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Eric D. Brown - One of the best experts on this subject based on the ideXlab platform.
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Crystallographic analysis of TarI and TarJ, a cytidylyltransferase and reductase pair for CDP-ribitol synthesis in Staphylococcus aureus wall Teichoic Acid biogenesis.
Journal of structural biology, 2021Co-Authors: Robert T. Gale, Eric D. Brown, Evgeniy V. Petrotchenko, Christoph H. Borchers, Natalie C. J. StrynadkaAbstract:Abstract The cell wall of many pathogenic Gram-positive bacteria contains ribitol-phosphate wall Teichoic Acid (WTA), a polymer that is linked to virulence and regulation of essential physiological processes including cell division. CDP-ribitol, the activated precursor for ribitol-phosphate polymerization, is synthesized by a cytidylyltransferase and reductase pair known as TarI and TarJ, respectively. In this study, we present crystal structures of Staphylococcus aureus TarI and TarJ in their apo forms and in complex with substrates and products. The TarI structures illustrate the mechanism of CDP-ribitol synthesis from CTP and ribitol-phosphate and reveal structural changes required for substrate binding and catalysis. Insights into the upstream step of ribulose-phosphate reduction to ribitol-phosphate is provided by the structures of TarJ. Furthermore, we propose a general topology of the enzymes in a heterotetrameric form built using restraints from crosslinking mass spectrometry analysis. Together, our data present molecular details of CDP-ribitol production that may aid in the design of inhibitors against WTA biosynthesis.
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characterization of wall Teichoic Acid degradation by the bacteriophage ϕ29 appendage protein gp12 using synthetic substrate analogs
Journal of Biological Chemistry, 2015Co-Authors: Cullen L Myers, Ronald G Ireland, Teresa A Garrett, Eric D. BrownAbstract:The genetics and enzymology of the biosynthesis of wall Teichoic Acid have been the extensively studied, however, comparatively little is known regarding the enzymatic degradation of this biological polymer. The GP12 protein from the Bacillus subtilis bacteriophage ϕ29 has been implicated as a wall Teichoic Acid hydrolase. We have studied the wall Teichoic Acid hydrolase activity of pure, recombinant GP12 using chemically defined wall Teichoic Acid analogs. The GP12 protein had potent wall Teichoic Acid hydrolytic activity in vitro and demonstrated ∼13-fold kinetic preference for glycosylated poly(glycerol phosphate) Teichoic Acid compared with non-glycosylated. Product distribution patterns suggested that the degradation of glycosylated polymers proceeded from the hydroxyl terminus of the polymer, whereas hydrolysis occurred at random sites in the non-glycosylated polymer. In addition, we present evidence that the GP12 protein possesses both phosphodiesterase and phosphomonoesterase activities.
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designing analogs of ticlopidine a wall Teichoic Acid inhibitor to avoid formation of its oxidative metabolites
Bioorganic & Medicinal Chemistry Letters, 2014Co-Authors: Maya A Farha, Robert T. Gale, Kalinka Koteva, Gerard D Wright, Edward W Sewell, Eric D. BrownAbstract:Abstract The thienopyridine antiplatelet agent, ticlopidine and its analog, clopidogrel, have been shown to potentiate the action of β-lactam antibiotics, reversing the methicillin-resistance phenotype of methicillin-resistant Staphylococcus aureus (MRSA), in vitro . Interestingly, these thienopyridines inhibit the action of TarO, the first enzyme in the synthesis of wall Teichoic Acid, an important cell wall polymer in Gram-positive bacteria . In the human body, both ticlopidine and clopidogrel undergo a rapid P450-dependent oxidation into their respective antiplatelet-active metabolites, resulting in very low plasma concentrations of intact drug. Herein, a series of analogs of ticlopidine and clopidogrel that would avoid oxidative metabolism were designed, prepared and evaluated as inhibitors of TarO. Specifically, we replaced the P450-labile thiophene ring of ticlopidine and clopidogrel to a more stable phenyl group to generate 2-(2-chlorobenzyl)-1,2,3,4-tetrahydro-isoquinoline) ( 6 ) and (2-chloro-phenyl)-(3,4-dihydro-1 H -isoquinolin-2-yl)-acetic Acid methyl ester ( 22 ), respectively. The latter molecules displayed inhibitory activity against TarO and formed the basis of a library of analogs. Most synthesized compounds exhibited comparable efficacy to ticlopidine and clopidogrel. So far, it was introduction of a trifluoromethyl group to compound 6 , to generate 2-(2-trifluoromethyl-benzyl)-1,2,3,4-tetrahydro-isoquinoline ( 13 ) that exhibited enhanced activity against TarO. Compound 13 represents a novel stable inhibitor of TarO with synergistic impact on β-lactam antibiotics against MRSA and low potential for P-450 metabolism.
