Mark P. Pereira

Lesions in Teichoic Acid Biosynthesis in Staphylococcus aureus Lead to a Lethal Gain of Function in the Otherwise Dispensable Pathway

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.

High-Throughput Screening Identifies Novel Inhibitors of the Acetyltransferase Activity of Escherichia coli GlmU

ABSTRACT The bifunctional GlmU protein catalyzes the formation of UDP-N-acetylglucosamine in a two-step reaction using the substrates glucosamine-1-phosphate, acetyl coenzyme A, and UTP. This metabolite is a common precursor to the synthesis of bacterial cell surface carbohydrate polymers, such as peptidoglycan, lipopolysaccharide, and wall teichoic acid that are involved in the maintenance of cell shape, permeability, and virulence. The C-terminal acetyltransferase domain of GlmU exhibits structural and mechanistic features unique to bacterial UDP-N-acetylglucosamine synthases, making it an excellent target for antibacterial design. In the work described here, we have developed an absorbance-based assay to screen diverse chemical libraries in high throughput for inhibitors to the acetyltransferase reaction of Escherichia coli GlmU. The primary screen of 50,000 drug-like small molecules identified 63 hits, 37 of which were specific to acetyltransferase activity of GlmU. Secondary screening and mode-of-inhibition studies identified potent inhibitors where compound binding within the acetyltransferase active site was requisite on the presence of glucosamine-1-phosphate and were competitive with the substrate acetyl coenzyme A. These molecules may represent novel chemical scaffolds for future antimicrobial drug discovery. In addition, this work outlines the utility of catalytic variants in targeting specific activities of bifunctional enzymes in high-throughput screens.

The Wall Teichoic Acid Polymerase TagF Efficiently Synthesizes Poly(glycerol phosphate) on the TagB Product Lipid III

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

The Wall Teichoic Acid Polymerase TagF Is Non-processive in Vitro and Amenable to Study Using Steady State Kinetic Analysis

Wall teichoic acids are a chemically diverse group of anionic polymers that constitute up to 50% of the Gram-positive cell wall. These polymers play a pivotal role in virulence and have been implicated in a diverse range of physiological functions. The TagF-like family of enzymes has been shown to be responsible for wall teichoic acid priming and polymerization events. Although many such enzymes are well validated therapeutic targets, a mechanistic understanding of this enzyme family has remained elusive. TagF is the prototypical teichoic acid polymerase and uses CDP-glycerol to catalyze synthesis of the linear (1,3)-linked poly(glycerol phosphate) teichoic acid in Bacillus subtilis 168. Here we used a synthetic soluble analog of the natural substrate of the enzyme, Lipid ϕ, to conduct the first detailed mechanistic investigation of teichoic acid polymerization. Through the use of a new high pressure liquid chromatography-based assay to monitor single glycerol phosphate incorporations into the Lipid ϕ analog, we conducted a detailed analysis of reaction product formation patterns and unequivocally showed TagF to be non-processive in vitro. Furthermore by monitoring the kinetics of polymerization, we showed that Lipid ϕ analog species varying in size have the same Km value of 2.6 μm and validated use of Bi Bi velocity expressions to model the TagF enzyme system. Initial rate analysis showed that TagF catalyzes a sequential Bi Bi mechanism where both substrates are added to the enzyme prior to product release consistent with a single displacement chemical mechanism.

Isolation of DNA Aptamers for CDP‐Ribitol Synthase, and Characterization of Their Inhibitory and Structural Properties

