Joel T. Weadge

Biosynthesis of the Pseudomonas aeruginosa Extracellular Polysaccharides, Alginate, Pel, and Psl

Pseudomonas aeruginosa thrives in many aqueous environments and is an opportunistic pathogen that can cause both acute and chronic infections. Environmental conditions and host defenses cause differing stresses on the bacteria, and to survive in vastly different environments, P. aeruginosa must be able to adapt to its surroundings. One strategy for bacterial adaptation is to self-encapsulate with matrix material, primarily composed of secreted extracellular polysaccharides. P. aeruginosa has the genetic capacity to produce at least three secreted polysaccharides; alginate, Psl, and Pel. These polysaccharides differ in chemical structure and in their biosynthetic mechanisms. Since alginate is often associated with chronic pulmonary infections, its biosynthetic pathway is the best characterized. However, alginate is only produced by a subset of P. aeruginosa strains. Most environmental and other clinical isolates secrete either Pel or Psl. Little information is available on the biosynthesis of these polysaccharides. Here, we review the literature on the alginate biosynthetic pathway, with emphasis on recent findings describing the structure of alginate biosynthetic proteins. This information combined with the characterization of the domain architecture of proteins encoded on the Psl and Pel operons allowed us to make predictive models for the biosynthesis of these two polysaccharides. The results indicate that alginate and Pel share certain features, including some biosynthetic proteins with structurally or functionally similar properties. In contrast, Psl biosynthesis resembles the EPS/CPS capsular biosynthesis pathway of Escherichia coli, where the Psl pentameric subunits are assembled in association with an isoprenoid lipid carrier. These models and the environmental cues that cause the cells to produce predominantly one polysaccharide over the others are subjects of current investigation.

Peptidoglycan O Acetylation and Autolysin Profile of Enterococcus faecalis in the Viable but Nonculturable State

The O acetylation of peptidoglycan occurs specifically at the C-6 hydroxyl group of muramoyl residues. Using a combination of high-performance liquid chromatography-based organic acid analysis and carbohydrate analysis by high-pH anion-exchange chromatography, we determined that strains of Entercoccus durans, E. faecalis, E. faecium, and E. hirae produce O-acetylated peptidoglycan. The levels of O acetylation ranged from 19% to 72% relative to the muramic acid content, and they were found to vary with the growth phase of the culture. Increases of 10 to 40% in O acetylation were observed with cultures entering the stationary phase. Cells of E. faecalis in the viable but nonculturable (VBNC) state had the highest levels of peptidoglycan O acetylation. The presence of this modification to peptidoglycan was shown to inhibit the action of hen egg white lysozyme in a concentration-dependent manner. Zymography using sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels containing either O-acetylated or chemically de-O-acetylated peptidoglycan was used to monitor the production of specific autolysins in E. faecalis. Differences in the expression of specific autolysins were observed with the age of the culture, and VBNC E. faecalis produced the highest levels of these enzymes. This technique also permitted classification of the enterococcal autolysins into enzymes that preferentially hydrolyze either O-acetylated or non-O-acetylated peptidoglycan and enzymes that show no apparent preference for either substrate type.

Immunologic Glycosphingolipidomics and NKT Cell Development in Mouse Thymus

Invariant NKT cells are a hybrid cell type of Natural Killer cells and T cells, whose development is dependent on thymic positive selection mediated by double positive thymocytes through their recognition of natural ligands presented by CD1d, a nonpolymorphic, non-MHC, MHC-like antigen presenting molecule. Genetic evidence suggested that beta-glucosylceramide derived glycosphingolipids (GSLs) are natural ligands for NKT cells. N-butyldeoxygalactonojirimycin (NB-DGJ), a drug that specifically inhibits the glucosylceramide synthase, inhibits the endogenous ligands for NKT cells. Furthermore, we and others have found a beta-linked glycosphingolipid, isoglobotriaosylceramide (iGb3), is a stimulatory NKT ligand. The iGb3 synthase knockout mice have a normal NKT development and function, indicating that other ligands exist and remain to be identified. In this study, we have performed a glycosphingolipidomics study of mouse thymus, and studied mice mutants which are deficient in beta-hexosaminidase b or alpha-galactosidase A, two glycosidases that are up- and downstream agents of iGb3 turnover, respectively. Our mass spectrometry methods generated a first database for glycosphingolipids expressed in mouse thymus, which are specifically regulated by rate-limiting glycosidases. Among the identified thymic glycosphingolipids, only iGb3 is a stimulatory ligand for NKT cells, suggesting that large-scale fractionation, enrichment and characterization of minor species of glycosphingolipids are necessary for identifying additional ligands for NKT cells. Our results also provide early insights into cellular lipidomics studies, with a specific focus on the important immunological functions of glycosphingolipids.

