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Cloning and characterization of a Moraxella bovis cytotoxin gene

John A. Angelos MS, DVM1, John F. Hess PhD2, and Lisle W. George DVM, PhD3
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  • 1 Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis, CA 95616.
  • | 2 Department of Cell Biology and Human Anatomy, School of Medicine, University of California, Davis, CA 95616.
  • | 3 Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis, CA 95616.

Abstract

Objective—To identify the Moraxella bovis cytotoxin gene.

Procedure—Hemolytic and nonhemolytic strains of M bovis were compared by use of western blotting to identify proteins unique to hemolytic strains. Oligonucleotide primers, designed on the basis of amino acid sequences of 2 tryptic peptides derived from 1 such protein and conserved regions of the C and 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 against these proteins were produced in rabbits.

Results—Several proteins ranging in molecular mass from 55 to 75 kd were unique to the hemolytic strain. An open reading frame encoding a 927-amino acid protein with a predicted molecular mass of 98.8 kd was amplified from M bovis genomic DNA. The deduced amino acid sequence encoded by this open reading frame was homologous to RTX toxins. Antisera against the recombinant carboxy terminus encoded by this open reading frame neutralized hemolytic and cytolytic activities of native M bovis cytotoxin.

Conclusions and Clinical Relevance—A gene was identified in M bovis that encodes a protein with sequence homology to other RTX toxins. Results of cytotoxin neutralization assays support the hypothesis that M bovis cytotoxin is encoded by this gene and belongs in the RTX family of bacterial exoproteins. Identification of this gene and expression of recombinant cytotoxin could facilitate the development of improved vaccines against infectious bovine keratoconjunctivitis. (Am J Vet Res 2001;62:1222–1228)

Abstract

Objective—To identify the Moraxella bovis cytotoxin gene.

Procedure—Hemolytic and nonhemolytic strains of M bovis were compared by use of western blotting to identify proteins unique to hemolytic strains. Oligonucleotide primers, designed on the basis of amino acid sequences of 2 tryptic peptides derived from 1 such protein and conserved regions of the C and 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 against these proteins were produced in rabbits.

Results—Several proteins ranging in molecular mass from 55 to 75 kd were unique to the hemolytic strain. An open reading frame encoding a 927-amino acid protein with a predicted molecular mass of 98.8 kd was amplified from M bovis genomic DNA. The deduced amino acid sequence encoded by this open reading frame was homologous to RTX toxins. Antisera against the recombinant carboxy terminus encoded by this open reading frame neutralized hemolytic and cytolytic activities of native M bovis cytotoxin.

Conclusions and Clinical Relevance—A gene was identified in M bovis that encodes a protein with sequence homology to other RTX toxins. Results of cytotoxin neutralization assays support the hypothesis that M bovis cytotoxin is encoded by this gene and belongs in the RTX family of bacterial exoproteins. Identification of this gene and expression of recombinant cytotoxin could facilitate the development of improved vaccines against infectious bovine keratoconjunctivitis. (Am J Vet Res 2001;62:1222–1228)