Synergistic and Additive Effects of Chromosomal and Plasmid-Encoded Hemolysins Contribute to Hemolysis and Virulence in Photobacterium damselae subsp. damselae

Abstract

Photobacterium damselae subsp. damselae causes infections and fatal disease in marine animals and in humans. Highly hemolytic strains produce damselysin (Dly) and plasmid-encoded HlyA (HlyA_pl). These hemolysins are encoded by plasmid pPHDD1 and contribute to hemolysis and virulence for fish and mice. In this study, we report that all the hemolytic strains produce a hitherto uncharacterized chromosome-encoded HlyA (HlyA_ch). Hemolysis was completely abolished in a single hlyA_ch mutant of a plasmidless strain and in a dly hlyA_pl hlyA_ch triple mutant. We found that Dly, HlyA_pl, and HlyA_ch are needed for full hemolytic values in strains harboring pPHDD1, and these values are the result of the additive effects between HlyA_pl and HlyA_ch, on the one hand, and of the synergistic effect of Dly with HlyA_pl and HlyA_ch, on the other hand. Interestingly, Dly-producing strains produced synergistic effects with strains lacking Dly production but secreting HlyA, constituting a case of the CAMP (Christie, Atkins, and Munch-Petersen) reaction. Environmental factors such as iron starvation and salt concentration were found to regulate the expression of the three hemolysins. We found that the contributions, in terms of the individual and combined effects, of the three hemolysins to hemolysis and virulence varied depending on the animal species tested. While Dly and HlyA_pl were found to be main contributors in the virulence for mice, we observed that the contribution of hemolysins to virulence for fish was mainly based on the synergistic effects between Dly and either of the two HlyA hemolysins rather than on their individual effects.

INTRODUCTION

The marine bacterium Photobacterium damselae subsp. damselae (formerly Vibrio damsela) is a primary pathogen for a variety of marine animals, such as sharks, dolphins, crustaceans, mollusks, and fish (1–3). In addition, this bacterium can cause a highly severe necrotizing fasciitis in humans that may lead to a fatal outcome (4–7).

Early studies described that P. damselae subsp. damselae isolates from marine animals and from human clinical cases exhibited hemolytic activity (8, 9). It was reported that a correlation between the ability of P. damselae subsp. damselae to cause disease in mice and the production of large amounts of a cytolytic toxin with hemolytic activity that was later named damselysin (Dly) existed (8). Dly was characterized as a cytolysin with phospholipase D activity against sphingomyelin, phosphatidylcholine, and phosphatidylethanolamine, and its main molecular activity consists of the removal of the polar choline groups of choline-containing membrane lipids (10, 11). The removal of the polar choline phosphate head group of sphingomyelin by Dly was found to enhance the hemolytic effect of staphylococcal delta-toxin, an observation that constituted the first evidence that Dly can act synergistically with hemolysins produced by other cells (11). The term “synergy” defines a process in which one or more factors in a system has intrinsic lytic activity and the combined factors cause erythrocyte lysis at a rate greater than the sum of the individual rates (12). One well-described synergistic effect is the so-called CAMP (Christie, Atkins, and Munch-Petersen) reaction, originally defined to be a synergistic hemolytic process produced by the interaction of a hemolysin (CAMP factor) of group B streptococci (Streptococcus agalactiae) with beta-toxin (a sphingomyelinase) of Staphylococcus aureus (13).

Recently, we found that Dly toxin is encoded by pPHDD1, a 153-kb plasmid that also encodes a homologue of HlyA (HlyA_pl), and demonstrated that synergistic effects between Dly and HlyA_pl contribute to hemolysis and virulence in pPHDD1-harboring P. damselae subsp. damselae strains (14). HlyA_pl shows a 50% identity to Vibrio cholerae VCC, a hemolysin of the pore-forming toxin (PFT) family. PFTs are released as water-soluble monomeric proteins that diffuse toward the target membrane, forming amphipathic noncovalently associated oligomers that render a ring-like structure called a prepore. After contact with the cell membrane, a subsequent conformational change leads to its insertion as a transmembrane channel and to pore formation (15, 16).

We observed that Δdly ΔhlyA_pl mutants retained hemolytic activity at levels similar to those for naturally occurring pPHDD1-negative strains, which suggested the presence of yet uncharacterized chromosome-encoded hemolysins. In the present study, we report the identification and characterization of a chromosome-encoded HlyA (HlyA_ch) in both pPHDD1-harboring and plasmidless strains that was highly similar (92% identity at the amino acid level) to pPHDD1-encoded HlyA_pl. We demonstrate here that hemolysis in strains containing pPHDD1 is due to the additive effect between HlyA_pl and HlyA_ch, on the one hand, and to the synergistic activity of Dly with HlyA_pl and HlyA_ch, on the other hand. Additional data on the interaction among these three hemolysins and with hemolysins from other species, their role in virulence, and their transcriptional regulation by environmental factors are discussed.

MATERIALS AND METHODS

Bacterial strains, plasmids, and culture conditions.

The bacterial strains and plasmids used here and those derived from this study are listed in Table 1. Additional P. damselae subsp. damselae isolates used in the PCR-based screening for the presence of hlyA_ch are described elsewhere (14). P. damselae subsp. damselae cells were routinely grown at 25°C on tryptic soy agar (TSA) supplemented with 1% NaCl (TSA-1) or tryptic soy broth supplemented with 1% NaCl (TSB-1). Sheep blood agar plates (Oxoid) were used for hemolysis assays and for conjugative matings. Mouse and turbot blood was aseptically collected and added to TSA-1 at a final concentration of 5% (vol/vol) to obtain mouse and turbot blood agar, respectively. For hemolysis assays on agar plates, a single colony of each strain grown on a TSA-1 plate was picked with the tip of a rounded wooden pick and seeded on the blood agar plate, and pictures were taken, unless otherwise stated, after 15 h of incubation at 25°C. Escherichia coli strains were routinely grown at 37°C in Luria-Bertani (LB) broth and LB agar supplemented with antibiotics when appropriate. Antibiotics were used at the following final concentrations: kanamycin at 50 μg ml⁻¹, ampicillin sodium salt at 50 μg ml⁻¹, gentamicin at 15 μg ml⁻¹, rifampin at 50 μg ml⁻¹, and chloramphenicol at 20 μg ml⁻¹.

Table 1.

