Application of CE-ESI-MS and LC-ESI-MSⁿ to profile glycoform expression during Haemophilus influenzae pathogenesis in the chinchilla model of experimental otitis media

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

^* To whom correspondence should be addressed. Email: elke.schweda{at}crc.ki.se.

	Abstract

Otitis media caused by non-typeable Haemphilus influenzae (NTHi) is a common and recurrent bacterial infection of childhood. Structural variability and diversity of H. influenzae lipopolysaccharide (LPS) glycoforms are known to play a significant role in the commensal and disease causing behaviour of this pathogen. In this study, we determine 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 et al. 2002. Eur J Biochem 269:808-818.], minor isomeric glycoform populations were determined by liquid chromatography multiple step tandem mass spectrometry (LC-ESI-MSⁿ). Using capillary electrophoresis mass spectrometry (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 media (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 post-infection 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 towards more truncated and less complex LPS glycoforms that lacked sialic acid was found as disease progressed.