Brucella abortus cyclic beta-1,2-glucan mutants have reduced virulence in mice and are defective in intracellular replication in HeLa cells.

Null cyclic beta-1,2-glucan synthetase mutants (cgs mutants) were obtained from Brucella abortus virulent strain 2308 and from B. abortus attenuated vaccinal strain S19. Both mutants show greater sensitivity to surfactants like deoxycholic acid, sodium dodecyl sulfate, and Zwittergent than the parental strains, suggesting cell surface alterations. Although not to the same extent, both mutants display reduced virulence in mice and defective intracellular multiplication in HeLa cells. The B. abortus S19 cgs mutant was completely cleared from the spleens of mice after 4 weeks, while the 2308 mutant showed a 1.5-log reduction of the number of brucellae isolated from the spleens after 12 weeks. These results suggest that cyclic beta-1,2-glucan plays an important role in the residual virulence of the attenuated B. abortus S19 strain. Although the cgs mutant was cleared from the spleens earlier than the wild-type parental strain (B. abortus S19) and produced less inflammatory response, its ability to confer protection against the virulent strain B. abortus 2308 was fully retained. Equivalent levels of induction of spleen gamma interferon mRNA and anti-lipopolysaccharide (LPS) of immunoglobulin G2a (IgG2a) subtype antibodies were observed in mice injected with B. abortus S19 or the cgs mutant. However, the titer of anti-LPS antibodies of the IgG1 subtype induced by the cgs mutant was lower than that observed with the parental S19 strain, thus suggesting that the cgs mutant induces a relatively exclusive Th1 response.

Molecular cloning and characterization of cgs, the Brucella abortus cyclic beta(1-2) glucan synthetase gene: genetic complementation of Rhizobium meliloti ndvB and Agrobacterium tumefaciens chvB mutants.

The animal pathogen Brucella abortus contains a gene, cgs, that complemented a Rhizobium meliloti nodule development (ndvB) mutant and an Agrobacterium tumefaciens chromosomal virulence (chvB) mutant. The complemented strains recovered the synthesis of cyclic beta(1-2) glucan, motility, virulence in A. tumefaciens, and nitrogen fixation in R. meliloti; all traits were strictly associated with the presence of an active cyclic beta(1-2) glucan synthetase protein in the membranes. Nucleotide sequencing revealed the presence in B. abortus of an 8.49-kb open reading frame coding for a predicted membrane protein of 2,831 amino acids (316.2 kDa) and with 51% identity to R. meliloti NdvB. Four regions of the B. abortus protein spanning amino acids 520 to 800, 1025 to 1124, 1284 to 1526, and 2400 to 2660 displayed similarities of higher than 80% with R. meliloti NdvB. Tn3-HoHo1 mutagenesis showed that the C-terminal 825 amino acids of the Brucella protein, although highly conserved in Rhizobium, are not necessary for cyclic beta(1-2) glucan synthesis. Confirmation of the identity of this protein as B. abortus cyclic beta(1-2) glucan synthetase was done by the construction of a B. abortus Tn3-HoHo1 insertion mutant that does not form cyclic beta(1-2) glucan and lacks the 316.2-kDa membrane protein. The recovery of this mutant from the spleens of inoculated mice was decreased by 3 orders of magnitude compared with that of the parental strain; this result suggests that cyclic beta(1-2) glucan may be a virulence factor in Brucella infection.

Characterization of the biosynthesis of beta(1-2) cyclic glucan in R. Fredii. Beta(1-2) glucan has no apparent role in nodule invasion of Mc Call and Peking soybean cultivars.

Three wild type strains of Rhizobium fredii, USDA 191, USDA 257 and HH 303, do not synthesize in vivo or in vitro beta(1-3), beta(1-6) cyclic glucans, all strains form in vitro and in vivo cyclic beta(1-2) glucans. Approximately 80% of the recovered R. fredii cellular cyclic beta(1-2) glucans were anionic and the substituent was identified as phosphoglycerol. Inner membranes prepared from these R. fredii strains have a beta(1-2) glucan-intermediate-protein with apparent molecular mass undistinguishable from Agrobacterium tumefaciens beta(1-2) glucan intermediate protein. Studies of the degree of polymerization of the oligosaccharides recovered from the protein-intermediate after short pulse incubations with UDP-14C-glucose suggested that the rate limiting step in the biosynthesis of cyclic glucan is cyclization. Kinetic studies revealed that the K(m) for UDP-glucose was 0.33 mM. No difference was detected between the K(m) for initiation/elongation and cyclization reactions. Nodulation studies of a ndvB R. fredii mutant with Mc Call and Peking soybean cultivars, revealed that beta(1-2) glucans do not seem to be required for normal nodule invasion of these soybean cultivars.

