INFECTION AND IMMUNITY, Apr. 1992, p. 1671-1676 0019-9567/92/041671-06$02.00/0 Copyright ©) 1992, American Society for Microbiology

Vol. 60, No. 4

Identification of a Second Hemolysin (HlyII) in Actinobacillus pleuropneumoniae Serotype 1 and Expression of the Gene in Escherichia coli JOACHIM FREY,l* HAN VAN DEN BOSCH,2 RUUD SEGERS,2 AND JACQUES NICOLET' Institute for Veterinary Bacteriology, University of Berne, Langgasstrasse 122, CH-3012 Berne, Switzerland, 1 and Intervet International B. V., NL-5830 AA Boxmeer, The Netherlands2 Received 9 October 1991/Accepted 3 January 1992

Hemolysin genes of the reference strains of ActinobaciUlus pkuropneumoniae serotypes 1 and 2 were identified, cloned, and expressed in Escherichia coli by using polymerase chain reaction amplification with oligonucleotides derived from the DNA sequence of the corresponding appA gene from A. pleuropneumoniae serotype 5. The three genes from serotypes 1, 2, and 5 have identical restriction maps and appear to encode a hemolysin which was previously identified in serotype 2 and designated HlyII. Gene appA is different from hlylA encoding the major hemolysin type I (HlyI) which was identified earlier in serotype 1. Polymerase chain reaction amplification with oligonucleotides derived from the DNA sequence of hIyIA of serotype 1 showed that the gene encoding HlyI is present in serotype 1 but not in serotype 2, in contrast to the gene encoding HlyII that was present in both serotypes. This was confirmed by Western blot (immunoblot) experiments using monoclonal antibodies specific for either recombinant HlyI or recombinant HlyII, which showed that A. pleuropneumoniae serotype 1 strain 4074 produces both HlyI and HlyII, whereas serotype 2 strain S1536 produces only HlyII. The expression of both hemolysins was investigated in all serotypes by the use of monoclonal antibodies. HlyI was shown to be expressed by the reference strains of serotypes 1, 5a, Sb, 9, 10, and 11, whereas HlyII was shown to be expressed by the reference strains of all 12 serotypes tested except serotype 10. A. pleuropneumoniae serotype 1 strain 4074 is the first bacterium which has been shown to contain two different actively expressed RTX toxin genes. Comparison of our data with those from other groups shows that the originally described strongly hemolytic hemolysin type I (HlyI) corresponds to cytolysin I (Clyl) which was recently described by others, while the weakly hemolytic hemolysin type II (HlyII) seems to be identical to ClylI and AppA. The hemolysins of Actinobacillus pleuropneumoniae, the causative agent of swine pleuropneumonia (34), are believed to play an important role in virulence (10, 11, 32). The hemolysin was identified as a secreted protein of 105 kDa (9). It is a major immunogenic factor and has been proposed to be an important component in vaccines to induce protective immunity (1, 5, 12, 22, 35). Among the 12 different serotypes of A. pleuropneumoniae, two different hemolysins with the same molecular weight have been distinguished on the basis of hemolytic activity, cofactor requirements, and regulation of expression. The first one is strongly active hemolysin type I (HlyI), which is induced by Ca2' and needs low concentrations of Ca2+ for its hemolytic activity. This hemolysin was isolated from serotype 1 type strain 4074 (10). The second, hemolysin type II (HlyII), has a much lower hemolytic activity and requires high concentrations of Ca2+ for activity, but its expression is not inducible by Ca2+. This hemolysin was detected in serotype 2 reference strain S1536 (7, 10). Both hemolysins HlyI and HlyII had the same apparent molecular size of approximately 105 kDa and apparently showed strong immunological cross-reactions but were identified as two different proteins by genetic analysis of their corresponding genes (7). Cloning and expression of the structural gene (hlyIA) of the 105-kDa HlyI protein in Escherichia coli revealed that HlyI is synthesized in the form of an inactive prohemolysin (HlyIA) which could be acti*

