Brucella abortus Cyclic β-1,2-Glucan Mutants Have Reduced Virulence in Mice and Are Defective in Intracellular Replication in HeLa Cells

Abstract

Null cyclic ␤-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 ␤-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.Brucella abortus is an intracellular pathogen that causes abortion in bovines and can infect humans. Abortion in cattle is the consequence of the tropism that the bacterium has for the placenta of pregnant animals, in which it multiplies intracellularly (10). Brucellosis in humans is primarily a disease of the reticuloendothelial system, in which the bacteria multiply inside the phagocytic cell; the intermittent release of bacteria from the cells into the bloodstream causes undulant fever (17, 29). Brucellosis does not spread among humans; consequently, eradication of the disease from the natural reservoirs, cattle, pigs, sheep, goats, and other susceptible animals, will lead to elimination of human infection. In regions with high prevalence of the disease, the only way of controlling and eventually eradicating this zoonosis is by vaccination of all susceptible hosts and elimination of infected animals.Vaccination represents an important tool for the control of bovine brucellosis. One of the most used vaccines is the attenuated strain B. abortus S19 obtained spontaneously from the virulent strain B. abortus 2308 (24, 25, 26, 29). Live attenuated B. abortus S19 has served for many years as an effective vaccine to prevent brucellosis in cattle (8,18). The genetic defect that leads to attenuation of this strain has not yet been defined. B. abortus S19 has lost some essential unknown mechanism of virulence. Despite this fact, the vaccinal strain conserves some degree of virulence, being pathogenic for humans (37), and...

Osmoregulated periplasmic glucans of Salmonella enterica serovar Typhimurium are required for optimal virulence in mice

“…No change in colony morphology or change in sensitivity towards bile salts was observed for strain SG111 (Fig S1a). OPG synthesis in Brucella sp., which are nonmotile, is not under osmotic control, but a defect in the opgG gene results in increased sensitivity to surfactants (SDS and deoxycholic acid) (Arellano-Reynoso et al, 2005;Briones et al, 2001), indicating cell-surface alterations, a phenotype also not observed in strain SG111 (Fig S2b). The other significant component of Gram-negative bacteria, the enterobacterial common antigen, has been proposed to play an important role in virulence by protecting the pathogen from bile salts (Ramos-Morales et al, 2003).…”

Study of free oligosaccharides derived from the bacterial N -glycosylation pathway

“…Although we cannot draw a conclusion about the exact release mechanism, the linear nature of C. jejuni fOS most likely excludes the mechanism known from cyclic glucan synthesis in other Proteobacteria that was suggested to be coupled to cyclization (10,35). The absence of a eukaryotic PNGaseF-like protein that would release fOS in C. jejuni, and the absence of a gene encoding a pyrophosphatase homolog protein (36) favors a mechanism proposed for the eukaryotic OTase that, in the absence of sufficient acceptor sequences, the OTase exhibits hydrolytic activity and transfers OSs from their lipid-linked intermediates to water (15,16).…”

“…While these glycans are comprised exclusively of glucose, they can be structurally diverse, and four distinct families were defined based on variations in chain length, glycosyl linkage, branching pattern, and presence of modifications (1). In addition, periplasmic glucans were reported to be important for biofilm development (3), antibiotic resistance (4), host-pathogen and plant-microbe interactions (5)(6)(7)(8)(9), as well as intracellular multiplication (10). Free oligosaccharides (fOS) have also been described in mammalian cells, yeast, plants, and fish (11).…”

A glycosyltransferase with a length-controlling activity as a mechanism to regulate the size of polysaccharides

“…Brucella controls the maturation of its vacuole to avoid innate immune host cell responses, such as lysosome fusion, and to reach its replicative niche (19). B. abortus C␤G-deficient mutants are unable to prevent phagosome-lysosome fusion and are significantly defective in intracellular multiplication in professional and nonprofessional phagocytic cells (7,9). C␤G play a major role in circumventing host cell defense by interacting with cholesterol and reorganizing lipids rafts of host cell membranes (9).…”

“…Cyclic ␤-1,2-glucan synthase (Cgs), the enzyme responsible for the synthesis of cyclic ␤-1,2-glucans (C␤G), is present in a restricted number of symbiotic or pathogenic bacteria, most of them belonging to the ␣-proteobacteria group, in which C␤G are a symbiotic or virulence factor required for successful host interaction (4)(5)(6)(7)(8)(9). Brucella abortus Cgs is a 320-kDa (2,867 amino acid residues) polytopic integral inner membrane protein with six transmembrane-spanning segments (TMSs) and with the N and C termini located on the cytoplasmic side of the membrane (10).…”