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Toxins are key virulence determinants of pathogens and can impair the function of host immune cells including platelets. Insights into pathogen toxin interference with platelets will be pivotal to improve treatment of patients with bacterial bloodstream infections. In this study, we deciphered the effects of Staphylococcus aureus toxins α-hemolysin, LukAB, LukDE and LukSF on human platelets and compared the effects with the pore forming toxin pneumolysin of Streptococcus pneumoniae. In contrast to pneumolysin, α-hemolysin initially activates platelets as indicated by CD62P and αIIbβ3 integrin expression, but the resulting pores also induce alterations in the phenotype of platelets and induce apoptosis of platelets. The presence of small amounts of α-hemolysin (0.2 µg/mL) in whole blood abrogates thrombus formation indicating that in systemic infections with S. aureus the stability of formed thrombi is impaired. This might be of high clinical relevance for S. aureus induced endocarditis of the aortic valves. Stabilizing the thrombi by inhibiting α-hemolysin induced impairment of platelets likely reduces the risk for septic (micro-)embolization. However, in contrast to pneumolysin, α-hemolysin induced platelets damage could not be neutralized by intravenous immune globulins. In contrast to α-hemolysin, S. aureus bi-component pore forming leukocidins LukAB, LukED and LukSF do not bind to platelets and had no significant effect on platelet activation and viability.Main point 1: α-hemolysin forms pores in platelets, which first activate but then result in apoptosis and impairs thrombus formation and stabilityMain point 2: Polyvalent immunoglobulins do not neutralize the mode of action of the toxin

Streptococcus pneumoniaeinfections are the leading cause of child mortality globally. Current vaccines fail to induce a protective immune response towards a conserved part of the pathogen, resulting in new serotypes causing disease. Therefore, new vaccine strategies are urgently needed. Described is a two-pronged approach combiningS.pneumoniaeproteins, pneumolysin and PspA, with a precisely defined synthetic oligosaccharide, whereby the carrier protein acts as a serotype-independent antigen to provide additional protection. Proof of concept in mice and swine models revealed that the conjugates inhibit colonization of the nasopharynx, decrease the bacterial load and reduce disease severity in the bacteria challenged model. Immunization of piglets provided the first evidence for the immunogenicity and protective potential of synthetic glycoconjugate vaccine in a large animal model. A combination of synthetic oligosaccharides with proteins from the target pathogen opens the path to create broadly cross-protective (“universal“) pneumococcal vaccines.

19 pags., 7 figs., Nucleotides are important for RNA and DNA synthesis and, despite a de novo synthesis by bacteria, uptake systems are crucial. Streptococcus pneumoniae, a facultative human pathogen, produces a surface-exposed nucleoside-binding protein, PnrA, as part of an ABC transporter system. Here we demonstrate the binding affinity of PnrA to nucleosides adenosine, guanosine, cytidine, thymidine and uridine by microscale thermophoresis and indicate the consumption of adenosine and guanosine by H NMR spectroscopy. In a series of five crystal structures we revealed the PnrA structure and provide insights into how PnrA can bind purine and pyrimidine ribonucleosides but with preference for purine ribonucleosides. Crystal structures of PnrA:nucleoside complexes unveil a clear pattern of interactions in which both the N- and C- domains of PnrA contribute. The ribose moiety is strongly recognized through a conserved network of H-bond interactions, while plasticity in loop 27–36 is essential to bind purine- or pyrimidine-based nucleosides. Further, we deciphered the role of PnrA in pneumococcal fitness in infection experiments. Phagocytosis experiments did not show a clear difference in phagocytosis between PnrA-deficient and wild-type pneumococci. In the acute pneumonia infection model the deficiency of PnrA attenuated moderately virulence of the mutant, which is indicated by a delay in the development of severe lung infections. Importantly, we confirmed the loss of fitness in co-infections, where the wild-type out-competed the pnrA-mutant. In conclusion, we present the PnrA structure in complex with individual nucleosides and show that the consumption of adenosine and guanosine under infection conditions is required for virulence., This work was supported by grants from the Deutsche Forschungsgemeinschaft DFG GRK1870 (to SH and ML), the Bundesministerium fu ̈rBildung und Forschung (BMBF) - Zwanzig20 -InfectControl 2020 - project VacoME - FKZ03ZZ0816A to SH, the Federal Excellence Initiative of Mecklenburg Western Pomerania and European Social Fund (ESF) Grant KoInfekt(ESF_14-BM-A55-0001_16) to SH and byfinancial support from the Spanish Ministry of Science, Innovation and Universities BFU2017-90030-P to JAH. We thankfully acknowledge thetechnical support provided by staff of thebeamline XALOC at the ALBA synchrotron facility(Barcelona, Spain) and Teresia Hallstro ̈m (NanoTemper, Munich, Germany).

