Molecular characterization of phosphorylcholine expression on the lipooligosaccharide of Histophilus somni

Abstract

Histophilus somni (Haemophilus somnus) is an important pathogen of cattle that is responsible for respiratory disease, septicemia, and systemic diseases such as thrombotic meningoencephalitis, myocarditis, and abortion. A variety of virulence factors have been identified in H. somni, including compositional and antigenic variation of the lipooligosaccharide (LOS). Phosphorylcholine (ChoP) has been identified as one of the components of H. somni LOS that undergoes antigenic variation. In this study, five genes (lic1ABCD(Hs) and glpQ) with homology to genes responsible for ChoP expression in Haemophilus influenzae LOS were identified in the H. somni genome. An H. somni open reading frame (ORF) with homology to H. influenzae lic1A (lic1A(Hi)) contained a variable number of tandem repeats (VNTR). However, whereas the tetranucleotide repeat 5'-CAAT-3' is present in lic1A(Hi), the VNTR in H. somni lic1A (lic1A(Hs)) consisted of 5'-AACC-3'. Due to the propensity of VNTR to vary during replication and cause the ORF to shift in and out of frame with the upstream start codon, the VNTR were deleted from lic1A(Hs) to maintain the gene constitutively on. This construct was cloned into Escherichia coli, and functional enzyme assays confirmed that lic1A(Hs) encoded a choline kinase, and that the VNTR were not required for expression of a functional gene product. Variation in the number of VNTR in lic1A(Hs) correlated with antigenic variation of ChoP expression in H. somni strain 124P. However, antigenic variation of ChoP expression in strain 738 predominately occurred through variable extension/truncation of the LOS outer core. These results indicated that the lic1(Hs) genes controlled expression of ChoP on the LOS, but that in H. somni there are two potential mechanisms that account for antigenic variation of ChoP.

Fig. 1

Electrophoretic profile of E. coli expressing the gene lic1A_Hs. The plasmid pSE1, which contained lic1A_Hs was transformed into E. coli. Transformed cells expressed a protein of the approximate molecular size to that of the predicated H. somni choline kinase (Lic1A). Lanes: 1, E. coli containing pSE1 pre-induced with IPTG; 2–4, E. coli containing pSE1 induced with IPTG after 1, 2, and 3 h; 5, Molecular size marker; 6, E. coli control lacking pSE1 pre-induced with IPTG; 7–9, E. coli control lacking pSE1 induced with IPTG after 1, 2, and 3 h.

Fig. 2

The choline kinase activity of H. somni Lic1A compared to the activity of yeast choline kinase CKI1. Lic1A catalyzed the production of 8.39 nmol ChoP/min/mg protein while CKI1 catalyzed the production of 11.86 mg ChoP/min/mg protein. The results are the average of three experiments.

Fig. 3

Electrophoretic profiles of LOS from ChoP⁺ and ChoP⁻ variants of H. somni strains. The ChoP⁺ variants contain more of the lower molecular size bands than LOS from ChoP⁻ variants and parent strains. Lanes: 1, parent strain 738; 2, 738P (a ChoP⁺ isolate from a previous study [27]); 3, 738⁺; 4, 738⁻; 5, parent strain 7735; 6, 7735⁺; 7, 7735⁻; 8, 124P⁺; 9, 124P⁻; 10, 2336; 11, 129Pt.