Cloning and characterization of a Moraxella bovis cytotoxin gene

Objective-To identify the Moraxella bovis cytotoxin gene. Procedure-Hemolytic and nonhemolytic strains of M bovis were compared by us e of western blotting to identify proteins unique to hemolytic strains. Oli gonucleotide primers, designed on the basis of amino acid sequences of 2 tr yptic peptides derived from 1 such protein and conserved regions of the C a nd B genes from members of the repeats in the structural toxin (RTX) family of bacterial toxins, were used to amplify cytotoxin-specific genes from M bovis genomic DNA. Recombinant proteins were expressed, and antisera agains t these proteins were produced in rabbits. Results Several proteins ranging in molecular mass from 55 to 75 kd were un ique to the hemolytic strain. An open reading frame encoding a 927-amino ac id protein with a predicted molecular mass of 98.8 kc! was amplified from M bovis genomic DNA. The deduced amino acid sequence encoded by this open re ading frame was homologous to RTX toxins. Antisera against the recombinant carboxy terminus encoded by this open reading frame neutralized hemolytic a nd cytolytic activities of native M bovis cytotoxin. Conclusions and Clinical Relevance-A gene was identified in M bovis that en codes a protein with sequence homology to other RTX toxins. Results of cyto toxin neutralization assays support the hypothesis that M bovis cytotoxin i s encoded by this gene and belongs in the RTX family of bacterial exoprotei ns. Identification of this gene and expression of recombinant cytotoxin cou ld facilitate the development of improved vaccines against infectious bovin e keratoconjunctivitis.