The in vitro biosynthesis of the exopolysaccharide produced by Rhizobium leguminosarum bv. trifolii, strain NA 30.

Rhizobium leguminosarum bv. trifolii, strain NA 30 nodulates both red (Trifolium pratense) and white (T. repens) clover and produces an acidic exopolysaccharide (EPS) containing glucose, galactose, glucuronic acid, acetate and ketalpyruvate residues in a 5:1:2:1:2 molar ratio. The in vitro synthesis of this EPS as well as the characterization of five structurally related lipid linked oligosaccharides is described employing EDTA treated cells as enzyme preparation and 14C-labelled UDP-Glc, UDP-GlcA, Acetyl CoA and phosphoenol pyruvate (PEP) and doubly labelled 32P UDP-14C-Glc as precursors. The lipidic derivatives, extracted with chloroform, methanol, water (1:2:0,3) had the properties expected for prenyl-diphospho-sugars, as judged by the pattern of labelling, DEAE cellulose column chromatography, catalytic reduction and acid lability, etc. The sugar moieties of these phosphoprenyl derivatives were identified as the acetylated octasaccharide repeating unit, its mono- and di-ketalpyruvate derivatives and two trisaccharides, one of them acetylated, on the basis of specific labelling, gel filtration, paper electrophoresis and chromatography, TLC, permethylation, etc. In vitro polymer synthesis was greatly increased when electroporated cells were substituted for EDTA treated cells as enzyme system.

New osmoregulated beta(1-3),beta(1-6) glucosyltransferase(s) in Azospirillum brasilense.

A linear beta(1-3),beta(1-6) glucan was detected in the periplasm of Azospirillum brasilense cells growing in a medium of low osmotic strength. This glucan was produced in vitro by purified bacterial inner membranes with UDP-glucose as the sugar donor in the presence of Mg2+. Growth in a high-osmotic-strength medium strongly reduced the amount of this glucan accumulated in the periplasmic space, and the inhibition was associated with a reduction in the enzymatic activity of the beta(1-3),beta(1-6) glucosyltransferase(s).

Expression of the Agrobacterium tumefaciens chvB virulence region in Azospirillum spp.

Inner membranes of Azospirillum brasilense incubated with UDP-glucose were unable to synthesize beta-(1-2) glucan and lacked the 235-kilodalton intermediate protein known to be involved in the synthesis of beta-(1-2) glucan in Agrobacterium tumefaciens and Rhizobium meliloti. Inner membranes of A. brasilense strains carrying a cosmid containing the chromosomal virulence genes chvA and chvB of Agrobacterium tumefaciens formed beta-(1-2) glucan in vitro and synthesized the 235-kilodalton intermediate protein. No DNA homology to the chvB region was found in different wild-type strains of A. brasilense, but the introduction of a cosmid containing the Agrobacterium tumefaciens chvA and chvB regions yielded strains in which DNA hybridization with the chvB region was detected, provided that the strains were grown under an antibiotic selective pressure.

A biochemical chimera suggesting the existence of at least two dolichol-P-glucose:dolichol-P-P-oligosaccharide glucosyltransferases.

As previously reported, incubation of liver dolichol-P, UDP-[14C]Gal, and a particulate preparation of Acetobacter xylinum leads to the synthesis of dolichol-P-[14C]Gal (P. Romero, R. Garcia, and M. Dankert (1977) Mol. Cell. Biochem. 16, 205-212). It is now reported that upon incubation of the latter with rat liver microsomes, [14C-galactose]-Gal1Man9GlcNAc2-P-P-dolichol and [14C-galactose]Gal1Glc1Man9GlcNAc2-P-P-dolichol are formed. The galactosyl residues appeared to be (1,3)-linked in the same positions as the glucose units in the respective physiological compounds. No lipid-linked Gal1Glc2Man9GlcNAc2 was formed, thus strongly suggesting the presence of at least two dolichol-P-Glc:dolichol-P-P-oligosaccharide glucosyltransferases, only one of which is able to use dolichol-P-Gal as substrate. Incubation of the galactosylated dolichol-P-P derivatives with rat liver microsomes led to the transfer of the oligosaccharides to microsomal proteins. No endogenous, membrane-bound glycosidases were able to remove the galactose residues but mannose units were excised by endogenous neutral mannosidases.