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Infection and Immunity, July 2004, p. 4249-4260, Vol. 72, No. 7
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.7.4249-4260.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Nontypeable Haemophilus influenzae Strain 2019 Produces a Biofilm Containing N-Acetylneuraminic Acid That May Mimic Sialylated O-Linked Glycans

L. L. Greiner,1,{dagger} H. Watanabe,1,{dagger},{ddagger} N. J. Phillips,2,{dagger} J. Shao,1 A. Morgan,1 A. Zaleski,1 B. W. Gibson,3 and M. A. Apicella1*

Department of Microbiology, University of Iowa, Iowa City, Iowa 52242,1 Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143-0446,2 Buck Institute for Age Research, Novato, California 949453

Received 26 November 2003/ Returned for modification 11 February 2004/ Accepted 19 March 2004

Previous studies suggested that nontypeable Haemophilus influenzae (NTHI) can form biofilms during human and chinchilla middle ear infections. Microscopic analysis of a 5-day biofilm of NTHI strain 2019 grown in a continuous-flow chamber revealed that the biofilm had a diffuse matrix interlaced with multiple water channels. Our studies showed that biofilm production was significantly decreased when a chemically defined medium lacking N-acetylneuraminic acid (sialic acid) was used. Based on these observations, we examined mutations in seven NTHI strain 2019 genes involved in carbohydrate and lipooligosaccharide biosynthesis. NTHI strain 2019 with mutations in the genes encoding CMP-N-acetylneuraminic acid synthetase (siaB), one of the three NTHI sialyltransferases (siaA), and the undecaprenyl-phosphate {alpha}-N-acetylglucosaminyltransferase homolog (wecA) produced significantly smaller amounts of biofilm. NTHI strain 2019 with mutations in genes encoding phosphoglucomutase (pgm), UDP-galactose-4-epimerase, and two other NTHI sialyltransferases (lic3A and lsgB) produced biofilms that were equivalent to or larger than the biofilms produced by the parent strain. The biofilm formed by the NTHI strain 2019pgm mutant was studied with Maackia amurensis fluorescein isothiocyanate (FITC)-conjugated and Sambucus nigra tetramethyl rhodamine isocyanate (TRITC)-conjugated lectins. S. nigra TRITC-conjugated lectin bound to this biofilm, while M. amurensis FITC-conjugated lectin did not. S. nigra TRITC-conjugated lectin binding was inhibited by incubation with {alpha}2,6-neuraminyllactose and by pretreatment of the biofilm with Vibrio cholerae neuraminidase. Matrix-assisted laser desorption ionization—time of flight mass spectometry analysis of lipooligosaccharides isolated from a biofilm, the planktonic phase, and plate-grown organisms showed that the levels of most sialylated glycoforms were two- to fourfold greater when the lipooligosaccharide was derived from planktonic or biofilm organisms. Our data indicate that NTHI strain 2019 produces a biofilm containing {alpha}2,6-linked sialic acid and that the sialic acid content of the lipooligosaccharides increases concomitant with the transition of organisms to a biofilm form.

* Corresponding author. Mailing address: Department of Microbiology, University of Iowa, 51 Newton Rd., Iowa City, IA 52242. Phone: (319) 335-7807. Fax: (319) 335-9006. E-mail: michael-apicella{at}uiowa.edu.

Editor: J. N. Weiser

{dagger} L.L.G., H.W., and N.J.P. contributed equally to work described in this paper.

{ddagger} Present address: Department of Internal Medicine, Institute of Tropical Medicine, Nagasaki University, Nagaski, Japan.

Infection and Immunity, July 2004, p. 4249-4260, Vol. 72, No. 7
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.7.4249-4260.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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