Identification of a new family of enzymes with potential O -acetylpeptidoglycan esterase activity in both Gram-positive and Gram-negative bacteria

BackgroundThe metabolism of the rigid bacterial cell wall heteropolymer peptidoglycan is a dynamic process requiring continuous biosynthesis and maintenance involving the coordination of both lytic and synthetic enzymes. The O-acetylation of peptidoglycan has been proposed to provide one level of control on these activities as this modification inhibits the action of the major endogenous lytic enzymes, the lytic transglycosylases. The O-acetylation of peptidoglycan also inhibits the activity of the lysozymes which serve as the first line of defense of host cells against the invasion of bacterial pathogens. Despite this central importance, there is a dearth of information regarding peptidoglycan O-acetylation and nothing has previously been reported on its de-acetylation.ResultsHomology searches of the genome databases have permitted this first report on the identification of a potential family of O-A cetylp eptidoglycan e sterases (Ape). These proteins encoded in the genomes of a variety of both Gram-negative and Gram-positive bacteria, including a number of important human pathogens such as species of Neisseria, Helicobacter, Campylobacter, and Bacillus anthracis, have been organized into three families based on amino acid sequence similarities with family 1 being further divided into three sub-families. The genes encoding these proteins are shown to be clustered with P eptidoglycan O-a cetylt ransferases (Pat) and in some cases, together with other genes involved in cell wall metabolism. Representative bacteria that encode the Ape proteins were experimentally shown to produce O-acetylated peptidoglycan.ConclusionThe hypothetical proteins encoded by the pat and ape genes have been organized into families based on sequence similarities. The Pat proteins have sequence similarity to Pseudomonas aeruginosa AlgI, an integral membrane protein known to participate in the O-acetylation of the exopolysaccaride, alginate. As none of the bacteria that harbor the pat genes produce alginate, we propose that the Pat proteins serve to O-acetylate peptidoglycan which is known to be a maturation event occurring in the periplasm. The Ape sequences have amino acid sequence similarity to the CAZy CE 3 carbohydrate esterases, a family previously known to be composed of only O-acetylxylan esterases. They are predicted to contain the α/β hydrolase fold associated with the GDSL and TesA hydrolases and they possess the signature motifs associated with the catalytic residues of the CE3 esterases. Specific signature sequence motifs were identified for the Ape proteins which led to their organization into distinct families. We propose that by expressing both Pat and Ape enzymes, bacteria would be able to obtain a high level of localized control over the degradation of peptidoglycan through the attachment and removal of O-linked acetate. This would facilitate the efficient insertion of pores and flagella, localize spore formation, and control the level of general peptidoglycan turnover.

Mechanism of Action of Neisseria gonorrhoeae O-Acetylpeptidoglycan Esterase, an SGNH Serine Esterase

