INFECTION AND IMMUNITY, Sept. 1987, p. 2274-22800019-9567/87/092274-07$02.00/0Copyright © 1987, American Society for Microbiology

Marker Exchange Mutagenesis of the Aerolysin Determinant inAeromonas hydrophila Demonstrates the Role of Aerolysin in

A. hydrophila-Associated Systemic InfectionsT. CHAKRABORTY,l* B. HUHLE,1 H. HOF,2 H. BERGBAUER,' AND W. GOEBEL1

Institut fur Genetik und Mikrobiologiel and Institut fur Hygiene und Mikrobiologie,2 University of Wurzburg, D 8700Wurzburg, Federal Republic of Germany

Received 28 January 1987/Accepted 28 May 1987

We report here on the isolation of isogenic strains of Aeromonas hydrophila AB3 deleted for a segment of theaerolysin gene. All aer mutants obtained lacked the 49-kilodalton aerolysin gene product and were neitherhemolytic 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 ofA. hydrophilainfections. The strain had a 50% lethal dose (LD50) of greater than 109 as compared with the parental strainwhich had an LD50 of 5 x 107. Reintegration of the deleted segment into AB3-5 resulted in an LD50 of 6 x 107cells for this revertant. Furthermore, all mice injected with a sublethal dose of the parental strains developednecrotic lesions; this was never obtained with the aerolysin-deficient strain AB3-5. More importantly, specificneutralizing antibody to aerolysin was detected in mice surviving A. hydrophila infection, demonstrating thataerolysin is produced during the course of systemic A. hydrophila infections.

Aeromonas hydrophila is a gram-negative, facultativelyanaerobic organism that is an autochthonous inhabitant offreshwater environments. Recent attention has focused onthese microorganisms because of an increasing awareness oftheir association with human infection (6, 11, 13). Theclinical manifestation of A. hydrophila infection ranges fromgastroenteritis to soft tissue infections, septicemia, andmeningitis (10, 16).Although a number of extracellular factors associated with

bacterial virulence have been described for this organism,their role in the pathogenesis of A. hydrophila infectionsremains unclear. A number of recent studies implicate thecytotoxic hemolysin aerolysin as an important virulencefactor (3, 9, 13). However, all reported studies have beenperformed with individual isolates that varied in their abilityto express hemolytic activity and probably in their expres-sion of other virulence phenotypes. This assessment isfurther complicated by the fact that many strains of A.hydrophila carry a second hemolytic factor (5, 16).To evaluate the role of aerolysin in A. hydrophila infec-

tions, we have sought to construct isogenic strains lackingthe aerolysin gene product to test them in a mouse model.Site-directed mutagenesis was used to construct isogenicaerolysin-deficient strains. The cloned aerolysin gene (4) wasinactivated in vitro by inserting a fragment carrying akanamycin resistance marker. Marker exchange was thenused to introduce the mutated aerolysin gene into the chro-mosome of A. hydrophila AB3. We report here on thevirulence properties of the parental strain and its isogenicmutant in a mouse model.

MATERIALS AND METHODSBacterial strains, plasmids, media, and culture conditions.

The bacterial strains and plasmids are listed in Table 1. Themedia for the growth and maintenance of A. hydrophila andEscherichia coli strains have been previously described (4).The 1.4-kilobase (kb) EcoRI fragment from Tn903 encoding

* Corresponding author.

resistance to kanamycin, flanked by the polylinker sequenceof plasmid pUC18 (23), has been cloned into the HindIl siteof plasmid pBR322 to give pRME1 (W. Messer, personalcommunication).DNA manipulations. Chromosomal and plasmid DNAs

were isolated as described previously (5). Restriction en-zyme cleavage, in vitro ligation, and transformation werecarried out as described in reference 4. Bacillus subtilisphage SPP1, cleaved with EcoRI, served as a molecularweight standard. Plasmids were labeled by nick translationwith [ot-32P]dATP as described by Rigby et al. (19) andpurified by ethanol precipitation.

Hybridization and autoradiography. The transfer of DNAfragments from agarose gels to nitrocellulose filters, wash-ing, and autoradiography have been previously described(5). Filters were hybridized in 50% formamide at 43°C for 15h. Stringent hybridization conditions were used for thewashing procedure.Immunoblots. Immunoblots were performed as described

in reference 4. Antisera obtained from mice infected with A.hydrophila and control mice were used at a 1:200 dilution.

