Engineering Escherichia coli for increased Und-P availability leads to material improvements in glycan expression technology.

Background: Bacterial surface glycans are assembled by glycosyltransferases (GTs) that transfer sugar monomers to long-chained lipid carriers. Most bacteria employ the 55-carbon chain undecaprenyl phosphate (Und-P) to scaffold glycan assembly. The amount of Und-P available for glycan synthesis is thought to be limited by the rate of Und-P synthesis and by competition for Und-P between phosphoglycosyl transferases (PGTs) and GTs that prime glycan assembly (which we collectively refer to as PGT/GTs).

Genetic synergy between undecaprenyl phosphate biosynthesis and the Mla system impacts cell envelope and antimicrobial resistance.

is a Gram-negative bacterial pathogen that poses a major health concern due to increasing multidrug resistance. The Gram-negative cell envelope is a key barrier to antimicrobial entry and includes an inner and outer membrane. The maintenance of lipid asymmetry (Mla) system is the main homeostatic mechanism by which Gram-negative bacteria maintain outer membrane asymmetry.

Metabolic Usage and Glycan Destinations of GlcNAz in .

Bacteria use a diverse range of carbohydrates to generate a profusion of glycans, with amino sugars, such as -acetylglucosamine (GlcNAc), being prevalent in the cell wall and in many exopolysaccharides. The primary substrate for GlcNAc-containing glycans, UDP-GlcNAc, is the product of the bacterial hexosamine pathway and a key target for bacterial metabolic glycan engineering. Using the strategy of expressing NahK, to circumvent the hexosamine pathway, it is possible to directly feed the analogue of GlcNAc, -azidoacetylglucosamine (GlcNAz), for metabolic labeling in .

Genetic synergy in undecaprenyl biosynthesis and maintenance of lipid asymmetry impacts outer membrane and antimicrobial resistance.

is a Gram-negative healthcare-associated pathogen that poses a major health concern due to increasing multidrug resistance. The Gram-negative cell envelope is a key barrier to antimicrobial entry and includes an inner and outer membrane. The outer membrane has an asymmetric composition that is important for structural integrity and barrier to the environment.

The Metabolic Usage and Glycan Destinations of GlcNAz in .

Bacteria use a diverse range of carbohydrates to generate a profusion of glycans, with amino sugars such as -acetylglucosamine (GlcNAc) being prevalent in the cell wall and in many exopolysaccharides. The primary substrate for GlcNAc-containing glycans, UDP-GlcNAc, is the product of the bacterial hexosamine pathway, and a key target for bacterial metabolic glycan engineering. Using the strategy of expressing NahK, to circumvent the hexosamine pathway, it is possible to directly feed the analogue of GlcNAc, -azidoacetylglucosamine (GlcNAz), for metabolic labelling in The cytosolic production of UDP-GlcNAz was confirmed using fluorescence assisted polyacrylamide gel electrophoresis.

Chemoenzymatic Preparation of a Lipid-Linked Heptasaccharide on an Azide-Linked Polyisoprenoid.

Complex poly- and oligosaccharides on the surface of bacteria provide a unique fingerprint to different strains of pathogenic and symbiotic microbes that could be exploited for therapeutics or sensors selective for specific glycans. To discover reagents that can selectively interact with specific bacterial glycans, a system for both the chemoenzymatic preparation and immobilization of these materials would be ideal. Bacterial glycans are typically synthesized in nature on the C55 polyisoprenoid bactoprenyl (or undecaprenyl) phosphate.

Lipopolysaccharide Is a 4-Aminoarabinose Donor to Exogenous Polyisoprenyl Phosphates through the Reverse Reaction of the Enzyme ArnT.

Modification of the lipid A portion of LPS with cationic monosaccharides provides resistance to polymyxins, which are often employed as a last resort to treat multidrug-resistant bacterial infections. Here, we describe the use of fluorescent polyisoprenoids, liquid chromatography-mass spectrometry, and bacterial genetics to probe the activity of membrane-localized proteins that utilize the 55-carbon lipid carrier bactoprenyl phosphate (BP). We have discovered that a substantial background reaction occurs when B-strain cell membrane fractions are supplemented with exogenous BP.

Tracking Colanic Acid Repeat Unit Formation from Stepwise Biosynthesis Inactivation in .

Colanic acid is a glycopolymer loosely associated with the outer membrane of that plays a role in pathogen survival. For nearly six decades since its discovery, the functional identities of the enzymes necessary to synthesize colanic acid have yet to be assessed in full. Herein, we developed a method for detecting the lipid-linked intermediates from each step of colanic acid biosynthesis in .

Making the Enterobacterial Common Antigen Glycan and Measuring Its Substrate Sequestration.

