The outer membrane autotransporters Fap2 and CmpA facilitate specific coaggregation between Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans serotypes b and d

Periodontal disease is a polymicrobial infection in which Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans have been identified as key pathogens associated with disease progression. F. nucleatum is capable of coaggregating with several oral bacterial species, including A. actinomycetemcomitans, and these coaggregations play crucial roles in dental plaque formation. In this study, we investigated the coaggregation properties of both bacteria and identified the molecules that mediate their interaction. Among A. actinomycetemcomitans serotypes a, b, c, d, and g, F. nucleatum strains coaggregated specifically with serotypes b and d. The coaggregation between F. nucleatum and A. actinomycetemcomitans serotype b was significantly suppressed by the addition of lipopolysaccharide (LPS) extracted from A. actinomycetemcomitans serotype b. Among the sugars comprising the O-polysaccharide (O-PS) of serotype b LPS, the addition of N-acetyl-D-galactosamine markedly inhibited their coaggregation. Similarly, the coaggregation between F. nucleatum and A. actinomycetemcomitans serotype d was strongly inhibited by the addition of either the LPS extracted from A. actinomycetemcomitans serotype d or the L-rhamnose present in serotype d O-PS. Furthermore, analysis of outer membrane autotransporter proteins as potential coaggregation factors of F. nucleatum revealed that mutants lacking functional fap2 or cmpA exhibited significantly reduced coaggregation with A. actinomycetemcomitans serotype b or d, respectively. Additionally, we found that these coaggregations significantly promoted biofilm formation during coculture. These findings suggest that the coaggregation between F. nucleatum and A. actinomycetemcomitans serotypes b and d is mediated through interactions between autotransporter proteins and LPSs, thereby contributing to the development of mature biofilms. Fusobacterium nucleatum is a periodontal pathogen that acts as a bridging organism by coaggregating with various oral microbes, including other periodontal pathogens. Recently, this bacterium has been reported to be associated with systemic diseases, including cardiovascular diseases, adverse pregnancy outcomes, and cancer. F. nucleatum possesses numerous adhesins, including autotransporter proteins that are considered to mediate colonization in human tissues. Our study demonstrated that F. nucleatum utilizes two autotransporter proteins (Fap2 and CmpA) to coaggregate with another periodontal pathogen, Aggregatibacter actinomycetemcomitans, through specific interactions with bacterial surface polysaccharides. In addition to the roles of these autotransporters in periodontitis through bacterial coaggregation with other oral bacterial species, our findings potentially provide new insights into the mechanism underlying systemic diseases caused by F. nucleatum binding to host cell carbohydrates and glycoproteins.