Background: O-Acetylpeptidoglycan esterase functions to release O-acetyl groups from peptidoglycan, thereby permitting the continued metabolism of this essential cell wall component. Results: Gly-236 and Asn-268 were identified as participating at the oxyanion hole to stabilize the tetrahedral species in the reaction mechanism. Conclusion: Ape1a functions as a classical serine esterase. Significance: Ape represents a novel target for the development of new antibiotics. O-Acetylpeptidoglycan esterase from Neisseria gonorrhoeae functions to release O-acetyl groups from the C-6 position of muramoyl residues in O-acetylated peptidoglycan, thereby permitting the continued metabolism of this essential cell wall heteropolymer. It has been demonstrated to be a serine esterase with sequence similarity to the family CE-3 carbohydrate esterases of the CAZy classification system. In the absence of a three-dimensional structure for any Ape, further knowledge of its structure and function relationship is dependent on modeling and kinetic studies. In this study, we predicted Neisseria gonorrhoeae Ape1a to be an SGNH hydrolase with an adopted α/β-hydrolase fold containing a central twisted four-stranded parallel β-sheet flanked by six α-helices with the putative catalytic triad, Asp-366, His-369, and Ser-80 appropriately aligned within a pocket. The role of eight invariant and highly conserved residues localized to the active site was investigated by site-directed replacements coupled with kinetic characterization and binding studies of the resultant engineered enzymes. Based on these data and theoretical considerations, Gly-236 and Asn-268 were identified as participating at the oxyanion hole to stabilize the tetrahedral species in the reaction mechanism, whereas Gly-78, Asp-79, His-81, Asn-235, Thr-267, and Val-368 are proposed to position appropriately the catalytic residues and participate in substrate binding.

Structural and functional characterization of Pseudomonas aeruginosa AlgX: role of AlgX in alginate acetylation

Background: AlgX is required for the biosynthesis and export of the exopolysaccharide alginate. Results: The structure of AlgX has been determined, and the functional characterization of AlgX and mutant variants has been performed. Conclusion: AlgX contains an SGNH hydrolase-like domain and carbohydrate-binding module. Mutation of the Ser-His-Asp triad in vivo results in non-acetylated alginate. Significance: This is the first structural characterization of a polysaccharide acetyltransferase. The exopolysaccharide alginate, produced by mucoid Pseudomonas aeruginosa in the lungs of cystic fibrosis patients, undergoes two different chemical modifications as it is synthesized that alter the properties of the polymer and hence the biofilm. One modification, acetylation, causes the cells in the biofilm to adhere better to lung epithelium, form microcolonies, and resist the effects of the host immune system and/or antibiotics. Alginate biosynthesis requires 12 proteins encoded by the algD operon, including AlgX, and although this protein is essential for polymer production, its exact role is unknown. In this study, we present the X-ray crystal structure of AlgX at 2.15 Å resolution. The structure reveals that AlgX is a two-domain protein, with an N-terminal domain with structural homology to members of the SGNH hydrolase superfamily and a C-terminal carbohydrate-binding module. A number of residues in the carbohydrate-binding module form a substrate recognition “pinch point” that we propose aids in alginate binding and orientation. Although the topology of the N-terminal domain deviates from canonical SGNH hydrolases, the residues that constitute the Ser-His-Asp catalytic triad characteristic of this family are structurally conserved. In vivo studies reveal that site-specific mutation of these residues results in non-acetylated alginate. This catalytic triad is also required for acetylesterase activity in vitro. Our data suggest that not only does AlgX protect the polymer as it passages through the periplasm but that it also plays a role in alginate acetylation. Our results provide the first structural insight for a wide group of closely related bacterial polysaccharide acetyltransferases.

P. aeruginosa SGNH hydrolase-like proteins AlgJ and AlgX have similar topology but separate and distinct roles in alginate acetylation.

The O-acetylation of polysaccharides is a common modification used by pathogenic organisms to protect against external forces. Pseudomonas aeruginosa secretes the anionic, O-acetylated exopolysaccharide alginate during chronic infection in the lungs of cystic fibrosis patients to form the major constituent of a protective biofilm matrix. Four proteins have been implicated in the O-acetylation of alginate, AlgIJF and AlgX. To probe the biological function of AlgJ, we determined its structure to 1.83 Å resolution. AlgJ is a SGNH hydrolase-like protein, which while structurally similar to the N-terminal domain of AlgX exhibits a distinctly different electrostatic surface potential. Consistent with other SGNH hydrolases, we identified a conserved catalytic triad composed of D190, H192 and S288 and demonstrated that AlgJ exhibits acetylesterase activity in vitro. Residues in the AlgJ signature motifs were found to form an extensive network of interactions that are critical for O-acetylation of alginate in vivo. Using two different electrospray ionization mass spectrometry (ESI-MS) assays we compared the abilities of AlgJ and AlgX to bind and acetylate alginate. Binding studies using defined length polymannuronic acid revealed that AlgJ exhibits either weak or no detectable polymer binding while AlgX binds polymannuronic acid specifically in a length-dependent manner. Additionally, AlgX was capable of utilizing the surrogate acetyl-donor 4-nitrophenyl acetate to catalyze the O-acetylation of polymannuronic acid. Our results, combined with previously published in vivo data, suggest that the annotated O-acetyltransferases AlgJ and AlgX have separate and distinct roles in O-acetylation. Our refined model for alginate acetylation places AlgX as the terminal acetlytransferase and provides a rationale for the variability in the number of proteins required for polysaccharide O-acetylation.

