Jonathan G. Swoboda

A regenerative approach to the treatment of multiple sclerosis

Progressive phases of multiple sclerosis are associated with inhibited differentiation of the progenitor cell population that generates the mature oligodendrocytes required for remyelination and disease remission. To identify selective inducers of oligodendrocyte differentiation, we performed an image-based screen for myelin basic protein (MBP) expression using primary rat optic-nerve-derived progenitor cells. Here we show that among the most effective compounds identifed was benztropine, which significantly decreases clinical severity in the experimental autoimmune encephalomyelitis (EAE) model of relapsing-remitting multiple sclerosis when administered alone or in combination with approved immunosuppressive treatments for multiple sclerosis. Evidence from a cuprizone-induced model of demyelination, in vitro and in vivo T-cell assays and EAE adoptive transfer experiments indicated that the observed efficacy of this drug results directly from an enhancement of remyelination rather than immune suppression. Pharmacological studies indicate that benztropine functions by a mechanism that involves direct antagonism of M1 and/or M3 muscarinic receptors. These studies should facilitate the development of effective new therapies for the treatment of multiple sclerosis that complement established immunosuppressive approaches.

Wall Teichoic Acid Function, Biosynthesis, and Inhibition

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] Figure 1 Simplified depiction of Gram-positive and Gram-negative bacterial cell envelopes. Gram-negative organisms have a distinct periplasm; Gram-positive organisms do not, but recent studies have suggested that they have a periplasmic-like compartment between ... 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-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.

Discovery of a Small Molecule that Blocks Wall Teichoic Acid Biosynthesis in Staphylococcus aureus

Both Gram-positive and Gram-negative bacteria contain bactoprenol-dependent biosynthetic pathways expressing non-essential cell surface polysaccharides that function as virulence factors. Although these polymers are not required for bacterial viability in vitro, genes in many of the biosynthetic pathways are conditionally essential: they cannot be deleted except in strains incapable of initiating polymer synthesis. We report a cell-based, pathway-specific strategy to screen for small molecule inhibitors of conditionally essential enzymes. The screen identifies molecules that prevent the growth of a wildtype bacterial strain but do not affect the growth of a mutant strain incapable of initiating polymer synthesis. We have applied this approach to discover inhibitors of wall teichoic acid (WTA) biosynthesis in Staphylococcus aureus. WTAs are anionic cell surface polysaccharides required for host colonization that have been suggested as targets for new antimicrobials. We have identified a small molecule, 7-chloro-N,N-diethyl-3-(phenylsulfonyl)-[1,2,3]triazolo[1,5-a]quinolin-5-amine (1835F03), that inhibits the growth of a panel of S. aureus strains (MIC = 1-3 microg mL(-1)), including clinical methicillin-resistant S. aureus (MRSA) isolates. Using a combination of biochemistry and genetics, we have identified the molecular target as TarG, the transmembrane component of the ABC transporter that exports WTAs to the cell surface. We also show that preventing the completion of WTA biosynthesis once it has been initiated triggers growth arrest. The discovery of 1835F03 validates our chemical genetics strategy for identifying inhibitors of conditionally essential enzymes, and the strategy should be applicable to many other bactoprenol-dependent biosynthetic pathways in the pursuit of novel antibacterials and probes of bacterial stress responses.

Late-Stage Polyribitol Phosphate Wall Teichoic Acid Biosynthesis in Staphylococcus aureus

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.

Small-Molecule Inducer of β Cell Proliferation Identified by High-Throughput Screening

The identification of factors that promote β cell proliferation could ultimately move type 1 diabetes treatment away from insulin injection therapy and toward a cure. We have performed high-throughput, cell-based screens using rodent β cell lines to identify molecules that induce proliferation of β cells. Herein we report the discovery and characterization of WS6, a novel small molecule that promotes β cell proliferation in rodent and human primary islets. In the RIP-DTA mouse model of β cell ablation, WS6 normalized blood glucose and induced concomitant increases in β cell proliferation and β cell number. Affinity pulldown and kinase profiling studies implicate Erb3 binding protein-1 and the IκB kinase pathway in the mechanism of action of WS6.

