Wall Teichoic Acid Function, Biosynthesis, and Inhibition

Wall Teichoic Acid Function, Biosynthesis, and Inhibition

Jonathan G. Swoboda, Jennifer Campbell, Timothy C. Meredith, and Suzanne Walker Department of Microbiology and Molecular Genetics Harvard Medical School, 200 Longwood Avenue Armenise 633, Boston, MA 02115 (USA) Fax: (+ 1) 617-738-7664

Keywords antibiotics; biosynthesis; conditionally essential enzymes; Gram-positive bacteria; wall teichoic acid (WTA)

Introduction One of the major differences between Gram-negative and Gram-positive organisms is the presence or absence of an outer membrane (Figure 1). In Gram-negative organisms, the outer membrane protects the organism from the environment. It filters out toxic molecules and establishes a compartment, the periplasm, which retains extracytoplasmic enzymes required for cell-wall growth and degradation. It also serves as a scaffold to which proteins and polysaccharides that mediate interactions between the organism and its environment are anchored.[1] In addition, in ways that are not completely understood, the outer membrane functions along with a thin layer of peptidoglycan to help stabilize the inner membrane so that it can withstand the high osmotic pressures within the cell.[2]

Gram-positive organisms, in contrast, lack an outer membrane and a distinct periplasm (Figure 1). The peptidoglycan layers are consequently very thick compared to those in Gram-negative organisms.[4] These thick layers of peptidoglycan stabilize the cell membrane and also provide many sites to which other molecules can be attached. Gram-positive peptidoglycan is heavily modified with carbohydrate-based anionic polymers that play an important role in membrane integrity.[5] These anionic polymers appear to perform some of the same functions as the outer membrane: they influence membrane permeability, mediate extracellular interactions, provide additional stability to the plasma membrane, and, along with peptidoglycan, act as scaffolds for extracytoplasmic enzymes required for cell-wall growth and degradation.

A major class of these cell surface glycopolymers are the teichoic acids (TAs), which are phosphate-rich molecules found in a wide range of Gram-positive bacteria, pathogens and nonpathogens alike. There are two types of TAs: the lipo-TAs (LTAs), which are anchored to the plasma membrane and extend from the cell surface into the peptidoglycan layer;[6] and the wall TAs (WTAs), which are covalently attached to peptidoglycan and extend through and beyond the cell wall (Figure 1).[7] Together, LTAs and WTAs create what has been aptly described as a “continuum of negative charge” that extends from the bacterial cell surface beyond the outermost layers of peptidoglycan.[5] Neuhaus and Baddiley comprehensively reviewed both LTAs and WTAs in 2003.[5] Since then, however, new functions for WTAs in pathogenesis have been uncovered and it has been suggested that the biosynthetic enzymes that make these polymers are targets for novel antibacterial agents.[8,9] Indeed, the first WTA-

© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim suzanne_walker@hms.harvard.edu.

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active antibiotic has just been reported.[10] This review will focus primarily on recent developments in the study of WTAs in Bacillus subtilis and Staphylococcus aureus, and will include a discussion of strategies for the discovery of WTA inhibitors and prospects for these inhibitors as antibiotics.

Wall Teichoic Acid Structure WTAs are anionic glycopolymers that are covalently attached to peptidoglycan via a phosphodiester linkage to the C6 hydroxyl of the N-acetyl muramic acid sugars.[5] They can account for as much as 60 % of the total cell wall mass in Gram-positive organisms. The chemical structures of WTAs vary among organisms, as described in detail by Neuhaus and Baddiley,[5] but the most common structures are composed of a ManNAc(β1→4)GlcNAc disaccharide with one to three glycerol phosphates attached to the C4 hydroxyl of the ManNAc residue (the “linkage unit”) followed by a much longer chain of glycerol- or ribitol phosphate repeats (the “main chain”; Figure 2).[11–18] B. subtilis, the Gram-positive model organism, makes poly(glycerol phosphate) or poly(ribitol phosphate) WTAs depending on the strain, [19] while S. aureus strains primarily make poly(ribitol phosphate) WTAs.[20–23] The hydroxyls on the glycerol- or ribitol phosphate repeats are tailored with cationic D-alanine esters and monosaccharides, such as glucose or N-acetylglucosamine.[24,25] The presence of WTAs and the particular tailoring modifications that are found on them have profound effects on the physiology of Gram-positive organisms, and impact everything from cation homeostasis to antibiotic susceptibility to survival in a host.

