The glycosylation of Dichelobacter nodosus fimbriae

Abstract

Footrot is a highly infectious and contagious disease of ruminants caused by the bacterium Dichelobacter nodosus, a gram negative and anaerobic bacterium that possesses fimbriae on its surface. This endemic and economically important disease is well recognized and investigated in New Zealand, Australia and throughout the sheep farming countries of the world.

Genetic variation in the fimbriae of D. nodosus from New Zealand isolates and the glycsosylation of fimbrial subunit proteins from those isolates were investigated in this thesis. The relationship between the calculated molecular weight (Mw) of some D. nodosus fimbrial subunit proteins and their apparent molecular weight (Mr) was investigated using SDS-PAGE electrophoresis. The discrepancy between the Mw and the Mr of fimbrial subunits suggested modification of the amino acid sequences of the fimA gene. In order to investigate these differences, rapid isolation, culturing and characterization techniques were developed for New Zealand footrot samples and several novel D. nodosus strains were isolated.

More than a thousand footrot samples were collected from four different farm areas in New Zealand. Thousands of primary plates were cultured anaerobicly and examined with gram staining in order to detect single colonies of D. nodosus. Approximately 500 plates that had potential D. nodosus colonies were subcultured several times to eliminate contaminating colonies until single colonies were obtained. No obvious relationship was observed between colony type, virulence and degree of piliation.

Both variable and a part of the conserved region of the fimbrial subunit gene (fimA) was amplified from bacterial DNA extracted either directly from footrot lesions or cultured D. nodosus isolates using the polymerase chain reaction (PCR). Different fimA amplimers were analysed using single-strand conformational polymorphism (SSCP) and by DNA sequencing.

On the basis of the PCR-SSCP pattern obtained and subsequent DNA sequencing, 16 new D. nodosus isolates belonging to eight different serogroups were identified from New Zealand. Serogroup D and H were not detected and serogroup E was the most common, followed by serogroups B, C, F and A. Sixteen new fimA gene sequences were identified indicating that PCR-SSCP is a very specific and useful tool for the identification and characterization of footrot cultures.

Agar plates cultured from single hoof swabs revealed the presence of multiple strains of D. nodosus on a single hoof. The PCR-SSCP patterns from those plates revealed that each plates contained at least two or three different D. nodosus strains.

Fimbrial subunit proteins from nine New Zealand and nine SPAHL vaccine strains were analyzed on SDS-PAGE. These had molecular weights in the range of ~15 to 20 kDa. The smallest (Mr ~16.5 kDa) were isolated from samples D-NZ1, D-NZ2 and D-SPAHL. The largest subunit (Mr 20 kDa) was isolated from samples F-NZ1 and F-SPAHL. All other isolates classified within serogroups A, B, C and E had fimbriae with a similar electrophoretic mobility (Mr ~17 to 18 kDa).

The calculated Mw of serogroups F-NZ1 and D-NZ1 did not correlate with the Mr determined from electrophoretic mobility.

What is more, DNA sequence analysis of fourteen fimbrial subunit genes suggested that D. nodosus fimbrial subunit proteins from serotypes A-NZ4, B₁-NZ2 and F-NZ3 have potential glycosylation sites.

PAS staining to detect carbohydrate on SDS-PAGE gels indicated that some of D. nodosus fimbrial subunit proteins from serotypes A-NZ4, B₁-NZ2 and F-NZ3 may be glycosylated.

Fourteen fimbrial subunit proteins previously stain with PAS were deglycosylated by enymatic deglycosylation enzymes. It was revealed that D. nodosus fimbrial proteins from B₁-NZ2 and F-NZ3 were N-glycosylated in variable regions of the fimA gene that are thought to be crucial in antigenicity.

However, further analysis of these fimbrial subunit proteins is needed to gain a better understanding the relationship between fimbriae and fimbrial modification, including glycosylation.