Membrane Topology Analysis of Cyclic Glucan Synthase, a Virulence Determinant of
            <i>Brucella abortus</i>

Ciocchini, Andrés E.; Roset, Mara S.; Iannino, Nora Iñón de; Ugalde, Rodolfo A.

doi:10.1128/jb.186.21.7205-7213.2004

Cited by 20 publications

(10 citation statements)

References 45 publications

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“…Conversely, a 9-kb gene encoding Cgs, the cyclic ␤1-2-glucan synthetase (BAB1_0108) known to be involved in virulence (5,8,21), is strictly conserved in the three species. Thus, it is tempting to speculate that the set of genes absent from B. abortus are involved in species-specific host recognition and dispensable in this species, while the cyclic glucan synthase, a protein required for the synthesis of a virulence FIG.…”

Section: Resultsmentioning

confidence: 99%

Whole-Genome Analyses of Speciation Events in Pathogenic Brucellae

et al. 2005

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Despite their high DNA identity and a proposal to group classical Brucella species as biovars of Brucella melitensis, the commonly recognized Brucella species can be distinguished by distinct biochemical and fatty acid characters, as well as by a marked host range (e.g., Brucella suis for swine, B. melitensis for sheep and goats, and Brucella abortus for cattle). Here we present the genome of B. abortus 2308, the virulent prototype biovar 1 strain, and its comparison to the two other human pathogenic Brucella species and to B. abortus field isolate 9-941. The global distribution of pseudogenes, deletions, and insertions supports previous indications that B. abortus and B. melitensis share a common ancestor that diverged from B. suis. With the exception of a dozen genes, the genetic complements of both B. abortus strains are identical, whereas the three species differ in gene content and pseudogenes. The pattern of species-specific gene inactivations affecting transcriptional regulators and outer membrane proteins suggests that these inactivations may play an important role in the establishment of host specificity and may have been a primary driver of speciation in the genus Brucella. Despite being nonmotile, the brucellae contain flagellum gene clusters and display species-specific flagellar gene inactivations, which lead to the putative generation of different versions of flagellum-derived structures and may contribute to differences in host specificity and virulence. Metabolic changes such as the lack of complete metabolic pathways for the synthesis of numerous compounds (e.g., glycogen, biotin, NAD, and choline) are consistent with adaptation of brucellae to an intracellular life-style.Brucellosis is a major infectious disease affecting animals and humans (11). Several Brucella species, such as Brucella abortus, Brucella melitensis, and Brucella suis, have been isolated from a variety of animals. Of these, B. abortus, the causative agent of bovine brucellosis, is the most widespread (11). All three Brucella species cause a severe human disease characterized in its acute phase by undulant fever and in its chronic phase by localization of the pathogen and damage of different organs (11,38). If localized in the brain or the heart, this can result in fatal meningitis or fatal endocarditis, respectively. Brucella infection is treated with a combination of antibiotics; however, in its chronic phase, eradication is difficult since Brucella spp. are intracellular pathogens, which puts them out of reach of humoral immunity and several antibiotics (25, 38). The lack of a safe and efficacious human vaccine underscores the importance of understanding the biology of brucellosis to develop human vaccines and effective therapeutic agents. Furthermore, all three Brucella species are listed as potential bioweapons by the Centers for Disease Control and Prevention (22,26). This is due to the highly infectious nature of all three species, the fact that they can be readily aerosolized, and the fact that an outbreak would be dif...

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Section: Resultsmentioning

confidence: 99%

Whole-Genome Analyses of Speciation Events in Pathogenic Brucellae

et al. 2005

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“…Whole-cell extracts of the corresponding strains were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (21), followed by electrophoretic transfer onto a nitrocellulose membrane (Inmobilon-NC; Millipore). Immunoblotting was performed as previously described (9) with a specific polyclonal antiserum against the Cgs region comprising amino acid residues 540 to 801 (13).…”

Section: Methodsmentioning

confidence: 99%

“…Brucella abortus Cgs is a 320-kDa (2,867-amino-acid-residue) polytopic integral inner membrane protein with six transmembrane-spanning segments (TMSs), which define three small periplasmic loops and four large cytoplasmic regions delimited by amino acid residues 1 to 418, 475 to 818, 870 to 938, and 991 to 2867 (13). Cgs is a polyfunctional modular protein in which two regions can be recognized: an N-terminal region (amino acid residues 1 to 1544) and a C-terminal region (residues 1545 to 2867) (11).…”

