title: Inactivation of two haemolytic toxin genes in Aeromonas hydrophila attenuates virulence in a suckling mouse model.
abstract: The contribution of two unrelated Aeromonas hydrophila beta-haemolytic toxins to virulence was assessed in a suckling mouse model. The first haemolysin gene, isolated from an A. hydrophila A6 cosmid bank, encoded a potential gene product of 621 amino acids and a predicted molecular size of 69.0 kDa. The inferred amino acid sequence showed 89% identity to the AHH1 haemolysin of A. hydrophila ATCC 7966, and 51% identity to the HlyA haemolysin of Vibrio cholerae EI Tor strain O17. The second haemolysin gene (designated aerA), which encodes aerolysin, a pore-forming toxin, was partially cloned by PCR for the purpose of mutant construction. This PCR product was a 1040 bp fragment from the C-terminal region of aerA. It is proposed that the 69.0 kDa V. cholerae-HlyA-like haemolysin gene be termed hlyA to contrast with the aerA terminology for the aerolysin. A suicide vector was used to inactivate both the hlyA and aerA genes in A. hydrophila A6. When assessed in the suckling mouse model, only the hlyA aerA double mutant showed a statistically significant reduction in virulence--a 20-fold change in LD50 (Scheffe test, P < 0.05). Cytotoxicity to buffalo green monkey kidney cell monolayers and haemolysis on horse blood agar were eliminated only in the hlyA aerA double mutants. This is the first report of cloning and mutagenesis of two unrelated haemolytic toxin genes in the same strain of a mesophilic aeromonad. For A. hydrophila, a two-toxin model provides a more complete explanation of virulence.
authors.affiliation: School of Pharmacy and Medical Sciences, University of South Australia, Adelaide, South Australia 5000, Australia.
authors.forename: Christopher Y F
authors.initials: CYF
authors.lastname: Wong
authors.affiliation: Infectious Diseases Laboratories, Institute of Medical and Veterinary Science, Adelaide, South Australia 5000, Australia.
authors.forename: Michael W
authors.initials: MW
authors.lastname: Heuzenroeder
authors.affiliation: School of Pharmacy and Medical Sciences, University of South Australia, Adelaide, South Australia 5000, Australia.
authors.forename: Robert L P
authors.initials: RLP
authors.lastname: Flower
mesh_terms: D016980:Aeromonas hydrophila / Q000235:genetics* / Q000378:metabolism* / Q000472:pathogenicity; D000483:Alleles; D000818:Animals; D001426:Bacterial Proteins; D001427:Bacterial Toxins / Q000235:genetics* / Q000378:metabolism*; D001681:Biological Assay; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D015870:Gene Expression; D016905:Gram-Negative Bacterial Infections / Q000235:genetics / Q000382:microbiology*; D006460:Hemolysin Proteins / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D052899:Pore Forming Cytotoxic Proteins; D017422:Sequence Analysis, DNA; D014774:Virulence
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004269:DNA, Bacterial; D006460:Hemolysin Proteins; D052899:Pore Forming Cytotoxic Proteins; C084287:hlyA protein, Vibrio cholerae; C032676:aerolysin