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. The objectives of this project are: 1. To determine if the hyaluronic acid synthesis genes (ORFs 93, 94 and 95) in the virulence plasmid pXO1 of Bacillus anthracis are functional genes. 2. To characterize the hyaluronic acid synthetase encoded by ORF 93 in the plasmid pXO1. 3. To determine whether hyaluronic acid synthetase is expressed in B. anthracis. We have PCR amplified the individual genes of the hyaluronic acid synthase gene cluster and cloned them separately into E. coli. 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. Although the inactive nature of the hyaluronic acid synthase is disappointing, upon further literature review we noticed that the only thing separating open reading frame 92 (hypothetical protein) and the hyaluronic acid synthase gene (open reading frame 93) is a frame shift in the nucleotide sequence. We also noticed that the C-terminus of the ORF92 protein showed some sequence similarities to the N-terminus (up to about 30 amino acid residues) of the Hyaluronic acid synthases from various Streptococci. 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. Interestingly, we find that this large protein does make a polysaccharide, although the polysaccharide appears to be a Chitin-like material (repeating units of b-1-4 N-acetyl glucosamine) and not hyaluronic acid. 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 as well as UDPgalactose as its substrate. Our results with the partially purified enzyme also suggests that UDPgalactose is a better substrate than UDPglucose. Therefore, we are currently investigating the substrate specificity of the dehydrogenase. Results of our studies also suggest that the synthase might indeed catalyze the formation of a polysaccharide composed of N-acetyl glucosamine and galactose or galacturonic acid. Therefore, we are utilizing 14C-labeled substrates to identify the substrate used by the synthase and to study the chemical nature the product. Polysaccharide made up of galactose and N-acetyl glucosamine has been observed to be a part of cell surface antigen of B. anthracis. Therefore, we are also investigating to see if the protein products of ORF 93 to 95 are capable of forming this polysaccharide.
