PROJECT SUMMARY Dental plaque represents one of the most complex microbial communities or biofilms known to affect humans. Oral biofilm-related diseases, e.g. dental carries, gingivitis, and periodontitis, impact a large population of all age groups and continue to impose a huge economic burden due to the lack of effective therapies. The development of dental plaque begins with the attachment of early colonizers to the tooth enamel, generating an adhesive matrix that attracts intermediate and late colonizers. Actinomyces spp. are key early colonizers that play a prominent role in biofilm development by their ability to interact directly with the tooth surface and both early and intermediate colonizers. Therefore, our studies have focused on dissecting the adhesive properties, i.e. pilus and non-pilus proteins, dictating these interactions and their assembly mechanism on the surface of Actinomyces oris, the most abundant Actinomyces species in the human oral cavity. During the past grant period, we identified a transmembrane protein named SafA in A. oris that is genetically linked to the housekeeping sortase SrtA (class E sortase), the conserved transpeptidase enzyme that is central to the morphogenesis and cell wall anchoring of pilus and non-pilus proteins. Specifically, safA deletion results in SrtA cleavage by signal peptidae LepB2 and extracellular excretion of SrtA, concomitant of secretion of non-pilus proteins and most pili, while the remaining cell-wall anchored pili are exceedingly long but unable to mediate bacterial coaggregation. Strikingly, the defects of ∆safA in SrtA membrane localization, cell wall anchoring of pili and surface proteins, and coaggregation can be rescued by ectopic expression of SafA homologs from Corynebacterium matruchotii and Corynebacterium diphtheriae, suggesting the conservation of SafA-associated mechanism in Actinobacteria. Remarkably, the ∆safA mutant is severely defective in coaggregation with Porphyromonas gingivalis (Pg) and in inhibition of Pg growth, in contrast to wild-type cells. Intriguingly, in Bifidobacterium the housekeeping sortase SrtE contains a C-terminal SafA domain, suggesting the co-evolutionary existence of class E sortases and SafA. Therefore, we propose that SafA is an evolutionarily conserved antagonist of signal peptidase that topologically modulates sortase function in polymicrobial interaction and biofilm formation. Using biochemical, genetic, and structural approaches, we aim to test this central hypothesis by elucidating the antagonistic mechanism of SafA-mediated topological modulation of sortase function in polymicrobial interaction and biofilm formation in A. oris, determining the conservation of this SafA-associated mechanism and the co-evolutionary existence of class E sortases and SafA in Actinobacteria, and examining how the SafA-associated mechanism modulates A. oris coaggregation with Pg and Pg virulence. The conservation of sortase-mediated surface assembly and SafA-ass...