Bacterial pathogens are often coated with polysaccharide virulence factors or produce toxins that bind to host oligosaccharides as receptors for cells entry. These polysaccharides and toxins continue to be excellent targets for prevention and controlling disease. Indeed inactivated toxins, polysaccharides, and polysaccharides conjugated to inactivated toxins form the basis for several of the currently licensed and future vaccines. The goals of this research program are to determine(a)the metabolic pathway for the synthesis of polysialic acids in gram negative pathogens and (b) the interaction of neurotoxins of Clostridium botulinum and C. tetani with the glycolipids found on nerve cells. As a part of the the counterterrorism effort at CBER we are also (c)developing an assay to determine antibodies to botulinum toxin and (d)characterizing the hyaluronic acid biosynthetic genes in Bacillus anthracis. Structure and function of enzymes involved the metabolism of polysialic acid. Polysialic acids are synthesized by pathogenic bacteria as capsular polysaccharides. These polymers have been implicated in the virulence of some strains of Escherichia coli, which cause neonatal meningitis and urinary tract infections. Polysialic acid is also present in the developing human brain. There has been significant progress in identifying the gene necessary for capsular polysaccharide biosynthesis in gram negative bacteria. The objective of this project is to determine the mechanism of capsular polysaccharide biosynthesis in virulent encapulated bacteria. Our approach is to characterize the structure and function of the enzymes in the pathway and to use them as tools for understanding sialylation in bacteria and humans. Much of the enzymology of polysialic acid capsular polysaccharide synthesis has been done with the a(2-8) polysialyltransferase complex of E. coli K1. Bacteria containing DNA fragments encoding several capsule related genes have been used as a source of enzyme activity. As a model system for investigating the mechanism of capsular glycosyltransferases we have chosen to investigate the K92 a(2-8)(2-9) polysialyltransferase in a genetic background lacking other capsule related genes. The neuS gene encodes this glycosyltransferase and is the only glycosyltransferase to date identified with synthesis of this polymer. We have shown that the K92 neuS gene product can synthesize both a(2,8) and a(2,9) neuNAc linkages in vitro in a background free of other capsule related gene products and confirmed in vivo synthesis of this polymer by 13C-NMR. We have shown that the acceptor specificity for polysialic acids is broad for thisenzyme. Gangliosides containing this disialylated oligosaccharide are elongated, while monsialylated gangliosides are not. Disialylgangliosides are better acceptors than short oligosaccharides suggesting a role for lipid in the elongation reaction. The idea of a lipid linked acceptor is further supported by the elongation of a disialyloligosaccharide possessing a hydrophobic aglycon. We have extracted membranes of strains defective in sialic acid and therefore polysaccharide synthesis and demonstrated the presence of an acceptor in these extracts. The acceptor is not a protein and behaves chromatographically like a glycolipid. This acceptor will support the formation of polymer by the K92 neuS polysialyltransferase. The lipid does not behave like an undecaprenol. Sialic acid is synthesized from N-acetylmannosamine and phosphoenol pyruvate. We have determine the sterochemistry of this condensation reaction. We have also shown that the source of the N-acetylmannosamine in the pathway is UDP-N-acetylglucosamine. The gene that encodes the enzyme catalyzing this conversion is neuC. Characterization of tetanus toxin structure and function. Clostridial neurotoxins (botulinum and tetanus toxins) are large proteins organized into three functional domains, an amino terminal proteolytic domain, central translocation domain, and a carboxyl terminal receptor binding region. The neurotoxic effects of clostridial neurotoxins results from binding to cell surface receptors, translocation into the neural cell, and the proteolytic cleavage of proteins essential for synaptic vesicle docking/fusion events (SNARE proteins) by the enzymatically active amino terminal domain. The subsequent block of neurotransmitter release at the neuromuscular junction by botulinum toxins or block of inhibitory neurotransmitter release within the central nervous system by tetanus toxin leads to flaccid or spasmodic paralysis of the victim, respectively. The focus of this project has been the characterization of the receptor binding domain of tetanus C-fragment. To determine which amino acids in tetanus toxin are involved in ganglioside binding, homology modeling was performed using recently resolved X-ray crystallographic structures