ABSTRACT
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). While decreasing Und-P availability disrupts glycan synthesis and promotes cell death, less is known about the effects of increased Und-P availability. RESULTS: To determine if cells can maintain higher Und-P levels, we first reduced intracellular competition for Und-P by deleting all known non-essential PGT/GTs in the Gram-negative bacterium Escherichia coli (hereafter called ΔPGT/GT cells). We then increased the rate of Und-P synthesis in ΔPGT/GT cells by overexpressing the Und-P(P) synthase uppS from a plasmid (puppS). Und-P quantitation revealed that ΔPGT/GT/puppS cells can be induced to maintain 3-fold more Und-P than wild type cells. Next, we determined how increasing Und-P availability affects glycan expression. Interestingly, increasing Und-P availability increased endogenous and recombinant glycan expression. In particular, ΔPGT/GT/puppS cells could be induced to express 7-fold more capsule from Streptococcus pneumoniae serotype 4 than traditional E. coli cells used to express recombinant glycans. CONCLUSIONS: We demonstrate that the biotechnology standard bacterium E. coli can be engineered to maintain higher levels of Und-P. The results also strongly suggest that Und-P pathways can be engineered to increase the expression of potentially any Und-P-dependent polymer. Given that many bacterial glycans are central to the production of vaccines, diagnostics, and therapeutics, increasing Und-P availability should be a foremost consideration when designing bacterial glycan expression systems.
Subject(s)
Escherichia coli , Polyisoprenyl Phosphates , Escherichia coli/genetics , Polysaccharides , BiotechnologyABSTRACT
Acinetobacter baumannii 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. Loss of the Mla system in A. baumannii results in attenuated virulence and increased susceptibility to membrane stressors and some antibiotics. We recently reported two strain variants of the A. baumannii type strain ATCC 17978: 17978VU and 17978UN. Here, ∆mlaF mutants in the two ATCC 17978 strains display different phenotypes for membrane stress resistance, antibiotic resistance, and pathogenicity in a murine pneumonia model. Although allele differences in obgE were previously reported to synergize with ∆mlaF to affect growth and stringent response, obgE alleles do not affect membrane stress resistance. Instead, a single-nucleotide polymorphism (SNP) in the essential gene encoding undecaprenyl pyrophosphate (Und-PP) synthase, uppS, results in decreased enzymatic rate and decrease in total Und-P levels in 17978UN compared to 17978VU. The UppSUN variant synergizes with ∆mlaF to reduce capsule and lipooligosaccharide (LOS) levels, increase susceptibility to membrane stress and antibiotics, and reduce persistence in a mouse lung infection. Und-P is a lipid glycan carrier required for the biosynthesis of A. baumannii capsule, cell wall, and glycoproteins. These findings uncover synergy between Und-P and the Mla system in maintaining the A. baumannii cell envelope and antibiotic resistance.IMPORTANCEAcinetobacter baumannii is a critical threat to global public health due to its multidrug resistance and persistence in hospital settings. Therefore, novel therapeutic approaches are urgently needed. We report that a defective undecaprenyl pyrophosphate synthase (UppS) paired with a perturbed Mla system leads to synthetically sick cells that are more susceptible to clinically relevant antibiotics and show reduced virulence in a lung infection model. These results suggest that targeting UppS or undecaprenyl species and the Mla system may resensitize A. baumannii to antibiotics in combination therapies. This work uncovers a previously unknown synergistic relationship in cellular envelope homeostasis that could be leveraged for use in combination therapy against A. baumannii.
