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Infection and Immunity, July 2008, p. 3255-3267, Vol. 76, No. 7
0019-9567/08/$08.00+0     doi:10.1128/IAI.01710-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Application of Capillary Electrophoresis Mass Spectrometry and Liquid Chromatography Multiple-Step Tandem Electrospray Mass Spectrometry To Profile Glycoform Expression during Haemophilus influenzae Pathogenesis in the Chinchilla Model of Experimental Otitis Media

Susanna L. Lundström,^1,# Jianjun Li,^2,# Martin Månsson,^1, Marisol Figueira,³ Magali Leroy,^3, Richard Goldstein,³ Derek W. Hood,⁴ E. Richard Moxon,⁴ James C. Richards,² and Elke K. H. Schweda^1*

Clinical Research Centre, Karolinska Institutet and University College of South Stockholm, NOVUM, S-141 86 Huddinge, Sweden,¹ Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada K1A OR6,² Division of Pediatric Infectious Diseases, BioSquare III, 670 Albany Street, Boston University Medical Center, Boston Medical Center, Boston, Massachusetts 02118,³ Molecular Infectious Diseases Group and Department of Paediatrics, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DS, United Kingdom⁴

Received 21 December 2007/ Returned for modification 27 February 2008/ Accepted 30 April 2008

Otitis media caused by nontypeable Haemophilus influenzae (NTHi) is a common and recurrent bacterial infection of childhood. The structural variability and diversity of H. influenzae lipopolysaccharide (LPS) glycoforms are known to play a significant role in the commensal and disease-causing behavior of this pathogen. In this study, we determined LPS glycoform populations from NTHi strain 1003 during the course of experimental otitis media in the chinchilla model of infection by mass spectrometric techniques. Building on an established structural model of the major LPS glycoforms expressed by this NTHi strain in vitro (M. Månsson, W. Hood, J. Li, J. C. Richards, E. R. Moxon, and E. K. Schweda, Eur. J. Biochem. 269:808-818, 2002), minor isomeric glycoform populations were determined by liquid chromatography multiple-step tandem electrospray mass spectrometry (LC-ESI-MSⁿ). Using capillary electrophoresis ESI-MS (CE-ESI-MS), we determined glycoform profiles for bacteria from direct middle ear fluid (MEF) samples. The LPS glycan profiles were essentially the same when the MEF samples of 7 of 10 animals were passaged on solid medium (chocolate agar). LC-ESI-MSⁿ provided a sensitive method for determining the isomeric distribution of LPS glycoforms in MEF and passaged specimens. To investigate changes in LPS glycoform distribution during the course of infection, MEF samples were analyzed at 2, 5, and 9 days postinfection by CE-ESI-MS following minimal passage on chocolate agar. As previously observed, sialic acid-containing glycoforms were detected during the early stages of infection, but a trend toward more-truncated and less-complex LPS glycoforms that lacked sialic acid was found as disease progressed.

Published ahead of print on 5 May 2008.

Editor: J. N. Weiser

^# S.L.L. and J.L. contributed equally to this work.

Present address: Alpharma AS, Harbitzalleen 3, Pb. 158 Skøyen, 0212 Oslo, Sweden.

Present address: Laboratory of Evolutionary, Molecular and Medical Genetics, INSERM U571, University Paris Descartes, Necker Children's Hospital School of Medicine, 75730 Paris Cedex 15, France.