mBio 11(5), e01612-20 (1-19) (2020). doi:10.1128/mBio.01612-20, Although it is normally an innocuous part of the human skin microbiota, Staphylococcus epidermidis has emerged as a major nosocomial pathogen, and implanted foreign materials are an essential risk factor for the development of an infection. The extraordinary efficiency of S. epidermidis to colonize artificial surfaces is particularly related to the ability to form biofilms. Biofilm formation itself critically depends on stable pathogen binding to extracellular host matrix components, e.g. fibronectin (Fn), covering inserted devices in vast amounts. Extracellular matrix binding protein (Embp) and its subdomains referred to as the F-repeat and the FG-repeat are critical for adherence of S. epidermidis to surface-immobilized Fn. Embp-Fn interactions preferentially occur with surface-bound, but not folded, globular Fn via binding to the F3 domain. High-resolution structure analysis of F- and FG-repeats revealed that both repeats are composed of two tightly connected triple α-helix bundles, exhibiting an elongated but rather rigid structural organization in solution. Both F- and FG-repeat possess Fn-binding capacity via interactions with type III subdomain FN12, involving residues within the C and F β-sheet. FN12 essentially supports stability of the globular Fn state, and thus these findings reasonably explain why Embp-mediated interaction of S. epidermidis necessitates Fn surface immobilization. Thus, Embp employs an uncharacterized bacterial Fn-binding mechanism to promote staphylococcal adherence., Published by American Society for Microbiology127930, Washington, DC

A successful colonization of different compartments of the human host requires multifactorial contacts between bacterial surface proteins and host factors. Extracellular matrix proteins and matricellular proteins such as thrombospondin-1 play a pivotal role as adhesive substrates to ensure a strong interaction with pathobionts like the Gram-positive Streptococcus pneumoniae and Staphylococcus aureus. The human glycoprotein thrombospondin-1 is a component of the extracellular matrix and is highly abundant in the bloodstream during bacteremia. Human platelets secrete thrombospondin-1, which is then acquired by invading pathogens to facilitate colonization and immune evasion. Gram-positive bacteria express a broad spectrum of surface-exposed proteins, some of which also recognize thrombospondin-1. This review highlights the importance of thrombospondin-1 as an adhesion substrate to facilitate colonization, and we summarize the variety of thrombospondin-1-binding proteins of S. pneumoniae and S. aureus.

Secreted antimicrobial peptides (AMPs) are an important part of the human innate immune system and prevent local and systemic infections by inhibiting bacterial growth in a concentration-dependent manner. In the respiratory tract, the cationic peptide LL-37 is one of the most abundant AMPs and capable of building pore complexes in usually negatively charged bacterial membranes, leading to the destruction of bacteria. However, the adaptation mechanisms of several pathogens to LL-37 are already described and are known to weaken the antimicrobial effect of the AMP, for instance, by repulsion, export or degradation of the peptide. This study examines proteome-wide changes in Streptococcus pneumoniae D39, the leading cause of bacterial pneumonia, in response to physiological concentrations of LL-37 by high-resolution mass spectrometry. Our data indicate that pneumococci may use some of the known adaptation mechanisms to reduce the effect of LL-37 on their physiology, too. Additionally, several proteins seem to be involved in resistance to AMPs which have not been related to this process before, such as the teichoic acid flippase TacF (SPD_1128). Understanding colonization- and infection-relevant adaptations of the pneumococcus to AMPs, especially LL-37, could finally uncover new drug targets to weaken the burden of this widespread pathogen.