Numerous studies in the past two decades have shown that nucleic acids have the ability to carry out more functions than simply acting as passive templates for genetic-information storage. For example, RNA has been shown to possess a great repertoire of activities in gene expression and regulation. The discovery of natural ribozymes (RNA catalysts) in the early 1980s, and extensive studies afterwards, have led to the invention of the “in vitro selection” or SELEX (systematic evolution of ligands by exponential enrichment) technique, which can be used to isolate nucleic-acid sequences with a desired function from large, random-sequence pools of RNA or DNA. When this function is molecular recognition, the isolated nucleic-acid molecules are called RNA or DNA “aptamers”. This invention has resulted in tremendous expansion of the number of functional ACHTUNGTRENNUNGnucleic acids in recent years. Since the first in vitro selection experiments in 1990, many aptamers have been identified for the recognition of diverse target molecules. Organic dyes, antibiotics and other drugs, amino acids, peptides, various proteins, nucleotides, and cellular cofactors, have all been successfully targeted by aptamer selection. For example, DNA aptamers have been identified for several protein targets, including human thrombin, l-selectin, immunoglobulin IgE, platelet-derived growth factor B-chain, ricin, a serine protease, and a tRNA synthetase. In recent years, aptamers have also been developed for the recognition of specific types of cells. In this study, we set out to isolate DNA aptamers for CDP-ribitol synthase—a bifunctional enzyme that catalyzes two consecutive chemical reactions that lead to the formation of CDPribitol : the reduction of ribulose 5-phosphate by using NADPH, and cytidylyltransfer by using CTP (Scheme 1A). CDP-ribitol is the activated form of ribitol 5-phosphate—a key building block of virulence-associated polyol phosphates found in the extracellular capsule of the Gram-negative pathogen Haemophilus influenzae, and in cell wall teichoic acid of Gram-positive pathogens, such as Staphylococcus aureus. Bcs1, the enzyme from H. influenzae, is a single polypeptide that harbors both ribulose 5-phosphate reductase activity (referred to as “reductase” activity in this report) and ribitol 5-phosphate cytidylyltransferase activity (referred to as “transferase” activity). We have previously reported that in Bcs1, reduction of ACHTUNGTRENNUNGribulose 5-phosphate to the intermediate ribitol 5-phosphate precedes cytidylyltransfer. In this study we set out to investigate whether we could isolate DNA aptamers for Bcs1, and

Biosynthesis of cell wall teichoic acid polymers

Publisher Summary In the model, Gram-positive organism Bacillus subtilis , the vegetative cell wall is primarily made up of a thick peptidoglycan layer, proteins, and covalently associated anionic polymers. The major anionic polymer is a phosphate-rich molecule termed wall teichoic acid (WTA) that can account for more than 50% of the cell wall dry weight. This chapter focuses on recent advances in the study of the biosynthesis of the most common and studied TA polymers, the major WTAs from B. subtilis, and Staphylococcus aureus . This in-depth review details synthesis of the required nucleotide-activated precursors as well as emerging understanding of priming and polymerization of teichoic acid polymers. This chapter discusses how availability of active recombinant proteins and soluble substrate analogues has facilitated some exciting advances in knowledge of TA biosynthesis. It also discusses the outstanding questions and hurdles that currently preclude a complete understanding of WTA biosynthesis.

Magnetic resonance tells microbiology where to go; bacterial teichoic acid protects liquid water at sub-zero temperatures

Numerous chemical additives lower the freezing point of water, but life at sub-zero temperatures is sustained by a limited number of biological cryoprotectants. Antifreeze proteins in fish, plants, and insects provide protection to a few degrees below freezing. Microbes have been found to survive at even lower temperatures, although, with a few exceptions, antifreeze proteins are missing. Survival has been attributed to external factors, such as high salt concentration (brine veins) and adhesion to particulates or ice crystal defects. Teichoic acid is a phosphodiester polymer ubiquitous in Gram positive bacteria, composing 50% of the mass of the bacterial cell wall and excreted into the extracellular space of biofilm communities. We have found that when bound to the peptidoglycan cell wall (wall teichoic acid) or as a free molecule (lipoteichoic acid), teichoic acid is surrounded by liquid water at temperatures significantly below freezing. Using solid-state NMR, we are unable to collect 31P CPMAS spectra for frozen solutions of lipoteichoic acid at temperatures above -60 °C. For wall teichoic acid in D2O, signals are not seen above -30 °C. These results can be explained by the presence of liquid water, which permits rapid molecular motion to remove 1H/31P dipolar coupling. 2H quadrupole echo NMR spectroscopy reveals that both liquid and solid water are present. We suggest that teichoic acids could provide a shell of liquid water around biofilms and planktonic bacteria, removing the need for brine veins to prevent bacterial freezing.