Subject(s)
Acinetobacter baumannii , Anti-Bacterial Agents , Polyisoprenyl Phosphates , Animals , Mice , Anti-Bacterial Agents/pharmacology , Drug Resistance, Bacterial , Cell Wall , Drug Resistance, Multiple, BacterialABSTRACT
Bacteria use a diverse range of carbohydrates to generate a profusion of glycans, with amino sugars, such as N-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, N-azidoacetylglucosamine (GlcNAz), for metabolic labeling in Escherichia coli. The cytosolic production of UDP-GlcNAz was confirmed by using fluorescence-assisted polyacrylamide gel electrophoresis. The key question of where GlcNAz is incorporated was interrogated by analyzing potential sites including peptidoglycan (PGN), the biofilm-related exopolysaccharide poly-ß-1,6-N-acetylglucosamine (PNAG), lipopolysaccharide (LPS), and the enterobacterial common antigen (ECA). The highest levels of incorporation were observed in PGN with lower levels in PNAG and no observable incorporation in LPS or ECA. The promiscuity of the PNAG synthase (PgaCD) toward UDP-GlcNAz in vitro and the lack of undecaprenyl-pyrophosphoryl-GlcNAz intermediates generated in vivo confirmed the incorporation preferences. The results of this work will guide the future development of carbohydrate-based probes and metabolic engineering strategies.
Subject(s)
Escherichia coli , Lipopolysaccharides , Escherichia coli/metabolism , Acetylglucosamine/metabolism , Polysaccharides, Bacterial , Peptidoglycan , Uridine DiphosphateABSTRACT
Acinetobacter baumannii 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. Therefore, Gram-negative bacteria have mechanisms to uphold this asymmetry such as the maintenance of lipid asymmetry system (Mla), which removes glycerophospholipids from the outer leaflet of the outer membrane and transports them to the inner membrane. Loss of this system in A. baumannii results in attenuated virulence and increased susceptibility to membrane stressors and some antibiotics. We recently reported two strain variants of the A. baumannii type strain ATCC 17978, 17978VU and 17978UN. We show here that ΔmlaF mutants in the two strains display different phenotypes for membrane stress resistance, antibiotic resistance, and pathogenicity in a murine pneumonia model. We used comparative genetics to identify interactions between ATCC 17978 strain alleles and mlaF to uncover the cause behind the phenotypic differences. Although allele differences in obgE were previously reported to synergize with ΔmlaF to affect growth and stringent response, we show that obgE alleles do not affect membrane stress resistance. Instead, a single nucleotide polymorphism (SNP) in the essential gene encoding undecaprenyl pyrophosphate (Und-PP) synthase, uppS, synergizes with ΔmlaF to increase susceptibility to membrane stress and antibiotics, and reduce persistence in a mouse lung infection. Und-P is a lipid glycan carrier known to be required for biosynthesis of A. baumannii capsule, cell wall, and glycoproteins. Our data suggest that in the absence of the Mla system, the cellular level of Und-P is critical for envelope integrity, antibiotic resistance, and lipooligosaccharide abundance. These findings uncover synergy between Und-P and the Mla system in maintaining the A. baumannii outer membrane and stress resistance.
ABSTRACT
Bacteria use a diverse range of carbohydrates to generate a profusion of glycans, with amino sugars such as N-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, N-azidoacetylglucosamine (GlcNAz), for metabolic labelling in E. coli. The cytosolic production of UDP-GlcNAz was confirmed using fluorescence assisted polyacrylamide gel electrophoresis. The key question of where GlcNAz is incorporated, was interrogated by analyzing potential sites including: peptidoglycan (PGN), the biofilm-related exopolysaccharide poly-ß-1,6-N-acetylglucosamine (PNAG), lipopolysaccharide (LPS) and the enterobacterial common antigen (ECA). The highest levels of incorporation were observed in PGN with lower levels in PNAG and no observable incorporation in LPS or ECA. The promiscuity of the PNAG synthase (PgaCD) towards UDP-GlcNAz in vitro and lack of undecaprenyl-pyrophosphoryl-GlcNAz intermediates generated in vivo confirmed the incorporation preferences. The results of this work will guide the future development of carbohydrate-based probes and metabolic engineering strategies.