Essentials Human platelets specifically interact with IgG opsonized bacteria through FcγRIIA. Platelet factor 4 (PF4) binds to polyanions (P) and undergoes a conformational change. Anti-PF4/P IgG opsonizes PF4-coated Gram-positive and Gram-negative bacteria. Platelets specifically kill E.coli opsonized with PF4 and human anti-PF4/P IgG. SUMMARY Background Activated platelets release the chemokine platelet factor 4 (PF4) stored in their granules. PF4 binds to polyanions (P) on bacteria, undergoes a conformational change and exposes neoepitopes. These neoepitopes induce production of anti-PF4/P antibodies. As PF4 binds to a variety of bacteria, anti-PF4/P IgG can bind and opsonize several bacterial species. Objective Here we investigated whether platelets are able to kill bacteria directly after recognizing anti-PF4/P IgG opsonized bacteria in the presence of PF4 via their FcγRIIA. Methods Using platelet-bacteria suspension co-culture experiments and micropatterns with immobilized viable bacteria, in combination with pharmacological inhibitors and human anti- PF4/P IgG we analyzed the role of platelet-mediated killing of bacteria. Results In the presence of PF4, human anti-PF4/P IgG and platelets, E. coli killing (> 50%) with colony forming units (CFU mL-1 ) 0.71 × 104 ± 0.19 was observed compared with controls incubated only with anti-PF4/P IgG (CFU mL-1 3.4 × 104 ± 0.38). Blocking of platelet FcγRIIA using mAb IV.3 (CFU mL-1 2.5 × 104 ± 0.45), or integrin αIIbβ3 (CFU mL-1 2.26 × 104 ± 0.31), or disruption of cytoskeletal functions (CFU mL-1 2.7 × 104 ± 0.4) markedly reduced E. coli killing by this mechanism. Our observation of E. coli killing by platelets on micropatterned arrays is compatible with the model that platelets kill bacteria by covering them, actively concentrating them into the area under their granulomere and then releasing antimicrobial substances of platelet α-granules site directed towards bacteria. Conclusion These findings collectively indicate that by bridging of innate and adaptive immune mechanisms, platelets and anti-PF4/polyanion antibodies cooperate in an antibacterial host response.

Summary Colonization of mucosal respiratory surfaces is a prerequisite for the human pathobiont Streptococcus pneumoniae (the pneumococcus) to cause severe invasive infections. The arsenal of pneumococcal adhesins interacts with a multitude of extracellular matrix proteins. A paradigm for pneumococci is their interaction with the adhesive glycoprotein fibronectin, which facilitates bacterial adherence to host cells. Here, we deciphered the molecular interaction between fibronectin and pneumococcal fibronectin-binding proteins (FnBPs) PavA and PavB, respectively. We show in adherence and binding studies that the pneumococcal interaction with fibronectin is a non-human specific trait. PavA and PavB target at least 13 out of 15 type III fibronectin domains as demonstrated in ligand overlay assays, surface plasmon resonance studies and SPOT peptide arrays. Strikingly, both pneumococcal FnBPs recognize similar peptides in targeted type III repeats. Structural comparisons revealed that the targeted type III repeat epitopes cluster on the inner strands of both β-sheets forming the fibronectin domains. Importantly, synthetic peptides of FnIII1, FnIII5 or FnIII15 bind directly to FnBPs PavA and PavB, respectively. In conclusion, our study suggests a common pattern of molecular interactions between pneumococcal FnBPs and fibronectin. The specific epitopes recognized in this study can potentially be tested as antimicrobial targets in further scientific endeavours. This article is protected by copyright. All rights reserved.

