Investigation of Haemophilus somnus Virulence Factors: Lipooligosaccharide Sialylation and Inhibition of Superoxide Anion Production

Virulent strains of the bovine opportunistic pathogen Haemophilus somnus (Histophilus somni) cause multi-systemic diseases in cattle. One of the reported virulence factors that H. somnus may use to persist in the host is resistance to intracellular killing. It is reported in this dissertation that H. somnus significantly (P <0.001) inhibited production of superoxide anion (O2-) by bovine mammary and alveolar macrophages as well as by polymorphonuclear leukocytes. Inhibition of O2- production was time- and dose-dependent and did not occur after incubation with Escherichia coli, H. influenzae, or Brucella abortus. Non-viable H. somnus, purified lipooligosaccharide (LOS), or cell-free supernatant from mid-log phase cultures did not inhibit O2- production, indicating that O2- inhibition required contact with live H. somnus. Commensal isolates of H. somnus were less capable or incapable of inhibiting macrophage O2- production compared to isolates tested from disease sites. H. somnus shares conserved epitopes in its LOS with Neisseria gonorrhoeae, N. meningitidis, and H. influenzae, and can also undergo structural phase variation of these LOS epitopes. Sialylation of the terminal galactose of H. somnus LOS is another reported virulence mechanism. Current sequencing of the genomes of H. somnus strains 2336 (pathogenic) and 129Pt (commensal) has enabled in silico identification of three open reading frames (ORFs) involved in sialylation. The ORFs-1 (hsst-I) and -2 (hsst-II) had BLASTx homology to sialyltransferases, while ORF-3 (neuAhs) had BLASTx homology to CMP-sialic acid synthetases. These ORFs were amplified by PCR and cloned into the expression vector pCWOri+. Thin layer chromatography of the hsst-I gene product showed this sialyltransferase exhibited preference for sialylation of terminal N-acetyllactosamine (LacNAc, beta-Gal-[1,4]-beta-GlcNAc-R). However, Hsst-II preferentially sialylated lacto-N-biose (LNB, beta-Gal-[1,3]-beta-GlcNAc-R). In this study, phase variation of the terminal linkage in isolate 738 from a 3 linked galactose (LNB) to a 4 linked galactose (LacNac) was demonstrated. Such variation of a glycose linkage appears to be a novel mechanism of LOS phase variation. Furthermore, the ability of sialylated strain 738 LOS vs de-sialylated strain 738 LOS to induce Toll-like receptor 4 signaling was decreased by 28%, as determined by ELISA for Macrophage Inflammatory Protein-2. Therefore, sialylated LOS may aid H. somnus to avoid host innate immunity.

Name thesis:degree_name

Ph. D.

Level thesis:degree_level

doctoral

Discipline thesis:degree_discipline

Veterinary Medical Sciences

Department dc:contributor.department

Veterinary Medical Sciences

Grantor dc:publisher

Virginia Tech

Year dc:date.issued

2005

Author dc:creator

• Howard, Michael D.

Chair dc:contributor.committeechair

• Inzana, Thomas J.

Committee members dc:contributor.committeemember

• Gregory, Eugene M.
• Buechner-Maxwell, Virginia A.
• Schurig, Gerhardt G.
• Toth, Thomas E.

• Histophilus somni
• Haemophilus somnus
• sialic acid
• sialylation
• microbial immunity
• virulence factors
• superoxide anion

Statement dc:rights

• In Copyright

Licence dc:rights.uri

• http://rightsstatements.org/vocab/InC/1.0/

Dc Identifier Other

etd-04142005-223651

OAI identifier oai:identifier

oai:vtechworks.lib.vt.edu:10919/26848