vated in trans by complementation with the E. coli or Proteus vulgaris activator gene hlyC, while secretion of the 105-kDa protein was achieved with the hlyB and hlyD genes from E. coli (13). DNA sequence analysis of the structural hemolysin gene hlyIA revealed strong similarities with the E. coli alpha-hemolysin and showed that HlyI belongs to the group of RTX (repeats in the structural toxin [28, 36]) toxins (8). Chang et al. (4) cloned and sequenced the structural appA gene and its activator gene appC of the hemolysin operon of an A. pleuropneumoniae serotype 5 strain, which also belongs to the RTX toxin gene family. Amino acid sequence analysis revealed that the protein derived from the sequence of gene appA contains a domain of 8 glycine-rich repeated sequences, whereas HlyI had a domain with 13 such sequences (4, 8). The glycine-rich repeated sequences have been demonstrated to be important for efficient binding of Ca2+ ions by another RTX toxin (2) and to play a crucial role in the level of Ca2+ that is required for maximal hemolytic activity (20, 21). Deletion of such repeats in the alpha-hemolysin of E. coli leads to a reduction of Ca2+ binding capacity (2) and a loss of hemolytic activity. This activity could be partially restored by the addition of relatively high concentrations of Ca2+ (30 mM) (20, 21). We therefore speculated that the appA gene (4) which contains only eight glycine-rich repeats encodes the weakly hemolytic HlyII, which requires high levels of Ca2+ for its activity. HlyII was postulated to be produced by A. pleuropneumoniae serotype 2 (10) and most other serotypes, including serotype 5 (11). In contrast, HlyI, which is expected to be

Corresponding author. Electronic mail address: JFREY@

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TABLE 1. A. pleuropneumoniae reference strains Strain

Serotype

Reference(s)

4074 1 18, 30 2 S1536 18 3 S1421 18 4 M62 18 K17 5a 18, 25 L20 5b 18, 25 femo 6 31 WF83 7 31 405 8 27 9 CVI 13261 23 13039 10 24 lla 56153 17 8329 12 26 a This strain was erroneously described as serotype 10 (17) and tentatively classified as serotype 11 (11).

able to bind Ca2+ more efficiently with its 13 glycine-rich repeated sequences, requires only a very low Ca2+ concentration for full hemolytic activity (8). This HlyI was postulated to be produced by serotypes 1, 5a, Sb, 9, 10, and 11 (11). In the present article, we show that the hemolysin gene appA which was cloned by Chang et al. (4) from a serotype 5 strain encodes the weakly active HlyII which was first detected in the serotype 2 reference strain (10). The gene appA is also present and expressed in A. pleuropneumoniae serotype 1 together with hlyL4, encoding the strongly active HlyI. Further, it was shown that A. pleuropneumoniae strains produce either HlyI or HlyII alone or HlyI and HlyII simultaneously.

MATERUILS AND METHODS Bacterial strains, plasmids, and growth conditions. The A. pleuropneumoniae strains used in this study are described in Table 1. A. pleuropneumoniae strains were grown in Columbia broth (BBL Microbiology Systems, Cockeysville, Md.) supplemented with 1% IsoVitaleX (BBL) and 10 ,ug of 1-NAD per ml (Sigma Chemical Co., St. Louis, Mo.). XL1-blue {endA hsdRJ7 supE44 lambda- recAl del(proABlac) [F' proAB lacIq ZdelM15 TnlO (Tcr)I} was used as the E. coli host strain in all experiments (3). Plasmid pBluescript SKII- (Stratagene, La Jolla, Calif.) was used for gene cloning and expression. Plasmid pJFF702 containing the hlyIA gene was described by Gygi et al. (13). Recombinant E. coli strains were grown in Luria-Bertani (LB) broth or agar (33) containing 25 ,ug of ampicillin per ml. Expression of the cloned hemolysin genes in pBluescript SKII- was obtained by the addition of 0.1 mM isopropyl-p-D-thiogalactopyranoside (IPTG) at mid-exponential growth phase at an A650 of 0.5 and subsequent incubation for 2 h at 37°C. Protein purifications and immunological methods. Hemolysin from A. pleuropneumoniae serotype 1 strain 4074 was purified as previously described (9). Crude hemolysin preparations were made from cell-free culture supernatants by precipitation with 50% saturated ammonium sulfate. HlyII was purified from A. pleuropneumoniae serotype 2 strain S1536 as previously described (7). Polyclonal antibodies were raised in rabbits against purified hemolysin from serotypes 1 and 2 by using standard procedures (14). Monoclonal antibodies (MAbs) Intl6-10, Int4O-3, and F14/4B3G9 were prepared by immunization of mice with purified hemolysin of A. pleuropneumoniae serotype 1 strain 4074. These MAbs