Strains and plasmids used and constructed in this study

Strain or plasmid	Description	Reference or source
Strains
P. damselae subsp. damselae
RM-71	Isolated from turbot (Psetta maxima), strongly hemolytic, carrying pPHDD1	30
AR57	RM-71 derivative, spontaneous rifampin-resistant mutant	14
AR64	AR57 with in-frame deletion of dly gene	14
AR133	AR57 with in-frame deletion of hlyApl gene	14
AR78	AR57 with in-frame deletion of dly and hlyApl genes	14
AR129	AR57 with in-frame deletion of hlyAch gene	This study
AR119	AR57 with in-frame deletion of dly and hlyAch genes	This study
AR158	AR57 with in-frame deletion of hlyApl and hlyAch genes	This study
AR89	AR57 with in-frame deletion of dly, hlyApl, and hlyAch genes	This study
LD-07	Isolated from seabream (Sparus aurata), weakly hemolytic	30
AR111	LD-07 derivative, spontaneous rifampin-resistant mutant	This study
AR112	AR111 with hlyAch gene disrupted by insertional mutation, Kmr	This study
E. coli
DH5α	Cloning strain, recA	Laboratory stock
XL1-Blue MR	Δ(mcrA)183 Δ(mcrCB-hsdSmr-mrr)173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac	Stratagene
S17-1-λpir	recA thi pro ΔhsdR-hsdM RP4-2 Tc::Mu Km::Tn7 λpir Tpr Smr	49
Streptococcus agalactiae AR170	Laboratory isolate from rat skin	This study
Vibrio cholerae N16961		23
Aeromonas schubertii TL3B0B	Laboratory isolate, sea bass (Dicentrarchus labrax)	This study
Rhodococcus equi CECT555		Spanish Type Culture Collection (CECT)
Plasmids
pWKS30	Low-copy-no. cloning vector, Apr	50
pNidKan	Suicide vector derived from pCVD442, Kmr	17
pHRP309	lacZ reporter plasmid, mob Gmr	20
pACYC184	Low-copy-no. cloning vector, Cmr	Stratagene
pAJR27	pWKS30 with hlyApl gene from RM-71	14
pAJR29	pACYC184 with dly gene from RM-71	14
pAJR55	pHRP309 with hlyAch gene from RM-71	This study
pAJR45	hlyAch::lacZ fusion in pHRP309	This study
pAJR51	dly::lacZ fusion in pHRP309	This study
pAJR53	hlyApl::lacZ fusion in pHRP309	This study

Mutant construction and gene complementation.

Nonpolar deletions were constructed by using PCR amplification of the amino- and carboxy-terminal fragments of each gene, which, when fused together, would result in an in-frame deletion of more than 90% of the coding sequence. Amplification was carried out using Hi-Fidelity Kapa Taq (Kapa). Allelic exchange was performed as previously described using the Km^r suicide vector pNidKan containing the sacB gene, which confers sucrose sensitivity, and R6K ori, which requires the pir gene product for replication (17). The plasmid constructions containing the deleted alleles were transferred from E. coli S17-1-λpir into a rifampin-resistant derivative (AR57) of P. damselae subsp. damselae RM-71. For conjugative matings, exponentially growing cells of donor and recipient strains were mixed, a drop (100 μl) was placed directly onto a sheep blood agar plate, and the plate was incubated at 25°C for 3 days. Cells were scraped off the plate and resuspended in TSB-1, and 100-μl aliquots of serial decimal dilutions were spread on TSA-1 plates, selecting for kanamycin resistance for plasmid integration and subsequently for sucrose resistance (15% [wt/vol]) for a second recombination event. This led to P. damselae subsp. damselae ΔhlyA_ch, Δdly ΔhlyA_ch, ΔhlyA_pl ΔhlyA_ch, and Δdly ΔhlyA_pl ΔhlyA_ch mutant strains (Table 1). The presence of the correct alleles was confirmed by PCR. The single hlyA_ch insertional mutation in strain LD-07 was generated by amplifying an internal fragment of 1,000 bp of hlyA_ch ligated into vector pWKS30 to generate pAJR37. The pAJR37 plasmid was mobilized from E. coli S17-1-λpir into the rifampin-resistant derivative (AR111) of P. damselae subsp. damselae LD-07. Insertion of the suicide vector into the chromosome by a single crossover resulted in a Km^r phenotype. The insertional mutants were selected on agar plates containing kanamycin (50 μg ml⁻¹) and rifampin (50 μg ml⁻¹), and the presence of the plasmid inserted into hlyA_ch gene was confirmed by PCR. Plasmids pAJR27, pAJR29, and pAJR55, containing the hlyA_pl, dly, and hlyA_ch, genes, respectively, were introduced into E. coli DH5α by electroporation.

Hemolytic assays with bacterial ECPs.

To obtain the extracellular products (ECPs), 100 μl of P. damselae subsp. damselae broth cultures adjusted to an optical density at 600 nm of 1 were spread over cellophane-covered TSA-1 plates with a sterile cotton swab as previously described (18). After 48 h of incubation at 25°C, cells were washed off the cellophane with a minimum volume of saline solution (0.85% [wt/vol] NaCl) and readjusted to an optical density at 600 nm of 1. The cell suspensions were centrifuged at 13,000 rpm for 5 min, and the supernatants were filtered through 0.22-μm-pore-size membranes and stored at −30°C until needed. For hemolytic liquid assays, a modification of the method of Bernheimer was used (19). Briefly, sheep erythrocytes (Oxoid) were washed with phosphate-buffered saline (PBS) and centrifuged at 3,000 rpm for 5 min at 4°C. Washes were repeated until the supernatant was visibly clear of hemoglobin. Different volumes of crude erythrocytes were washed and tested to assess the minimal volume necessary to get the maximal absorbance (the absorbance obtained by lysis of the erythrocytes with distilled water). Two hundred microliters of crude erythrocytes was enough to get the maximal absorbance, and consequently, the erythrocytes were adjusted with PBS to 0.5 ml per dilution tube assayed and 0.5 ml of serially diluted ECP samples was added. After 2 h of incubation at 25°C, the mixes were centrifuged at 3,000 rpm for 5 min at 4°C to remove undamaged erythrocytes. This assay is based on the measure of the released hemoglobin, whose concentration is estimated by reading the absorbance at 545 nm in a spectrophotometer and adjusted using nonlysed cells (0.5 ml of erythrocyte suspension with 0.5 ml of PBS) as a control. One unit of hemolytic activity was expressed as the amount of ECP which caused the release of 50% of the hemoglobin in the standardized erythrocyte suspension. All hemolytic assays were carried out in triplicate, and mean values with standard deviations are depicted.

Construction of lacZ transcriptional fusions and β-galactosidase assays.