ABSTRACT
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. However, this long-chain isoprenoid can be difficult to work with in vitro. Here, we describe the addition of a chemically functional benzylazide tag to polyisoprenoids. We have found that both the organic-soluble and water-soluble benzylazide isoprenoid can serve as a substrate for the well-characterized system responsible for Campylobacter jejuni N-linked heptasaccharide assembly. Using the organic-soluble analogue, we demonstrate the use of an N-acetyl-glucosamine epimerase that can be used to lower the cost of glycan assembly, and using the water-soluble analogue, we demonstrate the immobilization of the C. jejuni heptasaccharide on magnetic beads. These conjugated beads are then shown to interact with soybean agglutinin, a lectin known to interact with N-acetyl-galactosamine in the C. jejuni heptasaccharide. The methods provided could be used for a wide variety of applications including the discovery of new glycan-interacting partners.
ABSTRACT
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 E. coli cell membrane fractions are supplemented with exogenous BP. This reaction involves proteins associated with the arn operon, which is necessary for the covalent modification of lipid A with the cationic 4-aminoarabinose (Ara4N). Using a series of arn operon gene deletion mutants, we identified that the modification was dependent on ArnC, which is responsible for forming BP-linked Ara4N, or ArnT, which transfers Ara4N to lipid A. Surprisingly, we found that the majority of the Ara4N-modified isoprenoid was due to the reverse reaction catalyzed by ArnT and demonstrate this using heat-inactivated membrane fractions, isolated lipopolysaccharide fractions, and analyses of a purified ArnT. This work provides methods that will facilitate thorough and rapid investigation of bacterial outer membrane remodeling and the evaluation of polyisoprenoid precursors required for covalent glycan modifications.
ABSTRACT
Colanic acid is a glycopolymer loosely associated with the outer membrane of Escherichia coli 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 E. coli. The accumulation of each enzyme product was made possible by inactivating sequential genes involved in colanic acid biosynthesis and upregulating the colanic acid operon by inducing rcsA transcription. LC-MS analysis revealed that these accumulated materials were consistent with the well-documented composition analysis. Recapitulating the native bioassembly of colanic acid enabled us to identify the functional roles of the last two enzymes, WcaL and WcaK, associated with the formation of the lipid-linked oligosaccharide repeating unit of colanic acid. Importantly, biochemical evidence is provided for the formation of the final glycosylation hexasaccharide product formed by WcaL and the addition of a pyruvate moiety to form a pyruvylated hexasaccharide by WcaK. These findings provide insight into the development of methods for the identification of enzyme functions during cell envelope synthesis.
Subject(s)
Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , Polysaccharides/metabolism , Biosynthetic Pathways , Escherichia coli/genetics , Escherichia coli Proteins/genetics , Gene Expression Regulation, Bacterial , Polysaccharides/geneticsABSTRACT
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 in vitro 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. In this work, we have cloned and expressed each gene involved in ECA repeat unit biosynthesis and reconstituted the pathway in vitro, providing LC-MS characterized standards for the investigation of cellular glycan-linked intermediates and BP. We then generated ECA deficient mutants in genes associated with production of the polysaccharide, which we suspected would accumulate materials identical to our standards. We found that indeed accumulated products from these cells were indistinguishable from our enzymatically prepared standards, and moreover we observed a concomitant decrease in cellular BP levels with each mutant. This work provides the first direct biochemical evidence for the sequestration of BP upon the genetic disruption of glycan biosynthesis pathways in bacteria. This work also provides methods for the direct assessment of both the ECA glycan, and a new understanding of the dynamic interdependence of the bacterial polysaccharide repertoire.
Subject(s)
Antigens, Bacterial/metabolism , Chromatography, Liquid , Mass Spectrometry , Polysaccharides/metabolism , Substrate SpecificityABSTRACT
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. Because of the ubiquity of bactoprenyl phosphate in these critical processes, molecular probes appended to this lipid carrier simplify identification of enzymatic roles during polysaccharide bioassembly. A limited number of these probes exist in the literature or have been assessed with such pathways, and the limits of their use are not currently known. Herein, we devise an efficient method for producing fluorescently modified bactoprenyl probes. We further expand our previous efforts utilizing 2-nitrileaniline and additionally prepare nitrobenzoxadizol-tagged bactoprenyl phosphate for the first time. We then assess the enzyme promiscuity of these two probes utilizing four well-characterized initiating phosphoglycosyltransferases: CPS2E (Streptococcus pneumoniae), WbaP (Salmonella enterica), WecA (Escherichia coli), and WecP (Aeromonas hydrophilia). Both probes serve as substrates for these enzymes and could be readily used to investigate a wide range of bacterial glycoassembly pathways. Interestingly, we have also identified unique solubility requirements for the nitrobenzoxadizol moiety for efficient enzymatic utilization that was not observed for the 2-nitrileaniline.