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Taking aim at wall Teichoic Acid synthesis: new biology and new leads for antibiotics
The Journal of Antibiotics, 2014Co-Authors: Edward Wc Sewell, Eric D. BrownAbstract:Wall Teichoic Acids are a major and integral component of the Gram-positive cell wall. These structures are present across all species of Gram-positive bacteria and constitute roughly half of the cell wall. Despite decades of careful investigation, a definitive physiological function for wall Teichoic Acids remains elusive. Advances in the genetics and biochemistry of wall Teichoic Acid synthesis have led to a new understanding of the complexity of cell wall synthesis in Gram-positive bacteria. Indeed, these innovations have provided new molecular tools available to probe the synthesis and function of these cell wall structures. Among recent discoveries are unexpected roles for wall Teichoic Acid in cell division, coordination of peptidoglycan synthesis and β-lactam resistance in methicillin-resistant Staphylococcus aureus (MRSA). Notably, wall Teichoic Acid biogenesis has emerged as a bona fide drug target in S. aureus , where remarkable synthetic-viable interactions among biosynthetic genes have been leveraged for the discovery and characterization of novel inhibitors of the pathway.
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studies of the genetics function and kinetic mechanism of tage the wall Teichoic Acid glycosyltransferase in bacillus subtilis 168
Journal of Biological Chemistry, 2011Co-Authors: Sarah E Allison, Michael A Delia, Sharif Arar, Mario A Monteiro, Eric D. BrownAbstract:The biosynthetic enzymes involved in wall Teichoic Acid biogenesis in Gram-positive bacteria have been the subject of renewed investigation in recent years with the benefit of modern tools of biochemistry and genetics. Nevertheless, there have been only limited investigations into the enzymes that glycosylate wall Teichoic Acid. Decades-old experiments in the model Gram-positive bacterium, Bacillus subtilis 168, using phage-resistant mutants implicated tagE (also called gtaA and rodD) as the gene coding for the wall Teichoic Acid glycosyltransferase. This study and others have provided only indirect evidence to support a role for TagE in wall Teichoic Acid glycosylation. In this work, we showed that deletion of tagE resulted in the loss of α-glucose at the C-2 position of glycerol in the poly(glycerol phosphate) polymer backbone. We also reported the first kinetic characterization of pure, recombinant wall Teichoic Acid glycosyltransferase using clean synthetic substrates. We investigated the substrate specificity of TagE using a wide variety of acceptor substrates and found that the enzyme had a strong kinetic preference for the transfer of glucose from UDP-glucose to glycerol phosphate in polymeric form. Further, we showed that the enzyme recognized its polymeric (and repetitive) substrate with a sequential kinetic mechanism. This work provides direct evidence that TagE is the wall Teichoic Acid glycosyltransferase in B. subtilis 168 and provides a strong basis for further studies of the mechanism of wall Teichoic Acid glycosylation, a largely uncharted aspect of wall Teichoic Acid biogenesis.
Andreas Peschel - One of the best experts on this subject based on the ideXlab platform.
