Building a Bridge to New Antibiotics Nicola L. Pohl* Department of Chemistry and the Plant Sciences Institute, 2756 Gilman Hall, Iowa State University, Ames, Iowa 50011
A B S T R A C T Gram-positive bacteria modify their peptidoglycan layers with teichoic acid polymers via a highly conserved disaccharide bridge. Inhibition of the biosynthesis of this bridge is a potential antibiotic strategy that can be explored now that purified versions of the enzymes TagA and TagB and their substrates are accessible for the first time.
*To whom correspondence should be addressed. E-mail:
[email protected].
Published online February 17, 2006 10.1021/cb0600003 CCC: $33.50 © 2006 by American Chemical Society
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ram-positive and Gram-negative bacteria have substantial differences in the structures surrounding the cells (Figure 1). However, both types of bacteria have a strong, protective peptidoglycan layer. Many powerful anti biotics inhibit the biosynthesis of this protective layer and thereby weaken or destroy the bacterial protective coat. For example, penicillin and vancomycin both inhibit the biosynthesis of the peptidoglycan outer layer of Gram-positive bacteria. Unfortunately, bacterial strains resistant to these antibiotics are becoming ever more common (1). Many of these Gram-positive bacteria modify their peptidoglycan layers with anionic wall teichoic acid (WTA) polymers (Figure 2), which are also crucial for survival of the organism. Ideally, compounds that specifically target the biosynthesis of these anionic polymers could be developed as new antibiotics. However, the biochemical functions of the enzymes involved in WTA biosynthesis have been difficult to verify because of a lack of availability of the complex substrates required. On page 25 of this issue (2), the first chemical and enzymatic syntheses of key substrates for the first two committed steps in WTA biosynthesis are reported along with characterization of the two enzymes, TagA and TagB, that carry out this chemistry. Access to these critical proteins and their substrates now finally opens the door to structural, enzymatic, and inhibitor studies to explore the exciting possibility of new antibiotics that inhibit a different stage of bacterial cell wall biosynthesis and thereby treat currently resistant strains.
Teichoic acid polymers are linked to bacterial peptidoglycans via a disaccharide bridge that is made of N-acetylmannos amine linked to N-acetylglucosamine (Figure 2) (3). Although the polymers themselves can be modified with alanine or glucose and be made from glycerol phosphate and ribitol phosphate monomers, the disaccharide bridge is highly conserved. This bridge is biosynthesized by the enzyme TagA from UDP-N-acetylmannos amine and undecaprenyl-diphospho‑Nacetylglucosamine building blocks. TagB then forms a dimer or trimer of glycerol phosphate ester bonds attached to the mannosamine core before the polymer chain is extended by Tag F using CDP‑glycerol made by TagD; the polymer can also then be modified by additional proteins that add glucose or alanine, for example (3). The final cell wall structures trade the lipid-linked disaccharide core for peptidoglycan (Figure 2). Originally, teichoic acid polymers were thought to be dispensable and therefore not a viable antibiotic target. However, recently these polymers have been shown to be crucial for the survival of Bacillus subtilis, a model Grampositive organism, even under phosphatelimiting conditions in which bacteria can switch to synthesizing phosphate-free anionic teichuronic acid polymers (4, 5). The biochemical functions of the enzymes involved in WTA biosynthesis have not been verified previously because of a lack of availability of the complex substrates. The 55 carbon long “carrier” lipid attached to many of the cell wall precursor substrates make substrate isolation and w w w. a c s c h e m i ca l biology.org
Figure 1. The cell walls of bacteria. Unlike those of Gram-negative bacteria, the peptidoglycan layers of Gram-positive bacteria are often modified by teichoic acids. Penicillin inhibits the biosynthesis of the peptidoglycan layer of many Gram-positive bacteria and some Gramnegative bacteria, but resistance to this and related antibiotics makes inhibition of alternate biosynthetic targets very attractive.
into membranes suggest a mechanistic relationship between TagF and TagB, which shares about 30% pairwise sequence identity with the C-terminus of TagF (9). The work reported herein (2) finally nails down the role of TagB as a sort of primase for teichoic acid synthesis. Anionic wall teichoic acid polymers attached to the peptidoglycans of Grampositive bacteria not only protect the organism but also confer antigen specificity much like the lipopolysaccharides (LPS) that coat the surface of Gram-negative bacteria. Interestingly, patients with deepseated Staphylococcus aureus infections all contained IgG antibodies to teichoic acid antigens whereas only 40% of those patients with superficial infections did; however, the level of IgG antibodies against peptidoglycan ranged from 60 to 72% in both patient groups (10). The anionic WTA coat of S. aureus helps host colonization and serves as a virulence factor by allowing binding of the bacteria to host endothelial cells (11–14). Access to defined WTA fragments, especially as more modification enzymes are added to the core teichoic acid biosynthesis enzymes, opens the possibility of understanding molecular level structure and function relationships required for host interactions with these essential Grampositive bacterial cell wall components.
kinetic studies of this system particularly ment is only starting to emerge. Structures challenging. To circumvent this difficulty, a of the cytidylyltransferase enzyme that C13H27 hydrocarbon chain analogue was activates glycerol for polymerization are made by chemical synthesis and shown to now available (6, 7), but the structures be a substrate for the first enzyme, TagA, of other proteins in the WTA biosynthetic in the biosynthetic pathway. TagA forms pathways remain to be solved. Recent a ß-1,4-N-acetylmannosamine anomeric progress on two fronts promises elucibond—one of the most problematic dation of the structures and catalytic glycosidic linkages to make by chemical mechanisms of two of the WTA proteins means alone. Both the first enzyme and that are essentially black boxes now. The the second enzyme in the pathway, TagB, polymerization protein TagF has been are able to accept a substrate containing produced in purified recombinant form the shorter lipid. These in vitro studies (8). Initial studies with TagF assayed by demonstrate that long lipid chains are not incorporation of radioactive CDP-glycerol necessary for the enzymatic reactions and, in addition, even link age a membrane interface is not bios y nthe sized by Ta gB crucial for the activity of TagA OH and TagB. These exciting discovO O OH pep tidoglyc an NHA c eries suddenly make structural P P O HO O O O O O O OH and molecular studies of -O -O HO O OH HO OH OH these crucial WTA biosynthesis P m AcH N O O O O O enzymes less daunting. -O O HO te ic hoic a cid link ag e AcHN Although the basics of polym e r biosy nthe s ize d A cHN n O WTA biosynthesis have been by TagA elucidated by genetics experiPe ptid e ments and crude studies using membrane preps (3), a Figure 2. Teichoic acid polymer linked to peptidoglycan. TagA and TagB catalyze the biosynthesis of the molecular picture of the process highly conserved bridge between peptidoglycan and teichoic acid polymers. One potential antibiotic necessary for drug developstrategy that can be explored is to inhibit the reactions catalyzed by TagA and TagB. www.acschemicalbiolog y.o rg
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