Molecular characterization of <i>streptococcus uberis</i> CAMP factor, lactoferrin binding protein and their upstream genes

• Main Author: Jiang, Min
• Other Authors: Tabel, Henry
• Format: Others
• Language: en
• Published: University of Saskatchewan 1996
• Online Access: http://library.usask.ca/theses/available/etd-10202004-235803

The gene coding for the CAMP factor from a strain of <i>Streptococcus uberis</i> (<i><i>S. uberis </i></i>) was cloned in <i>E. coli</i>. Chromosomal DNA from <i>S. uberis </i> was used to construct a gene library in plasmid pTZ18R and six CAMP-reaction positive clones were obtained from a total of 10,000 transformants. One clone, pJLD21, was subcloned and the CAMP factor gene (cfu) was localized to a 3.2 kb BamHI fragment. The nucleotide sequence of cfu was determined and the deduced amino acid sequence shown to be homologous to the corresponding <i>Streptococcus agalactiae</i> (<i>S. agalactiae </i>) protein. Immunoblot analysis revealed that the recombinant strain containing pJLD21 expressed a protein with a molecular weight of 28,000. Antibodies raised against purified <i>S. uberis </i> CAMP factor cross-reacted with <i>S. agalactiae </i> protein B. Southern blot analysis demonstrated that the six CAMP-reaction positive E. coli clones contained the same CAMP factor gene, and this gene existed in three out of eight <i>S. uberis </i> strains. An ORF encoding a 277-residue protein was identified upstream of the CAMP factor gene. Sequence analysis indicated that the gene product is potentially a polar amino acid and opine binding protein of an ABC-type transport system. The interaction between <i>S. uberis </i> and bovine lactoferrin (bLf) has been characterized. Apo-bLf could inhibit ¹²⁵I-bLf binding as effectively as iron-saturated bLf. Bovine transferrin, human lactoferrin and human transferrin did not interfere with bLf binding. The Scatchard plot was linear and approximately 7800 binding sites were expressed by each bacterial cell, with an affinity of $1.0 10\sp{-7}$ M. Heat- or protease treatment of bacterial cells reduced bLf binding to a great degree. Two components with estimated molecular weights of 165,000 and 76,000 were originally identified from the cell wall as the functionally active bLf binding proteins. The gene coding for the bLf binding protein (Ibp) of <i>S. uberis </i> has been cloned and sequenced. A single ORF encoding 561 amino acid residues resulted in the presence of two proteins in the recombinant E. coli cell. These proteins were able to bind bovine lactoferrin and had molecular weights of 76,000 and 165,000, similar to those detected in <i>S. uberis </i>. A putative signal peptide was found at the N terminus of the deduced amino acid sequence and the C terminus had the features of the membrane anchor motif found in other surface proteins from Gram positive bacteria. Deletion analysis located the bLf binding domain to a 200 amino acid region at the N terminus of this protein. The vaccine potential of recombinant CAMP factor and lactoferrin binding protein has been evaluated. (Abstract shortened by UMI.)