Background Heparin induced thrombocytopenia (HIT) is likely a misdirected bacterial host defense mechanism. Platelet factor 4 (PF4) binds to polyanions on bacterial surfaces exposing neo‐epitopes to which HIT antibodies bind. Platelets are activated by the resulting immune complexes via FcγRIIA, release bactericidal substances, and kill Gram‐negative Escherichia coli. Objectives To assess the role of PF4, anti‐PF4/H antibodies and FcγRIIa in killing of Gram‐positive bacteria by platelets. Methods Binding of PF4 to protein‐A deficient Staphylococcus aureus (SA113Δspa) and non‐encapsulated Streptococcus pneumoniae (D39Δcps) and its conformational change were assessed by flow cytometry using monoclonal (KKO,5B9) and patient derived anti‐PF4/H antibodies. Killing of bacteria was quantified by counting colony forming units (cfu) after incubation with platelets or platelet releasate. Using flow cytometry, platelet activation (CD62P‐expression, PAC‐1 binding) and phosphatidylserine (PS)‐exposure were analyzed. Results Monoclonal and patient‐derived anti‐PF4/H antibodies bound in the presence of PF4 to both S. aureus and S. pneumoniae (1.6‐fold increased fluorescence signal for human anti‐PF4/H antibodies to 24.0‐fold increase for KKO). Staphylococcus aureus (5.5 × 104cfu/mL) was efficiently killed by platelets (2.7 × 104cfu/mL) or their releasate (2.9 × 104cfu/mL). Killing was not further enhanced by PF4 or anti‐PF4/H antibodies. Blocking FcγRIIa had no impact on killing of S. aureus by platelets. In contrast, S. pneumoniae was not killed by platelets or releasate. Instead, after incubation with pneumococci platelets were unresponsive to TRAP‐6 stimulation and exposed high levels of PS. Conclusions Anti‐PF4/H antibodies seem to have only a minor role for direct killing of Gram‐positive bacteria by platelets. Staphylococcus aureus is killed by platelets or platelet releasate. In contrast, S. pneumoniae affects platelet viability.

The bacterial lung pathogen Streptococcus pneumoniae has a unique nutritional requirement for exogenous choline and attaches phosphorylcholine (P-Cho) residues to the GalpNAc moieties of its teichoic acids (TAs) in its cell wall. Two phosphorylcholine transferases, LicD1 and LicD2, mediate the attachment of P-Cho to the O-6 positions of the two GalpNAc residues present in each repeating unit of pneumococcal TAs (pnTAs), of which only LicD1 has been determined to be essential. At the molecular level, the specificity of the P-Cho attachment to pnTAs by LicD1 and LicD2 remains still elusive. Here, using detailed structural analyses of pnTAs from a LicD2-deficient strain, we confirmed the specificity in the attachment of P-Cho residues to pnTA. LicD1 solely transfers P-Cho to α-d-GalpNAc moieties, whereas LicD2 attaches P-Cho to β-d-GalpNAc. Further, we investigated the role of the pneumococcal phosphorylcholine esterase (Pce) in the modification of the P-Cho substitution pattern of pnTAs. To clarify the specificity of Pce-mediated P-Cho hydrolysis, we evaluated different concentrations and pH conditions for the treatment of pneumococcal lipoteichoic acid with purified Pce. We show that Pce can hydrolyze both P-Cho residues of the terminal repeat of the pnTA chain and almost all P-Cho residues bound to β-d-GalpNAc in vitro. However, hydrolysis in vivo was restricted to the terminal repeat. In summary, our findings indicate that LicD1 and LicD2 specifically transfer P-Cho to α-d-GalpNAc and β-d-GalpNAc moieties, respectively, and that Pce removes distinct P-Cho substituents from pnTAs.

Staphylococcus aureus can cause bloodstream infections associated with infective endocarditis (IE) and disseminated intravascular coagulopathy (DIC). Both complications involve platelets. In view of an increasing number of antibiotic-resistant strains, new approaches to control systemic S. aureus infection are gaining importance. Using a repertoire of 52 recombinant S. aureus proteins in flow cytometry-based platelet activation and aggregation assays, we identified, in addition to the extracellular adherence protein Eap, three secreted staphylococcal proteins as novel platelet activating proteins. Eap and the chemotaxis inhibitory protein of S. aureus (CHIPS), the formyl peptide receptor-like 1 inhibitory protein (FLIPr) and the major autolysin Atl induced P-selectin expression in washed platelets and platelet-rich plasma. Similarly, AtlA, CHIPS and Eap induced platelet aggregation in whole blood. Fluorescence microscopy illustrated that P-selectin expression is associated with calcium mobilization and re-organization of the platelet actin cytoskeleton. Characterization of the functionally active domains of the major autolysin AtlA and Eap indicates that the amidase domain of Atl and the tandem repeats 3 and 4 of Eap are crucial for platelet activation. These results provide new insights in S. aureus protein interactions with platelets and identify secreted proteins as potential treatment targets in case of antibiotic-resistant S. aureus infection.

