Haemophilus influenzae and Moraxella catarrhalis intra- and interspecies quorum signaling

Otitis media (OM) is among the most common childhood diseases worldwide. OM infections often persist for long periods of time and can be highly resistant to antibiotic treatment due in part to persistence of bacteria within biofilms. The majority of OM infections also involve coinfection by multiple species. For instance, the leading causative agents Haemophilus influenzae and Moraxella catarrhalis are more frequently found to coexist than to be isolated individually. Polymicrobial infection and biofilm formation may have a dramatic impact on the severity and treatability of OM. Thus, there is a pressing need to investigate polymicrobial infection and to identify factors that contribute to the establishment of these infections. One factor that may contribute to the establishment of polymicrobial infection is bacterial communication via quorum signaling. Autoinducer-2 (AI-2) is termed an interspecies signal as the genetic determinant for AI-2 production (luxS) is highly conserved and many species possess the machinery to respond to AI-2. H. influenzae has a luxS homolog whereas M. catarrhalis does not but may still sense and respond to AI-2. Therefore, the focus of this project was to investigate the impact of AI-2 intra- and interspecies bacterial communication and H. influenzae and M. catarrhalis polymicrobial infection on biofilm formation, antibiotic resistance, and persistence in experimental OM.;For H. influenzae, a luxS mutant formed biofilms with decreased biomass and biofilm-associated lipooligosaccharide (LOS) glycoforms, and had a persistence defect during late stages of infection in the chinchilla model of OM. Internalization of and response to AI-2 was found to require NTHI_0632. Studies utilizing an NTHI_0632 mutant revealed accumulation of AI-2 in culture supernatants and altered LOS composition, biofilm formation, and persistence similar to that observed for a luxS mutant. Modulation of luxS expression was also found to be critical for nontypeable H. influenzae biofilm development and dispersal as interruption of luxS expression resulted in rapid dispersal of established biofilms while continuous expression prevented dispersal. Taken together, these data indicate that AI-2 represents a genuine signaling molecule in H. influenzae and support a model in which AI-2 signaling promotes biofilm maturation by modulating LOS composition, followed by decreased signaling to promote dispersal. Interfering with AI-2 signaling may therefore limit H. influenzae biofilm formation during infection and potentially force dispersal of already-established biofilms.;We next investigated H. influenzae and M. catarrhalis polymicrobial biofilm formation and interspecies bacterial communication. H. influenzae and M. catarrhalis formed polymicrobial biofilms in vitro with enhanced resistance to antibiotics, and production of AI-2 by H. influenzae contributed to the enhanced resistance. Incubation with synthetic AI-2 was sufficient to promote increased biofilm formation by M. catarrhalis and antibiotic resistance. H. influenzae and M. catarrhalis polymicrobial infection also promoted M. catarrhalis persistence in a luxS-dependent manner. We thus conclude that H. influenzae promotes M. catarrhalis persistence and antibiotic resistance within polymicrobial biofilms via AI-2 interspecies signaling. Disruption of AI-2 signaling during H. influenzae and M. catarrhalis polymicrobial infection may therefore disrupt polymicrobial biofilm formation and persistent infection. Moreover, successful vaccination against H. influenzae may also limit M. catarrhalis infectivity by removing a reservoir of the AI-2 signal that promotes M. catarrhalis persistence.