DNA fragments corresponding to P. damselae subsp. damselae dly, hlyA_pl, and hlyA_ch presumptive promoter regions were obtained by PCR. The PCR-amplified regions, extending from about 1 kb upstream of the ATG start codon to about 50 to 100 bp downstream of the start codon for each tested gene, were fused to a promoterless lacZ gene in the low-copy-number reporter plasmid pHRP309 (20). The resulting transcriptional fusion constructs, dly::lacZ (pAJR51), hlyA_pl::lacZ (pAJR53), and hlyA_ch::lacZ (pAJR45), were mobilized from E. coli S17-1-λpir into a P. damselae subsp. damselae RM-71 rifampin-resistant derivative (AR57) by conjugation. The P. damselae subsp. damselae strains carrying the promoter-lacZ fusion vectors or control plasmid pHRP309 were grown in LB broth alone and in LB broth supplemented with different concentrations of NaCl and of the iron chelator 2,2′-dipyridyl. The β-galactosidase activities were measured by the method of Miller (21). The data shown correspond to those from three independent experiments.

Mouse and fish virulence assays.

Virulence assays were carried out with BALB/c mice (age, 6 to 8 weeks; weight, 26 to 30 g), as well as with turbot (Psetta maxima) (average weight, 15 g), in three groups of five animals each per strain tested. The inoculum was prepared by suspending several colonies from a 24-h TSA-1 culture into saline solution to achieve the turbidity of a no. 2 McFarland standard. Mice were inoculated in the tail vein with 50 μl of a 2.5 μM hemoglobin solution (8 μg hemoglobin per mouse) 2 h before inoculation with the bacterial suspension, as previously described (22). Mice were inoculated intravenously in the tail vein, and turbot were inoculated intraperitoneally with 0.1 ml of 10-fold serial dilutions of the bacterial suspensions. The actual number of injected CFU was determined by plate count on TSA-1. The final doses assayed corresponded to 2.1 × 10⁶ bacterial cells per mouse and 2.1 × 10⁴ bacterial cells per fish. The mortalities were recorded daily for 3 days (mice) and 4 days (turbot), and the results from the three independent groups of five animals were pooled and expressed as mortality percentages. All the protocols of animal experimentation used in this study have been reviewed and approved by the Animal Ethic Committee of the University of Santiago de Compostela.

Statistical analysis.

The statistical analysis of the hemolytic activity data to assess the contributions of the three hemolysins was carried out using one-way and multifactor analysis of variance (ANOVA; full model, including interaction). For statistical analysis of differences between two groups, we used the Student t test. The β-galactosidase data (promoter expression levels) under changing concentrations of either NaCl or 2,2′-dipyridyl were subjected to a regression analysis. For the statistical analysis of the results of the fish and mouse virulence experiments, differences among data for live/dead animals for each pooled group of 15 animals were compared using a chi-square test. The SPSS statistical software package (version 20; IBM SPSS, Inc., Chicago, IL) was used for all statistical analyses. Adjusted P values of <0.05 were considered statistically significant.

RESULTS

A novel HlyA hemolysin is encoded in the chromosome of plasmidless and pPHDD1-harboring P. damselae subsp. damselae strains.

We searched the complete genome sequence of P. damselae subsp. damselae ATCC 33539 (GenBank accession no. ADBS00000000) for candidate hemolysin genes and found that the translated product of open reading frame VDA_002420 in chromosome I showed 92% identity to the previously described pPHDD1-encoded hemolysin HlyA. Here we refer to these two hemolysins as HlyA_pl (plasmid encoded) and HlyA_ch (chromosome encoded). Interestingly, while the similarity between the coding sequences of the two genes at the nucleotide sequence level was 91%, the 250-bp sequences upstream of the translational start codon containing the putative promoters showed only 72% identity (i.e., 28% divergence) between them, with three insertions of 14, 30, and 20 bp in hlyA_ch promoter which were absent in the hlyA_pl promoter sequence (data not shown). Nucleotide sequence substitutions between hlyA_ch- and hlyA_pl-coding sequences allowed the design of allele-specific PCR primers, and a PCR-based screening for specific hlyA_ch sequences demonstrated that all of a total of 16 hemolytic P. damselae subsp. damselae isolates (14) tested contained the hlyA_ch gene, including those harboring plasmid pPHDD1 (6 strains) and those lacking pPHDD1 (10 strains) (data not shown). We found that the genomes of several species of Vibrio whose genomes have been completely sequenced encode homologues of HlyA in chromosome II, with identity values at the amino acid level ranging from 47% to 50%. These Vibrio species include V. cholerae (23), V. anguillarum (24), V. fluvialis (25), V. furnissii (26), and V. harveyi (27), among others.

HlyA_ch contributes to hemolysis in plasmidless and in pPHDD1-harboring strains.

We previously reported that plasmidless P. damselae subsp. damselae strains produced hemolysis on sheep blood agar (14). In the present study, we observed that deletion of the hlyA_ch gene in naturally plasmidless strain AR111 (yielding strain AR112) caused a complete suppression of hemolysis on sheep blood agar (Fig. 1), suggesting that HlyA_ch is the only contributor of hemolysis in strains that lack pPHDD1. In addition, we had reported that a Δdly ΔhlyA_pl double mutant of a plasmid-bearing strain (AR78) maintained some hemolytic activity (14). Here, we demonstrate that deletion of hlyA_ch in AR78, yielding the triple mutant AR89, causes complete suppression of the hemolytic activity against sheep erythrocytes (Fig. 2). These results altogether demonstrate that HlyA_ch is a hemolytic factor in pPHDD1-harboring and also in plasmidless P. damselae subsp. damselae strains.

Fig 1.

HlyA_ch is necessary for hemolysis of sheep erythrocytes in plasmidless strain AR111. The respective insertional mutant derivative for the hlyA_ch gene (strain AR112) lacks detectable hemolytic activity.

Fig 2.

Hemolytic activity of the P. damselae subsp. damselae parental strain and mutants in sheep, mouse, and turbot blood agar plates. The genotype of the strain (either parental or deletion mutant) is given in parentheses. The hemolysins being produced are indicated below the strain name.