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the c type lectin receptor mgl senses n acetylgalactosamine on the unique staphylococcus aureus st395 wall Teichoic Acid
Cellular Microbiology, 2019Co-Authors: David Gerlach, Andreas Peschel, Guoqing Xia, Rob Van Dalen, Jos A. G. Van Strijp, Malgorzata Ewa Mnich, Astrid Hendriks, Nina M Van SorgeAbstract:Staphylococcus aureus is a common skin commensal but is also associated with various skin and soft tissue pathologies. Upon invasion, S. aureus is detected by resident innate immune cells through pattern-recognition receptors (PRRs), although a comprehensive understanding of the specific molecular interactions is lacking. Recently, we demonstrated that the PRR langerin (CD207) on epidermal Langerhans cells senses the conserved β-1,4-linked N-acetylglucosamine (GlcNAc) modification on S. aureus wall Teichoic Acid (WTA), thereby increasing skin inflammation. Interestingly, the S. aureus ST395 lineage as well as certain species of coagulase-negative staphylococci (CoNS) produce a structurally different WTA molecule, consisting of poly-glycerolphosphate with α-O-N-acetylgalactosamine (GalNAc) residues, which are attached by the glycosyltransferase TagN. Here, we demonstrate that S. aureus ST395 strains interact with the human Macrophage galactose-type lectin (MGL; CD301) receptor, which is expressed by dendritic cells and macrophages in the dermis. MGL bound S. aureus ST395 in a tagN- and GalNAc-dependent manner but did not interact with different tagN-positive CoNS species. However, heterologous expression of Staphylococcus lugdunensis tagN in S. aureus conferred phage infection and MGL binding, confirming the role of this CoNS enzyme as GalNAc-transferase. Functionally, the detection of GalNAc on S. aureus ST395 WTA by human monocyte-derived dendritic cells significantly enhanced cytokine production. Together, our findings highlight differential recognition of S. aureus glycoprofiles by specific human innate receptors, which may affect downstream adaptive immune responses and pathogen clearance.
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wall Teichoic Acid is a pathogen associated molecular pattern of staphylococcus aureus that is recognized by langerin cd207 on skin langerhans cells
bioRxiv, 2017Co-Authors: Rob Van Dalen, Matevž Rumpret, Felix F Fuchsberger, Jonas Hanske, Christoph Rademacher, Theunis B H Geijtenbeek, Christopher Weidenmaier, Nienke H. Van Teijlingen, Jos A. G. Van Strijp, Andreas PeschelAbstract:Staphylococcus aureus is a major cause of skin and soft tissue infections and aggravator of the inflammatory skin disease atopic dermatitis (AD). Langerhans cells (LCs) initiate a Th17 response upon exposure to S. aureus, which contributes to host defense but also to AD pathogenesis. However, the molecular mechanisms underlying the unique pro-inflammatory capacities of S. aureus remain unclear. We demonstrate that human LCs directly interact with S. aureus through the pattern-recognition receptor langerin (CD207), which specifically recognizes the conserved β-N-acetylglucosamine (GlcNAc) modifications of wall Teichoic Acid (WTA) that are not expressed by other staphylococcal species. The WTA glycoprofile strongly influences the production of Th1- and Th17-polarizing cytokines by LCs. Specifically, β-GlcNAc activates LCs, whereas co-decoration of WTA with α-GlcNAc through the enzyme TarM, uniformly present in the AD-associated CC1 lineage, attenuates LC immune activation. Our findings provide important mechanistic insights into the role of S. aureus in inflammatory skin disease.
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staphylococcus aureuswall Teichoic Acid is a pathogen associated molecular pattern that is recognized by langerin cd207 on skin langerhans cells
bioRxiv, 2017Co-Authors: Van Dalen R, Matevž Rumpret, Felix F Fuchsberger, Jonas Hanske, Christoph Rademacher, Theunis B H Geijtenbeek, Christopher Weidenmaier, De La Cruz Diaz Js, Andreas PeschelAbstract:Abstract Staphylococcus aureus is a major cause of skin and soft tissue infections and aggravator of the inflammatory skin disease atopic dermatitis (AD). Epicutaneous exposure to S. aureus induces Th17 responses through skin Langerhans cells (LCs), which paradoxically contribute to host defense but also to AD pathogenesis. The underlying molecular mechanisms of the association between S. aureus and skin inflammation are poorly understood. Here, we demonstrate that human LCs directly interact with S. aureus through the pattern-recognition receptor langerin (CD207). Human, but not mouse, langerin interacts with S. aureus through the conserved β-N-acetylglucosamine (GlcNAc) modifications on wall Teichoic Acid (WTA), thereby discriminating S. aureus from other staphylococcal species. Importantly, the specific S. aureus WTA glycoprofile strongly influences the level of Th1-and Th17-polarizing cytokines that are produced by in vitro generated LCs. Finally, in a murine epicutaneous infection model, S. aureus induced a more pronounced influx of inflammatory cells and pro-inflammatory cytokine transcripts in skin of human langerin transgenic mice compared to wild-type mice. Our findings provide molecular insight into the unique pro-inflammatory capacities of S. aureus in relation to inflammatory skin disease.