Development of Improved Inhibitors of Wall Teichoic Acid Biosynthesis with Potent Activity Against Staphylococcus aureus

A small molecule (1835F03) that inhibits Staphylococcus aureus wall teichoic acid biosynthesis, a proposed antibiotic target, has been discovered. Rapid, parallel, solution-phase synthesis was employed to generate a focused library of analogs, providing detailed information about structure-activity relationships and leading to the identification of targocil, a potent antibiotic.

An antibiotic that inhibits a late step in wall teichoic acid biosynthesis induces the cell wall stress stimulon in Staphylococcus aureus

ABSTRACT 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.

In Vitro Antimicrobial Activity of Wall Teichoic Acid Biosynthesis Inhibitors against Staphylococcus aureus Isolates

ABSTRACT Staphylococcus aureus is the leading cause of invasive and superficial human infections, is increasingly antibiotic resistant, and is therefore the target for the development of new antimicrobials. Compounds (1835F03 and targocil) were recently shown to function as bacteriostatic inhibitors of wall teichoic acid (WTA) biosynthesis in S. aureus. To assess the value of targeting WTA biosynthesis in human infection, it was therefore of interest to verify the involvement of WTA in bacterial binding to human corneal epithelial cells (HCECs) and to assess the activities of inhibitors of WTA biosynthesis against clinical isolates of methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) from cases of human keratitis. The 1835F03 MIC90s were 8 μg/ml for MSSA keratitis isolates and >32 μg/ml for MRSA keratitis isolates. The MIC90 for the analog of 1835F03, targocil, was 2 μg/ml for both MRSA and MSSA. Targocil exhibited little toxicity at concentrations near the MIC, with increased toxicity occurring at higher concentrations and with longer exposure times. Targocil activity was moderately sensitive to the presence of serum, but it inhibited extracellular and intracellular bacteria in the presence of HCECs better than vancomycin. Targocil-resistant strains exhibited a significantly reduced ability to adhere to HCECs.

Enabling Glycosyltransferase Evolution: A Facile Substrate‐Attachment Strategy for Phage‐Display Enzyme Evolution

