Genetic analysis of virulence factors of Mannheimia (Pasteurella) haemolytica A1.

Using a molecular genetic approach, the genes that code for the various virulence factors of Mannheimia haemolytica A1 have been cloned for detailed characterizations. These included analysis of the encoded proteins, their biological activities, secretion of the molecules from the bacterium as well as their use in a vaccine component. Two newly characterized antigens of M. haemolytica A1 have been identified. The first one is a TonB-dependent iron regulated outer-membrane receptor that is distinct from the transferrin binding proteins. The 84kDa Irp protein exhibits features including a TonB box and a 50 amino acid region that can adopt occluded beta-barrel structures similar to the "plug" domain of the Escherichia coli FhuA and FepA crystal structures. Homologues of Irp were identified by analysis of the genome sequences of a number of Gram negative mucosal pathogens, including Neisseria meningitidis and N. gonorrhoeae. The Neisserial irp genes were cloned by PCR and expressed the 84kDa protein as expected, demonstrating that they are functional genes. In addition to being regulated by iron and Fur, irp(Mh) undergoes phase variation by a slipped-strand mispairing mechanism and may represent a contingency locus for iron acquisition during an infection. Another locus that codes for a putative adhesin molecule has also been partially characterized. This putative adhesin protein is highly homologous with the high-molecular-weight adhesin proteins of non-piliated non-typable strains of Haemophilus influenzae (NTHi) including Hia, Hsf, HMW1, HMW2. Currently, we have cloned the DNA that codes for 2223 amino acids (225kDa) and is still missing the stop codon. It is anticipated that when complete, the protein could be close to 240kDa, similar to the molecular mass of Hsf. Though incomplete, analysis of the adhesin showed that it exhibits characteristics of autotransporter (AT) proteins. The role of this high-molecular-weight adhesin in infection is being investigated.