Functions of Teichoic Acids in Bacterial Physiology The functions of TAs in bacterial physiology are incompletely understood, but evidence for their importance is overwhelming. B. subtilis and S. aureus mutants deficient in LTA biosynthesis can be obtained but only if grown under a narrow range of conditions; they are temperature sensitive and exhibit severe growth defects.[26,27] Mutants deficient in WTA biosynthesis are also compromised and manifest increased sensitivity to temperature and certain buffer components, including citrate; they also tend to aggregate in culture.[26–31] In addition, B. subtilis strains that do not express WTAs show profound morphological aberrations. Bacterial strains in which both LTA and WTA expression are prevented are not viable, an observation suggesting that these polymers have overlapping functions and can partially compensate for one another.[26,27] Indeed, this might be expected for some functions since both polymers contain phosphate-linked repeat units with similar tailoring modifications. One of the tailoring modifications, D-alanylation, is accomplished by the same machinery, so there is even some overlap in the biosynthetic pathways. This fact makes dissecting the functions of the individual anionic glycopolymers difficult, but is consistent with the idea that LTAs and WTAs are partially redundant. Some of the functions attributed to WTAs are described in the following paragraphs. LTAs are beyond the scope of this review, but will be mentioned in cases where it is relevant to the discussion of WTAs. Morath et al. and Rahman et al. have each written recent reviews on LTA structure and biosynthesis.[6,32]

Cation binding functions WTAs form a dense network of negative charges on Gram-positive cell surfaces. To alleviate the resulting electrostatic repulsive interactions between neighboring phosphates, TAs bind cationic groups, including mono- and divalent metal cations. Networks of WTA-coordinated cations affect the overall structure of the polymers, and this in turn influences the porosity and rigidity of the cell envelope. WTAs are proposed to be important for cation homeostasis in Gram-positive organisms,[33,34] and provide a reservoir of ions close to the cell surface that might be required for enzyme activity. In addition, the gradient of ions could in some way mitigate the osmotic pressure change between the inside and outside of the cell. The amount

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of bound cations can be modulated by D-alanylation, a tailoring modification that introduces positively charged amines.[35] WTAs that lack D-alanyl esters can bind up to 60 % more Mg2+ ions than analogous polymers that contain this modification.[36] The importance of cation binding is highlighted by the observation that B. subtilis strains up-regulate their production of TAs in the presence of low Mg2+ concentrations, and produce other negatively charged polymers (teichuronic acid) in the presence of limiting phosphate concentrations. [37] Recent structural studies have been focused on elucidating modes of cation binding by WTA polymer phosphate groups, and researchers have suggested that a clear understanding of the three-dimensional structure of WTAs and their bound cation groups might provide insights that facilitate the design of novel antimicrobials.[38]

Scaffolding roles In addition to providing binding sites for cations, WTAs serve as scaffolds or receptors for a wide range of other molecules. In S. aureus, for example, they function as receptors that are required for phage infection.[39] Depending on their tailoring modifications (see below) they might also promote adhesion by lytic enzymes produced by neutrophils.[40] They are additionally thought to serve as scaffolds for endogenously produced cell wall hydrolases (autolysins) involved in cell growth and division.[41] In general, the molecular interactions between WTAs and other biomolecules are not well understood but could provide crucial insights into cell envelope function.

Tailoring modification-dependent functions The main chain hydroxyl groups on both glycerol- and ribitol phosphate WTA polymers are subject to further derivatization by tailoring enzymes (Figure 2). There are two classes of tailoring enzymes: those that catalyze the addition of D-alanyl esters, and those that append glycosyl groups. The extent to which these modifications occur on the TA polymers is strain dependent and can also be affected by environmental conditions. Efforts have been made to understand the role(s) of these modifications in bacterial physiology, and some of these studies are highlighted below.

The D-alanylation tailoring modification has been more extensively investigated than glycosylation and is far better understood at this point. Perego et al. were the first to characterize the genetic pathway responsible for this modification (dlt operon) in B. subtilis.[42] Briefly, the biosynthetic pathway begins intracellularly with the activation of D-alanine to its corresponding aminoacyl adenylate by DltA. This molecule is then covalently attached, as a thioester, to a cofactor bound to the D-Ala carrier protein, DltC. Although the precise roles of DltB and DltD have not been confirmed, it is believed that they facilitate the transport of DltC through the membrane and the incorporation of D-Ala onto both LTAs and WTAs.[43] It has been found that D-alanylation is affected by several factors, including growth media, pH and temperature.[5] The attachment of D-alanyl esters to the hydroxyls on TAs alters the net charge of the polymer by adding positively charged amines. This modification reduces the electrostatic repulsion between neighboring TA chains and possibly facilitates stabilizing ion-pair formation between the cationic esters and the anionic phosphate groups.[38]