Subject(s)
Bacterial Proteins/chemistry , Polyisoprenyl Phosphates/chemistry , Polyprenols/chemistry , Cloning, Molecular/methods , Escherichia coli/metabolism , Salmonella enterica/metabolism , Streptococcus pneumoniae/metabolism , SugarsABSTRACT
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. coli genome that encodes colanic acid biosynthesis has been reported, and the first enzyme in the biosynthesis pathway has been biochemically characterized. However, the specific roles of the remaining genes required for colanic acid biosynthesis have not been identified. Here we report the in vitro reconstitution of the next six steps in the assembly of the colanic acid repeating unit. To do this, we have cloned and overexpressed each gene within the colanic acid biosynthesis operon. We then tested the activity of the protein product of these genes using high-performance liquid chromatography analysis and a fluorescent analogue of the isoprenoid anchor bactoprenyl diphospho-glucose as a starting substrate. To ensure that retention time changes were associated with varying sugar additions or modifications, we developed a liquid chromatography-mass spectrometry method for analysis of the products produced by each enzyme. We have identified the function of all but one encoded glycosyltransferase and have identified the function of two acetyltransferases. This work demonstrates the centrality of acetylation in the biosynthesis of colanic acid and provides insight into the activity of key proteins involved in the production of an important and highly conserved bacterial glycopolymer.
Subject(s)
Bacterial Proteins/metabolism , Biosynthetic Pathways , Enterobacteriaceae/metabolism , Glycosyltransferases/metabolism , Polysaccharides/metabolism , Acetylation , Bacterial Proteins/genetics , Cloning, Molecular , Enterobacteriaceae/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Genes, Bacterial , Glycosyltransferases/genetics , Polysaccharides/geneticsABSTRACT
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 (1â»4) were used to evaluate the charge influence on the PDI of a Gram-negative bacteria, Escherichia coli (E. coli), and their interaction with the cell membrane. The dose-response PDI results confirm the relevance of the number of positive charges on the porphyrin molecule in the PDI of E. coli. The difference between the Hill coefficients of cationic porphyrins with 1â»3 positive charges and the tetra-cationic porphyrin (4) revealed potential variations in their mechanism of inactivation. Fluorescent live-cell microscopy studies showed that cationic porphyrins with 1â»3 positive charges bind to the cell membrane of E. coli, but are not internalized. On the contrary, the tetra-cationic porphyrin (4) permeates through the membrane of the cells. The contrast in the interaction of cationic porphyrins with E. coli confirmed that they followed different mechanisms of inactivation. This work helps to have a better understanding of the structure-activity relationship in the efficiency of the PDI process of cationic porphyrins against Gram-negative bacteria.
