipoteichoic acid biosynthesis did not confer Congo red susceptibility. Based on this finding, combination therapy was tested using the highly synergistic combination of Congo red plus tunicamycin at sub-MIC concentrations (to inhibit wall teichoic acid biosynthesis). The combination rescued Caenorhabditis elegans from a lethal challenge of S. aureus.
Our studies show that wall teichoic acid confers protection to S. aureus from anionic azo dyes and related compounds, and its inhibition raises the prospect of development of new combination therapies based on this inhibition.
bacteria; antibiotics; S. aureus
tagA, tagD, and tuaA operons are responsible for the synthesis of cell wall anionic polymer, teichoic acid, and teichuronic acid, respectively, in Bacillus subtilis. Under phosphate starvation conditions, teichuronic acid is synthesized while teichoic acid synthesis is inhibited. Expression of these genes is controlled by PhoP-PhoR, a two-component system. It has been proposed that PhoP∼P plays a key role in the activation of tuaA and the repression of tagA and tagD. In this study, we demonstrated the role of PhoP∼P in the switch process from teichoic acid synthesis to teichuronic acid synthesis, by using an in vitro transcription system. The results indicate that PhoP∼P is sufficient to repress the transcription of the tagA and tagD promoters and also to activate the transcription of the tuaA promoter.
Staphylococcus epidermidis ATCC 14990 produces a wall-associated glycerol teichoic acid which is chemically identical to the major wall-associated teichoic acid of Bacillus subtilis 168. The S. epidermidis tagF gene was cloned from genomic DNA and sequenced. When introduced on a plasmid vector into B. subtilis 1A486 carrying the conditionally lethal temperature-sensitive mutation tagF1 (rodC1), it expressed an 85-kDa protein which allowed colonies to grow at the restrictive temperature. This showed that the cloned S. epidermidis gene encodes a functional CDP-glycerol:poly(glycerophosphate) glycerophosphotransferase. An amino acid substitution at residue 616 in the recombinant TagF protein eliminated complementation. Unlike B. subtilis, where the tagF gene is part of the tagDEF operon, the tagF gene of S. epidermidis is not linked to any other tag genes. We attempted to disrupt the chromosomal tagF gene in S. epidermidis TU3298 by directed integration of a temperature-sensitive plasmid but this failed, whereas a control plasmid containing the 5′ end of tagF on a similarly sized DNA fragment was able to integrate. This suggests that the tagF gene is essential and that the TagF and other enzymes involved in teichoic acid biosynthesis could be targets for new antistaphylococcal drugs.
The cell envelopes of Gram-positive bacteria comprise two major constituents, peptidoglycan and teichoic acids. Wall teichoic acids (WTAs) are anionic glycophosphate polymers that play important roles in bacterial cell growth, division, and pathogenesis. They are synthesized intracellularly and exported by an ABC transporter to the cell surface, where they are covalently attached to peptidoglycan. We address here the substrate specificity of WTA transporters by substituting the Bacillus subtilis homologue, TagGHBs, with the Staphylococcus aureus homologue, TarGHSa. These transporters export structurally different substrates in their indigenous organisms, but we show that TarGHSa can substitute for the B. subtilis transporter. Hence, substrate specificity does not depend on the WTA main chain polymer structure, but may be determined by the conserved diphospholipid-linked disaccharide portion of the WTA precursor. We also show that the complemented B. subtilis strain becomes susceptible to a S. aureus-specific antibiotic, demonstrating that the S. aureus WTA transporter is the sole target of this compound.
Wall teichoic acids (WTAs) are phosphate-rich, sugar-based polymers attached to the cell walls of most Gram-positive bacteria. In Staphylococcus aureus, these anionic polymers regulate cell division, protect cells from osmotic stress, mediate host colonization, and mask enzymatically susceptible peptidoglycan bonds. Although WTAs are not required for survival in vitro, blocking the pathway at a late stage of synthesis is lethal. We recently discovered a novel antibiotic, targocil, that inhibits a late acting step in the WTA pathway. Its target is TarG, the transmembrane component of the ABC transporter (TarGH) that exports WTAs to the cell surface. We examined here the effects of targocil on S. aureus using transmission electron microscopy and gene expression profiling. We report that targocil treatment leads to multicellular clusters containing swollen cells displaying evidence of osmotic stress, strongly induces the cell wall stress stimulon, and reduces the expression of key virulence genes, including dltABCD and capsule genes. We conclude that WTA inhibitors that act at a late stage of the biosynthetic pathway may be useful as antibiotics, and we present evidence that they could be particularly useful in combination with beta-lactams.
