IAI Accepts, published online ahead of print on 29 June 2009

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Infect. Immun. doi:10.1128/IAI.00320-09
Copyright (c) 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

LuxS promotes biofilm maturation and persistence of nontypeable Haemophilus influenzae in vivo via modulation of lipooligosaccharides on the bacterial surface

Chelsie E. Armbruster, Wenzhou Hong, Bing Pang, Kristin E. Dew, Richard A. Juneau, Matthew S. Byrd, Cheraton F. Love, Nancy D. Kock, and W. Edward Swords^*

Departments of Microbiology and Immunology, Pathology, and Comparative Medicine Wake Forest University Health Sciences

^* To whom correspondence should be addressed. Email: wswords{at}wfubmc.edu.

Nontypeable Haemophilus influenzae (NTHi) is an extremely common airway commensal, which can cause opportunistic infections that are usually localized to airway mucosal surfaces. During many of these infections, NTHi forms biofilm communities that promote persistence in vivo. For many bacterial species, density-dependent quorum signaling networks can affect biofilm formation and/or maturation. Mutation of luxS, a determinant of the autoinducer-2 (AI-2) quorum signal pathway, increases NTHi virulence in the chinchilla model for otitis media infections. For example, bacterial counts within middle-ear fluids and the severity of the host inflammatory response were increased in luxS mutants as compared with parental strains. As these phenotypes are consistent with those we have observed for biofilm-defective NTHi mutants, we hypothesized that luxS may affect NTHi biofilms. A luxS mutant was generated in the well-characterized NTHi 86-028NP strain and tested for effects of the mutation on biofilm phenotypes in vitro and bacterial persistence and disease severity during experimental otitis media. Quantitation of biofilm structure by confocal microscopy and COMSTAT analysis revealed significantly reduced biomass for NTHi 86-028NP luxS biofilms, which was restored by a soluble mediator in NTHi 86-028NP supernatants. Analysis of lipooligosaccharide moieties by ELISA and immunoblot showed decreased levels of biofilm-associated glycoforms in NTHi 86-028NP luxS. Infection studies showed that NTHi 86-028NP luxS had a significant persistence defect in vivo during chronic otitis media infection. Based on these data, we conclude that a luxS-dependent soluble mediator modulates the composition of the NTHi lipooligosaccharides, with consequential effects on biofilm maturation and bacterial persistence in vivo.