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

Abstract

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-MS(n)). 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-MS(n) 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.

FIG. 1.

Schematic representation of major LPS glycoforms reported for NTHi strain 1003 (20). A triheptosyl inner-core unit is substituted at the O-4 position of HepI by a β-d-Glcp residue (GlcI) which, in turn, is linked to PCho at O-6. HepIII is chain elongated at O-2 by either a β-d-Glcp residue (Hex2 glycoform), lactose (Hex3 glycoform), or sialyllactose [minor, i.e., α-Neu5Ac-(2→3)-β-d-Galp-(1→4)-β-d-Glcp]. GlcI has been found to carry an O-acetyl substituent at the O-4 position; in some glycoforms, a second O-acetyl group is located at O-3 of HepIII, and a third minor acetylation site was identified at the glucose residue off HepIII. Glycine is linked to HepIII and Kdo. Dashed lines indicate the various truncated Hex2 and Hex3 glycoforms.

FIG. 2.

Isomeric glycoforms observed in NTHi strain 1003 as identified by tandem ESI-MSⁿ. Glycoform B2 is only indicated in chinchilla sample 25 and evidenced by MS² spectra showing ions m/z 1,205.4 and 957.3 due to consecutive losses of tHep-Hep. Glycoform E was not observed in vitro but was detected in chinchilla samples 22 to 24, as described in the text.

FIG. 3.

CE-ESI-MS analysis (negative mode) of LPS-OH isolated from animal 6. The spectra correspond to LPS-OH in MEF (A), LPS-OH isolated after ex vivo growth in sBHI (B), and LPS-OH obtained from cells grown ex vivo on uCA (C).

FIG. 4.

HPLC-ESI-MS² spectra (positive mode) obtained for the ions m/z 1,264.0 (A), 1,468.0 (B), and 1,672.0 (C) of permethylated sample 3 (animal 3), taken directly from the MEF.

FIG. 5.

Total ion chromatogram of the ions m/z 1,264.0, 1,468.0, 1,672.0, 1,876.0, and 2,121.0 obtained on permethylated oligosaccharide from sample 23 (animal 3) after passage on uCA. The chromatograms of each single ion are shown in insets A (m/z 1,264.0), B (m/z 1,468.0), C (m/z 1,672.0), D (m/z 1,876.0), and E (m/z 2,121.0).

FIG. 6.

HPLC-ESI-MS² spectra (positive mode) of the Hex2 glycoform (m/z 1,468.0) of sample 23 from animal 3 (A) and sample 25 from animal 5 (B), both cultured ex vivo on uCA.

FIG. 7.

HPLC-ESI-MS² spectra (positive mode) of the Hex4 (A) and Hex4HexNAc (B) glycoforms (m/z 1,876.0 and 2,121.0) of sample 23 from animal 3. MS³ spectra of the fragment ions at m/z 1,145.4 (Aa) and 1,001.4 (Ab), as well as at m/z 1,391.7 (Ba) and 1,002.3 (Bb), are shown.

FIG. 8.

Histogram showing relation of glycoform compositions from bacteria isolated from animal 11 at day 5 following one to four passages on uCA and on CA supplemented with sialic acid. +, present; −, absent.

FIG. 9.

CE-ESI-MS analysis (negative mode) of LPS-OH isolated from animal 11 after 2 days (A), 5 days (B), and 9 days (C). (D) Precursor ion spectrum (negative mode) using m/z 290 as the fragment ion for identification of sialylated components after 2 days.

FIG. 10.

Histogram showing changes in LPS glycoform populations of bacteria isolated from animal 11 after 2, 5, and 9 days following passage on uCA. Data were obtained by CE-ESI-MS analysis of LPS-OH (see Fig. 9). Glycoforms are depicted in structural model of NTHi 1003 LPS.

FIG. 11.

CE-ESI-MS and tandem MSⁿ analysis (positive mode) of LPS-OH from animal 15 showing extracted mass spectrum (A), tandem MS spectrum of ion at m/z 1,141 ([M + 2]²⁺) (B), and MS³ spectrum of ion at m/z 1,328 ([M + 1]⁺) derived from m/z 1,141 (C). Assignments of major fragment ions are indicated.