Teichoic acid (TA), a crucial cell wall constituent of the pathobiont Streptococcus pneumoniae, is bound to peptidoglycan (wall teichoic acid, WTA) or to membrane glycolipids (lipoteichoic acid, LTA). Both TA polymers share a common precursor synthesis pathway, but differ in the final transfer of the TA chain to either peptidoglycan or a glycolipid. Here, we show that LTA exhibits a different linkage conformation compared to WTA, and identify TacL (previously known as RafX) as a putative lipoteichoic acid ligase required for LTA assembly. Pneumococcal mutants deficient in TacL lack LTA and show attenuated virulence in mouse models of acute pneumonia and systemic infections, although they grow normally in culture. Hence, LTA is important for S. pneumoniae to establish systemic infections, and TacL represents a potential target for antimicrobial drug development., Teichoic acid is bound to peptidoglycan (wall teichoic acid, WTA) or to membrane glycolipids (lipoteichoic acid, LTA) in most Gram-positive bacteria. Here, the authors identify a putative ligase required for the assembly of LTA, but not WTA, and important for Streptococcus pneumoniae virulence in mouse models.

Dermcidin (DCD) is an antimicrobial peptide which is constitutively expressed in eccrine sweat glands. By postsecretory proteolytic processing in sweat, the DCD protein gives rise to anionic and cationic DCD peptides with a broad spectrum of antimicrobial activity. Many antimicrobial peptides induce membrane permeabilization as part of their killing mechanism, which is accompanied by a loss of the bacterial membrane potential. In this study we show that there is a time-dependent bactericidal activity of anionic and cationic DCD-derived peptides which is followed by bacterial membrane depolarization. However, DCD-derived peptides do not induce pore formation in the membranes of gram-negative and gram-positive bacteria. This is in contrast to the mode of action of the cathelicidin LL-37. Interestingly, LL-37 as well as DCD-derived peptides inhibit bacterial macromolecular synthesis, especially RNA and protein synthesis, without binding to microbial DNA or RNA. Binding studies with components of the cell envelope of gram-positive and gram-negative bacteria and with model membranes indicated that DCD-derived peptides bind to the bacterial envelope but show only a weak binding to lipopolysaccharide (LPS) from gram-negative bacteria or to peptidoglycan, lipoteichoic acid, and wall teichoic acid, isolated from Staphylococcus aureus . In contrast, LL-37 binds strongly in a dose-dependent fashion to these components. Altogether, these data indicate that the mode of action of DCD-derived peptides is different from that of the cathelicidin LL-37 and that components of the bacterial cell envelope play a role in the antimicrobial activity of DCD.

Most of the severe bacterial infections originate from the endogenous microflora of human body surfaces. However, the molecular basis of colonization, e.g. of the human nose by Staphylococcus aureus, has remained incompletely understood. Several surface-exposed proteins and wall teichoic acid (WTA) polymers have previously been implicated in S. aureus attachment to nasal epithelial cells. Here we dissect the role of these molecules in colonization using S. aureus sortase A (srtA) and tagO mutants deficient in surface protein and WTA display, respectively. Although the two mutants were similarly affected in attachment to nasal cells they were abrogated in binding to different types of epithelial ligands. Surface protein sorting, but not WTA, were required for keratin- or fibronectin-mediated interactions while only WTA-mediated binding to nasal cells was effectively inhibited by polyinosinic acid, indicating a possible role of scavenger receptor-like molecules in WTA-dependent epithelial interactions. Both mutants exhibited profound colonization defects in a cotton rat nasal colonization model, albeit at different stages of colonization (>90% reduced bacterial counts at 24 h or several days after inoculation with the tagO or srtA mutant, respectively). These data indicate that S. aureus nasal colonization is a multifactorial process with various ligands affecting initial colonization and prolonged persistence in different ways. Our studies should be useful in the development of new preventive and therapeutic strategies.