did not neutralize the hemolytic activity produced by this strain. MAb 2D1F6D5 was raised against hemolysin of anA. pleuropneumoniae serotype 1 strain, possesses neutralizing activity, and was purchased from S. Rosendal, University of Guelph, Guelph, Ontario, Canada (6). The MAbs were used as purified antibodies from mouse ascitic fluid. The protocols for protein gel electrophoresis and immunoblot analysis were described previously (33). Oligonucleotides, polymerase chain reaction (PCR) amplification, and gene cloning. The following oligonucleotides produced by Microsynth, Windisch, Switzerland, were used: HLYIA-L (5'-TGGCTAACTCTCAGCTCG-3'), corresponding to the sequence of the sense strand at the beginning of the hlyIA gene, and HLYIA-R (5'-ATAGACTA ACGGTCCGCC-3'), corresponding to the antisense strand at the end of the hlyIA gene (8). Oligonucleotides APPSA-L (5'-GTCATCATTAAAATCGTCC-3'), corresponding to the sense strand at the beginning of the appA gene, and APP5AR (5'-AATATTAAGCGGCTCTAGC-3'), corresponding to the antisense strand at the end of the appA gene (4), were also used. For the PCR amplification and subsequent cloning of the appA gene from serotypes 1 and 2 into the expression vector pBluescript SKII-, the oligonucleotide APP5A-LT

(5'-CCCATATGGATCCGTCAAAAATCACTTTGTCATCA TT-3'), which corresponds to nucleotides 4 to 26 of the coding sequence of the appA gene preceded by 14 nucleotides comprising the BamHI site and sequences permitting the production of a gene fusion with the beginning of the 1galactosidase gene on pBluescript SKII-, and APP5A-RT (5'-

TCCGGAATTCAAGCGGCTCTAGCTAATrGA-3'), comprising the last 19 oligonucleotides of the antisense strand of the appA gene followed by nucleotides forming a translational stop codon and an EcoRI site for cloning into the pBluescript SKII-, were used. PCR amplification was performed in a thermal cycler (Perkin-Elmer Cetus, Norwalk, Conn.) with the following parameters: denaturation at 94'C for 1 min, annealing at 54'C for 1 min, elongation at 74WC for 3 min, total of 35 cycles, by using the protocol of Innis et al. (15). Genomic DNA fromA. pleuropneumoniae was isolated by a guanidine-thiocyanate method (29). Purification of plasmids and DNA fragments from agarose gels, digestion by restriction enzymes, analysis by agarose and polyacrylamide gels, ligation, and selection for recombinant clones are described by Sambrook et al. (33). Screening for clones expressing HlyII was done by the following protocol. Isolated clones were grown on nitrocellulose filters that were placed on LB agar plates overnight. They were then placed on LB agar plates containing 0.1 mM IPTG for 2 h at 37°C. The cells were then lysed by chloroform vapor for 20 min and then incubated in blocking solution for immunoblots (33), which was supplemented with 0.1 mg of lysozyme per ml, for an additional 20 min. The filters were then treated like immunoblot filters with polyclonal anti-HlyII antibodies (33). Colonies that showed a strong reaction were retained for further analysis of their plasmids. RESULTS PCR amplification of the hemolysin genes. PCR amplification with the oligonucleotides HLYIA-L and HLYIA-R, derived from the DNA sequence of hlyIA, with genomic DNA from A. pleuropneumoniae serotypes 1 and 2 as templates and analysis by agarose gel electrophoresis showed that a 3.05-kbp fragment was amplified with serotype 1 DNA. No amplification with serotype 2 DNA was

IDENTIFICATION OF HlyII IN A. PLEUROPNEUMONIAE

VOL. 60, 1992

HIy I

Hly 11

FIG. 1. PCR amplification of hlyIA and appA genes in A. pleuropneumoniae serotypes 1 and 2. Agarose gel electrophoresis of the PCR products produced with the oligonucleotides HLYIA-L and HLYIA-R (sector marked HlyI) and with oligonucleotides APP5A-L and APP5A-R (sector marked HlyII) is shown. The templates were genomic DNA from serotype 1 strain 4074 (lane 1), genomic DNA from serotype 2 strain S1536 (lane 2), and plasmid pJFF702 (cloned hlyI4 gene) (lane P). Lane S shows the molecular size standards of bacteriophage lambda DNA digested by HindIll corresponding to 23.1, 9.4, 6.6, 4.3, 2.2, and 2.0 kbp (0.56 kbp, not visible).

