AI-2 Sensing Without Production: A Study of luxS-Deficient F. nucleatum

Fusobacterium nucleatum is a Gram-negative, spindle-shaped anaerobe that plays a central role in oral biofilm development by bridging early and late colonizers. It is also associated with systemic diseases, including preterm birth and colorectal cancer. It consists of four main subspecies: nucleatum (FNN), polymorphum (FNP), animalis (FNA), and vincentii (FNV). AI-2, a conserved signaling molecule involved in interspecies communication, is synthesized by LuxS, encoded by the luxS gene. While FNA strains produce AI-2, FNN and FNV strains lack luxS. In FNP, luxS is present but truncated by a transposon, rendering it nonfunctional. To test whether FNN can respond to AI-2 despite lacking luxS, we used strain ATCC 23726 and constructed a chromosomal insertion strain (WT::luxS) by integrating the luxS gene from FNA ATCC 51191. Three experiments were performed: (1) comparing WT and WT::luxS strains under anaerobic growth, followed by RNA extraction; (2) treating WT cells at mid-log phase (OD600 ~0.4) with 20 uM synthetic AI-2 or vehicle control for 1.5 hours before RNA isolation; and (3) exposing WT cells to culture supernatants from either WT or luxS mutant F. periodonticum ATCC 33693 and collecting RNA after 1.5 hours. These studies aim to determine whether FNN can sense AI-2 and to characterize the transcriptional effects of AI-2 exposure or luxS expression.