To better understand the contribution of HlyA_ch to hemolysis, we generated hlyA_ch mutants under different genetic contexts (Fig. 2). On sheep blood, the ΔhlyA_ch ΔhlyA_pl double mutant (AR158), which only secretes Dly, produced an almost unperceivable and turbid hemolytic halo. This observation, together with the previously reported weak halo produced by the Δdly ΔhlyA_pl mutant (AR78) (14), which produces only HlyA_ch, clearly suggests that the strong hemolytic halo of AR133 (ΔhlyA_pl) on sheep blood agar is the result of a synergistic effect between Dly and HlyA_ch rather than an additive sum of the individual effects of these two hemolysins. Interestingly, we observed on sheep blood agar that the single ΔhlyA_ch mutant (AR129) lacked the hemolytic halo that would be expected by the action of HlyA_pl. Most surprisingly, deletion of dly in AR129 (yielding AR119) caused a recovery of hemolytic activity on sheep blood (Fig. 2), which suggests that repression or inhibition of HlyA_pl activity is mediated, directly or indirectly, by Dly on a ΔhlyA_ch background (see below). Altogether, the results using sheep blood agar indicate that Dly is unable by itself to produce complete lysis of sheep erythrocytes but is necessary to produce synergistic effects with either of the two HlyA hemolysins in this erythrocyte source.

On mouse and turbot blood, the ΔhlyA_ch ΔhlyA_pl double mutant (AR158) showed only a slight reduction of hemolytic activity compared to AR57. However, deletion of dly in strain AR129, resulting in the Δdly ΔhlyA_ch double mutant (AR119), caused a reduction of the hemolytic halo of >80%. The results using mouse and turbot blood agar confirm the main role of Dly in hemolysis of these two erythrocyte species.

Hemolysis in strains harboring pPHDD1 is the result of a synergistic effect between Dly and either of the two HlyA hemolysins (HlyA_pl and HlyA_ch) and of additive effects between HlyA_pl and HlyA_ch.

In order to obtain a quantitative measure of the contribution of each of the three hemolysins to the hemolytic phenotype, we carried out assays with sheep erythrocyte suspensions and bacterial extracellular products (ECPs) obtained under standard TSA growth conditions (Fig. 3A). We found significant differences in the hemolytic activities obtained for each assayed strain (P = 6.6 × 10⁻¹⁹ by ANOVA). The results showed that HlyA_pl was the most active (75 hemolytic units [HU]), followed by HlyA_ch (33 HU), while Dly alone did not produce hemolysis (0 HU), demonstrating that the presence of at least one of the two HlyA hemolysins is necessary and sufficient to produce detectable hemolysis of sheep erythrocytes (P < 0.001 by Student's t test). No significant differences (P = 0.0841 by Student's t test) between the sum of the individual values obtained for the HlyA_pl- and the HlyA_ch-producing mutants (75 and 33 units, respectively) and the value obtained with the mutant that produced the two HlyA (96 units) were detected, suggesting that the contribution of HlyA_pl and HlyA_ch to hemolysis constitutes an additive effect (the multifactor ANOVA analysis [P = 0.15] did not detect an interaction between HlyA_pl and HlyA_ch).

Fig 3.

Synergistic and additive effects among the Dly, HlyA_pl, and HlyA_ch hemolysins are demonstrated by calculation of the hemolytic activity of the ECPs of P. damselae subsp. damselae on sheep erythrocytes. (A) ECP of individual strains; (B) combinations of ECPs from different pairs of strains mixed in a 1:1 ratio. The release of hemoglobin in the supernatant was measured at A₅₄₀. One hemolytic unit is defined as the amount of hemolysin which lyses 50% of sheep erythrocytes. All assays were carried out in triplicate, and mean values with standard deviations are shown. The results for bars with different letters are significantly different from each other (one-way ANOVA).

In addition, we found that the full hemolytic activity of the parental strain did not result from a mere sum of the individual activities of each hemolysin. Mutation of dly strongly prevented the values obtained with the parental strain from being achieved. The Δdly mutant producing the two HlyA hemolysins achieved 96 units, whereas in the presence of Dly (i.e., in the parental strain), this value increased to 316 units. In the multifactor ANOVA analysis, this fact was demonstrated by a significant strong interaction between Dly and either of the two HlyA hemolysins (the interaction of Dly, HlyA_ch, and HlyA_pl yielded a P value of 1.22 × 10⁻⁸ by ANOVA). This observation, together with the data obtained with the Δdly ΔhlyA_pl (33 units) and the ΔhlyA_pl (196 units) strains (the interaction between Dly and HlyA_ch yielded a P value of 8.70 × 10⁻¹³ by ANOVA), clearly demonstrates that Dly interacts synergistically with either of the two HlyA hemolysins.

The synergistic and additive effects among hemolysins are reproduced in an Escherichia coli background.

In order to gain additional evidence of the synergistic and additive effects between hemolysins, we assayed the hemolytic phenotype conferred by each gene individually and by combinations of two or three genes to Escherichia coli (Fig. 4). Cells of E. coli harboring the dly gene showed an almost undetectable hemolytic halo compared to the halo observed in E. coli cells harboring either hlyA_pl or hlyA_ch, confirming again the low activity of Dly against sheep erythrocytes and confirming that either of the two HlyA hemolysins is necessary and sufficient to cause hemolysis of sheep erythrocytes. The halo shown by E. coli harboring the hlyA_pl gene was wider than that shown by E. coli harboring the hlyA_ch gene, consistent with the 2-fold higher values obtained with HlyA_pl than HlyA_ch in the liquid hemolytic assays (see above). This differential hemolytic activity of the two HlyA toxins was also visible when each was combined with Dly. In addition, the existence of synergistic effects between Dly and each HlyA was demonstrated by the much stronger haloes observed in their different combinations compared with the haloes produced by the individual hemolysins. The widest and most translucent halo was observed in E. coli harboring the three hemolysin genes. Introduction of the two distinct hlyA genes together produced a halo which did not differ substantially from the halo seen with introduction of hlyA_pl alone, indicating an additive rather than a synergistic effect between HlyA_pl and HlyA_ch. Altogether, these results support the hypothesis that in strains harboring plasmid pPHDD1, hemolysis is due to the synergistic activity of Dly with HlyA_pl and HlyA_ch, on the one hand, and to the additive effect between HlyA_pl and HlyA_ch, on the other hand.

Fig 4.

Transformation of Escherichia coli DH5α with different combinations of the P. damselae subsp. damselae hemolysin genes dly, hlyA_pl, and hlyA_ch demonstrates the synergistic and additive effects of hemolysins against sheep erythrocytes on sheep blood agar plates.

The synergistic effect between Dly and HlyA constitutes a case of the CAMP reaction.