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an accessory wall Teichoic Acid glycosyltransferase protects staphylococcus aureus from the lytic activity of podoviridae
Scientific Reports, 2015Co-Authors: David Gerlach, Jesper Larsen, Marc Stegger, Petra Kuhner, Andreas Peschel, Guoqing Xia, Volker WinstelAbstract:Many Staphylococcus aureus have lost a major genetic barrier against phage infection, termed clustered regularly interspaced palindromic repeats (CRISPR/cas). Hence, S. aureus strains frequently exchange genetic material via phage-mediated horizontal gene transfer events, but, in turn, are vulnerable in particular to lytic phages. Here, a novel strategy of S. aureus is described, which protects S. aureus against the lytic activity of Podoviridae, a unique family of staphylococcal lytic phages with short, non-contractile tails. Unlike most staphylococcal phages, Podoviridae require a precise wall Teichoic Acid (WTA) glycosylation pattern for infection. Notably, TarM-mediated WTA α-O-GlcNAcylation prevents infection of Podoviridae while TarS-mediated WTA β-O-GlcNAcylation is required for S. aureus susceptibility to podoviruses. Tracking the evolution of TarM revealed an ancient origin in other staphylococci and vertical inheritance during S. aureus evolution. However, certain phylogenetic branches have lost tarM during evolution, which rendered them podovirus-susceptible. Accordingly, lack of tarM correlates with podovirus susceptibility and can be converted into a podovirus-resistant phenotype upon ectopic expression of tarM indicating that a “glyco-switch” of WTA O-GlcNAcylation can prevent the infection by certain staphylococcal phages. Since lytic staphylococcal phages are considered as anti-S. aureus agents, these data may help to establish valuable strategies for treatment of infections.
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wall Teichoic Acid glycosylation governs staphylococcus aureus nasal colonization
Mbio, 2015Co-Authors: Volker Winstel, Jesper Larsen, Petra Kuhner, Andreas Peschel, Ferdinand Salomon, Robert Skov, W Hoffmann, Christopher WeidenmaierAbstract:ABSTRACT Nasal colonization by the human pathogen Staphylococcus aureus is a major risk factor for hospital- and community-acquired infections. A key factor required for nasal colonization is a cell surface-exposed zwitterionic glycopolymer, termed wall Teichoic Acid (WTA). However, the precise mechanisms that govern WTA-mediated nasal colonization have remained elusive. Here, we report that WTA GlcNAcylation is a pivotal requirement for WTA-dependent attachment of community-acquired methicillin-resistant S. aureus (MRSA) and emerging livestock-associated MRSA to human nasal epithelial cells, even under conditions simulating the nutrient composition and dynamic flow of nasal secretions. Depending on the S. aureus strain, WTA O-GlcNAcylation occurs in either α or β configuration, which have similar capacities to mediate attachment to human nasal epithelial cells, suggesting that many S. aureus strains maintain redundant pathways to ensure appropriate WTA glycosylation. Strikingly, a lack of WTA glycosylation significantly abrogated the ability of MRSA to colonize cotton rat nares in vivo . These results indicate that WTA glycosylation modulates S. aureus nasal colonization and may help to develop new strategies for eradicating S. aureus nasal colonization in the future. IMPORTANCE Nasal colonization by the major human pathogen Staphylococcus aureus is a risk factor for severe endogenous infections and contributes to the spread of this microbe in hospitals and the community. Here, we show that wall Teichoic Acid (WTA) O-GlcNAcylation is a key factor required for S. aureus nasal colonization. These data provide a mechanistic explanation for the capacity of WTA to modulate S. aureus nasal colonization and may stimulate research activities to establish valuable strategies to eradicate S. aureus nasal colonization in high-risk hospitalized patients and in the general community.
Sophia Kathariou - One of the best experts on this subject based on the ideXlab platform.
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Teichoic Acid glycosylation mediated by gtca is required for phage adsorption and susceptibility of listeria monocytogenes serotype 4b
Applied and Environmental Microbiology, 2008Co-Authors: Ying Cheng, Nattawan Promadej, Jaewon Kim, Sophia KathariouAbstract:An insertion mutant of gtcA, responsible for serotype-specific glycosylation of the cell wall Teichoic Acid in serotype 4b strains of Listeria monocytogenes, was also resistant to both Listeria genus- and serotype 4b-specific phages. The sugar substituents on Teichoic Acid appeared essential for the adsorption of phages A500 (serotype 4b specific) and A511 (Listeria genus specific) to serotype 4b L. monocytogenes.
