Gram-positive bacteria assemble a unique class of covalently-linked protein polymers known as sortase-assembled pili or fimbriae that are important for polymicrobial interactions (or coaggregation), adhesion, and bacterial virulence. Sortase-mediated pilus assembly is a two-step process: polymerization of pilus proteins catalyzed by pilus-specific sortase is followed by cell wall anchoring of pilus polymers catalyzed by the housekeeping sortase. How these sortases coordinate their enzymatic activities to assemble pili with an optimal length on the cell surface is not well understood. In the oral bacterium Actinomyces oris, a key colonizer in the development of oral biofilms, type 2 fimbriae, made of the fimbrial shaft FimA and the tip coaggregation factor CafA, are essential for Actinomyces coaggregation with oral streptococci. The pilus-specific sortase SrtC2 polymerizes fimbrial polymers that are then anchored to the cell wall by the housekeeping sortase SrtA. Importantly, mutant cells lacking srtA produce exceedingly long pili but fail to mediate bacterial coaggregation. To elucidate a mechanism of SrtA modulation of pilus length, we sought to characterize safA, a gene immediately downstream of srtA, since functionally related genes tend to cluster in the bacterial genome. Remarkably, deletion of safA causes altered cell morphology, abolishment of polymicrobial interactions, and production of exceedingly long pili, all phenotypes consistent with deletion of srtA. These defective phenotypes were rescued with ectopic expression of safA from Corynebacterium diphtheriae and Corynebacterium matruchotii in the A. oris safA mutant, suggesting a common mechanism in Actinobacteria. Bacterial two-hybrid analysis demonstrated that SafA interacts with the housekeeping sortase SrtA. Intriguingly, in the oral bacterium Bifidobacterium dentium, the housekeeping sortase SrtE contains a fused homologous SafA sequence at its C-terminus. Altogether, it is hypothesized that SafA maintains membrane homeostasis of SrtA through an evolutionarily conserved mechanism across Actinobacteria to modulate pilus assembly and pilus length. Three specific aims are proposed to test this hypothesis. By genetic and biochemical approaches, we aim to elucidate the mechanism of SrtA membrane homeostasis by the evolutionary conserved protein SafA (Aim 1) and investigate the conservation of SafA and its co-evolution with the housekeeping sortase SrtA (Aim 2). Using nematode and guinea pig models of infection, we aim to examine the role of SafA in bacterial virulence (Aim 3). Besides providing a predoctoral training vehicle, this proposed study will not only advance our understanding of surface assembly of bacterial virulence factors, but also may provide attractive targets for anti-virulence strategies.