An extensive literature has established that the synthesis of wall teichoic acid in Bacillus subtilis is essential for cell viability. Paradoxically, we have recently shown that wall teichoic acid biogenesis is dispensable in Staphylococcus aureus (M. A. D'Elia, M. P. Pereira, Y. S. Chung, W. Zhao, A. Chau, T. J. Kenney, M. C. Sulavik, T. A. Black, and E. D. Brown, J. Bacteriol. 188:4183-4189, 2006). A complex pattern of teichoic acid gene dispensability was seen in S. aureus where the first gene (tarO) was dispensable and later acting genes showed an indispensable phenotype. Here we show, for the first time, that wall teichoic acid synthesis is also dispensable in B. subtilis and that a similar gene dispensability pattern is seen where later acting enzymes display an essential phenotype, while the gene tagO, whose product catalyzes the first step in the pathway, could be deleted to yield viable mutants devoid of teichoic acid in the cell wall.
The biosynthesis of the linkage region between peptidoglycan and the ribitol teichoic acid was investigated in the bacteriophage-resistant, teichoic acid-less mutant Staphylococcus aureus 52A5 (Chatterjee et al., J. Bacteriol. 100:846--853, 1969). Membrane preparations of this strain were found to be incapable of forming the first intermediate of the biosynthetic pathway, namely, the transfer of N-acetyl-D-glucosamine (GlcNAc) from UDP-GlcNAc to the acceptor molecule, which presumbably is undecaprenol phosphate (R. Bracha and L. Glaser, Biochem. Biophys. Res. Commun. 72:1091--1098, 1976). The addition of heat-inactivated membrane preparations of S. aureus 52A2 (which normally has ribitol teichoic acid) that had been preincubated with UDP-GlcNAc to membranes of strain 52A5 enabled the synthesis of teichoic acid. These data suggest that the mutational defect in the teichoic acid-less organism is in the synthesis of the first compound of the linkage unit, and this is apparently the reason for its absence in the cell walls.
The structure of the linkage regions between ribitol teichoic acids and peptidoglycan in the cell walls of Staphylococcus aureus H and 209P and Bacillus subtilis W23 and AHU 1390 was studied. Teichoic acid-linked saccharide preparations obtained from the cell walls by heating at pH 2.5 contained mannosamine and glycerol in small amounts. On mild alkali treatment, each teichoic acid-linked saccharide preparation was split into a disaccharide identified as N-acetylmannosaminyl beta(1----4)N-acetylglucosamine and the ribitol teichoic acid moiety that contained glycerol residues. The Smith degradation of reduced samples of the teichoic acid-linked saccharide preparations from S. aureus and B. subtilis gave fragments characterized as 1,2-ethylenediol phosphate-(glycerolphosphate)3-N-acetylmannosaminyl beta(1----4)N- -acetylxylosaminitol and 1,2-ethylenediolphosphate-(glycerol phosphate)2-N-acetylmannosaminyl beta(1----4)N-acetylxylosaminitol, respectively. The binding of the disaccharide unit to peptidoglycan was confirmed by the analysis of linkage-unit-bound glycopeptides obtained from NaIO4 oxidation of teichoic acid-glycopeptide complexes. Mild alkali treatment of the linkage-unit-bound glycopeptides yielded disaccharide-linked glycopeptides, which gave the disaccharide and phosphorylated glycopeptides on mild acid treatment. Thus, it is concluded that the ribitol teichoic acid chains in the cell walls of the strains of S. aureus and B. subtilis are linked to peptidoglycan through linkage units, (glycerol phosphate)3-N-acetylmannosaminyl beta(1----4)N-acetylglucosamine and (glycerol phosphate)2-N-acetylmannosaminyl beta(1----4)N-acetylglucosamine, respectively.