Bacterial matings. The principle and method of mobiliza-tion of mob-based vectors have been described by Simon etal. (21, 22). Matings between E. coli S17-1 and A. hydrophilaAB3 were done on filters. Cultures containing 5 x 107recipient and 2 x 108 donor cells were mated on a membranefilter (0.45-,um pore size, 25-mm diameter; Millipore Corp.,Bedford, Mass.). The filter was placed on solid agar mediumand incubated at 37°C for 4 h. The cultures were washedtwice in sterile phosphate-buffered saline and spread ontoblood agar plates supplemented with the appropriate antibi-otics.Animal assays. Cells from overnight cultures grown in L

broth were harvested, washed twice in phosphate-bufferedsaline, and resuspended in phosphate-buffered saline to givea concentration of approximately 1010 cells per ml. FemaleNMRI mice, 6 to 10 weeks old and weighing approximately25 g each (Zentral Institut fur Versuchstiere, Hannover,Federal Republic of Germany), were injected either intra-

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TABLE 1. Bacterial strains and plasmids

Strain Relevant genotype References and comments

A. hydrophila AH2 Prototrophic Hly+ (5)

AH2 derivativesAB3 nal-i Spontaneous Nalr mutant of AH2; Hly+AB3-1 pHPC3-702 nal-I From AB3 by integration of pHPC3-702; Hly+ TcrAB3-2 pHPC3-702 nal-i From AB3 by integration of pHPC3-702; Hly+ TcrAB3-3 pHPC3-702 AaerB2::nptll nal-i From AB3 by integration of HPC3-702 and subsequent loss

of aerA+B+C+: Hly- Kmr TcsAB3-5 AaerAB2::nptll nal-I From AB3 by marker exchange with pHPC3-702; Hly- KmrAB3-9 AaerAB2::nptII nal-I From AB3 by marker exchange with pHPC3-702; Hly- Kmr;

reduced protease productionAB3-13 AaerAB2::nptll nal-i From AB3 by marker exchange with pHPC3-702; Hly- Kmr;

reduced protease production

E. coliLE 392 F hsdR514 hsdR hsdM supE44 supF58 lacYI 4

galK galT22 metBi trpR55S17-1 thi pro hsdR hsdM recA RP4-2-Tc::Mu-kan::Tn7 22

PlasmidspRME1 pBR22 nptII W. Messer; Apr KmrpSUP205 pBR325 mob Cmr Tcr (22)pHPC3-1 aerA+B+C+ Apr Hly+ (5)pHPC3-700 pSUP205 aerA+B+C+ Tcr Hly+ (this work)pHPC3-702 pSUP205 aerAB2::nptII Tcr Kmr Hly- (this work)

peritoneally (i.p.) or subcutaneously (s.c.) with 0.2 ml of anappropriate concentration of A. hydrophila bacteria. Micewere placed in cages with unrestricted food and water andobserved for death at intervals up to 72 h. Median (50%)lethal doses (LD50s) were calculated by the Spearman-Karber method (8). Mice infected with sublethal doses ofbacteria were monitored for a period of 1 month postinfec-tion. Multiplication of bacteria in animals was tested byhomogenizing spleens and livers of individual mice andplating dilutions onto LB plates with the appropriate antibi-otic selection.

Tissue culture assays. Tissue culture assays were per-formed as described in reference 5. CHO cells were grown inEagle minimal medium (Serva, Heidelberg, Federal Repub-lic of Germany) with 10% fetal calf serum and 50 ,ug ofpolymyxin B per ml (Sigma Chemical Co., St. Louis, Mo.).The cytotoxic activity of supernatant fluids was expressed inunits as the reciprocal of the highest dilution resulting in 50%of cells killed.

Hemolytic activity. Cell-free culture supernatant wastested for hemolytic activity after samples were diluted with0.01 M Tris hydrochloride buffer (pH 7.2) containing 0.9%NaCl. Portions of 0.1 ml from each dilution were transferredinto the wells of polystyrene trays to which 0.02-ml amountsof washed human erythrocyte suspension were added in thesame buffer. The concentration of human erythrocyte sus-pension was adjusted to give an A540 of 0.8 at complete lysis.The hemolytic activity of a sample was expressed in units asthe reciprocal of the highest dilution of the sample resultingin a 50% hemolysis.