The enterobacterial common antigen (ECA), a three-sugar repeat unit polysaccharide produced by Enterobacteriaceae family members, impacts bacterial outer membrane permeability, and its biosynthesis affects the glycan landscape of the organism. ECA synthesis impacts the production of other polysaccharides by reducing the availability of shared substrates, the most notable of which is the 55-carbon polyisoprenoid bactoprenyl phosphate (BP), which serves as a carrier for the production of numerous bacterial glycans including ECA, peptidoglycan, O-antigen, and more. Here, using a combination of enzymatic synthesis and liquid chromatography-mass spectrometry (LC-MS) analysis of bacterial lysates, we provide biochemical evidence for the effect on endogenous polyisoprenoid pools from cell culture that arises from glycan pathway disruption.

Diffusible Signal Factors Act through AraC-Type Transcriptional Regulators as Chemical Cues To Repress Virulence of Enteric Pathogens.

Successful colonization by enteric pathogens is contingent upon effective interactions with the host and the resident microbiota. These pathogens thus respond to and integrate myriad signals to control virulence. Long-chain fatty acids repress the virulence of the important enteric pathogens and by repressing AraC-type transcriptional regulators in pathogenicity islands.

General Utilization of Fluorescent Polyisoprenoids with Sugar Selective Phosphoglycosyltransferases.

The protective surfaces of bacteria are comprised of polysaccharides and are involved in host invasion and colonization, host immune system evasion, and antibacterial resistance. A major barrier to our fundamental understanding of these complex surface polysaccharides lies in the tremendous diversity in glycan composition among bacterial species. The polyisoprenoid bactoprenyl phosphate (or undecaprenyl phosphate) is an essential lipid carrier necessary for early stages of glycopolymer assembly.

Identification of the Functional Roles of Six Key Proteins in the Biosynthesis of Enterobacteriaceae Colanic Acid.

When subjected to harsh conditions such as low pH, pathogenic Escherichia coli can secrete colanic acid to establish a protective barrier between the organism and the acidic environment. The colanic acid consists of a six-sugar repeating unit polymer comprised of glucose, fucose, galactose, and glucuronic acid. The region of the E.

Influence of Cationic -Substituted Porphyrins on the Antimicrobial Photodynamic Efficacy and Cell Membrane Interaction in .

Photodynamic inactivation (PDI) is a non-antibiotic option for the treatment of infectious diseases. Although Gram-positive bacteria have been shown to be highly susceptible to PDI, the inactivation of Gram-negative bacteria has been more challenging due to the impermeability properties of the outer membrane. In the present study, a series of photosensitizers which contain one to four positive charges (⁻) were used to evaluate the charge influence on the PDI of a Gram-negative bacteria, (), and their interaction with the cell membrane.

A Defective Undecaprenyl Pyrophosphate Synthase Induces Growth and Morphological Defects That Are Suppressed by Mutations in the Isoprenoid Pathway of Escherichia coli.

The peptidoglycan exoskeleton shapes bacteria and protects them against osmotic forces, making its synthesis the target of many current antibiotics. Peptidoglycan precursors are attached to a lipid carrier and flipped from the cytoplasm into the periplasm to be incorporated into the cell wall. In , this carrier is undecaprenyl phosphate (Und-P), which is synthesized as a diphosphate by the enzyme undecaprenyl pyrophosphate synthase (UppS).

Complete Tetrasaccharide Repeat Unit Biosynthesis of the Immunomodulatory Bacteroides fragilis Capsular Polysaccharide A.

Capsular polysaccharide A (CPSA) is a four-sugar repeating unit polymer found on the surface of the gut symbiont Bacteroides fragilis that has therapeutic potential in animal models of autoimmune disorders. This therapeutic potential has been credited to its zwitterionic character derived from a positively charged N-acetyl-4-aminogalactosamine (AADGal) and a negatively charged 4,6-O-pyruvylated galactose (PyrGal). In this report, using a fluorescent polyisoprenoid chemical probe, the complete enzymatic assembly of the CPSA tetrasaccharide repeat unit is achieved.

Tuning the production of variable length, fluorescent polyisoprenoids using surfactant-controlled enzymatic synthesis.

Bactoprenyl diphosphate (BPP), a two-E eight-Z configuration C55 isoprenoid, serves as a critical anchor for the biosynthesis of complex glycans central to bacterial survival and pathogenesis. BPP is formed by the polymerase undecaprenyl pyrophosphate synthase (UppS), which catalyzes the elongation of a single farnesyl diphosphate (FPP) with eight Z-configuration isoprene units from eight isopentenyl diphosphates. In vitro analysis of UppS and other polyprenyl diphosphate synthases requires the addition of a surfactant such as Triton X-100 to stimulate the release of the hydrophobic product from the enzyme for effective and efficient turnover.

Species differences in alternative substrate utilization by the antibacterial target undecaprenyl pyrophosphate synthase.