detected (Fig. 1), even when a 20-fold amount of PCR product was analyzed on a gel or when the PCR amplification was performed at a lower stringency, by annealing at 42°C. As a positive control template DNA, plasmid pJFF702 containing the hlyIA gene was used, which also showed amplification of a 3.05-kbp fragment as expected (Fig. 1). When the primers APP5A-L and APP5A-R, derived from the sequence of the appA gene, were used, a 2.8-kbp DNA fragment was amplified by using chromosomal DNA from A. pleuropneumoniae serotype 1, 2, 5a, and Sb strains as the template, whereas no amplification was observed with plasmid pJFF702 as the template. This shows that these two pairs of primers were specific for two different hemolysin genes (Fig. 1). The length of the amplified fragment in all serotypes tested corresponds to the 2,858-bp length expected from the sequence of the appA gene (4). Probably, the serotype 1 and 2 reference strains contain hemolysin genes homologous, and possibly identical, to the appA gene of serotype 5. After digestion with the enzymes BglII, ClaI, Hinfl, PstI, RsaI, and SspI, identical restriction maps, which correspond to the map of the appA gene, were obtained for all four serotypes tested, which is further evidence for this conclusion. Cloning and expression of the structural gene for HlyII. The genes homologous to the serotype 5 appA gene, from A. pleuropneumoniae serotypes 1 and 2, were amplified by PCR with oligonucleotides APP5A-LT and APP5A-RT as primers and chromosomal DNA from the reference strains as the template. The resulting 2.8-kbp fragments were cloned after digestion with BamHI and EcoRI in cloning vector pBlue-

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script SKII- and transformed into E. coli XL1-blue. Clones were screened with polyclonal anti-HlyII antibodies. Plasmids extracted from the positive clones had the 2.8-kbp fragment inserted in pBluescript SKII- in the correct orientation for gene fusion, while negative clones had no insert DNA in the plasmids. Plasmid pJFF727 containing the 2.8-kbp fragment from serotype 1 was retained. Plasmid pJFF728 obtained from cloning the 2.8-kbp fragment amplified from A. pleuropneumoniae serotype 2 seemed to be identical to pJFF727. E. coli strains harboring either plasmid pJFF727 or plasmid pJFF728 both expressed a protein with an apparent molecular mass of 105 kDa after induction with IPTG. This protein reacts strongly with anti-HlyII antibodies. The cloned appA genes from serotype 1 on plasmid pJFF727 and from serotype 2 on plasmid pJFF728 were shown to be identical by restriction enzyme analysis. To be able to express the hlyLA gene from the same expression vector as appA cloned from serotypes 1 and 2, we isolated the hlyL4 gene from plasmid pJFF702 (a pUC19based clone) (13) as a 3.7-kbp PvuII-EcoRI fragment and cloned it into the SmaI and EcoRI sites of plasmid pBluescript SKII-. The resulting plasmid pJFF729 was introduced into E. coli XL1-blue for the expression of the HlyIA protein. Serological comparison of HlyI and HlyII. The antigenic relatedness and serological cross-reaction of the recombinant structural proteins HlyIA and HlyIIA (which are the prohemolysin forms of HlyI and HlyII, respectively) and the expression of their corresponding genes in the reference strains of serotypes 1 and 2 were investigated by immunoblot analysis by using several monoclonal and polyclonal antisera with different specificities. The immunoblots presented in Fig. 2 show that polyclonal antibodies raised against hemolysin purified from serotype 2 also react with the hemolysin from serotype 1 and with recombinant HlyIIA but not with recombinant HlyIA. This indicates that this serum is specific for HlyII and that serotype 1 expresses the appA gene which was identified by PCR. Similar results were obtained with the nonneutralizing MAbs Intl6-10 (Fig. 2) and F14/4B3G9 (data not shown), which were both raised against hemolysin purified from serotype 1 and specific for HlyII. Polyclonal antibodies raised against hemolysin that was purified from culture supernatant of A. pleuropneumoniae serotype 1 reacts with the hemolysins produced by serotypes 1 and 2, as well as with both recombinant HlyIA and HlyIIA proteins. This is further proof of the concomitant expression of HlyI and HlyII in serotype 1. The neutralizing MAb 2D1F6D5 reacts with hemolysin purified from serotype 1 and with recombinant HlyIA but not with hemolysin from serotype 2 or recombinant HlyII (Fig. 2). Therefore, HlyI does not seem to be expressed by the serotype 2 strain. This corresponds to the results from the PCR experiments which indicated that the hlyLA gene is absent from this strain. Similar results were obtained with MAb Int4O-3, which specifically reacts with HlyI also (data not shown). No reaction is seen with E. coli XL1-blue harboring pBluescript SKII- with any of the above-mentioned antibodies (Fig. 2). Activation of the recombinant 105-kDa prohemolysins HlyIA and HlyIIA to active hemolysin HlyI and HlyII by HlyC expressed from plasmid pEK70 (13) did not change the specificity of the antibodies (data not shown). Therefore, artifacts resulting from heterologous gene expression of prohemolysins rather than processed active hemolysins can be excluded. Expression of HlyI and HlyII in different serotypes of A. pleuropneumoniae. Immunoblot experiments of crude hemo-