RESULTS

Construction of deletion-substitution derivatives of theaerolysin determinant. Effective marker exchange mutagen-esis requires the delivery of the mutated gene on an unstablereplicon. In initial experiments, using a transformation sys-tem, we found that although plasmid pBR322 could be

introduced into A. hydrophila AH2, it was rapidly lost evenunder selective conditions (data not shown). The transfor-mation rates obtained were low, and we therefore turned tothe pBR325-based mob vectors (pSUP series) (21, 22) toefficiently mobilize the plasmid into a spontaneously derivednalidixic acid resistance derivative of A. hydrophila AH2,designated AB3.

In heterospecific mating experiments performed betweenAB3 and E. coli S17-1 derivatives harboring the plasmidpSUP205, stable transconjugants harboring the plasmid-mediated markers for chloramphenicol and tetracyclineresistance were detected at a rate greater than 10-7 perdonor. We then cloned the 5.8-kb EcoRI fragment carryingthe aerolysin gene from pHPC3-1 (4) into the mob vectorpSUP205 to give the plasmid pHPC3-700 (Fig. 1). Inheterospecific matings between E. coli S17-1 harboringpHPC3-700 and AB3, transmission frequencies of about 10-3per donor were obtained upon selection for the plasmid-mediated antibiotic resistance marker. The results demon-strated that, provided homology to the A. hydrophila chro-mosome is present on the incoming pBR-based plasmid,relatively high rates of transfer can be obtained in hetero-specific matings. Moreover, no plasmid DNA could beisolated from strains harboring plasmid antibiotic resistancemarkers.Our strategy in obtaining aerolysin-negative mutants was

therefore to introduce a deletion within the aerolysin deter-minant cloned onto the vector pSUP205 (pHPC3-700). Todetect the mutant, association of a selectable marker withthe cloned DNA sequence was required. We chose theneomycin phosphotransferase II gene of transposon Tn9O3(18) cloned into plasmid pRME1, since it does not carrysequences homologous to the A. hydrophila AB3 chromo-some.A 1.8-kb KpnI fragment of A. hydrophila DNA from

pHPC3-700 (Fig. 1) was replaced by a 1.4-kb KpnI fragmentcarrying the kanamycin resistance gene of Tn9O3. The loss ofthe 1.8-kb KpnI fragment results in a deletion at the C-

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2276 CHAKRABORTY ET AL.

E K K E H

I I l I I

IC A

K

iL. AB 3 chromosome

B \E E K K E

I I I I E

E E K K E

I I1 I I I

pHPC 3-700

pHPC 3-702

1kb

FIG. 1. Physical map of pHPC3-700 and pHPC3-702. Chromosomal regions flanking the aerolysin determinant in A. hydrophila AB3 are

also depicted. Vector pSUP205 DNA is indicated by closed bars, open bars represent A. hydrophila DNA sequences, and the neomycinphosphotransferase gene of Tn9O3 is shown as a hatched box. Abbreviations: A, aerA; B, aerB; C, aerC; E, EcoRI; H, Hindlll; K, KpnI.

terminal end of aerA and removes the promoter and theN-terminal end of the aerB gene product. This aerA aerBdeletion present on the resulting plasmids is a deletion-substitution since the DNA encoding resistance to kanamy-cin is inserted at a position formerly occupied by the deletedaerA aerB DNA. Plasmids were obtained with the kana-mycin gene in both orientations. E. coli recombinants har-boring these plasmids were completely devoid of bothhemolytic and cytotoxic activities. One such plasmid,pHPC3-702, harboring the kanamycin resistance gene in theclockwise orientation within the aer determinant, was cho-sen for further manipulations.The marker exchange procedure of Ruvkun and Ausubel