Human sera were found to contain antibodies precipitating with each of two samples of teichoic acid of Staphylococcus aureus prior to immunization; these antibodies were probably formed as a result of contact or infection with this microorganism. Injection of teichoic acid into two individuals resulted in a rise in circulating antibody to teichoic acid; a third subject probably had a primary response to α-teichoic acid. Quantitative precipitin and agar diffusion studies revealed the presence of two distinct antibodies in the sera and showed that each specimen of teichoic acid was a mixture of two polymers an α-linked N-acetylglucosaminyl-ribitol polymer and a β-linked N-acetylglucosaminyl-ribitol polymer, termed α- and β-teichoic acids respectively. The α-teichoic acid anti-α-teichoic acid system was inhibited best by α-linked glucosaminides and the β-anti-β-teichoic acid system was inhibited best by a β-linked glucosaminide. The α- and (β-teichoic acids could be separated from each other by specific precipitation under appropriate conditions and recovered from the washed specific precipitates. The existence of two distinct teichoic acid polymers raises important questions as to cell wall structure and the biosynthesis of the teichoic acids.
Specific strains of Lactobacillus plantarum are marketed as health-promoting probiotics. The role and interplay of cell-wall compounds like wall- and lipo-teichoic acids (WTA and LTA) in bacterial physiology and probiotic-host interactions remain obscure. L. plantarum WCFS1 harbors the genetic potential to switch WTA backbone alditol, providing an opportunity to study the impact of WTA backbone modifications in an isogenic background.
Through genome mining and mutagenesis we constructed derivatives that synthesize alternative WTA variants. The mutants were shown to completely lack WTA, or produce WTA and LTA that lack D-Ala substitution, or ribitol-backbone WTA instead of the wild-type glycerol-containing backbone. DNA micro-array experiments established that the tarIJKL gene cluster is required for the biosynthesis of this alternative WTA backbone, and suggest ribose and arabinose are precursors thereof. Increased tarIJKL expression was not observed in any of our previously performed DNA microarray experiments, nor in qRT-PCR analyses of L. plantarum grown on various carbon sources, leaving the natural conditions leading to WTA backbone alditol switching, if any, to be identified. Human embryonic kidney NF-κB reporter cells expressing Toll like receptor (TLR)-2/6 were exposed to purified WTAs and/or the TA mutants, indicating that WTA is not directly involved in TLR-2/6 signaling, but attenuates this signaling in a backbone independent manner, likely by affecting the release and exposure of immunomodulatory compounds such as LTA. Moreover, human dendritic cells did not secrete any cytokines when purified WTAs were applied, whereas they secreted drastically decreased levels of the pro-inflammatory cytokines IL-12p70 and TNF-α after stimulation with the WTA mutants as compared to the wild-type.
The study presented here correlates structural differences in WTA to their functional characteristics, thereby providing important information aiding to improve our understanding of molecular host-microbe interactions and probiotic functionality.
Lactobacillus plantarum; Probiotic; Wall teichoic acid; Lipoteichoic acid; tag and tar genes; Immunomodulation
Methicillin resistance in Staphylococcus aureus depends on the production of mecA, which encodes penicillin-binding protein 2A (PBP2A), an acquired peptidoglycan transpeptidase (TP) with reduced susceptibility to beta-lactam antibiotics. PBP2A crosslinks nascent peptidoglycan when the native TPs are inhibited by beta-lactams. Although mecA expression is essential for beta-lactam resistance, it is not sufficient. Here we show that blocking the expression of wall teichoic acids (WTAs) by inhibiting the first enzyme in the pathway, TarO, sensitizes MRSA strains to beta-lactams even though the beta-lactam-resistant transpeptidase, PBP2A, is still expressed. The dramatic synergy between TarO inhibitors and beta-lactams is noteworthy not simply because strategies to overcome methicillin-resistant S. aureus (MRSA) are desperately needed, but because neither TarO nor the activities of the native TPs are essential in MRSA strains. The “synthetic lethality” of inhibiting TarO and the native TPs suggests a functional connection between ongoing WTA expression and peptidoglycan assembly in S. aureus. Indeed, transmission electron microscopy shows that S. aureus cells blocked in WTA synthesis have extensive defects in septation and cell separation, indicating dysregulated cell wall assembly and degradation. Our studies imply that WTAs play a fundamental role in S. aureus cell division and raise the possibility that synthetic lethal compound combinations may have therapeutic utility for overcoming antibiotic resistant bacterial infections.