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Functional Mapping of Brucella abortus Cyclic β-1,2-Glucan Synthase: Identification of the Protein Domain Required for Cyclization

et al. 2009

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Cyclic ␤-1,2-glucans (C␤G) are periplasmic homopolysaccharides that have been shown to play an important role in several symbiotic and pathogenic relationships. Cyclic ␤-1,2-glucan synthase (Cgs), the enzyme responsible for the synthesis of C␤G, is an integral membrane polyfunctional protein that catalyzes the four enzymatic activities (initiation, elongation, phosphorolysis, and cyclization) required for the synthesis of C␤G. Recently, we have identified the glycosyltransferase and the ␤-1,2-glucooligosaccharide phosphorylase domains of Brucella abortus Cgs. In this study, we performed large-scale linker-scanning mutagenesis to gain further insight into the functional domains of Cgs. This analysis allowed us to construct a functional map of the enzyme and led to the identification of the minimal region required for the catalysis of initiation and elongation reactions. In addition, we identified the Cgs region (residues 991 to 1544) as being the protein domain required for cyclization and demonstrated that upon cyclization and releasing of the C␤G, one or more glucose residues remain attached to the protein intermediate that serves as a primer for the next round of C␤G synthesis. Finally, our results indicate that the overall control of the degree of polymerization of C␤G is the result of a balance between elongation, phosphorolysis, and cyclization reactions.Osmoregulated periplasmic glucans are cyclic, branched cyclic, or branched linear oligosaccharides present in the periplasm of certain gram-negative bacteria. Common features of these oligosaccharides are the presence of glucose as the sole sugar constituent and the regulation of their synthesis by the osmolarity of the growth medium (5). Agrobacterium, Rhizobium, Sinorhizobium, and Brucella species synthesize osmoregulated periplasmic glucans of family II. Glucans of this family have 17-to 25-glucose-residue cyclic ␤-1,2-backbones substituted with sn-1-phosphoglycerol, succinic acid, methylmalonic acid, or a combination of them (5,6,25).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 alphaproteobacteria, in which C␤G are a symbiotic or virulence factor required for successful host interactions (4,17,24). In Brucella abortus, the etiological agent of bovine brucellosis, the synthesis and transport of C␤G to the periplasmic space are required for the complete expression of virulence (7,19,25,26). Furthermore, C␤G plays a major role in circumventing host cell defense. Brucella C␤G act on host cell membranes at the level of lipid rafts, controlling vacuole maturation, avoiding lysosome fusion, and allowing Brucella to reach an endoplasmic reticulum-derived vacuole permissive for bacterial replication (4). Thus, C␤G is a Brucella virulence factor required for intracellular survival; accordingly, Cgs may be a good target for developing new chemotherapy alternatives against this pathogen.Cgs catalyzes the...

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“…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). Cgs, an enzyme using UDP-glucose as sugar donor and Mg 2ϩ as cofactor, functions as an inverting processive ␤-1,2-glucosyltransferase that catalyzes the three enzymatic activities (initiation, elongation, and cyclization) required for synthesis of C␤G.…”

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

Ciocchini

Guidolin

Casabuono

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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Cyclic ␤-1,2-glucans (C␤G) are osmolyte homopolysaccharides with a cyclic ␤-1,2-backbone of 17-25 glucose residues present in the periplasmic space of several bacteria. Initiation, elongation, and cyclization, the three distinctive reactions required for building the cyclic structure, are catalyzed by the same protein, the C␤G synthase. The initiation activity catalyzes the transference of the first glucose from UDP-glucose to a yet-unidentified amino acid residue in the same protein. Elongation proceeds by the successive addition of glucose residues from UDP-glucose to the nonreducing end of the protein-linked ␤-1,2-oligosaccharide intermediate. Finally, the protein-linked intermediate is cyclized, and the cyclic glucan is released from the protein. These reactions do not explain, however, the mechanism by which the number of glucose residues in the cyclic structure is controlled. We now report that control of the degree of polymerization (DP) is carried out by a ␤-1,2-glucan phosphorylase present at the C␤G synthase C-terminal domain. This last activity catalyzes the phosphorolysis of the ␤-1,2-glucosidic bond at the nonreducing end of the linear protein-linked intermediate, releasing glucose 1-phosphate. The DP is thus regulated by this ''lengthcontrolling'' phosphorylase activity. To our knowledge, this is the first description of a control of the DP of homopolysaccharides.cyclic ␤-1,2-glucan ͉ phosphorylase ͉ size control O smoregulated periplasmic glucans are oligosaccharides present in the periplasm of certain Gram-negative bacteria. Common features of these oligosaccharides are the presence of glucose as the sole sugar constituent and the regulation of their synthesis by the osmolarity of the growth media. Osmoregulated periplasmic glucans may be cyclic, branched cyclic, or branched linear and, depending on the species, may be substituted with a variety of nonglycosidic residues (1, 2). Agrobacterium, Rhizobium, Sinorhizobium, and Brucella species synthesize osmoregulated periplasmic glucans of family II. Glucans of this family have 17-25 glucose residue cyclic ␤-1,2-backbones substituted with sn-1-phosphoglycerol, succinic acid, methylmalonic acid, or a combination of them (1-3).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 (4-9). 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). Cgs, an enzyme using UDP-glucose as sugar donor and Mg 2ϩ as cofactor, functions as an inverting processive ␤-1,2-glucosyltransferase that catalyzes the three enzymatic activities (initiation, elongation, and cyclization) required for synthesis of C␤G. Synthesis is initiated...

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Membrane Topology Analysis of Cyclic Glucan Synthase, a Virulence Determinant of Brucella abortus

Cited by 20 publications

References 45 publications

Whole-Genome Analyses of Speciation Events in Pathogenic Brucellae

Whole-Genome Analyses of Speciation Events in Pathogenic Brucellae

Functional Mapping of Brucella abortus Cyclic β-1,2-Glucan Synthase: Identification of the Protein Domain Required for Cyclization

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

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