DNA sequences corresponding to the aerolysin gene (aer) of Aeromonas hydrophila AH2 DNA were identified by screening a cosmid gene library for hemolytic and cytotoxic activities. A plasmid containing a 5.8-kilobase EcoRI fragment of A. hydrophila DNA was required for full expression of the hemolytic and cytotoxic phenotype in Escherichia coli K-12. Deletion analysis and transposon mutagenesis allowed us to localize the gene product to 1.4 kilobases of Aeromonas DNA and define flanking DNA regions affecting aerolysin production. The reduced hemolytic activity with plasmids lacking these flanking regions is associated with a temporal delay in the appearance of hemolytic activity and is not a result of a loss of transport functions. The aerolysin gene product was detected as a 54,000-dalton protein in E. coli maxicells harboring aer plasmids and by immunoblotting E. coli whole cells carrying aer plasmids. We suggest that the gene coding aerolysin be designated aerA and that regions downstream and upstream of aerA which modulate its expression and activity be designated aerB and aerC, respectively.

We report here on the isolation of isogenic strains of Aeromonas hydrophila AB3 deleted for a segment of the aerolysin gene. All aer mutants obtained lacked the 49-kilodalton aerolysin gene product and were neither hemolytic for blood erythrocytes nor cytotoxic for Chinese hamster ovary tissue culture cells. One such mutant, AB3-5, was used in a mouse toxicity model to evaluate the role of aerolysin in the pathogenesis of A. hydrophila infections. The strain had a 50% lethal dose (LD50) of greater than 109 as compared with the parental strain which had an LD50 of 5 x 107. Reintegration of the deleted segment into AB3-5 resulted in an LD50 of 6 x 107 cells for this revertant. Furthermore, all mice injected with a sublethal dose of the parental strains developed necrotic lesions; this was never obtained with the aerolysin-deficient strain AB3-5. More importantly, specific neutralizing antibody to aerolysin was detected in mice surviving A. hydrophila infection, demonstrating that aerolysin is produced during the course of systemic A. hydrophila infections.

The nucleotide sequence of a 2510 base pair chromosomal fragment containing the aerolysin gene aerA, and its regulatory region aerC, from a clinical isolate of Aeromonas sobria was determined. The aerolysin gene coded for a 54.5 kD polypeptide and had a G + C content of 59%, indicating that it is endogenous to the genus Aeromonas. In contrast, the aerC region was characterized by its high A + T content (61%) and the presence of a core motif, aATAAAa, repeated eight times within 300 base pairs. A 12 base pair repeat, 5'AATAAAACCGGG3', present within this region occurred as a direct repeat 544 base pairs away, within the coding region of aerolysin. RNA polymerase binding studies and S1 mapping allowed the detection of two divergent non-overlapping promoters within aerC. Despite having identical transcriptional start sites in both A. sobria and Escherchia coli, the amount of aerolysin transcript produced in E. coli is 30-40 times less than that found in A. sobria. The signal peptide of preproaerolysin was shown by deletion to be essential for export of the toxin to the external medium. The mature toxin is a hydrophilic protein with no hydrophobic stretches long enough to cross a membrane. A search for similarities to the primary sequence of aerolysin revealed that the toxin may share a functional similarity to haemolysin (hlyA) of E. coli.

Results of molecular and pathogenic studies of three different bacterial hemolysins (cytolysins) are presented. These exoproteins derive from the two gram-negative bacteria Escherichia coli and Aeromonas hydrophila and from the gram-positive pathogen Listeria monocytogenes. The hemolysin of E. coli is determined by an 8-kilobase (kb) region that includes four clustered genes (hlyC, hlyA, hlyB, and hlyD). This hemolysin determinant is part either of large transmissible plasmids or of the chromosome. The genes located chromosomally are found predominantly in E. coli strains that can cause pyelonephritis and/or other extraintestinal infections. A detailed analysis of the chromosomal hly determinants of one nephropathogenic E. coli strain revealed the existence of specific, large chromosomal insertions 75 kb and lOO kb in size that carry the hly genes but that also influence the expression of other virulence properties, i.e., adhesion and serum resistance. The direct involvement of E. coli hemolysin in virulence could be demonstrated in several model systems. The genetic determinants for hemolysin (cytolysin) formation in , A. hydrophila (aerolysin) and L. monocytogenes (listeriolysin) are less complex. Both cytolysins seem to be encoded by single genes, although two loci (aerB and aerC) that affect the expression and activity of aerolysin have been identified distal and proximal to the structural gene for aerolysin (aerA). Cytolysin-negative mutants of both bacteria were obtained by site-specific deletion and/or transposon mutagenesis. These mutants show a drastic reduction in the virulence of the respective bacteria.

The cytotoxic haemolysin, aerolysin, has been implicated as an important virulence factor in Aeromonas-associated infections t'5'6. Wild type isolates vary widely in their ability to produce the toxin, and toxicity assays performed with mice show a correlation between amount of toxin produced and mortality 4. More recently, a study of 686 isolates from various geographical locations has shown a strong correlation between hemolysis and enterotoxicity 2. In order to study the role of the toxin in Aeromonas-associated infections, we have cloned the corresponding gene from a clinical isolate. The aerolysin gene (aer A) was identified from a gene bank carrying DNA from strain AH 2, by screening for hemolytic activity on blood agar plates and cytotoxicity to Chinese Hamster ovary cells. Genetic mapping of the cloned fragment by transposon mutagenesis with Tn lO00 and deletion analysis with restriction endonucleases allowed us to define flanking regions which modulate the synthesis (aer B) and expression (aer C) of the aerolysin gene (aer A) 3.The pathogenic potential of aerolysin was evaluated by constructing isogenic mutants of AH 2 which carry specific deletions within the aer determinant on the chromosome. Our strategy for obtaining such mutants was to introduce deleted derivatives of the aer determinant, cloned onto pMBl-based mobilizable vectors, into A. hydrophila AH 2. These vectors are unable to repli-cate stably in AH 2 and are rapidly lost by segregation. In order to detect mutants, association of a selectable marker with the cloned DNA sequence was required. The kanamycin resistance gene from the transposon Tn 903 was used for this purpose.The plasmid pHPC3-702 carries a 1.4 kilobase(kb) Kpn I fragment encoding resistance to kanamycin in place of the 1.8 kb fragment from the aer determinant on plasmid pHPC3-700. E. coli recombinants harboring pHPC3-702 are completely devoid of both hemolytic and cytotoxic activities. A marker exchange procedure was used to recombine the deletion-kanr-substitution on pHPC3-702 into the chromosome of a spontaneously derived nalidixic acid-resistant strain of AH 2 (AB3). Subsequent in vivo recombination and segregation of the plasmid produced genetic recombinants that had the resident aerA aerB region replaced by the substitution mutation carried on pHPC3-702 ( fig. 1, A and B). One such recombinant, AB3 aer-5 was used in subseqeunt studies. DNA hybridization was used to confirm the genetic structure of AB3 aer-5. Immunoblots performed with specific antisera directed against aerolysin showed total absence of aerolysin in both cell lysates and supernatant fluids of AB3 aer-5. We next determined the 50% lethal dose (LDs0) of both the parental strain and its isogenic aerolysin-negative derivative.The results are depicted in the table. Intraperitoneal injection of mice with the parental strain AB3 showed a LDs0 of around 5 x 107 cells, while the aerolysin-negative strain had a LDs0 of more than 8 x 108 cells. To exclude the possibility that unlinked mutations affecting toxicity had...