The D-alanine modification modulates interactions between the cell envelope and the environment and has been implicated in many of the known scaffolding/receptor functions of WTAs.[5,44] For example, it has been shown that the absence of D-alanyl esters on the TA polymers increases susceptibility to cationic antimicrobial peptides, possibly by increasing the negative charge density on the cell surface.[45,46] Removing the alanine residues also increases bacterial sensitivity to glycopeptide antibiotics and to the lytic activity of enzymes produced by neutrophils during host infection.[40,41] In contrast, the activity of autolytic enzymes is decreased, suggesting a role for TAs in scaffolding and/or activating bacterial

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enzymes involved in the processes of cell-wall synthesis and degradation.[41] Removal of D- alanyl esters from TAs has also been shown to attenuate the binding of S. aureus to artificial surfaces as well as host tissue. A recent study has illustrated the importance of the charge balance of WTAs in adhesion to artificial surfaces, such as glass and polystyrene.[44]

Since D-alanylation promotes better adhesion to host tissue and confers some resistance to lytic enzymes produced by the host, mutant strains lacking this modification have been studied in animal infection models. For example, in a mouse tissue cage infection model, bacterial strains lacking D-alanylation were more susceptible to Toll-like receptor 2-dependent host defenses; [46] in a septicemia model, such strains were attenuated in their ability to establish an infection, possibly because they were more readily killed by neutrophils.[40] Based on these and other studies, it was proposed that the D-alanine modification is a putative target for novel antimicrobials that function by attenuating virulence. In 2005, May et al. reported the synthesis and evaluation of a nonhydrolysable analogue of D-Ala aminoacyl adenylate as the first designed inhibitor of DltA, the enzyme that activates D-Ala. The compound enhanced the activity of vancomycin against B. subtilis.[43] This result is consistent with inhibition of DltA, and supports the idea that small molecules that interfere with D-alanylation might provide a novel strategy for antimicrobials.

Glycosylation is a ubiquitous tailoring modification of WTAs but its functions are not well understood. Glucose is commonly added to the WTA polymers in B. subtilis, whereas N-acetyl glucosamine (GlcNAc) is added in S. aureus (Figure 2).[5] Depending on the bacterial strain, the stereochemistry of the glycosidic linkage may be β-, α-, or a mixture of the two anomers. All sequenced B. subtilis and S. aureus strains contain one or more putative glycosyltransferase genes clustered with the WTA biosynthetic genes (Figure 3). For example, B. subtilis 168 contains a gene for a putative retaining glycosyltransferase that might add a-Glu to the glycerol phosphate polymers. S. aureus strains contain two genes encoding putative inverting glycosyltransferases that might transfer β-GlcNAc to the poly(ribitol phosphate) polymers. Although some S. aureus strains have been shown to contain α-glycosidically linked WTAs, there are no genes yet identified for any glycosyltransferases that can carry out this tailoring modification. Furthermore, no studies have confirmed the enzymatic functions of any of the putative WTA glycosyltransferases or have explored the effects of preventing WTA glycosylation on bacterial cell growth, division, intercellular interactions, or pathogenesis. In fact, as far as we know there is only one piece of data pertaining to the functions of WTA glycosyltransferases in the literature: a transposon mutant in a putative glycosyltransferase in the S. aureus strain Newman showed attenuated virulence in a nematode killing assay, suggesting that glycosylation might play a role in pathogenesis in S. aureus.[47] If glycoslyation proves important for bacterial pathogenesis, the glycosyltransferase tailoring enzymes, like the enzymes involved in D-alanylation (see above) would be possible targets for antimicrobials.

Roles in cell elongation and division Recent studies have implicated LTAs and WTAs in cell growth, division, and morphogenesis. In the rod-shaped organism B. subtilis, TAs have been shown to play distinct roles in bacterial morphogenesis. Preventing WTA expression results in the production of round, severely defective progeny, while preventing LTA biosynthesis causes major defects in septum formation and cell separation.[27,49] It is known that there are separate multiprotein complexes involved in septation and elongation in B. subtilis, and Errington and co-workers have suggested (based on localization studies using fluorescently tagged enzymes) that the WTA biosynthetic enzymes associate with the machinery involved in elongation, while the LTA enzymes might associate with machinery involved in septation and cell division.[27,50] It was suggested that the spatial distribution of these two anionic glycopolymers determines their

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