We saw, as indicated above, that hemolysis in the parental strain is the result of synergistic effects between Dly and HlyA. This phenomenon might constitute a CAMP reaction. This is a two-step process where a first-step agent modifies the erythrocyte membrane in a nonlytic or sublytic fashion, making it vulnerable to the action of the second-step agent (the so-called CAMP factor). The CAMP reaction was described to be a synergistic hemolytic process produced by the interaction of the beta-toxin sphingomyelinase of Staphylococcus aureus with a hemolysin (CAMP factor) of Streptococcus agalactiae, causing increased hemolysis where the two toxins converge (13).

To assess whether synergy in P. damselae subsp. damselae hemolysis constitutes a case of the CAMP reaction, we cultured Dly-producing strains (the parental strain and the double mutant for the two hlyA genes, AR158) surrounded by non-Dly-producing strains on a sheep blood agar plate. The hemolytic halo of two non-Dly-producing strains was increased and acquired a half-moon shape at the area that intersects with the halo of partial hemolysis produced by Dly both in the parental strain (Fig. 5A) and in the AR158 mutant (Fig. 5B). Mutation of the dly gene prevented us from observing the CAMP reaction (Fig. 5C), demonstrating that Dly acts as the first-step agent. CAMP reactions between two parental strains were not observed (Fig. 5D) because the factor limiting the size of the hemolytic halo is the extent of the HlyA diffusion zone rather than that of the Dly diffusion zone. Hence, the translucent hemolytic halo produced by each parental strain is not susceptible to further improvement when additional Dly is supplied. This phenomenon can also be observed when the Dly halo produced by AR158 overlaps the hemolytic halo produced by AR64 (Fig. 5E).

Fig 5.

Evidence of synergistic CAMP reactions among P. damselae subsp. damselae strains. The hemolysins being produced are indicated below the strain name. Arrows, half-moon-shaped areas where CAMP reactions are observed due to the confluence of Dly (first-step agent) and one or two HlyA hemolysins (second-step agent). (A, B) Dly-producing strains AR57 (the parental that produces the three hemolysins) and AR158 generate CAMP reactions with the non-Dly-producing strains AR78 and AR111 (plasmidless strain); (C) the non-Dly-producing strain AR64 is unable to produce CAMP reactions; (D) CAMP reactions are not observed between two Dly-producing strains; (E) CAMP reactions are produced only when a second-step agent (HlyA_pl or HlyA_ch) is secreted.

CAMP reactions can therefore be observed in plasmidless (AR111) and in Δdly P. damselae subsp. damselae strains. HlyA (in either of the two forms, HlyA_ch or HlyA_pl) is present in the two types of strains, making this toxin a good candidate to act as a CAMP factor. To prove this hypothesis, different hlyA mutants were tested for CAMP reactions in the presence of the Dly-producing strain AR158 (Fig. 5E). A strain producing only HlyA_ch (AR78) showed a strong CAMP reaction at the intersection with the zone of Dly diffusion. However, as expected, the triple mutant AR89 as well as the hlyA_ch mutant (AR112) of a pPHDD1-negative strain yielded no CAMP reactions (Fig. 5E). We therefore propose that Dly acts as a first-step agent and the pore-forming toxin HlyA acts as a CAMP factor in the CAMP reactions observed in Photobacterium damselae subsp. damselae strains.

Since previous studies reported the existence of CAMP reactions in Streptococcus agalactiae and Rhodococcus equi (13, 28), we also tested whether the hemolytic activity of these two species is enhanced by the interaction with P. damselae subsp. damselae Dly. As a result, we observed the existence of synergistic effects between Dly and these two species (Fig. 6). We also found that Dly produces CAMP reactions with Vibrio cholerae and with Aeromonas schubertii (Fig. 6). Taken together, these results indicate that Dly is capable of producing CAMP reactions with a variety of species that produce CAMP factors.

Fig 6.

Synergistic CAMP reactions between P. damselae subsp. damselae parental strain AR57 (which produces the three hemolysins) and four different bacterial species: Vibrio cholerae, Streptococcus agalactiae, Aeromonas schubertii, and Rhodococcus equi. Arrows, areas where CAMP reactions are observed due to the confluence of P. damselae subsp. damselae Dly (first-step agent) and the second-step agent produced by each tested species. The S. agalactiae and R. equi plates were cultured at 37°C, and the V. cholerae and A. schubertii plates were cultured at 25°C.

Dly exerts an inhibitory effect on HlyA_pl activity against sheep erythrocytes in a ΔhlyA_ch background.

We observed, as indicated above, that the single hlyA_ch mutant (AR129) undergoes a drastic decrease in its hemolytic activity compared with that of the parental strain and that the dly mutation in this strain to yield AR119 Δdly ΔhlyA_ch causes a significant recovery of the hemolytic activity (P = 4.5 × 10⁻⁷ by Student's t test) (Fig. 3A). This suggests that upon deletion of hlyA_ch from the genome, Dly plays a role in the repression or inhibition of HlyA_pl activity. In order to assess at which level this inhibition occurs, we tested two different hypotheses: (i) whether it occurs in a bacterial cell-independent manner or (ii) whether it occurs in a bacterial cell-dependent manner.

To test whether inhibition occurred in a cell-independent fashion by means of protein-protein interactions in the extracellular medium, we mixed ECPs from strain AR158 ΔhlyA_pl ΔhlyA_ch, producing only Dly, with ECPs of strains that produce either both of the HlyA hemolysins (the Δdly strain) or only one of the two HlyA hemolysins (the Δdly ΔhlyA_pl and Δdly ΔhlyA_ch strains) and conducted liquid hemolytic assays. Since, as said above, the ECPs of strain AR158 alone yield no hemolytic values (Fig. 3A), we can attribute the hemolytic values of the combinations containing AR158 ECPs to the synergistic effects between extracellular products. As a result, we found that all the combinations of mixed ECPs from different strains yielded values of hemolytic units similar to those yielded by a single strain producing the same combination of hemolysins (Fig. 3A and B), with the notable exception of ECPs from strains AR158 and AR119. The definite demonstration that the inhibition of HlyA_pl does not occur via extracellular interactions between hemolysins comes from the comparative study of the hemolytic values of the ΔhlyA_ch mutant (1.8 units) (Fig. 3A) and of the combination of ECPs from AR158 and AR119 (223 units) (Fig. 3B) (P = 4.29 × 10⁻⁶ by Student's t test). In the two cases, the hemolysins that are present are the same. However, the ECP mix behaves as if all the hemolysins are being normally produced and functional, whereas in the ΔhlyA_ch mutant, there is a phenomenon of inhibition/repression. Additional confirmation that repression/inhibition of HlyA_pl activity is not due to protein-protein interactions between hemolysins at the extracellular level can be drawn from the results obtained with E. coli strains harboring different gene combinations (see above and Fig. 4).