Undecaprenyl pyrophosphate synthase (UPPS) is a critical enzyme required for the biosynthesis of polysaccharides essential for bacterial survival. In this report, we have tested the substrate selectivity of UPPS derived from the mammalian symbiont Bacteroides fragilis, the human pathogen Vibrio vulnificus, and the typically benign but opportunistic pathogen Escherichia coli. An anthranilamide-containing substrate, 2-amideanilinogeranyl diphosphate (2AA-GPP), was an effective substrate for only the B.

Functional identification of a galactosyltransferase critical to Bacteroides fragilis Capsular Polysaccharide A biosynthesis.

Capsular Polysaccharide A (CPSA), a polymer of a four-sugar repeating unit that coats the surface of the mammalian symbiont Bacteroides fragilis, has therapeutic potential in animal models of Multiple Sclerosis and other autoinflammatory diseases. Genetic studies have demonstrated that CPSA biosynthesis is dependent primarily on a single gene cluster within the B. fragilis genome.

Fluorescent probes for investigation of isoprenoid configuration and size discrimination by bactoprenol-utilizing enzymes.

Undecaprenyl Pyrophosphate Synthase (UPPS) is an enzyme critical to the production of complex polysaccharides in bacteria, as it produces the crucial bactoprenol scaffold on which these materials are assembled. Methods to characterize the systems associated with polysaccharide production are non-trivial, in part due to the lack of chemical tools to investigate their assembly. In this report, we develop a new fluorescent tool using UPPS to incorporate a powerful fluorescent anthranilamide moiety into bactoprenol.

Biosynthetic assembly of the Bacteroides fragilis capsular polysaccharide A precursor bactoprenyl diphosphate-linked acetamido-4-amino-6-deoxygalactopyranose.

The sugar capsule capsular polysaccharide A (CPSA), which coats the surface of the mammalian symbiont Bacteroides fragilis, is a key mediator of mammalian immune system development. In addition, this sugar polymer has shown therapeutic potential in animal models of multiple sclerosis and other autoimmune disorders. The structure of the CPSA polymer includes a rare stereoconfiguration sugar acetamido-4-amino-6-deoxygalactopyranose (AADGal) that we propose is the first sugar linked to a bactoprenyl diphosphate scaffold in the production of CPSA.

Farnesyl diphosphate analogues with aryl moieties are efficient alternate substrates for protein farnesyltransferase.

Farnesylation is an important post-translational modification essential for the proper localization and function of many proteins. Transfer of the farnesyl group from farnesyl diphosphate (FPP) to proteins is catalyzed by protein farnesyltransferase (FTase). We employed a library of FPP analogues with a range of aryl groups substituting for individual isoprene moieties to examine some of the structural and electronic properties of the transfer of an analogue to the peptide catalyzed by FTase.

Chemoenzymatic synthesis of an isoprenoid phosphate tool for the analysis of complex bacterial oligosaccharide biosynthesis.

Undecaprenyl Pyrophosphate Synthase (UPPS) is a key enzyme that catalyzes the production of bactoprenols, which act as membrane anchors for the assembly of complex bacterial oligosaccharides. One of the major hurdles in understanding the assembly of oligosaccharide assembly is a lack of chemical tools to study this process, since bactoprenols and the resulting isoprenoid-linked oligosaccharides lack handles or chromophores for use in pathway analysis. Here we describe the isolation of a new UPPS from the symbiotic microorganism Bacteroides fragilis, a key species in the human microbiome.

Development of a multicomponent kinetic assay of the early enzymes in the Campylobacter jejuni N-linked glycosylation pathway.

The human pathogen Campylobacter jejuni possesses a general N-linked glycosylation system that is known to play a role in pathogenicity; however, a detailed understanding of this role remains elusive. A considerable hindrance to studying bacterial N-glycosylation in vivo is the absence of small molecule inhibitors to reversibly control the process. This report describes a pathway-screening assay that targets the early enzymes of C.

Anticancer activity of novel unnatural synthetic isoprenoids.

Background: The KRAS oncogene has a high prevalence in solid malignancies. Targeting KRAS and inappropriate activation of the MAPK pathway with novel drugs is of interest. This study developed and screened a library of compounds designed to inhibit KRAS signaling by altering prenyl function.

Campylobacter jejuni PglH is a single active site processive polymerase that utilizes product inhibition to limit sequential glycosyl transfer reactions.

Asparagine-linked protein glycosylation is essential for the virulence of the human gut mucosal pathogen Campylobacter jejuni . The heptasaccharide that is transferred to proteins is biosynthesized via the glycosyltransferase-catalyzed addition of sugar units to an undecaprenyl diphosphate-linked carrier. Genetic studies on the heptasaccharide assembly enzymes have shown that PglH, which transfers three terminal N-acetyl-galactosamine (GalNAc) residues to the carrier polyisoprene, is essential for chick colonization by C.