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Polycional anti HIy serotype I

Serotype

Polyclonal anti Hly serotype 2

1

S

1

2

IA

hIA

E

S

1

1

2 IA IIA E

IIA E

IA

2 3 4 5a5b 6 7 8 9 10 11 12

A

105 kD

B

105 kO

Monoclonal antilbody

Monoclonal antiboy Int 16.10

S

2

2D1F6D5 S

1

2

IA

IIA

E

FIG. 2. Immunoblot analysis of HlyI and HlyII. Four identical immunoblots were made containing 100 ng of purified hemolysin from A. pleuropneumoniae serotype 1 (lanes 1), 100 ng of purified hemolysin from A. pleuropneumoniae serotype 2 (lanes 2), proteins from E. coli XL1-blue containing plasmid pJFF729 (cloned hlyL4 gene) (lanes IA), proteins from E. coli XL1-blue containing plasmid pJFF727 (cloned appA gene) (lanes IIA), and proteins from E. coli XL1-blue containing vector pBluescript SKII- (lanes E). The E. coli strains were induced with IPTG for expression of the cloned genes as described in Materials and Methods. The immunoblots were incubated with polyclonal antibodies made against purified hemolysin from serotype 1 in rabbits (anti-Hly serotype 1), with polyclonal antibodies made against purified hemolysin from serotype 2 in rabbits (anti-Hly serotype 2), with nonneutralizing MAb made against hemolysin from serotype 1 (Intl6.10), and with neutralizing MAb made against hemolysin from serotype 1 (2D1F6D5). Lane S contains prestained molecular size standards (Bio-Rad, Richmond, Calif., product no. 161-0305) with the apparent molecular sizes of 110, 84, 47, 33, 24, and 16 kDa.

lysin preparations with MAbs showed that Intl6-10, which specifically reacts with HlyII, reacted with all serotypes except serotype 10, which only showed a faint reaction with MAb Intl6-10. Therefore, A. pleuropneumoniae reference strains from all serotypes except serotype 10 seem to contain and express the appA gene encoding HlyII (Fig. 3A). MAbs 2D1F6D5 (Fig. 3B) and Int4O-3 (data not shown), which both specifically react with HlyI, only react with the type strains for serotypes 1, 5a, 5b, 9, 10, and 11. Therefore, these strains seem to contain an actively expressed hlyIA gene.

DISCUSSION We have cloned and expressed in E. coli the genes encoding the weakly hemolytic hemolysin, HlyII, from A. pleuropneumoniae serotypes 1 and 2 by using PCR amplification with oligonucleotides derived from the DNA sequence of the hemolysin gene appA from an A. pleuropneumoniae serotype 5 strain (4). The genes cloned from

FIG. 3. Immunoblot analysis of crude hemolysin preparations from all A. pleuropneumoniae reference strains incubated with MAb Intl610, specific for HlyII (A), or MAb 2D1F6D5, specific for HlyI (B).

