Glycosylation of the collagen adhesin EmaA of Aggregatibacter actinomycetemcomitans is dependent upon the lipopolysaccharide biosynthetic pathway

Abstract

The human oropharyngeal pathogen Aggregatibacter actinomycetemcomitans synthesizes multiple adhesins, including the nonfimbrial extracellular matrix protein adhesin A (EmaA). EmaA monomers trimerize to form antennae-like structures on the surface of the bacterium, which are required for collagen binding. Two forms of the protein have been identified, which are suggested to be linked with the type of O-polysaccharide (O-PS) of the lipopolysaccharide (LPS) synthesized (G. Tang et al., Microbiology 153:2447-2457, 2007). This association was investigated by generating individual mutants for a rhamnose sugar biosynthetic enzyme (rmlC; TDP-4-keto-6-deoxy-d-glucose 3,5-epimerase), the ATP binding cassette (ABC) sugar transport protein (wzt), and the O-antigen ligase (waaL). All three mutants produced reduced amounts of O-PS, and the EmaA monomers in these mutants displayed a change in their electrophoretic mobility and aggregation state, as observed in sodium dodecyl sulfate (SDS)-polyacrylamide gels. The modification of EmaA with O-PS sugars was suggested by lectin blots, using the fucose-specific Lens culinaris agglutinin (LCA). Fucose is one of the glycan components of serotype b O-PS. The rmlC mutant strain expressing the modified EmaA protein demonstrated reduced collagen adhesion using an in vitro rabbit heart valve model, suggesting a role for the glycoconjugant in collagen binding. These data provide experimental evidence for the glycosylation of an oligomeric, coiled-coil adhesin and for the dependence of the posttranslational modification of EmaA on the LPS biosynthetic machinery in A. actinomycetemcomitans.

FIG. 1.

(A) O-PS structure of serotype b A. actinomycetemcomitans. (B) Silver-stained 5 to 15% polyacrylamide-SDS gel of serotype b LPS. A total of 1.0 ml of mid-logarithmic-phase cells were collected and lysed. Three lysates from each strain were combined and treated with proteinase K at 60°C for 60 min before electrophoresis, followed by silver staining. C, control: whole-cell lysate without proteinase K digestion; WT, wild type (VT1169); emaA, extracellular matrix protein adhesin A mutant; rmlC, rhamnose epimerase mutant; wzt, ATP-binding cassette sugar transport mutant. The dark brown staining of the high molecular weight (75,000 to 250,000) corresponds to polymerized O-PS.

FIG. 2.

Analyses of purified LPS from O-PS mutants by ELISA. The phenol-water-extracted LPS samples were adsorbed to wells of 96-well microtiter plates and detected using purified rabbit anti-A. actinomycetemcomitans antibodies. (A) WT, wild type (VT1169); rmlC, rhamnose epimerase mutant; rmlC/rlmC⁺, rhamnose epimerase complemented. (B) waaL, O-antigen ligase mutant; waaL/waaL⁺, O-antigen ligase complemented.

FIG. 3.

LPS immunoblot using an anti-A. actinomycetemcomitans antibody. A total of 500 ng of phenol-water-purified LPS was resolved on 4 to 15% polyacrylamide Tris-HCl Ready Gels. The separated carbohydrate molecules were transferred to a nitrocellulose membrane and probed with purified rabbit anti-A. actinomycetemcomitans immunoglobulins. WT, wild type (VT1169); rmlC, rhamnose epimerase mutant; rmlC/rlmC⁺, rhamnose epimerase complemented; waaL, O-antigen ligase mutant; waaL/waaL⁺, O-antigen ligase complemented; wzt, ATP-binding cassette sugar transport mutant.

FIG. 4.

Characterization of EmaA in the O-PS mutants. Equivalent amounts of membrane protein from each strain was prepared and separated by electrophoresis using 4 to 15% gradient polyacrylamide Tris-HCl gels. The proteins were transferred to nitrocellulose and probed with a monoclonal antibody specific for EmaA. The solid arrow indicates the electrophoretic mobility of the EmaA monomers associated the wild type and complemented strains in the separating gel. The dashed arrow corresponds to the mobility of the EmaA monomers associated with the rmlC mutant strain in the separating gel. The immunoreactive material at the top of the immunoblot corresponds to EmaA aggregates associated with the stacking gel. WT, wild type (VT1169); rmlC, rhamnose epimerase mutant; rmlC/rlmC⁺, rhamnose epimerase complemented; wzt, ABC sugar transport mutant; waaL, O-antigen ligase mutant; waaL/waaL⁺, O-antigen ligase complemented.

FIG. 5.

Fucose-specific Lens culinaris agglutinin (LCA) blots of membrane proteins from EmaA-producing and emaA mutant strains. Equivalent amounts of membrane proteins from the EmaA-overproducing strain VT1169 (pKM2/emaA) and the emaA mutant (emaA) were prepared, loaded into the 4 to 15% polyacrylamide Tris-HCl gel, and transferred to nitrocellulose membranes. (A) The same protein-transferred membrane was probed with anti-EmaA monoclonal antibody (panel 1); biotinylated LCA, with different exposure times of the film (panels 2 and 3); and avidin alone (nonlectin control) (panel 4). Antibody binding was detected using goat anti-mouse antibodies, and lectin binding was detected using avidin. The solid arrow at ∼200 kDa corresponds to the EmaA monomer. The dashed arrow corresponds to EmaA aggregates associated with the stacking gel. (B) Colloidal blue stain of membrane proteins. Equivalent amounts of membrane proteins from the EmaA-overproducing strain VT1169(pKM2/emaA) and the emaA mutant strain were separated in a 5 to 15% polyacrylamide-SDS gel with a 3% stacking gel. Following electrophoresis, the gel was stained with colloidal blue. The region of the gel corresponding to the EmaA aggregates, shown with the square bracket ([) in the stacking gel, and a similar region of the gel in the emaA mutant ([) were excised and analyzed using LC/MS (Table 4).

FIG. 6.

Assessment of collagen binding activities using trypsin-treated rabbit heart valves. Rabbit heart valves were surgically removed and treated with trypsin to remove the endothelia. Equal CFU numbers of the wild-type (WT) and the rmlC (rhamnose epimerase mutant) bacteria were added to the treated valves and incubated. The competitive index (CI) was calculated as the ratio of the numbers of mutant to wild-type CFU in the cardiac valve samples divided by the ratio of the numbers of mutant to wild-type CFU in the inoculum (CI, 0.33; Paired t test; P = 0.008), which was similar to that of the emaA mutant (CI, 0.27).

FIG. 7.

Hypothetical pathway for LPS and EmaA biosynthesis in A. actinomycetemcomitans. The proposed LPS biosynthetic pathway is based on enzymatic reactions that are known or proposed, based on protein homology, for other Gram-negative bacteria. We propose that EmaA monomers are translocated across the inner membrane (IM) using the Sec translocon mediated by the signal sequence (data not shown). Once in the periplasmic space, the O-PS sugars are transferred to the individual EmaA monomers by the O-antigen ligase (WaaL) before translocation through the outer membrane (OM) and presentation on the bacterial surface.