The next hypothesis that we tested was whether Dly-mediated inhibition of HlyA_pl occurs at cellular levels, i.e., whether the presence of the P. damselae subsp. damselae cells rather than their ECPs is necessary to produce the repression/inhibition phenomenon. We therefore investigated whether mutation of the hlyA_ch gene had any effect on the transcriptional activity of hlyA_pl expression. We could not find significant differences (P = 0.0791 by Student's t test) in the β-galactosidase activities of the hlyA_pl promoter in the parental and the ΔhlyA_ch genetic backgrounds tested (17,450 versus 16,183 units), clearly indicating that the inhibition of HlyA_pl in a ΔhlyA_ch background does not occur at the transcriptional level.

We know that inhibition of HlyA_pl activity is evidenced when Dly is being produced in the same cell that undergoes the inhibition (Fig. 2 and 3A). We therefore wanted to investigate whether Dly-mediated inhibition of HlyA_pl can also be observed in a cell-dependent manner in cells that are exposed to exogenous Dly supplied in the extracellular environment, even in those cells that do not produce Dly. For this purpose, we cultured (i) parental strain AR57, (ii) the AR158 ΔhlyA_ch ΔhlyA_pl double mutant (which produces only Dly), and (iii) mutant AR64 Δdly on the center of sheep blood agar plates. After 3 days of growth (enabling Dly to be produced and diffused through the agar plate), we streaked five different mutants radially from the plate edge toward the center of the plate (Fig. 7). As a result, we found that the radially streaked double Δdly ΔhlyA_pl mutant and the single Δdly mutant underwent an enhancement of their hemolytic activity by synergistic effects in the presence of Dly (Fig. 7A and B) which was not observed if Dly was absent (Fig. 7C). The slight hemolysis produced by the ΔhlyA_ch mutant was totally inhibited in the presence of exogenous Dly (Fig. 7A and B). Most interestingly, Dly-mediated inhibition was clearly visible in the Δdly ΔhlyA_ch double mutant, where hemolysis was produced only in the absence of exogenous Dly, whereas cells growing on the Dly diffusion area were unable to produce hemolysis. Interestingly, a synergistic effect was observed at the edge of the Dly diffusion area in the Δdly ΔhlyA_ch mutant, with this synergistic effect likely being derived from the interaction of Dly with HlyA_pl molecules which were secreted before the repression/inhibition started. As expected, no HlyA_pl inhibition could be observed in any of the strains assayed in experiments with the dly mutant growing on the plate center (Fig. 7C).

Fig 7.

Demonstration of Dly-mediated inhibition of HlyA_pl activity in P. damselae subsp. damselae. The hemolysins being produced are indicated below the strain name. (A, B) The presence of a Dly halo in the two Dly-producing strains AR57 (A) and AR158 (B) causes an inhibition of the otherwise HlyA_pl-mediated hemolysis in the ΔhlyA_ch and ΔhlyA_ch Δdly backgrounds. Arrows, areas where the disappearance of hemolysis occurs. (C) When Dly disappears from the extracellular environment due to dly mutation in AR64, neither inhibition nor synergistic effects are observed. The parental strain (AR57), the ΔhlyA_ch ΔhlyA_pl double mutant (AR158), and the Δdly mutant (AR64) were each cultured in the center of a sheep agar plate and left to grow for 3 days. Subsequently, tester strains were streaked radially from the plate edge to the center. Pictures were taken after 15 h of incubation.

From these results and considering the results presented above, we propose that inhibition of HlyA_pl activity does not occur under bacterial cell-free conditions. Rather, this inhibition is evidenced at cellular levels in a ΔhlyA_ch background, and it occurs not only when Dly is produced inside the cell but also when Dly is provided in the extracellular environment.

Expression of the three hemolysins is regulated at the transcriptional level by iron and sodium chloride concentrations.

Many Vibrionaceae species can live both in a free-living form and in a pathogenic lifestyle, and P. damselae subsp. damselae is not an exception, since it is known to survive in seawater microcosms in a culturable stage for at least 1 year, maintaining its infective potential for fish (29). It would be expected that expression of the P. damselae subsp. damselae hemolysins does not occur while the bacterium is in a free-living style in the ocean but, rather, occurs when the bacterium has established contact with a host. We examined the changes in promoter activity for the three hemolysin genes when the cell faced two situations that mimic entry into a host: (i) low iron availability and (ii) a decrease in the sodium chloride concentration. As a result, we found a positive correlation between the β-galactosidase activity values of the three promoters and the concentrations of the iron chelator 2,2′-dipyridyl (which causes a decrease in iron availability) (Fig. 8A).

Fig 8.

Transcriptional activity (β-galactosidase units) of the lacZ fusions to the dly (white circles), hlyA_pl (black triangles), and hlyA_ch (white triangles) promoters are correlated with the concentrations of the iron-chelator 2,2′-dipyridyl (A) and NaCl (B) in the culture medium. Transcriptional fusions were carried out in P. damselae subsp. damselae parental strain AR57. The significance of the correlation test was determined with the F statistic, and P and r values are shown.

To test whether salt concentration acts as an environmental regulatory signal, we measured promoter expression in the presence of three increasing concentrations of NaCl in LB medium. We found that the β-galactosidase activity of the three promoters decreased as the salt concentration was increased (Fig. 8B), denoting a negative correlation between the transcriptional activity of the three hemolysin genes and salt concentration. Interestingly, we found that in an E. coli XL1-Blue MR background, there was no transcriptional response of the three promoters to changes in NaCl concentration (data not shown), suggesting that this regulation is mediated by a P. damselae subsp. damselae mechanism which is absent in E. coli. Moreover, we found a transcriptional response to salinity when the three promoters were introduced in plasmidless strain AR111 (data not shown), which suggests that the genetic determinants that mediate the salinity-dependent regulation are not encoded by pPHDD1 but, rather, are encoded by the chromosomes of P. damselae subsp. damselae.

Contribution of P. damselae subsp. damselae hemolysins to virulence for mice and fish.