serotypes 1 and 2 and the appA gene were shown to have the same restriction map and are probably the same. These genes are clearly different from the hlyLA gene encoding the strongly hemolytic HlyI on the basis of different restriction maps and serological comparison of the expression products and DNA sequence comparison (7, 8). Recombinant E. coli strains that express either HlyIA or HlyIIA and MAbs allowed us to demonstrate that both hemolysins are immunologically distinct and can be separately demonstrated. Moreover, polyclonal antibodies that were made against native HlyII from A. pleuropneumoniae serotype 2 were specific for HlyIIA and showed no obvious cross-reactions with the recombinant HlyIA protein. Since serotype 1 of A. pleuropneumoniae produces two different hemolysins with similar electrophoretic properties in sodium

dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) (9), culture supernatants or even purified hemolysin of serotype 1 contains both proteins. Consequently, the polyclonal antibodies made against hemolysin from serotype 1 detect both proteins. We further demonstrated that the reference strain of serotype 1 of A. pleuropneumoniae possesses both the hlyL4 and appA genes and expresses both hemolysins HlyI and HlyII, whereas the reference strain of serotype 2 possesses only the appA gene expressing HlyII. The two different hemolysins ofA. pleuropneumoniae were postulated earlier on the basis of biological and genetic properties (7, 10, 11). The fact that the presence of HlyII in serotype 1 has not been previously recognized may be explained by the much stronger hemolytic activity of HlyI, which masks the activity of HlyII, the high levels of Ca2" required for HlyII activity, and the similar apparent molecular masses of HlyI and HlyII in SDS-PAGE (10). This explains why sera raised against hemolysin from serotype 1 react with hemolysin from serotype 2. On the basis of these results, hemolysins HlyI and HlyII were claimed to be serologically related (5-7, 11). Although Kamp and coworkers (16) isolated a MAb which reacts with both hemolysins, the absence of a detectable cross-reaction of HlyI with polyclonal serum raised against hemolysin from serotype 2 (Fig. 2) suggests that the presence of cross-reacting epitopes must be very limited. In the present article, we showed that two MAbs, specifically reacting with recombinant HlyI, react in immunoblot experiments with a protein with an apparent molecular mass

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IDENTIFICATION OF HlyII IN A. PLEUROPNEUMONIE

of 105 kDa in the supernatants from the reference strains of serotypes 1, 5a, 5b, 9, 10, and 11. On the other hand, a MAb specific for HlyII reacts with a protein with a similar apparent molecular mass in the supernatant of reference strains from all serotypes with the exception of serotype 10. This suggests that HlyI is expressed by serotypes 1, 5a, 5b, 9, 10, and 11 only, whereas HlyII is expressed by the reference strains of all serotypes except serotype 10. The presently available MAbs against HlyII, however, do not permit us to exclude the possible presence of a variant type of HlyII in the serotype 10 reference strain, since supematants of this strain show faint reactions with the HlyIIspecific MAb IntlO-16. These results are generally in agreement with a recent report of Kamp et al. (16), who identified a 105-kDa strongly hemolytic toxin (Clyl), assumed to be identical to HlyI, in serotypes 1, 5, 9, 10, and 11, and a 103-kDa cytolytic toxin (ClyII), probably identical to HlyII, in all serotypes except serotype 10. The results also agree in general (with the exception of serotype 1 for the reasons named above) with the earlier predicted distribution of the two hemolysins among the different serotypes which was based on cofactor requirements and expression studies (11). Preliminary observations made from genetic experiments sustain the suggested presence of HlyI and/or HlyII in the different serotype reference strains but also indicate that the two hemolysin genes are not fully conserved throughout all different serotypes and might exist as variants in some A. pleuropneumoniae serotypes. Classification of A. pleuropneumoniae is based on the antigenic similarity of the polysaccharide capsule, and, so far, no correlation between this classification, the genetic relatedness, and the presence of different hemolysin genes has been established. Possibly, strains of the same serotype may contain different hemolysin genes. This will have to be established by further research. A. pleuropneumoniae serotype 1 strain 4074 is the first gram-negative bacterium known to contain and express genes for two different RTX toxins: a strongly hemolytic HlyI that resembles the E. coli alpha-hemolysin and a weakly active HlyII which is much more closely related to the Pasteurella haemolytica leukotoxin (8). A. pleuropneumoniae serotype 2, on the other hand, expresses only the weakly active HlyII. The finding that A. pleuropneumoniae serotype 1 and some other A. pleuropneumoniae serotypes possess and express genes for two different RTX toxins sheds a new light on speculations about the origin of these toxins, which are spread among a broad range of pathogenic gram-negative bacteria. It is interesting to note that the serotypes that possess the strongly hemolytic HlyI toxin seem to belong to a group of more virulent serotypes (19) that are frequently involved in A. pleuropneumoniae outbreaks. Therefore, further investigations about the specific lytic, and also nonlytic, activities of these RTX toxins (36) should be performed to gain a better understanding of the role of these toxins in pathogenicity. It must be noted that the strong hemolysin type I, originally named HlyI (10), corresponds to ClyI described recently by Kamp et al. (16). The weak hemolysin type II, originally named HlyII (10), is identical to ClylI (16) and to the gene product of appA (4). ACKNOWLEDGMENTS We are grateful to C. Lozano and T. Loeffen for their excellent technical assistance in the production of MAbs. This work was partly supported by grant 31-28401.90 from the Swiss National Science Foundation.