of tetanus toxin HC fragment. Based on these analyses, two regions in tetanus toxin that share structural homology with the binding domains of other sialic acid and galactose-binding proteins were targeted for mutagenesis. Specific amino acids within these regions were altered using site-directed mutagenesis. The amino acid residue tryptophan-1288 was found to be critical for binding of the HC fragment to ganglioside GT1b. Docking of GD1b within this region of the toxin suggested that histidine-1270 and aspartate-1221 were within hydrogen bonding distance of the ganglioside. These two residues were mutagenized and found to also be important for the binding of tetanus toxin HC fragment to ganglioside GT1b. In addition, the HC fragments mutagenized at these residues have reduced levels of binding to neurites of differentiated PC-12 cells. These studies indicate that the amino acids tryptophan-1288, histidine-1270, and aspartate-1221 are components of the GT1b-binding site on the tetanus toxin HC fragment. Biosynthesis of polysaccharides in Bacillus anthracis. Bacillus anthracis is a gram-positive bacterium that causes Anthrax. Two megaplasmids pXO1 and pXO2 in B. anthracis have been shown to encode for the virulence factors of this bacteria. Biosynthesis of the anthrax toxin requires the presence of the pXO1 plasmid (110 Mda), since it contains the genes encoding for the proteins protective antigen, lethal factor and edema factor. The other plasmid pXO2 (60 Mda) contains the genes capB, capC, capA and dep that are required for the synthesis of poly-D-glutamic acid capsule. Both of these plasmids are required for the full virulence of B. anthracis. In spite of the importance of these two plasmids for the virulence, many of the genes in these plasmids are yet to be studied in detail. Recently, the sequence and organization of the plasmid pXO1 has been reported by Okinaka et al and has been shown to contain a total of 143 ORFs. Functions for the proteins encoded by 35 of these ORFs have been assigned based on their similarities to proteins from other organisms. All the toxin genes in the pXO1 plasmid are contained in a 44.8-kb region designated as pathogenecity island (PAI). Interestingly, ORFs 93, 94 and 95 which encode for proteins with sequence similarity to hyaluronic acid synthetase (hasA), UDP-glucose-pyrophosphorylase (hasC) and UDP-glucose-dehydrogenase (hasB) of Streptococcus are present immediately adjacent to the PAI. Hyaluronic acid is capsular polysaccharide and a virulence factor for Streptococcus pyogenes and Pasteurella multocida. There is no precedence for the presence of a polysaccharide capsule in B. anthracis. Therefore, it is not known if these genes (ORFs 93, 94 and 95) are even functional and even if they express to form the corresponding proteins, their importance in terms of virulence is unknown. We have amplified and express 2 of the putative hyaluronic acid synthesis genes. Western blot analysis of the culture lysates of these E. coli cells indicated that both the UDP-glucose dehydrogenase and the hyaluronic acid synthase were expressed in this system. Recombinant B. anthracis hyaluronic synthase did not appear to catalyze the formation of hyaluronic acid in vitro. Closer examination f the sequence of this gene cluster revealed a frame shift mutation. We corrected this frame shift by site directed mutagenesis. The mutated protein product obtained from these E. coli cells was assayed for their ability to form hyaluronic acid. We find that this large protein incorporates glcNAc into high MW material. We are currently studying the biochemical characteristics of this mutant protein as well as the polysaccharide that is formed by this protein. Our preliminary studies with UDP-glucose dehydrogenase showed that this enzyme is active and that it can utilize both UDP-glucose . Determination of Antibody of Botulinum toxins. Assay development for antibody against botulinum toxin will contribute to effective testing of regulated products by developing in vitro tests of toxin presence and potency. Current assays for botulinum toxin are based on mouse lethality and are therefore lengthy, expensive and imprecise. Most neutralizing antibody induced by botulinum toxoid vaccine is against the receptor binding domain. We propose to develop a convenient and sensitive method to measure this type of antibody. We have reinvestigated the parameters used for measuring the binding of tetanus Hc fragment to GT1b coated Biacore chip and determine binding constants for tetanus Hc to GT1b. We have constructed chimeras of tetanus Hc and indicator fragments that will be adaptable to ELISA assays. We have prepared derivatized GT1b oligosaccharides to facilitate coating to microtiter plates. This project incorporates FY2002 projects 1Z01BJ004001-12, 1Z01BJ004005-08, 1Z01BJ004006-02, and 1Z01BJ004010-01.