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a novel serotype specific gene cassette glta gltb is required for expression of Teichoic Acid associated surface antigens in listeria monocytogenes of serotype 4b
Journal of Bacteriology, 2001Co-Authors: Xianghe Lei, Zheng Lan, Franz Fiedler, Sophia KathariouAbstract:Listeria monocytogenes serotype 4b strains account for about 40% of sporadic cases and many epidemics of listeriosis. Mutations in a chromosomal locus resulted in loss of reactivity with all three monoclonal antibodies (MAbs) which were specific to serotype 4b and the closely related serotypes 4d and 4e. Here we show that this locus contains a serotype 4b-4d-4e-specific gene cassette (3,071 bp) which consists of two genes, gltA and gltB, and is flanked by palindromic sequences (51 and 44 nucleotides). Complete loss of reactivity with the three serotype-specific MAbs resulted from insertional inactivation of either gltA or gltB. The gltA and gltB mutants were characterized by loss and severe reduction, respectively, of glucose in the Teichoic Acid, whereas galactose, the other serotype-specific sugar substituent in the Teichoic Acid, was not affected. Within L. monocytogenes, only strains of serotypes 4b, 4d, and 4e harbored the gltA-gltB cassette, whereas coding sequences on either side of the cassette were conserved among all serotypes. Comparative genomic analysis of a serotype 1/2b strain showed that the 3,071-bp gltA-gltB cassette was replaced by a much shorter (528-bp) and unrelated region, flanked by inverted repeats similar to their counterparts in serotype 4b. These findings indicate that in the evolution of different serotypes of L. monocytogenes, this site in the genome has become occupied by serotype-specific sequences which, in the case of serotype 4b, are essential for expression of serotype-specific surface antigens and presence of glucose substituents in the Teichoic Acids in the cell wall.
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a sheep in wolf s clothing listeria innocua strains with Teichoic Acid associated surface antigens and genes characteristic of listeria monocytogenes serogroup 4
Journal of Bacteriology, 2000Co-Authors: Zheng Lan, Franz Fiedler, Sophia KathariouAbstract:Listeria monocytogenes serotype 4b has been implicated in numerous food-borne epidemics and in a substantial fraction of sporadic listeriosis. A unique lineage of the nonpathogenic species Listeria innocua was found to express Teichoic Acid-associated surface antigens that were otherwise expressed only by L. monocytogenes of serotype 4b and the rare serotypes 4d and 4e. These L. innocua strains were also found to harbor sequences homologous to the gene gtcA, which has been shown to be essential for Teichoic Acid glycosylation in L. monocytogenes serotype 4b. Transposon mutagenesis and genetic studies revealed that the gtcA gene identified in this lineage of L. innocua was functional in serotype 4b-like glycosylation of the Teichoic Acids of these organisms. The genomic organization of the gtcA region was conserved between this lineage of L. innocua and L. monocytogenes serotype 4b. Our data are in agreement with the hypothesis that, in this lineage of L. innocua, gtcA was acquired by lateral transfer from L. monocytogenes serogroup 4. The high degree of nucleotide sequence conservation in the gtcA sequences suggests that such transfer was relatively recent. Transfer events of this type may alter the surface antigenic properties of L. innocua and may eventually lead to evolution of novel pathogenic lineages through additional acquisition of genes from virulent listeriae.
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cell wall Teichoic Acid glycosylation in listeria monocytogenes serotype 4b requires gtca a novel serogroup specific gene
Journal of Bacteriology, 1999Co-Authors: Nattawan Promadej, Franz Fiedler, Pascale Cossart, Shaynoor Dramsi, Sophia KathariouAbstract:We have identified a novel gene, gtcA, involved in the decoration of cell wall Teichoic Acid of Listeria monocytogenes serotype 4b with galactose and glucose. Insertional inactivation of gtcA brought about loss of reactivity with the serotype 4b-specific monoclonal antibody c74.22 and was accompanied by a complete lack of galactose and a marked reduction in the amounts of glucose on Teichoic Acid. Interestingly, the composition of membrane-associated lipoTeichoic Acid was not affected. Complementation of the mutants with the cloned gtcA in trans restored galactose and glucose on Teichoic Acid to wild-type levels. The complemented strains also recovered reactivity with c74.22. Within L. monocytogenes, sequences homologous to gtcA were found in all serogroup 4 isolates but not in strains of any other serotypes. In serotype 4b, gtcA appears to be the first member of a bicistronic operon which includes a gene with homology to Bacillus subtilis rpmE, encoding ribosomal protein L31. In contrast to gtcA, the latter gene appears conserved among all screened serotypes of L. monocytogenes.
