Research Spotlight: Dissecting the pathway of EmaA glycosylation in an oral pathogen

By Matt Wargo • March 24th, 2010

Citation: Tang, G. and Mintz, K.P. 2010. Glycosylation of the collagen adhesin EmaA of Aggregatibacter actinomycetemcomitans is dependent upon the lipopolysaccharide biosynthetic pathway. Journal of Bacteriology 192(5):1395-1405.

Direct link to the article

Authors’ Association with MMG:
Gaoyan Tang – is a postdoctoral associate in the MMG Department
Keith P. Mintz – is an Associate Professor in the MMG Department

Impact and Significance:
Aggregatibacter actinomycetemcomitans is an oropharyngeal, Gram-negative human pathogen associated with periodontitis (gum disease) and non-oral infections. A. actinomycetemcomitans also belongs to the group of HACEK microorganisms, which are slow-growing fastidious organisms that have an enhanced capacity for the initiation of infective endocarditis (heart valve infection). The ability of pathogens to bind to extracellular matrix proteins, e.g. collagen, fibronectin, secreted by a variety of cell types, is considered to be important for the pathogenicity of these microorganisms. It is our hypothesis that the binding of A. actinomycetemcomitans to collagen is a tropic mechanism for both periodontal disease and infective endocarditis. Therefore, the dissection of the mechanism(s) this pathogen uses for the interaction with collagen and other ECM proteins will provide valuable insights into the prevention and treatment of infectious diseases.

Summary:
We identified an extracellular matrix protein adhesin A (EmaA), which belongs to the oligomeric coiled-coil or trimeric autotransporter protein family of adhesins. EmaA monomers trimerize to form antennae-like structures on the surface of the bacterium, which are required for A. actinomycetemcomitans binding to collagen. Two forms of the EmaA protein have been identified, which are suggested to be linked with the O-polysaccharide (O-PS) of lipopolysaccharide (LPS) covering the surface of the bacterium. This association with LPS was investigated by constructing genetic mutants for genes encoding enzymes of the O-PS biosynthetic pathway: 1) a cytoplasmic rhamnose sugar biosynthetic enzyme (rmlC), 2) a ATP binding cassette (ABC) sugar transport protein (wzt) located in the inner membrane, and 3) the periplasmic O-antigen ligase (waaL). All three mutants synthesized reduced amounts of O-PS, as determined by GCMS, and displayed a change in the electrophoretic mobility of the EmaA monomer, as well as the aggregation state, as observed in sodium dodecyl sulfate (SDS)-polyacrylamide gels. The change in the electrophoretic mobility of the EmaA monomer suggested an alteration in the glycosylation state of the protein. This data was supported by the positive staining of the EmaA protein with a lectin specific for one of the sugars of the O-PS. This staining was absent in the emaA mutant strain. The rmlC mutant strain expressing the modified EmaA protein demonstrated reduced collagen adhesion in a rabbit heart valve model. These data provide, for the first time, experimental evidence for the glycosylation of an oligomeric, coiled-coil adhesin and supports a role for the glycoconjugant in collagen binding. Additionally, in A. actinomycetemcomitans a novel pathway for the posttranslational modification of EmaA is suggested, which diverges from the known paradigms for protein glycosylation found for other Gram-negative bacteria.

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