(20) as modified by Simon et al. (22) was used to recombineto deletion-Kanr_substitution of pHPC3-702 into strain AB3.Heterospecific mating with E. coli S17-1(pHPC3-702) and A.hydrophila AB3 was performed for 4 h on filters, after whichthe organisms were plated out at various dilutions on bloodagar plates supplemented with the antibiotics nalidixic acidand kanamycin. The rate of conjugal transfer was approxi-mately 10-4 transconjugants per donor. Of all transconju-gants obtained, 90% were nonhemolytic, resistant to nali-dixic acid and kanamycin, and sensitive to tetracycline(which is a plasmid-coded resistance). This is expected ifrecombination of the plasmid with the desired marker ex-

change reaction between the wild-type sequence and themutated sequence carried on pHPC3-702 occurs with subse-quent loss of vector sequences. The remaining 10% oftransconjugants were hemolytic and resistant to both tetra-cycline and kanamycin. These strains were merogenotes,harboring both the mutated and wild-type alleles ofaerolysin, the result of a single crossover event leading tointegration of the plasmid pHPC3-702. Interestingly, thesestrains were less hemolytic than the parental strain AB3. Asingle nonhemolytic transconjugant, resistant to kanamycin,tetracycline, and nalidixic acid, was also found.To ascertain that the nonhemolytic transconjugants ob-

tained were not merely mutants defective in transport, 100independently derived transconjugants were streaked onto

DNase and protease indicator plates. After overnight incu-bation at 37°C, all strains were found to secrete DNase;however, 92 strains also secreted protease. The remainingeight strains showed delayed zones of lysis on proteaseindicator plates, usually after 2 to 3 days of growth at 37°C.

Characterization of A. hydrophila AB3 aerolysin-defectivemutants. A selected number of mutants were then picked forfurther characterization. All nonhemolytic strains were neg-ative (even at the lowest dilution) in a cytotoxicity assayusing Chinese hamster ovary cells. Two mutant strains,AB3-1 and AB3-2, both harboring a copy each of thewild-type and mutated alleles, were clearly less cytotoxicthan the parental strain AB3. These strains were furtherchecked for the presence of aerolysin in both cell lysates andsupernatant fluids by using an immunoblot assay. Further-more, the predicted deletions and rearrangements in thechromosomal DNA of the mutants were detected by DNAhybridization using the plasmids pHPC3-1 and pRME1 as

gene probes (Fig. 2).No aerolysin was detected in either culture supernatants

or supernatant fluids of strains harboring the deletion-Kanr-substitution of the aerolysin determinant (Fig. 3C). StrainAB3-1 (described above) produced aerolysin detectable insupernatant fluids, albeit in lesser quantity than the parentalstrain.Genomic DNA from AB3 and six mutants was isolated

and cleaved with the restriction endonuclease KpnI. Theabsence of the 1.8-kb KpnI A. hydrophila DNA was detectedby using plasmid pHPC3-1 (aerA+B+C+) as a probe; simi-larly, the presence of the substituted 1.4-kb kanamycinresistance fragment was detected using plasmid pRME1(Fig. 2).

Strains AB3-1 and -2, which carry the wild-type andmutated aerolysin alleles, show the presence of the flankingKpnI fragments proximal (11.4 kb) and distal (13.6 kb) to the1.8-kb KpnI fragment. The fragment hybridizing at 11.4 kb,however, appears to contain more than one copy of theflanking sequence. An 11.6-kb fragment consisting of se-

quences from plasmid pHPC3-702 was expected to hybridize

K H

I I

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AEROLYSIN IN A. HYDROPHILA-ASSOCIATED INFECTIONS

rQ ) f)U C)'IN r.3'IC-) coI 'cl ') r)

en

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FIG. 2. Hybridization pattern of KpnI-cleaved chromosomal DNA from strains AB3, AB3-1, AB3-2, AB3-3, AB3-5, AB3-9, and AB3-13with nick-translated oa-32P-labeled plasmid probes pHPC3-1 (A) and pRME1 (B). BglII fragments of lambda DNA and EcoRI fragments ofSPP1 DNA were used as size markers.

with the pHPC3-1 and pRME1 plasmid probes (both arepBR-based plasmids). This occurred as predicted and al-lowed us to locate the site of integration of the plasmidpHPC3-702 upstream of the chromosomal aerolysin gene. Asexpected, strains AB3-1 and -2 harbor the 1.4-kb fragmentcoding for resistance to kanamycin.

Strains AB3-3, -5, -9, and -13 were all aerolysin negative.