Rising drug resistance is limiting treatment options
by methicillin-resistant Staphylococcus aureus (MRSA).
Herein we provide new evidence that wall teichoic acid (WTA) biogenesis
is a remarkable antibacterial target with the capacity to destabilize
the cooperative action of penicillin-binding proteins (PBPs) that
underlie β-lactam resistance in MRSA. Deletion of gene tarO, encoding the first step of WTA synthesis, resulted
in the restoration of sensitivity of MRSA to a unique profile of β-lactam
antibiotics with a known selectivity for penicillin binding protein
2 (PBP2). Of these, cefuroxime was used as a probe to screen for previously
approved drugs with a cryptic capacity to potentiate its activity
against MRSA. Ticlopidine, the antiplatelet drug Ticlid, strongly
potentiated cefuroxime, and this synergy was abolished in strains
lacking tarO. The combination was also effective
in a Galleria mellonella model of infection. Using
both genetic and biochemical strategies, we determined the molecular
target of ticlopidine as the N-acetylglucosamine-1-phosphate
transferase encoded in gene tarO and provide evidence
that WTA biogenesis represents an Achilles heel supporting the cooperative
function of PBP2 and PBP4 in creating highly cross-linked muropeptides
in the peptidoglycan of S. aureus. This approach
represents a new paradigm to tackle MRSA infection.
The effect of various salts on the autolysis of cell wall of a ribitol teichoic acid-deficient mutant of Staphylococcus aureus H (strain 52A5 carrying tar-1) was compared with the parent strain. In the presence of high concentrations of certain salts such as 1.0 m NaCl, the mutant undergoes autolysis with the release of osmotically sensitive spheroplasts. The parent strain is not affected by these conditions. The stimulation of lysis is related to an activation of N-acylmuramyl-l-alanine amidase.
Wild-type Staphylococcus aureus rapidly expands on the surface of soft agar plates. The rates of expansion and the shapes of the resultant giant colonies were distinct for different strains of laboratory stocks and clinical isolates. The colony spreading abilities did not correlate with the biofilm-forming abilities in these strains. Insertional disruption of the dltABCD operon, which functions at the step of d-alanine addition to teichoic acids, and of the tagO gene, which is responsible for the synthesis of wall teichoic acids, decreased the colony spreading ability. The results indicate that wall teichoic acids and d-alanylation of teichoic acids are required for colony spreading.
Antisera, prepared against formalin-killed cells of Staphylococcus aureus, strain Copenhagen, agglutinated the cell walls of this strain. The agglutination was inhibited by the teichoic acid from the cell wall of this strain, by any degradation product of this teichoic acid which contained the α-acetylglucosaminyl-ribitol unit, by α-phenyl-acetylglucosaminide, and by N-acetylglucosamine, but not by a large number of other haptens related to the cell wall. In quantitative experiments, however, only 40 to 50 per cent of antibody adsorption to cell wall could be inhibited by teichoic acid or by N-acetylglucosamine. The α-acetylglucosaminyl-ribitol unit in the teichoic acid is, therefore, an important immunological determinant in the cell wall of this strain, although other immunological specificities may also exist. The cell walls were also agglutinated by heterologous antisera prepared against streptococcal Group A carbohydrate or against horse serum azophenyl-β-acetylglucosaminide. The heterologous agglutination, however, was specific for the β-acetylglucosaminyl-ribitol units in the teichoic acid.
Resistance has emerged to every family of clinically used antibiotics, and there is a pressing need to explore novel antibacterial targets. Wall teichoic acids (WTAs) are anionic polymers that coat the cell walls of many Gram-positive bacteria. Because WTAs play an essential role in Staphylococcus aureus colonization and infection, the enzymes involved in WTA biosynthesis are proposed to be targets for antibiotic development. To facilitate the discovery of WTA inhibitors, we have reconstituted the intracellular steps of S. aureus WTA biosynthesis. We show that two intracellular steps in the biosynthetic pathway are different from what was proposed. The work reported here lays the foundation for the discovery and characterization of inhibitors of wall teichoic acid biosynthetic enzymes to assess their potential for treating bacterial infections.