Autoinducer 2 (AI-2) production by nontypeable Haemophilus influenzae 86-028NP promotes expression of a predicted glycosyltransferase that is a determinant of biofilm maturation, prevention of dispersal, and persistence in vivo.

Nontypeable Haemophilus influenzae (NT Hi ) is an extremely common human pathobiont that persists on the airway mucosal surface within biofilm communities, and our prior work has shown that NT Hi biofilm maturation is coordinated by production and uptake of AI-2 quorum signals. To directly test roles for AI-2 in maturation and maintenance of NT Hi biofilms, we generated an NT Hi 86-028NP mutant in which luxS transcription was under control of the xylA promoter (NT Hi 86-028NP luxS xylA :: luxS ), rendering AI-2 production inducible by xylose. Comparison of biofilms under inducing and noninducing conditions revealed a biofilm defect in the absence of xylose, whereas biofilm maturation increased following xylose induction. Removal of xylose resulted in interruption of luxS expression and biofilm dispersal. Measurement of luxS transcript by real-time RT-PCR showed that luxS expression peaked as biofilms matured, and waned before dispersal. Transcript profiling revealed significant changes following induction of luxS , including increased transcript for a predicted family 8 glycosyltransferase (NTHI1750, designated gstA ); this result was confirmed by real-time RT-PCR. An isogenic NT Hi 86-028NP gstA mutant had a biofilm defect, including decreased level of sialylated matrix and significantly altered biofilm structure. In experimental chinchilla infections we observed a significant decrease in bacteria in the biofilm population (but not in effusions) for NT Hi 86-028NP gstA as compared to the parental strain. Therefore, we conclude that AI-2 promotes NT Hi biofilm maturation and maintenance of biofilm integrity, due at least in part to expression of a probable glycosyltransferase that is potentially involved in synthesis of the biofilm matrix.