1 2 3 4 1 2 3 4 1 2 3 4

__ -_

It was therefore not surprising that these strains lacked theparental 1.8-kb KpnI fragment (Fig. 2A) and harbored the1.4-kb KpnI kanamycin resistance fragment from Tn9O3 (seeFig. 2B) instead. The presence of the 11.4- and 13.6-kbflanking KpnI fragments were also detected in these strains.The mutant strain AB3-3 was tetracycline resistant andnonhemolytic; the wild-type allele had probably been lost by

1 2 3 4 1 2 3 4Hp Al I

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FIG. 3. Immunoblots of total lysates and concentrated supernatant fluids of strains AB3 and AB3-5 with antisera obtained from controland infected mice. Lanes 1, AB3-5 total cell lysate; lanes 2, AB3 total cell lysate; lanes 3, AB3 supernatant fluid; lanes 4, AB3-5 supernatantfluid. Blots were reacted with the respective antiserum, treated with peroxidase-labeled second antibody, and developed with 4-chloro-1-naphthol. Antisera from two or three mice were pooled and obtained 14 to 16 days after the last injection. All infected mice received 108bacteria either i.p. or s.c. Antiserum was obtained from (A) uninfected mice, (B) mice injected s.c. once with AB3-5, (C) mice injected s.c.once with AB3, (D) mice injected s.c. with AB3 on day 0 and again on day 16, and (E) mice injected s.c. with AB3 on day 0 and i.p. on day16. The 49-kilodalton (kd) protein indicated is mature aerolysin.

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2278 CHAKRABORTY ET AL.

TABLE 2. Mortality of mice after i.p. injection of strainsAB3, AB3-5, and AB3-5 rev-i

Strain Approximate No. of cells No. of miceStrain LD50 (cells) injected dead/no. tested

AB3 7 x 107 8 x 108 5/56 x 108 9/94 x 108 5/52 x 108 5/51 X 108 3/55 x 107 1/5

AB3-5 8 x 108 8 x 108 0/56 x 108 1/94 x 108 0/52 x 108 0/51 x 108 0/55 x 107 0/5

AB3-5 rev-i 5 x 107 8 x 108 5/56 x 108 5/54 x 108 5/52 x 108 5/51 x 108 3/55 x 107 2/5

recombination between homologous A. hydrophila DNAsegments after integration of plasmid pHPC3-702. StrainsAB3-9 and AB3-13 were additionally defective in the pro-

duction of extracellular protease(s). The present analysis,however, revealed no apparent changes in the sizes of thehybridizing DNA fragment; a finer hybridization analysis ofthis region is required to define the lesion (if it is locatedthere). The hybridization was repeated, using the same

genomic DNAs restricted with BamHI and the same twoprobes, with identical results (data not shown).

Virulence of AB3 and AB3-5 in a mouse model. Previousstudies on the virulence of Aeromonas sp. (7, 14) havesuggested that mice lethality assays may be an appropriatemodel for the study of invasive disease. We thereforedetermined the LD50 of the parental strain AB3 and that ofan isogenic aerolysin-negative derivative, AB3-5. Mice in-jected i.p. with strain AB3 showed an LD50 of around 7 x 107cells, whereas the aerolysin-negative derivative had an LD50of >8 x 108 cells (Table 2). All mouse deaths were recorded30 h postinoculation, with the majority of mice succumbingwithin 18 h of i.p. infection. Pure cultures of A. hydrophilaAB3 could be isolated from individually homogenized livers(5 x 107 cells per g) and spleens (3 x 107 cells per g) of deadmice. High concentrations (2 x 105 cells per ml) of bacteriawere also detected in blood samples from these mice. Noloss of antibiotic markers coded by AB3 (Nalr) or AB3-5(Nalr Kanr) was detected upon plating on selective andnonselective plates.Although the genetic structure, immunoblot analysis, and

phenotype of strain AB3-5 confirm the deletion-substitutionand the absence of active hemolysin, we were concernedthat unlinked mutations affecting toxicity had been intro-duced during its construction. We therefore reintroduced thewild-type aer determinant on pHPC3-700 into AB3-5. Inte-gration of the wild-type allele on pHPC3-700 into the chro-mosome of AB3-5 by homogenization was detected byplating on hlood agar plates. Stable transconjugants showinghemolysis and sensitivity to kanamycin were obtained at a

