Inactivation of two haemolytic toxin genes in Aeromonas hydrophila attenuates virulence in a suckling mouse model

Abstract

The contribution of two unrelated Aeromonas hydrophila fihaemolytic 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 690 kDa. The inferred amino acid sequence showed 89% identity to the AHHI haemolysin of A. hydrophila ATCC 7966, and 51 O/ O identity to the HlyA haemolysin of Vibrio cholerae E l Tor strain 017. 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 LD,, (Scheffe test, P < 0 0 5 ) . Cytotoxicity t o buffalo green monkey kidney cell monolayers and haemolysis on horse blood agar were eliminated only in the hlyA aerA double mutants. This is the f i r s t 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.

Type III Secretion System Genes in Clinical Aeromonas Isolates

“…In addition to several virulence factors' having been investigated in Aeromonas (7,8,16,19,24,27,31,32), genes for a putative type III secretion system (TTSS) were recently identified in this genus (4,5,25). The TTSS is common in pathogenic strains of gram-negative bacteria (enteropathogenic Escherichia coli, Salmonella enterica, Shigella flexneri, Yersinia spp., and Pseudomonas aeruginosa), and the cluster of genes encoding it is frequently included in genomic regions called pathogenicity islands (13,30).…”

Detection and Characterization of the Hemolysin Genes in Aeromonas hydrophila and Aeromonas sobria by Multiplex PCR

“…A majority of the A. hydrophila and A. sobria isolates were highly hemolytic, whereas only 11% of the A. caviae isolates were capable of lysing sheep erythrocytes (30). Mutagenesis of A. hydrophila isolates that carry two hemolysin genes (hlyA and aerA) revealed that the hemolytic activity of the isolate on horse blood agar was eliminated only following double mutations in the hlyA and aerA genes (44). A subsequent study suggested that all virulent A. hydrophila isolates carried both the hlyA and the aerA genes (19).…”

Identification and Characterization of Putative Virulence Genes and Gene Clusters in Aeromonas hydrophila PPD134/91

“…Group I includes genes expressing hemolysin (hlyA) and aerolysin (aerA), which have been reported to be associated with the virulence of A. hydrophila (1,42). Group II includes genes expressing histone-like protein (hup1 for AH-1 and hup3 for AH-3) (36); opdA, expressing ligopeptidase A (27); bvgA, bvgS, and ascN (46); and ompAI, expressing an outer membrane protein (40).…”

“…LD 50 s of both insertion and deletion mutants of ascN in AH-1 were determined to be 10 6.3 . Wong and coworkers (42) reported that inactivation of hlyA or aerA alone showed no statistically significant attenuation in a suckling mouse model when compared to the wild type. Our virulence assay showed similar results for these genes, and extended this finding to most of the other virulence genes which we analyzed.…”