Suzanne Walker - One of the best experts on this subject based on the ideXlab platform.
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Teichoic Acid biosynthesis as an antibiotic target
Current Opinion in Microbiology, 2013Co-Authors: Lincoln Pasquina, John Santa P Maria, Suzanne WalkerAbstract:The relentless spread of antibiotic-resistant pathogens makes it imperative to develop new chemotherapeutic strategies to overcome infection. The bacterial cell wall has served as a rich source for both validated and unexploited pathways that are essential for virulence and survival. LipoTeichoic Acids (LTAs) and wall Teichoic Acids (WTAs) are cell wall polymers that play fundamental roles in Gram-positive bacterial physiology and pathogenesis, and both have been proposed as novel antibacterial targets. Here we describe recent progress toward the discovery of Teichoic Acid biosynthesis inhibitors and their potential as antibiotics to combat Staphylococcus aureus infections.
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wall Teichoic Acid protects staphylococcus aureus from inhibition by congo red and other dyes
Journal of Antimicrobial Chemotherapy, 2012Co-Authors: Takashi Suzuki, Jonathan G Swoboda, Suzanne Walker, Jennifer Campbell, Younghoon Kim, Eleftherios Mylonakis, Michael S GilmoreAbstract:Methods: We studied antimicrobial activity of azo dyes against S. aureus strains with or without inhibition of Teichoic Acid in vitro and in vivo. Results: 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. Conclusions: 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.
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in vitro antimicrobial activity of wall Teichoic Acid biosynthesis inhibitors against staphylococcus aureus isolates
Antimicrobial Agents and Chemotherapy, 2011Co-Authors: Takashi Suzuki, Jonathan G Swoboda, Suzanne Walker, Jennifer Campbell, Michael S GilmoreAbstract: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.
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development of improved inhibitors of wall Teichoic Acid biosynthesis with potent activity against staphylococcus aureus
Bioorganic & Medicinal Chemistry Letters, 2010Co-Authors: Kyungae Lee, Jonathan G Swoboda, Jennifer Campbell, Gregory D Cuny, Suzanne WalkerAbstract: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.
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discovery of a small molecule that blocks wall Teichoic Acid biosynthesis in staphylococcus aureus
ACS Chemical Biology, 2009Co-Authors: Jonathan G Swoboda, Timothy C Meredith, Takashi Suzuki, Jennifer Campbell, Michael S Gilmore, Stephanie Brown, Tobias Bollenbach, Amy J Malhowski, Roy Kishony, Suzanne WalkerAbstract: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.
Michael A Delia - One of the best experts on this subject based on the ideXlab platform.
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studies of the genetics function and kinetic mechanism of tage the wall Teichoic Acid glycosyltransferase in bacillus subtilis 168
Journal of Biological Chemistry, 2011Co-Authors: Sarah E Allison, Michael A Delia, Sharif Arar, Mario A Monteiro, Eric D. BrownAbstract:The biosynthetic enzymes involved in wall Teichoic Acid biogenesis in Gram-positive bacteria have been the subject of renewed investigation in recent years with the benefit of modern tools of biochemistry and genetics. Nevertheless, there have been only limited investigations into the enzymes that glycosylate wall Teichoic Acid. Decades-old experiments in the model Gram-positive bacterium, Bacillus subtilis 168, using phage-resistant mutants implicated tagE (also called gtaA and rodD) as the gene coding for the wall Teichoic Acid glycosyltransferase. This study and others have provided only indirect evidence to support a role for TagE in wall Teichoic Acid glycosylation. In this work, we showed that deletion of tagE resulted in the loss of α-glucose at the C-2 position of glycerol in the poly(glycerol phosphate) polymer backbone. We also reported the first kinetic characterization of pure, recombinant wall Teichoic Acid glycosyltransferase using clean synthetic substrates. We investigated the substrate specificity of TagE using a wide variety of acceptor substrates and found that the enzyme had a strong kinetic preference for the transfer of glucose from UDP-glucose to glycerol phosphate in polymeric form. Further, we showed that the enzyme recognized its polymeric (and repetitive) substrate with a sequential kinetic mechanism. This work provides direct evidence that TagE is the wall Teichoic Acid glycosyltransferase in B. subtilis 168 and provides a strong basis for further studies of the mechanism of wall Teichoic Acid glycosylation, a largely uncharted aspect of wall Teichoic Acid biogenesis.
