Post-assembly Modification of Bordetella bronchiseptica O Polysaccharide by a Novel Periplasmic Enzyme Encoded by wbmE

Abstract

Bordetella bronchiseptica is a pathogen of humans and animals that colonizes the respiratory tract. It produces a lipopolysaccharide O antigen that contains a homopolymer of 2,3-dideoxy-2,3-diacetamido-L-galacturonic acid (L-GalNAc3NAcA). Some of these sugars are found in the uronamide form (L-GalNAc3NAcAN), and there is no discernible pattern in the distribution of amides along the chain. A B. bronchiseptica wbmE mutant expresses an O polysaccharide unusually rich in uronamides. The WbmE protein localizes to the periplasm and catalyzes the deamidation of uronamide-rich O chains in lipopolysaccharide purified from the mutant, to attain a wildtype uronamide/uronic acid ratio. WbmE is a member of the papain-like transglutaminase superfamily, and this categorization is consistent with a deamidase role. The periplasmic location of WbmE and its acceptance of complete lipopolysaccharide as substrate indicate that it operates at a late stage in lipopolysaccharide biosynthesis, after polymerization and export of the O chain from the cytoplasm. This is the first report of such a modification of O antigen after assembly. The expression of wbmE is controlled by the Bordetella virulence gene two-component regulatory system, BvgAS, suggesting that this deamidation is a novel mechanism by which these bacteria modify their cell surface charge in response to environmental stimuli.Bordetella bronchiseptica is a Gram-negative coccobacillus, which colonizes the mammalian respiratory tract. It has a broad host range and is commonly associated with atrophic rhinitis in pigs (1) and infectious tracheobronchitis (kennel cough) in dogs (2). Most of the genes implicated in host colonization and virulence are under the transcriptional control of the two-component regulatory system BvgAS (reviewed in Ref. One of the Bvg-regulated bacterial structures is lipopolysaccharide (LPS).3 LPS is the major component of the outer leaflet of the outer membrane. LPS has three domains: first, Lipid A is the lipophilic domain that anchors LPS into the outer membrane; second, a complex, branched-chain oligosaccharide known as core is attached to lipid A and in Bordetella the lipid A-core structure is known as B-band LPS; third, a domain distal to the membrane consisting of saccharide repeats may be present, which is commonly called O antigen. In a proportion of B. bronchisepica LPS molecules the lipid A core is substituted with a trisaccharide, and this species is known as A-band LPS. The O polysaccharide consists of a homopolymer of 2,3-dideoxy-2,3-diacetamido-L-galacturonic acid (L-GalNAc3NAcA) (7) capped at the nonreducing terminus with a complex 2,3,4-trideoxy-2,3,4-triamino galacturonamide (GalN3N4NAN) derivative (8), and in B. bronchiseptica is attached to the A-band trisaccharide via a pentasaccharide linker (Fig. 1) (9). Expression of O antigen by B. bronchiseptica is required for full virulence in animal models of infection and for resistance to complement-mediated killing (10).A proportion of the O polysaccharide repeating units are present as ...

TgpA, a Protein with a Eukaryotic-Like Transglutaminase Domain, Plays a Critical Role in the Viability of Pseudomonas aeruginosa

“…Catalysis of this reaction involves a catalytic triad consisting of cysteine, histidine and aspartate residues that are well-conserved in eukaryotic and prokaryotic TGases [17]. First of all, we verified the conservation of the catalytic triad in the PA2873 TG domain by sequence alignment with characterized members of the TGase superfamily: the human coagulation factor XIII conserved domain [26], TGase from red sea bream liver (fish-derived transglutaminase, FTG) [27] and WbmE, a periplasmic TGase of Bordetella bronchiseptica [28] involved in the post-assembly modification of LPS O-antigen. As shown in Figure 2A, the catalytic triad of PA2873 TG domain appeared to be well-conserved.…”

“…In the Methanothermobacter species, prophage proteins PeiW and PeiP act as pseudomurein endoisopeptidases [33], [34]. In the periplasm of B. bronchiseptica , WbmE protein catalyzes the deamidation of uronamide-rich O chains of lipopolysaccharide (LPS) [28]. It is conceivable that TgpA activity can participate in cell wall functions such as i) assembly of peptidoglycan structures ii) maturation/secretion of key periplasmic proteins iii) assembly of surface polypeptide structures iv) biogenesis/maturation of LPS.…”

BpsR Modulates Bordetella Biofilm Formation by Negatively Regulating the Expression of the Bps Polysaccharide

“…In Bordetella, the expression of lipopolysaccharide (LPS) and a putative type II capsular operon is regulated by the BvgAS signal transduction system (34,44). Many other bacterial species have also evolved complex multifactorial mechanisms to regulate various exopolysaccharides, which are often crucial for an organism's transition from planktonic to biofilm growth and for its pathogenesis (3,26,46,50,59,62).…”

Bordetella parapertussis PagP Mediates the Addition of Two Palmitates to the Lipopolysaccharide Lipid A

“…1, lane 1). In the Bvg Ϫ phase, the O PS migrated as a compacted series of bands due to the action of the Bvg Ϫ -phase enzyme WbmE, which deaminates some of the O PS uronamide sugars (27). In addition, the core-lipid A LPS was present as a single intense band and just one or two other very faint bands, suggesting that a restricted number of core-lipid A structures were expressed in the minus phase compared to the plus phase (Fig.…”