Method for overexpression of zwitterionic polysaccharides

#The invention relates to compositions and methods for the production and isolation of capsular polysaccharides which have been reported to have immunomodulatory effects. Specifically, the invention relates to compositions and methods for the production and isolation of capsular polysaccharide A (PSA) of Bacteroides fragilis (B. fragilis).

#Capsular polysaccharide A (PSA) of Bacteroides fragilis NCTC9343 has been reported to be an immunomodulator with therapeutic and preventative applications. U.S. Pat. Nos. 5,679,654 and 5,700,787; Tzianabos A O et al. (2000) J Biol Chem 275:6733-40. It was recently reported that in addition to PSA, B. fragilis NCTC9343 synthesizes at least seven other capsular polysaccharide (PSB-PSH). Krinos C M et al. (2001) Nature 414:555-558. It has also recently been reported that expression of seven of these eight capsular polysaccharides of B. fragilis are variable due to phase variation dictated by inversion of DNA segments containing the promoters of each of the polysaccharide biosynthesis loci. Krinos C M et al., supra. The fact that this strain synthesizes so many polysaccharides makes purification of PSA from this strain very laborious. This, coupled with the variable expression of PSA due to its phase variation, often results in a very low yield of PSA following extensive procedures for its purification. Scaled-up purification of PSA for preventative or therapeutic applications thus presents technical obstacles.

#The invention arises in part from the discovery by the present inventors of methods for controlling the phase variation of seven of the eight known capsular polysaccharides of B. fragilis, PSA, PSB, PSD, PSE, PSF, PSG, and PSH. It was surprisingly discovered that inactivation of a novel gene, res02 (also denoted mpi, multiple promoter invertase), has the effect of locking the invertible promoters of each of the capsular polysaccharide biosynthesis loci in either an “on” orientation or an “off” orientation. When an invertible promoter of a capsular polysaccharide is in an on orientation, the promoter is transcriptionally active with respect to the polysaccharide biosynthesis locus associated with the biosynthesis of the capsular polysaccharide. Conversely, when an invertible promoter of a capsular polysaccharide is in an off orientation, the promoter is transcriptionally inactive with respect to the polysaccharide biosynthesis locus associated with the biosynthesis of the capsular polysaccharide. By locking the promoters and then selecting for bacterial cells constitutively expressing only a particular capsular polysaccharide, or expressing only a restricted set of capsular polysaccharides including the particular capsular polysaccharide, it was discovered according to the instant invention that it was now possible to increase the yield of the particular capsular polysaccharide. Thus, it was discovered according to the instant invention that it is now possible to generate and select mutant strains of B. fragilis that constitutively express any combination of selected capsular polysaccharides and not others. Furthermore, using this method it is now possible to generate and select mutant strains of B. fragilis that constitutively express only one particular capsular polysaccharide, e.g., PSA, and no other capsular polysaccharide. Because each selected mutant strain is rendered incapable of phase variation, the amount of selected polysaccharide, e.g., PSA, made by each cell is increased compared to wild type. In addition, because a selected mutant strain expresses only a single capsular polysaccharide, or it expresses only a desired combination of capsular polysaccharides, purification of capsular polysaccharide is greatly simplified and more efficient.

#FIG. 1 is the nucleotide sequence of res02 (SEQ ID NO:1).

#FIG. 2 is the deduced amino acid sequence of res02 (SEQ ID NO:2).

#FIG. 3 is the nucleotide sequence of inv19 (SEQ ID NO:3).

#The technology described in this application has utilized the genetic information that the inventors have discovered about B. fragilis NCTC9343 to circumvent both the difficulty of purification of one capsular polysacharide from the other seven polysaccharides, and the low yield of a given capsular polysaccharide due to the phase variation in its expression. Importantly, the technology described in this application circumvents both the difficulty of purification of PSA from the other seven polysaccharides, and the low yield of PSA due to the phase variation in its expression.

#In featured aspects of the invention, res02 is inactivated so that phase variation of the capsular polysaccharides is eliminated. The lack of res02 activity renders the invertible promoters for the polysaccharide biosynthesis loci locked in either the on or the off orientation. Selection of particular phenotypes then permits selection of mutants with the promoter of a selected capsular polysaccharide locked on and the promoters of other capsular polysaccharides locked off.

#The terms “res02”, “res02 gene”, and “res02 nucleic acid molecule” refer to a nucleic acid molecule comprising or consisting of a nucleotide sequence as set forth in SEQ ID NO:1, a nucleotide sequence encoding the polypeptide as set forth in SEQ ID NO:2, and nucleic acid molecules as defined herein.