Many Gram-positive bacteria produce lipoteichoic acid (LTA) polymers whose physiological roles have remained a matter of debate because of the lack of LTA-deficient mutants. The ypfP gene responsible for biosynthesis of a glycolipid found in LTA was deleted in Staphylococcus aureus SA113, causing 87% reduction of the LTA content. Mass spectrometry and nuclear magnetic resonance spectroscopy revealed that the mutant LTA contained a diacylglycerol anchor instead of the glycolipid, whereas the remaining part was similar to the wild-type polymer except that it was shorter. The LTA mutant strain revealed no major changes in patterns of cell wall proteins or autolytic enzymes compared with the parental strain indicating that LTA may be less important in S. aureus protein attachment than previously thought. However, the autolytic activity of the mutant was strongly reduced demonstrating a role of LTA in controlling autolysin activity. Moreover, the hydrophobicity of the LTA mutant was altered and its ability to form biofilms on plastic was completely abrogated indicating a profound impact of LTA on physicochemical properties of bacterial surfaces. We propose to consider LTA and its biosynthetic enzymes as targets for new antibiofilm strategies.

Streptococcus pneumoniae is a Gram-positive human pathogen with a complex lipoteichoic acid (pnLTA) structure. Because the current structural model for pnLTA shows substantial inconsistencies, we reinvestigated purified and, more importantly, O-deacylated pnLTA, which is most suitable for NMR spectroscopy and electrospray ionization-MS spectrometry. We analyzed pnLTA of nonencapsulated pneumococcal strains D39Δcps and TIGR4Δcps, respectively. The data obtained allowed us to (re)define (i) the position and linkage of the repeating unit, (ii) the putative α-GalpNAc substitution at the ribitiol 5-phosphate (Rib-ol-5-P), and (iii) the length of (i.e. the number of repeating units in) the pnLTA chain. We here also describe for the first time that the terminal sugar residues in the pnLTA (Forssman disaccharide; α-D-GalpNAc-(1→3)-β-D-GalpNAc-(1→)), responsible for the cross-reactivity with anti-Forssman antigen antibodies, can be heterogeneous with respect to its degree of phosphorylcholine substitution in both O-6-positions. To assess the proinflammatory potency of pnLTA, we generated a (lipopeptide-free) Δlgt mutant of strain D39Δcps, isolated its pnLTA, and showed that it is capable of inducing IL-6 release in human mononuclear cells, independent of TLR2 activation. This finding was quite in contrast to LTA of the Staphylococcus aureus SA113Δlgt mutant, which did not activate human mononuclear cells in our experiments. Remarkably, this is also contrary to various other reports showing a proinflammatory potency of S. aureus LTA. Taken together, our study refines the structure of pnLTA and indicates that pneumococcal and S. aureus LTAs differ not only in their structure but also in their bioactivity.

Staphylococcal cell separation depends largely on the bifunctional autolysin Atl that is processed to amidase-R(1,2) and R(3)-glucosaminidase. These murein hydrolases are targeted via repeat domains (R) to the septal region of the cell surface, thereby allowing localized peptidoglycan hydrolysis and separation of the dividing cells. Here we show that targeting of the amidase repeats is based on an exclusion strategy mediated by wall teichoic acid (WTA). In Staphylococcus aureus wild-type, externally applied repeats (R(1,2)) or endogenously expressed amidase were localized exclusively at the cross-wall region, while in Delta tagO mutant that lacks WTA binding was evenly distributed on the cell surface, which explains the increased fragility and autolysis susceptibility of the mutant. WTA prevented binding of Atl to the old cell wall but not to the cross-wall region suggesting a lower WTA content. In binding studies with ConcanavalinA-fluorescein (ConA-FITC) conjugate that binds preferentially to teichoic acids, ConA-FITC was bound throughout the cell surface with the exception of the cross wall. ConA binding suggest that either content or polymerization of WTA gradually increases with distance from the cross-wall. By preventing binding of Atl, WTA directs Atl to the cross-wall to perform the last step of cell division, namely separation of the daughter cells.