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probing Teichoic Acid genetics with bioactive molecules reveals new interactions among diverse processes in bacterial cell wall biogenesis
Chemistry & Biology, 2009Co-Authors: Michael A Delia, Kathryn E Millar, Amit P Bhavsar, Ana M Tomljenovic, Bernd Hutter, Christoph Schaab, Gabriel Morenohagelsieb, Eric D. BrownAbstract:Summary The bacterial cell wall has been a celebrated target for antibiotics and holds real promise for the discovery of new antibacterial chemical matter. In addition to peptidoglycan, the walls of Gram-positive bacteria contain large amounts of the polymer Teichoic Acid, covalently attached to peptidoglycan. Recently, wall Teichoic Acid was shown to be essential to the proper morphology of Bacillus subtilis and an important virulence factor for Staphylococcus aureus . Additionally, recent studies have shown that the dispensability of genes encoding Teichoic Acid biosynthetic enzymes is paradoxical and complex. Here, we report on the discovery of a promoter (P ywaC ), which is sensitive to lesions in Teichoic Acid synthesis. Exploiting this promoter through a chemical-genetic approach, we revealed surprising interactions among undecaprenol, peptidoglycan, and Teichoic Acid biosynthesis that help explain the complexity of Teichoic Acid gene dispensability. Furthermore, the new reporter assay represents an exciting avenue for the discovery of antibacterial molecules.
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the wall Teichoic Acid polymerase tagf efficiently synthesizes poly glycerol phosphate on the tagb product lipid iii
ChemBioChem, 2008Co-Authors: Mark P. Pereira, Michael A Delia, Jefferey W Schertzer, Kalinka Koteva, Donald W Hughes, Gerard D Wright, Eric D. BrownAbstract:Our understanding of the function of cell-wall Teichoic Acid polymerases such as TagF from Bacillus subtilis has been limited by the tools available for a functional assay. Teichoic Acid polymerase activity has previously been studied by using crude membrane preparations as a source of substrate(s). Thus, an understanding of the most basic features of the Teichoic Acid polymerization has eluded characterization. Here we make use of a soluble synthetic glycolipid to provide the first demonstration that TagF polymerizes glycerol phosphate directly on the product of TagB—Teichoic Acid lipid III—at a rate approximately 100 times higher than observed with crude membrane preparations. Interestingly, polymer length was determined by the ratio of glycolipid acceptor to CDP-glycerol, implying that polymerization occurs in a distributive manner. This work provides new insights into the reaction catalyzed by TagF, a prototypic Teichoic Acid polymerase. The bacterial cell wall has been a popular target for the design of antibacterial agents. Nevertheless, cell wall-active antibiotics have exclusively targeted peptidoglycan synthesis and thus overlook other cell wall components. In Gram-positive bacteria, cell wall Teichoic Acids are a chemically diverse group of phosphate-rich polymers that are covalently linked to peptidoglycan. Wall Teichoic Acid accounts for up to 60 % of the Gram-positive cell-wall dry weight. [1] Indeed, wall Teichoic Acid has recently been shown to be essential to the proper rodshaped morphology of Bacillus subtilis [2] and a key virulence determinant for the human pathogen Staphylococcus aureus. [3, 4] Wall Teichoic Acid synthesis is thus an emerging
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wall Teichoic Acid polymers are dispensable for cell viability in bacillus subtilis
Journal of Bacteriology, 2006Co-Authors: Michael A Delia, Terry J. Beveridge, Kathryn E Millar, Eric D. BrownAbstract: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.
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lesions in Teichoic Acid biosynthesis in staphylococcus aureus lead to a lethal gain of function in the otherwise dispensable pathway
Journal of Bacteriology, 2006Co-Authors: Michael A Delia, Mark P. Pereira, Yu Seon Chung, Wenjun Zhao, Andrew S Chau, Teresa J Kenney, Mark C Sulavik, Todd A Black, Eric D. BrownAbstract: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.