In a previous work, we found evidence that Dly and HlyA_pl contribute to the virulence of P. damselae subsp. damselae for mice and fish (14). Following the discovery of the hlyA_ch gene in the present study, we wanted to evaluate the contribution of the three hemolysins to P. damselae subsp. damselae virulence. For this purpose, we conducted virulence tests in mice, inoculating the parental strain as well as the seven possible combinations of hemolysin gene mutants. Statistically significant differences were observed among the assayed strains (P = 1.444 × 10⁻⁸ by chi-square test) (Fig. 9A). The parental strain caused death in the 15 animals inoculated, whereas no deaths were recorded among animals inoculated with the triple mutant (P = 4.32 × 10⁻⁸), which indicates that at least one of the hemolysins is required for virulence. As a general observation, we found that strains producing any combination of two hemolysins caused higher mortality ratios than a strain producing only one of those two hemolysins, with the exception of the hlyA_pl mutant, which yielded a slightly lower mortality value than the hlyA_pl hlyA_ch double mutant. By comparing the three different double mutants (AR158, AR119, and AR78, strains producing only one of the three individual hemolysins) with the triple mutant (AR89), we observed that although each of the three hemolysins individually is sufficient to cause death in mice, each one contributes to the virulence to a different degree. The mutant AR158, which produces only Dly, killed 12 animals (P = 7.744 × 10⁻⁶), whereas the mutants producing HlyA_pl (AR119) and HlyA_ch (AR78) killed 9 (P = 3.362 × 10⁻⁴) and 3 (P = 0.06789) animals, respectively. These results suggest that the contribution of HlyA_ch to virulence is the lowest among the three toxins. In this regard, we found that although the strain with deletion of hlyA_ch (AR129) (P = 0.1432) alone or in combination with deletion of hlyA_pl (AR158) (P = 0.06789) caused a lower percentage of dead mice than the parental strain, the differences were found not to be significant (Fig. 9A). The virulence data altogether suggest that albeit the highest values of mortality for mice (death of all the animals tested) are achieved only when the three hemolysins are being produced, Dly and HlyA_pl are the main contributors to the virulence of this bacterium for mice.

Fig 9.

Results of mouse (A) and turbot (B) virulence assays with P. damselae subsp. damselae parental strain and mutants. The results are expressed as percent mortality (number of dead animals/number of animals assayed). Numbers inside the bars denote the number of dead animals. Results from three independent groups of 5 animals each per strain tested were pooled and compared using a chi-square test. Asterisks denote statistically significant differences between strains: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Because P. damselae subsp. damselae is a primary pathogen for fish, we judged it to be of maximum interest to assess the role of each of the three hemolysins in virulence for turbot (Fig. 9B). Statistically significant differences were evidenced among the strains tested (P = 6.587 × 10⁻¹⁴). Most interestingly, of the three single hemolysin-producing mutants, only the Dly-producing strain (AR158) was able to cause death in fish (P 0.01431). The observation that no fish deaths were recorded with either the ΔhlyA_ch Δdly (AR119) or the ΔhlyA_pl Δdly (AR78) double mutants demonstrates that either of the two HlyA hemolysins alone does not cause death in turbot but, rather, that one HlyA hemolysin needs the presence of either Dly or the other HlyA hemolysin to cause death. The production of Dly in combination with either of the two HlyA hemolysins causes an increase in the number of animals killed with respect to the number of animals killed by strains that produce Dly alone, and this increase is particularly significant (P = 0.0006501) when Dly is combined with HlyA_ch. This clearly suggests that, unlike what is observed in mice, the contribution of hemolysins to virulence for fish is not so much based on the individual effects of each hemolysin but, rather, on the synergistic effects between Dly and HlyA.

DISCUSSION

P. damselae subsp. damselae is a hemolytic bacterium that causes disease and death in a variety of marine animals and also in humans. Tissue damage and hemorrhagic areas are characteristics of P. damselae subsp. damselae infections (6, 30), suggesting that membrane-damaging factors and particularly hemolysins contribute to pathogenesis. We previously reported that the phospholipase D Dly and the pore-forming toxin HlyA_pl encoded by plasmid pPHDD1 are two main contributors to the hemolytic activity and virulence in this species (14). Strains lacking pPHDD1 as well as a Δdly ΔhlyA_pl mutant are hemolytic, pointing out the production of other hemolysins. After searching the complete genome sequence of P. damselae subsp. damselae ATCC 33539, we identified HlyA_ch, a novel hemolysin encoded by chromosome I of this bacterium. Its 92% amino acid sequence identity to pPHDD1-encoded HlyA_pl and its 50% identity to V. cholerae VCC suggest that HlyA_ch belongs to the family of pore-forming toxins. Early studies had demonstrated the existence of one major and two minor components with hemolytic activity in the supernatants of P. damselae subsp. damselae strains (31), and we propose that they correspond to Dly, HlyA_pl, and HlyA_ch.

We have demonstrated here that HlyA_ch is responsible for hemolysis in P. damselae subsp. damselae strains that lack pPHDD1. Since hlyA_ch is located on chromosome I and is also present in plasmidless strains, it is likely that hlyA_pl has its evolutionary origin in an hlyA_ch-like gene. In line with this, many species of Vibrio harbor hlyA_ch homologues in their chromosomes. The two HlyA hemolysins showed differential activity against sheep erythrocytes. Transcriptional lacZ fusions revealed that the two genes have similar expression levels, which suggests that their differential hemolytic activity is due to punctual amino acid substitutions. We demonstrated that HlyA_pl and HlyA_ch contribute to hemolysis of sheep erythrocytes in an additive manner, and their high sequence similarities suggest that their additive effect in hemolysis is due to a dosage effect.

It was previously reported that Dly is strongly active against mouse and fish erythrocytes but weakly active against sheep erythrocytes (31), and this is consistent with the results obtained with the ΔhlyA_ch ΔhlyA_pl double mutant. The differential sphingomyelin content among erythrocyte membranes of mammal species (32) might explain the different susceptibilities of sheep and mouse erythrocytes to the action of Dly.