rate of 10-6 per transconjugant. One such transconjugant,designated AB3-5 rev-I, was checked for hemolytic andcytotoxic activity. Full hemolytic and cytotoxic activity wasregained with this strain (hemolytic and cytotoxic activities,

respectively, were 512 and 1,024 units for AB3, 4 and 4 unitsfor AB3-5, and 512 and 1,024 units for AB3-5 rev-I).Reintegration of the deleted segment was confirmed byDNA-DNA hybridization (Fig. 2A). The LD50 of strainAB3-5 rev-i was around 5 x 107 cells, demonstrating that theloss of virulence in the mouse model is a result of the aerA-Bdeletion present on the chromosome of AB3-5.

It has been previously shown for Vibrio vulnificus, arelated bacterial species that causes wound infections andsepticemia, that s.c. injection of mice mimics the course ofsepticemic infections observed during human disease (2).We therefore injected mice s.c. with different amounts of theaerolysin-producing and aerolysin-deficient strains. TheLD50 of strain AB3 was 4 x 108 cells, approximately 10 timeshigher than that observed with i.p. injections. The LD50 ofthe aerolysin-negative strains was found to be greater than109 bacteria (data not shown). In all mice surviving s.c.infection with the aerolysin-positive strain AB3, dermone-crotic lesions developed at the site of infection. Necrotizinglesions became noticeable 30 h postinfection and grew to 1 to2 cm in diameter at around 11 days; lesions generally healed14 to 16 days postinfection. Such lesions were never ob-tained with the isogenic aerolysin-negative strains, even atthe highest dose tested. At all stages of infection, purecultures of AB3 could be cultivated from the site of thelesion; large numbers of bacteria could be cultured from theblood, livers, and spleens of sacrificed mice. In contrast, theaerolysin-negative strain AB3-5 could no longer be culti-vated from sacrificed mice 1 to 2 days postinfection. Thedata point to a decisive role for aerolysin at the onset ofsepticemic infection; strains lacking active hemolysin areclearly unable to initiate systemic infection in mice.

If aerolysin is important in the initiation of tissue damageand subsequent septicemia, it should be produced in vivo,and its production should be indirectly detected by usingantisera from infected mice in immunoblot assays. Wetherefore used antisera from mice that had survived an s.c.injection after healing of the lesion on day 14. Control seraincluded those from uninfected mice and mice infected withthe aerolysin-negative mutant AB3-5; these mice showed nodetectable antibody to aerolysin (Fig. 3).

Antisera obtained from mice surviving infection with theparental strain AB3 showed a strong reaction to aerolysin inthe immunoblot assay, where it was the major reactiveprotein species.

Antibodies against aerolysin were detected in mice thathad been injected either s.c. or i.p. injected with strain AB3(see Fig. 3). Therefore, antibody to aerolysin was present inthe infected mice independent of the route of delivery of theorganism. Subsequent s.c. rechallenge of mice that hadsurvived a first s.c. or i.p. infection did not always result ina reduction in the lesion produced at the site of injection.Antisera obtained from mice that had been reinfectedshowed a vigorous response to aerolysin. In addition, anti-body to additional antigens of A. hydrophila AB3 were nowdetectable in the sera of these mice. We observed qualitativeand quantitative differences in the antisera, depending uponthe route of infection.The rapid appearance of antibody to aerolysin points to a

role for aerolysin in the infection process. We found theantihemolytic activity of sera obtained from mice infectedwith AB3 to be consistently higher than that of serum fromuninfected mice. Sera obtained from mice that had subse-quently been infected s.c. with a sublethal dose of AB3showed an even further increase in antihemolysin activity as

compared with uninfected control sera.