Subject(s)
Anti-Bacterial Agents/pharmacology , Escherichia coli/drug effects , Photosensitizing Agents/pharmacology , Porphyrins/pharmacology , Quantitative Structure-Activity Relationship , Anti-Bacterial Agents/chemistry , Biological Transport , Cations/chemistry , Cell Membrane/drug effects , Escherichia coli/radiation effects , Photosensitizing Agents/chemistry , Photosensitizing Agents/radiation effects , Porphyrins/chemistry , Porphyrins/radiation effects , Static Electricity , Ultraviolet RaysABSTRACT
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 Escherichia coli, this carrier is undecaprenyl phosphate (Und-P), which is synthesized as a diphosphate by the enzyme undecaprenyl pyrophosphate synthase (UppS). E. coli MG1655 exhibits wild-type morphology at all temperatures, but one of our laboratory strains (CS109) was highly aberrant when grown at 42°C. This strain contained mutations affecting the Und-P synthetic pathway genes uppS, ispH, and idi Normal morphology was restored by overexpressing uppS or by replacing the mutant (uppS31) with the wild-type allele. Importantly, moving uppS31 into MG1655 was lethal even at 30°C, indicating that the altered enzyme was highly deleterious, but growth was restored by adding the CS109 versions of ispH and idi Purified UppSW31R was enzymatically defective at all temperatures, suggesting that it could not supply enough Und-P during rapid growth unless suppressor mutations were present. We conclude that cell wall synthesis is profoundly sensitive to changes in the pool of polyisoprenoids and that isoprenoid homeostasis exerts a particularly strong evolutionary pressure.IMPORTANCE Bacterial morphology is determined primarily by the overall structure of the semirigid macromolecule peptidoglycan. Not only does peptidoglycan contribute to cell shape, but it also protects cells against lysis caused by excess osmotic pressure. Because it is critical for bacterial survival, it is no surprise that many antibiotics target peptidoglycan biosynthesis. However, important gaps remain in our understanding about how this process is affected by peptidoglycan precursor availability. Here, we report that a mutation altering the enzyme that synthesizes Und-P prevents cells from growing at high temperatures and that compensatory mutations in enzymes functioning upstream of uppS can reverse this phenotype. The results highlight the importance of Und-P metabolism for maintaining normal cell wall synthesis and shape.
Subject(s)
Alkyl and Aryl Transferases/genetics , Escherichia coli Proteins/genetics , Escherichia coli/genetics , Mutation , Polyisoprenyl Phosphates/biosynthesis , Biosynthetic Pathways , Escherichia coli/enzymology , Escherichia coli/growth & development , Peptidoglycan/biosynthesisABSTRACT
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. The proposed pyruvyltransferase, WcfO; galactopyranose mutase, WcfM; and glycosyltransferases, WcfP and WcfN, encoded by the CPSA biosynthesis gene cluster were heterologously expressed and functionally characterized. Pyruvate modification, catalyzed by WcfO, was found to occur on galactose of the polyisoprenoid-linked disaccharide (AADGal-Gal), and did not occur on galactose linked to uridine diphosphate (UDP) or a set of nitrophenyl-galactose analogues. This pyruvate modification was also found to be required for the incorporation of the next sugar in the pathway N-acetylgalactosamine (GalNAc) by the glycosyltransferase WcfP. The pyruvate acetal modification of a galactose has not been previously explored in the context of a polysaccharide biosynthesis pathway, and this work demonstrates the importance of this modification to repeat unit assembly. Upon production of the polyisoprenoid-linked AADGal-PyrGal-GalNAc, the proteins WcfM and WcfN were found to work in concert to form the final tetrasaccharide, where WcfM formed UDP-galactofuranose (Galf) and WcfN transfers Galf to the AADGal-PyrGal-GalNAc. This work demonstrates the first enzymatic assembly of the tetrasaccharide repeat unit of CPSA in a sequential single pot reaction.
Subject(s)
Bacteroides fragilis/enzymology , Bacteroides fragilis/metabolism , Biosynthetic Pathways , Polysaccharides, Bacterial/metabolism , Acetylgalactosamine/genetics , Acetylgalactosamine/metabolism , Aldehyde-Ketone Transferases/genetics , Aldehyde-Ketone Transferases/metabolism , Animals , Bacteroides fragilis/chemistry , Bacteroides fragilis/genetics , Gene Expression , Glycosyltransferases/genetics , Glycosyltransferases/metabolism , Multigene Family , Polysaccharides, Bacterial/chemistry , Polysaccharides, Bacterial/geneticsABSTRACT
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. Here using a fluorescent 2-nitrileanilinogeranyl diphosphate analogue of FPP, we have found that a wide range of surfactants can stimulate release of product from UppS and that the structure of the surfactant has a major impact on the lengths of products produced by the protein. Of particular importance, shorter chain surfactants promote the release of isoprenoids with four to six Z-configuration isoprene additions, while larger chain surfactants promote the formation of natural isoprenoid lengths (8Z) and larger. We have found that the product chain lengths can be readily controlled and coarsely tuned by adjusting surfactant identity, concentration, and reaction time. We have also found that binary mixtures of just two surfactants can be used to fine-tune isoprenoid lengths. The surfactant effects discovered do not appear to be significantly altered with an alternative isoprenoid substrate. However, the surfactant effects do appear to be dependent on differences in UppS between bacterial species. This work provides new insights into surfactant effects in enzymology and highlights how these effects can be leveraged for the chemoenzymatic synthesis of otherwise difficult to obtain glycan biosynthesis probes. This work also provides key reagents for the systematic analysis of structure-activity relationships between glycan biosynthesis enzymes and isoprenoid structure.
