The serotype a-EmaA adhesin of Aggregatibacter actinomycetemcomitans does not require O-PS synthesis for collagen binding activity

Abstract

Aggregatibacter actinomycetemcomitans, a causative agent of periodontitis and non-oral diseases, synthesizes a trimeric extracellular matrix protein adhesin A (EmaA) that mediates collagen binding and biofilm formation. EmaA is found as two molecular forms, which correlate with the serotype of the bacterium. The canonical protein (b-EmaA), associated with serotypes b and c, has a monomeric molecular mass of 202 kDa. The collagen binding activity of b-EmaA is dependent on the presence of O-polysaccharide (O-PS), whereas biofilm activity is independent of O-PS synthesis. The EmaA associated with serotype a strains (a-EmaA) has a monomeric molecular mass of 173 kDa and differs in the amino acid sequence of the functional domain of the protein. In this study, a-emaA was confirmed to encode a protein that forms antenna-like appendages on the surface of the bacterium, which were found to be important for both collagen binding and biofilm formation. In an O-PS-deficient talose biosynthetic (tld) mutant strain, the electrophoretic mobility of the a-EmaA monomers was altered and the amount of membrane-associated EmaA was decreased when compared to the parent strain. The mass of biofilm formed remained unchanged. Interestingly, the collagen binding activity of the mutant strain was similar to the activity associated with the parent strain, which differs from that observed with the canonical b-EmaA isoform. These data suggest that the properties of the a-EmaA isoform are like those of b-EmaA, with the exception that collagen binding activity is independent of the presence or absence of the O-PS.

Keywords: Gram-negative bacterium; O-polysaccharides; adhesin; collagen; endocarditis; glycosylation; periodontal disease.

Fig. 1.

Amino acid sequence comparison of the functional domain of the b- and a-EmaA isoforms. Serotype b: the canonical b-EmaA derived from A. actinomycetemcomitans strain VT1169. Serotype a: the a-EmaA isoform derived from A. actinomycetemcomitans strain KM708. The first 510 amino acids of each isoform were aligned using the NCBI blast-P suite (BLASTP, RRID:SCR_001010). ‘+’ represents a conservative amino acid substitution. Pentameric repeats crucial for adhesin function are marked in bold and underlined.

Fig. 2.

Transmission electron micrographs of whole mount, negatively stained preparations of A. actinomycetemcomitans . (a) Strain ATCC 29523, serotype a, mutant for EmaA. (b) Strain ATCC 29523 transformed with a plasmid expressing a-emaA (pKM753). Arrows indicate antenna-like EmaA adhesins. Bar, 100 nm.

Fig. 3.

Collagen binding activity of serotype a strains in the presence or absence of the a-emaA gene. Light grey: strain ATCC 29523 (mutant for emaA); dark grey: strain ATCC 29523 transformed with a plasmid expressing a-emaA (pKM753). Collagen binding activity was normalized to strain ATCC 29523 and was set at 100%; a minimum of three biological experiments were performed. Asterisks indicate significance, **P<0.01.

Fig. 4.

Characterization of O-PS biosynthesis of wild-type, isogenic tld mutant and transformed serotype a A. actinomycetemcomitans strains. Anti-serotype a concentrated antiserum was used to detect O-PS differences between strains. Wild-type: strain KM354; tld^- : KM354 containing a disrupted GDP-6-deoxy-d-talose-4-dehydrogenase (tld) gene; complement: tld mutant strain transformed with a plasmid expressing tld. Immunoreactivity was normalized to the wild-type and was set at 100 %; a minimum of three biological experiments were performed. Asterisks indicate significance, *P<0.05.

Fig. 5.

Characterization of EmaA monomers isolated from wild-type and tld mutant strains. (a) Equivalent amounts of membrane protein from each strain were prepared and separated by electrophoresis using 4–15 % gradient polyacrylamide-SDS 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 with the wild-type strain. The dashed arrow corresponds to the mobility of the EmaA monomers associated with the tld mutant strain. The immunoreactive material at the top of the immunoblot corresponds to EmaA aggregates associated with the stacking gel. WT: wild type (KM354); tld^- : O-PS mutant strain; emaA ^-: strain ATCC 29523 mutant for emaA. (b) The membrane and cytosolic fractions were prepared from strain ATCC 29523 expressing emaA and analysed as described in (a). The solid arrow indicates membrane-localized EmaA, and the dashed arrow indicates cytosol-localized EmaA. Right-most lane: membrane fraction proteins isolated from strain ATCC 29523 and transformed with an empty plasmid. (c) Immuno-dot blot of equivalent amounts of membrane protein from the wild-type strain, O-PS mutant and O-PS mutant strain transformed with plasmid expressing a-emaA, probed with the same antibody as in (a).

Fig. 6.

Characterization of biofilm formation in the O-PS-deficient tld mutant strain. Wild type: strain KM354; tld^- : the O-PS biosynthesis-deficient mutant; tld^-/a-emaA⁺ : O-PS mutant strain transformed with a plasmid expressing a-emaA. The assay was for 24 h; biofilm formation was normalized to the wild-type strain and was set at 100 %, and a minimum of three biological replicates were performed. Asterisks indicate significance, *P<0.05.

Fig. 7.

O-PS synthesis and collagen binding activity of A. actinomycetemcomitans . Serotype a and b strains deficient for O-PS synthesis were transformed with a plasmid expressing a-emaA and were tested for collagen binding activity. (a) Serotype a; an anti-serotype b polyclonal antibody was used. Wild-type: strain KM354; emaA^- : isogenic emaA mutant; tld ^-: isogenic O-PS mutant strain; tld^-/a-emaA⁺ : O-PS mutant strain transformed with plasmid expressing a-emaA. (b) Serotype b; an anti-serotype a polyclonal antibody was used. Wild type: strain KM733; rmlC^- : O-PS mutant strain; rmlC^- /b-emaA⁺ : O-PS mutant strain transformed with plasmid expressing b-emaA; rmlC^- /a-emaA⁺ : O-PS mutant strain transformed with plasmid expressing a-emaA. Collagen binding activity was normalized to the wild-type strains and was set at 100%; a minimum of three biological replicates were performed for each. Asterisks indicate statistical significance (*P<0.05).