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DISCUSSION

Our previous attempts to isolate aerolysin-negative mu-tants by using chemical and transposon mutagenesis havebeen unsuccessful. The molecular cloning and identificationof the gene allowed us to construct specific deletions withinthe aerolysin determinant of A. hydrophila. We have usedsite-directed mutagenesis to replace deleted regions on thechromosome of A. hydrophila with an antibiotic resistancemarker. The experimental protocol of Simon et al. (22) wasused to construct these mutants, and we have been aided inour studies by the finding that pMB-based vectors are unableto replicate in A. hydrophila.Mutants were shown to lack aerolysin both within the cell

and in culture supernatants by using antisera to aerolysin inan immunoblot assay. Strain AB3 has a single copy of theaerolysin gene on its chromosome, and all mutants wereshown to lack the chromosomal 1.8-kb KpnI fragment and tohave the 1.4-kb KpnI fragment of Tn9O3 in its place. Whilemost strains were unimpaired in their export of protease(s)and DNase, some mutants were defective in the productionof protease(s). These strains did not show gross deletions orrearrangements around the region of the deletion-substi-tution. In any case, small changes would go undetected inthe protocols described herein. We have also isolated andidentified meroploids and have located the site of integrationof the mutated allele as proximal to the chromosomalaerolysin gene. As noted above, these strains produced lessaerolysin (as detected by hemolytic activity) than the paren-tal strains. The results suggest that an activator protein maybe titrated away by the presence of a site on the mutatedaerolysin allele. Experiments, currently in progress, areaimed at testing this hypothesis.

In a mouse toxicity model, aerolysin-deficient strains weresignificantly less toxic than the parental strain. More than80% of mice died 22 h after inoculation, and the mice diedwithin a 30-h period. The mouse model therefore reflectswell the rapidly progressing wound infection and septicemiaseen with A. hidrophila infections. Reinsertion of the de-leted DNA segment into the mutant AB3-5 restored the LD50value of the strain to that of the parental type.The parental strain AB3 is able to cause necrotizing

lesions emanating from the site of injection; this has neverbeen observed with the aerolysin-deficient strain. Further-more, while the parental strain could be detected in theblood, liver, spleen, and kidneys 14 days postinfection,mutant AB3-5 was no longer cultivable 24 h postinfection.To show that aerolysin was produced during the infection

process, we obtained antisera from mice surviving infectionwith both the parental strain AB3 and its isogenic derivativeAB3-5. Noninfected mice served as controls. Irrespective ofthe route of infection, antibody to aerolysin could always bedetected in mice surviving infection. Indeed, it is the majorprotein species detected in an immunoblot assay. The neu-tralizing capacity of the antibody produced can be boostedby subsequent reinfection of the mice with the parentalstrain.We are unable to distinguish in our present study whether

aeerA or aerB is responsible for the effects described above.We have constructed strains lacking either (aerA or lerB toaddress this question; results similar to those described herehave been obtained with strains deleted only for aerA (T.Chakraborty, B. Huhle, H. Hof, H. Bergbauer, and W.Goebel, manuscript in preparation).The data discussed above point to a decisive role for

aerolysin at the onset of systemic infection; the absence of

an active gene product renders the mutant strain greatlyattenuated. We envisage aerolysin as a factor required veryearly in the establishment and subsequent maintenance ofinfection. The presence of specific antibodies to aerolysin inmice surviving infection can be interpreted as evidence forthis hypothesis. Many species of the genus Vibrio have adisease spectrum not unlike that produced by A. hydrophila(17). Potent cytotoxins have been demonstrated and isolatedin Vibrio fluvialis (24), V. v'ulnifiCu(s (12), and some non-01Vibrio eholerae (15, 17). It remains to be shown whether therole of these toxins is similar if not identical to that ofaerolysin in Vibrio-associated septicemic infections (1).

ACKNOWLEDGMENTS

We thank R. Simon (University of Bielefeld) and W. Messer (MaxPlanck Institute of Molecular Genetics, West Berlin) for strains andplasmids, Brigitte Gopfert for invaluable technical assistance, andEllen Appel for help in preparing the manuscript.This work was supported by a grant from the Deutsche

Forschungsgemeinschaft (SFB 105-A-12).

LITERATURE CITED

1. Blake, P. A., R. E. Weaver, and D. G. Hollis. 1980. Diseases ofhumans (other than cholera) caused by vibrios. Annu. Rev.Microbiol. 34:341-367.

2. Bowdre, J. H., M. D. Poole, and J. D. Oliver. 1981. Edema andhemoconcentration in mice experimentally infected with Vibriovidnificius. Infect. Immun. 32:1193-1199.

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