Subject(s)
Alkyl and Aryl Transferases/chemistry , Bacterial Proteins/chemistry , Fluorescent Dyes/chemical synthesis , Surface-Active Agents/chemistry , Terpenes/chemical synthesis , Bacteroides fragilis/enzymology , Stereoisomerism , Time FactorsABSTRACT
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. fragilis UPPS protein, yet replacing the amide with a nitrile [2-nitrileanilinogeranyl diphosphate (2CNA-GPP)] led to a compound that was fully functional for UPPS from all three target organisms. These fluorescent substrate analogues were also found to undergo increases in fluorescence upon isoprenoid chain elongation, and this increase in fluorescence can be utilized to monitor the activity and inhibition of UPPS in 96-well plate assays. The fluorescence of 2CNA-GPP increased by a factor of 2.5-fold upon chain elongation, while that of 2AA-GPP increased only 1.2-fold. The 2CNA-GPP compound was therefore more versatile for screening the activity of UPPS from multiple species of bacteria and underwent a larger increase in fluorescence that improved its ability to detect increases in chain length. Overall, this work describes the development of new assay methods for UPPS and demonstrates the difference in substrate utilization between forms of UPPS from different species, which has major implications for UPPS inhibitor development, assay construction, and the development of polysaccharide biosynthesis probes.
Subject(s)
Alkyl and Aryl Transferases/chemistry , Anti-Bacterial Agents/chemistry , Drug Delivery Systems/trends , Alkyl and Aryl Transferases/antagonists & inhibitors , Anti-Bacterial Agents/administration & dosage , Bacteroides fragilis/enzymology , Escherichia coli/enzymology , Humans , Molecular Probes/chemistry , Species Specificity , Spectrometry, Mass, Electrospray Ionization , Substrate Specificity/drug effects , Substrate Specificity/physiology , Vibrio vulnificus/enzymologyABSTRACT
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. However, the precise functions of the individual glycosyltransferases encoded by this cluster have not been identified. In this report each of these glycosyltransferases (WcfQ, WcfP, and WcfN) have been expressed and tested for their function in vitro. Using a reverse phase high performance liquid chromatography (HPLC) assay, WcfQ and WcfP were found to transfer galactose from uridine diphosphate (UDP)-linked galactose (Gal) to N-acetyl-4-amino-6-deoxy-galactosamine (AADGal) linked to a fluorescent mimic of bactoprenyl diphosphate, the native isoprenoid anchor for bacterial polysaccharide biosynthesis. The incorporation of galactose to form a bactoprenyl-linked disaccharide was confirmed by radiolabel incorporation and mass spectrometry (MS) of purified product. Using varying concentrations of UDP-Gal and enzyme, WcfQ was found to be the most effective protein at transferring galactose, and is the most likely candidate for in vivo incorporation of the sugar. WcfQ also cooperated in the presence of three preceding biosynthetic enzymes to form an isoprenoid-linked disaccharide in a single-pot reaction. This work represents a critical step in understanding the biosynthetic pathway responsible for the formation of CPSA, an unusual and potentially therapeutic biopolymer.