The incorporation of characteristic carbohydrate-based polymers into the cell wall is a general trait of most Gram-positive bacteria. These cell wall glycopolymers (CWGs) are attached to the membrane or the peptidoglycan and enclose, for example, the teichoic acids, which belong to the most prevalent types of CWG. The structure, function, and biosynthesis of CWGs are only superficially understood. CWG composition and structure is highly variable and often strain- and species-specific. Recent studies have yielded a more precise picture of the biosynthetic pathway for the wall teichoic acid (WTA) polymers, which are covalently anchored in the cell wall, and the lipoteichoic acid (LTA) polymers, which are attached to the cell membrane of Bacillus subtilis and Staphylococcus aureus. Some Gram-positive bacteria have other CWGs, aside from WTA and LTA, such as polyanionic teichuronic acids or uncharged lipoglycans. CWGs are important in bacterial physiology and various potential functions such as the control of autolytic enzymes, regulation of divalent cations, attachment of surface proteins, or protection against antibacterial molecules have been described. For those bacteria that colonize or infect the host organism, certain CWGs have been implicated in the adherence to host cells and the activation of the immune responses, for example through Toll-like receptors (TLRs). Additionally, CWGs are important targets for vaccines, antimicrobials and diagnostics. Thus CWGs represent an important field for scientific research.

Most Gram-positive bacterial cell walls are composed of two types of structural components, the peptidoglycan (PG) with highly conserved composition and additional cell wall glycopolymers (CWGs), which vary extensively between species and even between individual strains. This chapter describes the structures, biosynthesis, and functions of different CWGs, including teichoic acids (TAs), lipoteichoic acid (LTA), and others CWGs. \{CWG\} polymers are composed of repeating units containing one or more sugar building blocks plus nonsugar residues such as phosphate, alanine, succinate, pyruvate, choline, or mycolic acids to name a few. Those \{CWGs\} containing phosphate in the polymer backbone are traditionally referred to as TAs. Many Gram-positive bacteria produce \{CWGs\} without phosphate groups in repeating units. Such polymers are either uncharged, e.g., in many actinobacteria and certain bacilli, or are anionic because of the presence of uronic acid, pyruvyl, or succinyl groups. The \{CWGs\} play important roles in bacterial physiology and various potential functions, such as control of autolytic enzymes, regulation of divalent cations, attachment of surface proteins, or protection against antibacterial molecules, have been described. In those bacteria that colonize or infect animal hosts, \{CWGs\} have been implicated in adherence to host cells and activation of immune responses, e.g., via the toll-like receptors. In addition, \{CWGs\} are very important targets for antimicrobials, vaccines, and diagnostics.

Staphylococci and most other Gram-positive bacteria incorporate complex teichoic acid (TA) polymers into their cell envelopes. Several crucial roles in Staphylococcus aureus fitness and cell wall maintenance have been assigned to these polymers, which are either covalently linked to peptidoglycan (wall teichoic acid, WTA) or to the cytoplasmic membrane (lipoteichoic acid, LTA). However, the exact TA structures, functions, and biosynthetic pathways are only superficially understood. Recently, most of the enzymes mediating TA biosynthesis have been identified and mutants lacking or with defined changes in WTA or LTA have become available. Their characterization has revealed crucial roles of TAs in protection against harmful molecules and environmental stresses; in control of enzymes directing cell division or morphogenesis and of cation homeostasis; and in interaction with host or bacteriophage receptors and biomaterials. Accordingly, several in vivo studies have demonstrated the importance of WTA and LTA in S. aureus colonization, infection, and immune evasion. TAs and enzymes required for TA biosynthesis represent attractive candidates for novel vaccines and antibiotics and are targeted by recently developed antibacterial therapeutics.

Skin-colonizing gram-positive bacteria produce wall teichoic acids (WTAs) or related glycopolymers for unclear reasons. Using a WTA-deficient Staphylococcus aureus mutant, we demonstrated that WTA confers resistance to antimicrobial fatty acids from human sebaceous glands by preventing fatty acid binding. Thus, WTA is probably important for bacterial skin colonization.