Incorporation of N-acetylneuraminic acid into Haemophilus somnus lipooligosaccharide (LOS): enhancement of resistance to serum and reduction of LOS antibody binding

Abstract

Haemophilus somnus isolates from cases of thrombotic meningoencephalitis, pneumonia, and other disease sites are capable of undergoing a high rate of phase variation in the oligosaccharide component of their lipooligosaccharides (LOS). In contrast, the LOS of commensal strains isolated from the normal reproductive tract phase vary little or not at all. In addition, the LOS of H. somnus shares conserved epitopes with LOS from Neisseria gonorrhoeae, Haemophilus influenzae, and other species that can incorporate sialic acid into their LOS. We now report that growth of disease isolates of H. somnus with CMP-N-acetylneuraminic acid (CMP-NeuAc) or NeuAc added to the medium resulted in incorporation of NeuAc into the LOS. However, NeuAc was not incorporated into the LOS of commensal isolates and one disease isolate following growth in medium containing CMP-NeuAc or NeuAc. Sialylated LOS was detected by an increase in the molecular size or an increase in the amount of the largest-molecular-size LOS electrophoretic bands, which disappeared following treatment with neuraminidase. Sialylated LOS could also be detected by reactivity with Limax flavus agglutinin lectin, which is specific for sialylated species, by dot blot assay; this reactivity was also reversed by neuraminidase treatment. H. somnus strain 2336 LOS was found to contain some sialic acid when grown in medium lacking CMP-NeuAc or NeuAc, although supplementation enhanced NeuAc incorporation. In contrast strain 738, an LOS phase variant of strain 2336, was less extensively sialylated when the growth medium was supplemented with CMP-NeuAc or NeuAc, as determined by electrophoretic profiles and electrospray mass spectrometry. The sialyltransferase of H. somnus strain 738 was confirmed to preferentially sialylate the Gal(beta)-(1-3)-GlcNAc component of the lacto-N-tetraose structure by capillary electrophoresis assay. Enhanced sialylation of the strain 2336 LOS inhibited the binding of monoclonal antibodies to LOS by enzyme immunoassay and Western blotting. Furthermore, sialylation of the LOS enhanced the resistance of H. somnus to the bactericidal action of antiserum to LOS. Sialylation and increased resistance to killing by normal serum also occurred in a deletion mutant that was deficient in the terminal Gal-GlcNAc disaccharide. LOS sialylation may therefore be an important virulence mechanism to protect H. somnus against the host immune system.

FIG. 1.

Dot blot of extracted LOS from H. somnus strain 738 grown with or without CMP-NeuAc or fresh bovine serum with peroxidase-conjugated LFA. One microgram of each LOS was blotted onto nitrocellulose, and reactivity with LFA was determined as described in Materials and Methods. 1, LOS from bacteria grown in fresh bovine serum; 2, LOS from bacteria grown in CMP-NeuAc; 3, LOS from bacteria grown in CTT only; 4, LOS from bacteria grown in CMP-NeuAc and treated with V. cholerae neuraminidase.

FIG. 2.

Electrophoretic profiles of LOS from H. somnus strains 738, 738-lob2A1:: Km, 1P, and 127P grown in the presence or absence of CMP-NeuAc (A) and from strain 2336 grown in the presence or absence of CMP-NeuAc or NeuAc (B). (A) Lanes: 1, LOS from strain 738 grown in CTT only; 2, LOS from strain 738 grown in CTT plus CMP-NeuAc and treated with neuraminidase; 3, LOS from strain 738 grown in CTT plus CMP-NeuAc; 4 and 5, LOS from strain 738-lob2A1::Km grown in CTT only or in CTT plus CMP-NeuAc, respectively; 6 and 7, LOS from strain 1P grown in CTT only or in CTT plus CMP-NeuAc, respectively; 8 and 9, LOS from strain 127P grown in CTT only or in CTT plus CMP-NeuAc, respectively. Bands marked with asterisks on the right have increased in molecular mass, putatively due to the addition of NeuAc. (B) Lanes: 1, LOS from strain 2336 grown in CTT only; 2, LOS from strain 2336 grown in CTT and treated with neuraminidase; 3, LOS from strain 2336 grown in CTT plus CMP-NeuAc; 4, LOS from strain 2336 grown in CTT plus CMP-NeuAc and treated with neuraminidase; 5, LOS from strain 2336 grown in CTT plus NeuAc; 6, LOS from strain 2336 grown in CTT plus NeuAc and treated with neuraminidase.

FIG. 3.

Negative-ion ES-MS of OdA LOS from H. somnus strain 2336 (A) or 738-lob2A1::Km (B). Addition of glycose units is indicated on the spectra. OdA LOS was isolated from cells grown on fresh sheep's blood agar plates supplemented with 50 μg of CMP-NeuAc per ml and analyzed as described in Materials and Methods.

FIG. 4.

Reactivity of H. somnus strain 2336-R and phase variant 738 to LOS MAb determined by ELISA following growth with or without CMP-NeuAc. Bacteria were grown to 10⁹ CFU/ml in CTT, diluted 1:10 in fresh CTT containing 50 μg of CMP-NeuAc per ml, and shaken for 3 h at 37°C. LOS was extracted, and 1 μg/well was used as antigen in the ELISA with MAb 5F5.9 (strain 738) or 3F11 (strain 2336-R). For some assays, 5 μg of LOS was pretreated with 5.5 U of V. cholerae neuraminidase for 1 h at 37°C. +S, LOS from cells grown with CMP-NeuAc; +N, LOS treated with neuraminidase; +S +N, LOS from cells grown with CMP-NeuAc and treated with neuraminidase. Error bars indicate standard deviations.

FIG. 5.

Electrophoretic profiles (A) and Western blots with MAb 3F11 (B) of LOS from H. somnus strain 2336-R grown in the absence (lanes 1) or presence (lanes 2) of CMP-NeuAc. There is no change in the profile, but note the increased quantity of the 4.5-kDa band when H. somnus is grown in the presence of CMP-NeuAc and the lack of reactivity of MAb 3F11 with the 4.5-kDa band.

FIG. 6.

Bactericidal activity of normal bovine serum against H. somnus strain 738-lob2A1::Km, which is deficient in the terminal Galβ-(1-3)-GlcNAc disaccharide on its LOS. Log-phase bacteria were incubated for 3 h with (solid line) or without (dashed line) CMP-NeuAc and then incubated with various concentrations of normal calf serum for 1 h, as described in Materials and Methods. Bactericidal activity was determined by viable plate count at time zero and 60 min after incubation. Error bars indicate standard deviations.

FIG. 7.

Capillary electrophoresis assay of sialyltransferase activity in H. somnus strain 738. The enzyme activity was measured with lacto-N-tetraose or lacto-N-neo-tetraose, which were labeled with APTS. (A) Electropherogram from the reaction of H. somnus strain 738 extract with lacto-N-tetraose-APTS. Trace 1 is from the complete enzyme reaction, and trace 2 is from a reaction without the donor sugar nucleotide CMP-NeuAc. (B) The same type of analysis with lacto-N-neo-tetraose-APTS. Trace 3 is from the complete reaction, and trace 4 is from the incomplete reaction. RFU, relative fluorescence units. The arrows show the product formed in the reaction.