Characterization and identification of the immunoreactive 35 kilodalton periplasmic iron-regulated protein of Mannheimia (Pasteurella) haemolytica

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Belzer, Carol
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Louisa B. Tabatabai
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Veterinary Microbiology and Preventive Medicine
Our faculty promote the understanding of causes of infectious disease in animals and the mechanisms by which diseases develop at the organismal, cellular and molecular levels. Veterinary microbiology also includes research on the interaction of pathogenic and symbiotic microbes with their hosts and the host response to infection.
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Mannheimia (Pasteurella) haemolytica A1 is a bacterial pathogen associated with bovine respiratory disease (BRD) also known as shipping fever. Most bacterial pathogens have developed one of two iron acquisition systems, either a high affinity siderophore acquisition system or an outer membrane transferrin/lactoferrin receptor system. The iron acquisition system allows the pathogenic bacteria to obtain iron from the host's iron binding proteins. Pasteurella haemolytica A1 uses the latter mechanism. When P. haemolytica is grown under iron-deficient conditions, three periplasmic iron-regulated proteins (PIRPs) are expressed. The expressed PIRPs had molecular weights of 31, 35, and 45 kilodalton (kDa). The 35-kDa PIRP from P. haemolytica serovar A1 has been isolated from osmotic shock fluids with a two step ammonium sulfate precipitation procedure. The purified and characterized protein had an isoelectric pH (pI) of 6.8 and a molecular weight of 35,822. Equilibrium velocity ultracentrifugation established that the protein existed as a monomer under native conditions with a Mr of 33,795. The secondary structure revealed the protein contained 34.1% alpha helical structure, 16.8% beta sheet, 31.1% beta turns, and 17.9% random structure. The N-terminal sequence of the protein was ANEVNVYSYRQPYLIEPMLK. The 35-kDa protein was identified from the N-terminal sequence as the FbpA homologue of the Haemophilus influenzae iron transport protein. Expression of the iron-regulated protein was inhibited by Fe3+ and by Fe2+, but not by Cu2+ , Co2+, Ni2+, Mn2+, and Zn2+. P. haemolytic up-regulated and expressed receptors for transferrin, hemoglobin, and lactoferrin to obtain iron, but did not produce siderophores to acquire iron. Receptors for hemoglobin and lactoferrin have not been described previously for P. haemolytica A1. Most cattle had low antibody levels to the 35-kDa protein. Therefore, the 35-kDa FpbA would not be a good candidate for a serologic test for detection of antibody to P. haemolytica. The results also showed that convalescent cattle had high IgG antibody levels to the 35-kDa FpbA, suggesting the 35-kDa protein could be a potential component for an improved vaccine.

Mon Jan 01 00:00:00 UTC 2001