Enzymatic synthesis of Gb3 and iGb3 ceramides

Gb3 and iGb3 are physiologically important trihexosylceramides with a terminal alpha-d-Galp-(1-->4)-beta-d-Galp- and alpha-d-Galp-(1-->3)-beta-d-Galp sequence, respectively. In particular iGb3 is attracting considerable attention as it is believed to serve as a ligand for natural killer T cells. Whether or not iGb3 is present in humans and which enzyme might be responsible for its synthesis is at present a matter of lively debate. In the current investigation we evaluated human blood group B galactosyltransferase (GTB) for its ability to catalyze the formation of iGb3 from lactosylceramide and UDP-Galp. GTB is a retaining glycosyltransferase that in vivo catalyzes the transfer of galactose from UDP-Galp donors to OH-3 of Galp on the H-antigen (alpha-l-Fucp-(1-->2)-beta-d-Galp) acceptor forming the blood group B antigen. GTB tolerates modifications in donor and acceptor substrates and its ability to accept lactosides as acceptors makes it a possible candidate for iGb3 production in humans. For comparison iGb3 and Gb3 were also synthesized from the same acceptor using an alpha-(1-->3)- and alpha-(1-->4)-specific galactosyltransferase, respectively. All the enzymes tested catalyzed the desired reactions. Product characterization by NMR analysis clearly differentiated between the alpha-Galp-(1-->3)-Galp and alpha-Galp-(1-->4)-Galp product, with the GTB product being identical to that of the alpha-(1-->3)-GalT-catalyzed reaction. The rate of transfer by GTB however was very low, only 0.001% of the rate obtained with a good substrate, H antigen disaccharide (octyl alpha-l-Fucp-(1-->2)-beta-d-Galp). This is too low to account for the possible formation of the iGb3 structure in humans in vivo.

A Syst-OMICS Approach to Ensuring Food Safety and Reducing the Economic Burden of Salmonellosis

The Salmonella Syst-OMICS consortium is sequencing 4,500 Salmonella genomes and building an analysis pipeline for the study of Salmonella genome evolution, antibiotic resistance and virulence genes. Metadata, including phenotypic as well as genomic data, for isolates of the collection are provided through the Salmonella Foodborne Syst-OMICS database (SalFoS), at https://salfos.ibis.ulaval.ca/. Here, we present our strategy and the analysis of the first 3,377 genomes. Our data will be used to draw potential links between strains found in fresh produce, humans, animals and the environment. The ultimate goals are to understand how Salmonella evolves over time, improve the accuracy of diagnostic methods, develop control methods in the field, and identify prognostic markers for evidence-based decisions in epidemiology and surveillance.

Purification, N-terminal sequencing and partial characterization of a novel aspartic proteinase from the leaves of Medicago sativa L. (alfalfa)

A novel aspartic proteinase (EC 3.4.23) from Medicago sativa L. (alfalfa) was purified to homogeneity using Source Q ion-exchange, concanavalin-A Sepharose and pepstatin-A agarose affinity chromatography. The enzyme, Mr=33.5 kDa, is monomeric and catalyzes the cleavage of a broad spectrum of peptide bonds of hydrophobic amino acids from pH 2.6 to 6.4. The enzyme is inhibited by pepstatin-A and is consistent with the properties of an aspartic proteinase. The N-terminal amino acid sequence of the protein shows 50 and 40% similarity with the cyprosin and barley aspartic proteinases, respectively.