Albeit Dly alone is weakly active against sheep erythrocytes, Dly is essential to yield the highest hemolytic values due to a synergistic effect with either of the two HlyA hemolysins. This synergistic reaction can be considered a typical CAMP reaction (13). Previous work demonstrated that Dly shows sphingomyelinase activity and can act synergistically with staphylococcal delta-toxin on sheep erythrocytes (11). Sphingomyelinases are capable of interacting with outer leaflet membrane phospholipids and are responsible for the incomplete hemolytic areas observed in sheep blood agar plates (33, 34). The group of second-step agents, the so-called CAMP factors, is more heterogeneous and includes nonenzymatic bacterial exoproteins as pore-forming toxins, as well as phospholipases, lipases, and cholesterol oxidases (35). The umbrella model (36, 37) provides an explanation of how Dly and HlyA might cooperate synergistically to produce cell lysis. It has been demonstrated that cholesterol is required for different pore-forming toxins to induce cytolysis, as is the case for V. vulnificus cytolysin (16, 38), staphylococcal alpha-toxin (39), and V. cholerae VCC (36, 40). Hydrolysis of choline head groups in membranes by sphingomyelinases has been demonstrated to facilitate access to cholesterol and elicit destruction of the membrane by second-step agents (37, 41). We demonstrated in our study that the action of Dly as a first-step agent allows the second-step agents HlyA_pl and HlyA_ch to enhance their hemolytic activity and to produce CAMP reaction. In addition, we observed CAMP reactions between Dly and V. cholerae, S. agalactiae, R. equi, and Aeromonas schubertii CAMP factors. In S. agalactiae the second-step agent has been reported to be a ceramide-binding protein (42), whereas in R. equi it is a cholesterol oxidase (41). Since it has been demonstrated that deletion of the V. cholerae VCC gene leads to a total suppression of hemolysis (43), we suggest that the unknown CAMP factor produced by V. cholerae is indeed VCC. Albeit A. schubertii has been reported to be hemolytic (44), the molecular basis of hemolysis in this species remains uncharacterized.

The drastic hemolytic reduction observed in the ΔhlyA_ch mutant in sheep erythrocytes is likely due to a repression/inhibition of HlyA_pl hemolytic activity mediated by Dly. It is interesting to note that the predominant role of Dly in the lysis of mouse and turbot erythrocytes prevents us from observing whether this repression also occurs in these two blood types. Recently, it was reported that V. cholerae HlyA hemolysin is negatively regulated via the quorum-sensing-regulated transcription factor HapR, and this negative regulation is exerted at the transcriptional and posttranslational levels (45). We demonstrated, using transcriptional lacZ fusions, that inhibition of HlyA_pl in P. damselae subsp. damselae does not occur at the transcriptional level, and liquid hemolytic assays demonstrated that it is not due to interactions among the hemolysins and does not take place under bacterial cell-free conditions. Rather, we propose that HlyA_pl inhibition occurs in a bacterial cell-dependent manner in the presence of intra- or extracellular Dly. The exact mechanism by which this inhibition takes place remains to be elucidated and might be related to inhibition of maturation and/or secretion of HlyA_pl or to HlyA_pl degradation in a ΔhlyA_ch mutant background in the presence of Dly molecules.

We have demonstrated that iron-deficient conditions enhance expression of the dly, hlyA_pl, and hlyA_ch genes. Since the ability to obtain iron from host tissues is crucial for most pathogenic bacteria in order to establish an infection (46), hemolysin production is expected to facilitate hemoglobin release as a source of iron. Indeed, hemoglobin availability and the susceptibility to bacterial infections are directly related (47), and a functional heme uptake system has been described in P. damselae subsp. damselae (48). Previous studies reported that P. damselae subsp. damselae cultures containing Na⁺ ion concentrations of ≥0.8% underwent a considerable reduction in Dly toxin levels (8). Our results show that increasing sodium chloride concentrations induced a decrease in expression of the three hemolysin promoters. It is unlikely that P. damselae subsp. damselae cells secrete the three hemolysins while in a free-living stage in seawater. Interestingly, the environmental conditions in which higher transcriptional expression values were obtained (iron deficiency and low salt concentration) simulate the conditions that the bacterium encounters upon entry into a host. Therefore, low iron and low salinity might act as molecular signals that trigger expression of Dly and HlyA hemolysins. The fact that the influence of salinity in the transcription of the hemolysin genes was not reproduced in an E. coli background suggests that this regulation might be controlled by a mechanism which is specific to marine bacteria or even specific to P. damselae subsp. damselae. Studies to assess the molecular basis of this salinity-mediated regulation are under way.

In a previous work, we demonstrated that plasmid pPHDD1 is necessary for the full virulence of P. damselae subsp. damselae for mice and fish (14). Here we demonstrate that virulence for mice resides mainly in pPHDD1-encoded Dly and HlyA_pl, since deletion of hlyA_ch did not produce a statistically significant decrease in virulence for mice. The virulence data for mice clearly indicated that Dly is highly toxic for this species, in agreement with the findings of previous studies (11). In addition, we found that the two double mutants AR119 and AR78, which produce only HlyA_pl and HlyA_ch, respectively, are avirulent for fish. Thus, we can conclude that either of the two HlyA hemolysins alone does not cause death in turbot, with the presence of either Dly (synergistic effect) or the other HlyA hemolysin (additive effect) being necessary to cause death. It was somehow surprising that HlyA_ch, with homologues in several Vibrio species, some of them potential fish pathogens, was not toxic for fish in our virulence model unless it was accompanied by Dly. However, Dly alone was able to cause death in turbot, and its virulence was enhanced by the presence of either HlyA_ch or HlyA_pl. Thus, we can conclude that while Dly and HlyA_pl are the main contributors to the virulence for mice, the contribution of hemolysins to virulence for fish is very much based on the synergistic effects between Dly and either of the two HlyA hemolysins rather than on their individual effects. The reason for the differential behavior of HlyA toxins for mice and for fish is currently unknown.

Altogether, our results demonstrate that, depending on the erythrocyte and animal source, the outcomes achieved with the individual hemolysins and of the interactions among the three hemolysins are very different. We found a good correlation between hemolysis and virulence data in mice and fish. As an example, the parental strain and the mutants showing wide hemolytic haloes on either mouse or turbot blood agar plates (Fig. 2) broadly corresponded to the ones that caused higher percentages of mortality for the cognate species (Fig. 9). An exception to this was the observation that HlyA_pl alone was weakly hemolytic in mouse blood agar, while a strain producing only HlyA_pl caused death in 9 of 15 mice inoculated. However, we can guess from the results of previous studies that the role of the P. damselae subsp. damselae hemolysins is not necessarily reduced to lysis of erythrocytes (9). Rather, these three proteins could be exerting effects on other cell types in the animal host that would pass unadvertised in the blood agar plates.

To date, the great majority of synergistic effects reported between hemolysins are between toxins produced by two different bacterial species, with the only exception being Bacillus cereus, which produces synergistic effects with hemolysins produced simultaneously by the same cell (12). Therefore, to our knowledge, P. damselae subsp. damselae represents the first report in a Gram-negative bacterium of synergistic hemolysis occurring within the same species. This synergistic process between Dly and either of the two HlyA forms might be of special relevance in the severity of P. damselae subsp. damselae pathogenesis and highlights the importance of synergistic interactions among virulence factors in pathogenic bacteria.