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

Abstract

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...

Interaction Network and Localization of Brucella abortus Membrane Proteins Involved in the Synthesis, Transport, and Succinylation of Cyclic β-1,2-Glucans

“…S3A in the supplemental material). In agreement with these results, we previously obtained and characterized Cgs mutants with in-frame pentapeptide insertions at positions 160 and 302, flanking the coiled-coil domain identified in this work, that also produced C␤G with a high DP (14). These results suggest that the Cgs amino-terminal region may interact with the cyclization and/or phosphorylase domains located at the carboxy terminus, thus allowing the enzyme to acquire a proper spatial conformation suitable for catalysis and control of C␤G DP.…”

“…The Cgs region from amino acid residues 1 to 1544 constitutes the minimal region required for catalysis of the initiation, elongation, and cyclization reactions; thus, it is a polyfunctional region by itself in which each reaction is catalyzed by a different discrete domain (14). In this region, we previously identified the glycosyltransferase domain GT-84 (Cgs positions 475 to 818), which catalyzes the initiation and elongation reactions (12), and the cyclization domain (Cgs positions 991 to 1544) (14). The carboxylterminal region (residues 1545 to 2867) displays the ␤-1,2-glucooligosaccharide phosphorylase activity through which Cgs controls the DP of the glucooligosaccharide protein-linked intermediate (13).…”

“…The glucose-removing activity catalyzed by the ␤-1,2-glucooligosaccharide phosphorylase domain (Cgs residues 1545 to 2867) counteracts the Cgs elongation reaction, thus controlling the length of the ␤-1,2-glucoologosaccharide protein-linked intermediate (13). The cyclization reaction catalyzed by the Cgs region from positions 991 to 1544 puts an end to the balance between the elongation and phosphorolysis reactions, the linear ␤-1,2-glucooligosaccharide protein-linked intermediate is cyclized, and C␤G (with the appropriate ring size) are released from the protein to the cytoplasm (14). Once in the cytoplasm, C␤G are transported to the periplasm by the cyclic glucan transporter Cgt, an ABC transporter of 66 kDa with six predicted TMSs (9).…”

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

“…It will be interesting to determine whether all Proteobacteria with orthologous N-glycan pathways also produce fOS and whether this unique mechanism to cope with salt stress has provided evolutionary pressure to maintain the N-glycosylation pathway in these organisms. Also, comparisons are warranted between the N-glycosylation pathway and OPG biosynthesis in some Proteobacteria that use a remarkable mechanism of cyclic glucan production in which one polyfunctional protein of Ϸ320 kDa is responsible for covalent attachment of nucleotide-activated Glc onto itself, elongation, polymerization control, flipping, cyclization, and release (49).…”

Cyclic β-glucans at the bacteria-host cells interphase: One sugar ring to rule them all

“…The cyclization leaves one or more glucose residues linked to Cgs serving as a primer for the next round of CβG synthesis (Guidolin et al, 2009). Once the cyclization and the release of CβG from the synthase have occurred, the newly formed CβG is transported to the periplasmic space by an ABC type transporter that uses ATP hydrolysis as a driving force.…”