Directed enzyme evolution has the potential to generate novel catalysts able to compete with chemical methodologies in the synthesis of complex biomolecules. Directed evolution is often carried out in cells, but for many classes of enzymes, such as glycosyltransferases, it is difficult to couple enzymatic activity to a cell-based selection. M13 phage display is an in vitro methodology that potentially enables the selection of enzymes that do not provide cell-based phenotypes. In order to select phage-bound enzymes based on catalytic activity, the desired substrate needs to be immobilized near the displayed enzyme to allow for affinity capture of the desired product. Schultz and co-workers developed the first catalysis-based display method, in which the substrate molecule was attached next to the displayed enzyme by a coiled-coil interaction. Although this approach is promising, only a handful of enzymes have been displayed on phage in an active form, and there are still no facile, general methods for substrate immobilization on the phage (vide infra). Here we introduce a chemically straightforward method for substrate attachment using selenocysteine (Sec) residues. Implementation of this method required construction of a phagemid/helper phage system that allows the display of pIII fusions bearing Sec residues adjacent to a displayed enzyme. We also report the first display of an active glycosyltransferase on phage. This work lays the foundation for evolving glycosyltransferases to make novel glycoconjugates and should also enable the directed evolution of other enzyme classes for which ex vivo selection strategies are desirable. Phage display is a convenient strategy to link phenotype and genotype; it enables amplification and identification of peptides and proteins following an in vitro selection. In phage-display enzyme evolution, proximal substrate attachment can be accomplished by using an M13 phagemid/helper phage system in which two different types of pIII fusions are presented on the phage surface (see Scheme 1). The enzyme is displayed on a pIII fusion expressed from the phagemid; the adjacent pIII peptides expressed from the helper phage genome provide potential sites for substrate attachment. Widespread implementation of phage-display enzyme evolution requires a method of modifying a uniquely reactive handle with a functionalized substrate that can be accessed with minimal synthesis. One of us (C.J.N.) has shown recently that selenocysteine (Sec), which is encoded by the TGA codon, can be expressed on phage by fusing a cassette consisting of a TGA codon followed by a selenocysteine insertion sequence (SECIS) to the 5’ end of gIII. Because Sec is considerably more nucleophilic than cysteine and reacts at lower pH (pKa of 5.2 versus 8.1 for Cys), Sec-bearing phage can be derivatized selectively in the presence of other potential side-chain nucleophiles. Encouraged by these results, we wanted to evaluate selenocysteine as a handle for substrate immobilization on phage displaying a pIII-enzyme fusion. We prepared helper phage encoding pIII bearing an N-terminal Sec residue by performing a vector swap between a M13KE vector containing the SECIS insert and M13KO7 using the PacI and BsrGI sites. We also prepared a phagemid (pMurG-pIII) encoding the E. coli glycosyltransferase (Gtf) MurG, an enzyme essential in bacterial cell-wall biosynthesis. The murG gene was subACHTUNGTRENNUNGcloned from a pET-21a expression vector (Novagen) into the pFAB5cHis.TT.HUI phagemid vector as a fusion to the 3’-end of a pelB leader sequence and the 5’-end of a truncated gIII. MurG was chosen as starting point to explore enzyme evolution by phage display because it is a member of the GT-B superfamily of Gtfs that have related three dimensional structures but different substrate selectivity. Many members of the GT-B superfamily are involved in antibiotic biosynthesis, and the ability to alter the substrate selectivity of these Gtfs could enable the production of new biologically active glycoconjugates that are synthetically difficult to access. Infection of cultures containing the pMurG-pIII phagemid with the Sec-expressing helper phage resulted in the production of phage bearing both the enzyme and the Sec handle on the same end of the phage particle (Scheme 1). As a negative control for Sec modification, we also prepared phage expressing wild-type pIII by infecting bacteria containing the pMurGpIII phagemid with the commercially available M13KO7 helper phage. All phage were purified by using CsCl gradient centrifugation. The activity of phage-bound MurG was established by incubating phage with UDP-[C]-GlcNAc and a biotin-labeled lipid I analogue. Product formation was monitored by spotting the reaction mixtures onto streptavidin membranes at various time points (Scheme 2). The membranes were washed and counted to evaluate the product formation. Radioactive counts above background were detected on the membranes within a few minutes and continued to increase for approximately 1 h before leveling off (Figure 1). Using the specific activity of the radiolabeled UDP-GlcNAc (30 mCi mmol ), we estimate that 0.1 mm product was formed within an hour; this means that each phage-bound MurG performed between 10 and 10 turnovers. Only a few phage-bound enzymes reported have been [a] Dr. K. R. Love, J. G. Swoboda, Prof. S. Walker Department of Microbiology and Molecular Genetics Harvard Medical School Boston, MA 02115 (USA) Fax: (+1)617-738-7664 E-mail : suzanne_walker@hms.harvard.edu [b] Dr. C. J. Noren New England Biolabs Ipswich, MA 01938 (USA) [] These authors contributed equally to this work. Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author.

Role of Wall Teichoic Acids in Staphylococcus aureus Endophthalmitis

PURPOSE Wall teichoic acids (WTAs) are major polyanionic polymer components of the cell wall of Staphylococcus aureus. However, little is known about their role at the host-pathogen interface, especially in endophthalmitis. This study was designed to investigate the extent to which WTAs contribute to the pathogenicity of S. aureus in models of endophthalmitis and to determine whether there would be value in targeting their biosynthesis as a new therapeutic approach. METHODS S. aureus RN6390 and its isogenic WTA-null mutant (RN6390ΔtarO) were used to evaluate the role of WTAs in endophthalmitis. RN6390 and RN6390ΔtarO were cultured in bovine vitreous humor (VH) in vitro or inoculated into the vitreous chamber of C57B6 mice. Changes in the number of bacteria, organ function as determined by electroretinography (ERG), and histopathologic changes were assessed throughout the course of infection. In addition, the efficacy of WTA biosynthesis inhibitors in VH in vitro was examined. RESULTS It was observed that a component of VH synergized with WTA biosynthesis inhibitors in vitro and killed the S. aureus. This effect was also seen when mutants incapable of expressing WTA were exposed to VH. The killing activity of VH was lost on treatment with a protease inhibitor. RN6390ΔtarO could not survive in mouse eyes and did not affect organ function, nor was it able to establish endophthalmitis. CONCLUSIONS WTAs are essential cellular constituents for the manifestation of virulence by S. aureus in endophthalmitis, and appears to be a viable target for treating the endophthalmitis caused by S. aureus strains.