Subject(s)
Bacterial Proteins/genetics , Bacteroides fragilis/enzymology , Genome, Bacterial , Glycosyltransferases/genetics , Polysaccharides, Bacterial/biosynthesis , Bacterial Capsules/chemistry , Bacterial Proteins/metabolism , Bacteroides fragilis/chemistry , Bacteroides fragilis/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Galactose/analogs & derivatives , Galactose/metabolism , Gene Expression , Glycosyltransferases/metabolism , Multigene Family , Polysaccharides, Bacterial/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Terpenes/metabolism , Uridine Diphosphate/analogs & derivatives , Uridine Diphosphate/metabolismABSTRACT
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. The activity of this analogue in polysaccharide biosynthesis is then tested with the initiating hexose-1-phosphate transferases involved in Capsular Polysaccharide A biosynthesis in the symbiont Bacteroides fragilis and the asparagine-linked glycosylation system of the pathogenic Campylobacter jejuni. In addition, it is shown that the UPPS used to make this probe is not specific for E-configured isoprenoid substrates and that elongation by UPPS is required for activity with the downstream enzymes.
Subject(s)
Alkyl and Aryl Transferases/metabolism , Fluorescent Dyes/chemistry , Terpenes/chemistry , Bacteroides/metabolism , Campylobacter jejuni/enzymology , Campylobacter jejuni/metabolism , Glycosylation , Polysaccharides/biosynthesis , Polysaccharides/chemistry , Stereoisomerism , Substrate Specificity , Terpenes/metabolism , ortho-Aminobenzoates/chemistryABSTRACT
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. In this report, we have utilized a heterologous system to reconstitute bactoprenyl diphosphate-linked AADGal production. Construction of this system included a previously reported Campylobacter jejuni dehydratase, PglF, coupled to a B. fragilis-encoded aminotransferase (WcfR) and initiating hexose-1-phosphate transferase (WcfS). The function of the aminotransferase was confirmed by capillary electrophoresis and a novel high-performance liquid chromatography (HPLC) method. Production of the rare uridine diphosphate (UDP)-AADGal was confirmed through a series of one- and two-dimensional nuclear magnetic resonance experiments and high-resolution mass spectrometry. A spectroscopically unique analogue of bactoprenyl phosphate was utilized to characterize the transfer reaction catalyzed by WcfS and allowed HPLC-based isolation of the isoprenoid-linked sugar product. Importantly, the entire heterologous system was utilized in a single-pot reaction to biosynthesize the bactoprenyl-linked sugar. This work provides the first critical step in the in vitro reconstitution of CPSA biosynthesis.
Subject(s)
Acetylgalactosamine/analogs & derivatives , Bacteroides fragilis/enzymology , Bacteroides fragilis/metabolism , Polysaccharides, Bacterial/metabolism , Uridine Diphosphate/metabolism , Acetylgalactosamine/chemistry , Acetylgalactosamine/metabolism , Animals , Bacteroides Infections/microbiology , Bacteroides fragilis/chemistry , Humans , Phosphotransferases/metabolism , Polysaccharides, Bacterial/chemistry , Transaminases/metabolism , Uridine Diphosphate/chemistryABSTRACT
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. Analysis of steady-state kinetics for modification of peptide substrates revealed that the multiple-turnover activity depends on the analogue structure. Analogues in which the first isoprene is replaced with a benzyl group and an analogue in which each isoprene is replaced with an aryl group are good substrates. In sharp contrast with the steady-state reaction, the single-turnover rate constant for dansyl-GCVLS alkylation was found to be the same for all analogues, despite the increased chemical reactivity of the benzyl analogues and the increased steric bulk of other analogues. However, the single-turnover rate constant for alkylation does depend on the Ca(1)a(2)X peptide sequence. These results suggest that the isoprenoid transition-state conformation is preferred over the inactive E·FPP·Ca(1)a(2)X ternary complex conformation. Furthermore, these data suggest that the farnesyl binding site in the exit groove may be significantly more selective for the farnesyl diphosphate substrate than the active site binding pocket and therefore might be a useful site for the design of novel inhibitors.