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ADDENDUM IN PROOF

After this paper had been submitted, it was reported that A. pleuropneumoniae serotype 9 also contains two hemolysin genes, like serotype 1. However, hemolysin type I (HlyI) was renamed cytolysin type I (ClyI), and its corresponding gene, which was originally named hlyIA for serotype 1 (13), was called clyL4; HlyII was renamed ClyII, and its gene was called clyIL4 (M. A. Smits, J. Briaire, R. Jansen, H. E. Smith, E. M. Kamp, and A. L. J. Gielkens, Infect. Immun. 59:4497-4504, 1991). REFERENCES 1. Bhatia, B., K. R. Mittal, and J. Frey. 1991. Factors involved in the immunity againstActinobacilluspleuropneumoniae in mice. Vet. Microbiol. 28:147-158. 2. Boehm, D. F., R. A. Welch, and I. S. Snyder. 1990. Domains of Escherichia coli hemolysin (HlyA) involved in binding of calcium and erythrocyte membranes. Infect. Immun. 58:19591964. 3. Bullock, W. O., J. M. Fernandez, and J. M. Short. 1978. XL1-blue: a high efficiency plasmid transforming recA E. coli strain with a beta-galactosidase selection. BioTechniques 5:376379. 4. Chang, Y.-F., R. Young, and D. K. Struck. 1989. Cloning and characterization of a hemolysin gene from Actinobacillus (Haemophilus) pleuropneumoniae. DNA 8:635-647. 5. Devenish, J., S. Rosendal, J. T. Bosse, B. N. Wilkie, and R. Johnson. 1990. Prevalence of seroreactors to the 104-kilodalton hemolysin of Actinobacillus pleuropneumoniae in swine herds. J. Clin. Microbiol. 28:789-791. 6. Devenish, J., S. Rosendal, R. Johnson, and S. Hubler. 1989. Immunoserological comparison of 104-kilodalton proteins associated with hemolysis and cytolysis in Actinobacillus pleuropneumoniae, Actinobacillus suis, Pasteurella haemolytica, and

Escherichia coli. Infect. Immun. 57:3210-3213.

7. Frey, J., J.-B. Deillon, D. Gygi, and J. Nicolet. 1991. Identification and partial characterization of the hemolysin (HlyII) of Actinobacillus pleuropneumoniae serotype 2. Vet. Microbiol.

28:303-312. 8. Frey, J., R. Meier, D. Gygi, and J. Nicolet. 1991. Nucleotide sequence of the hemolysin I gene from Actinobacillus pleuro-

pneumoniae. Infect. Immun. 59:3026-3032. 9. Frey, J., and J. Nicolet. 1988. Purification and partial characterization of a hemolysin produced by Actinobacillus pleuropneumoniae type strain 4074. FEMS Microbiol. Lett. 55:41-46. 10. Frey, J., and J. Nicolet. 1988. Regulation of hemolysin expression in Actinobacillus pleuropneumoniae serotype 1 strain by Ca2 . Infect. Immun. 57:2570-2575. 11. Frey, J., and J. Nicolet. 1990. Hemolysin patterns of Actinobacillus pleuropneumoniae. J. Clin. Microbiol. 28:232-236. 12. Frey, J., and J. Nicolet. 1991. Immunological properties of Actinobacillus pleuropneumoniae hemolysin I. Vet. Microbiol. 28:61-73. 13. Gygi, D., J. Nicolet, J. Frey, M. Cross, V. Koronakis, and C. Hughes. 1990. Isolation of theActinobacilluspleuropneumoniae haemolysin gene and the activation and secretion of the prohaemolysin by the HlyC, HlyB and HlyD proteins of Escherichia coli. Mol. Microbiol. 4:123-128. 14. Harlow, E., and D. Lane. 1988. Antibodies. A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 15. Innis, M. A., D. H. Gelfand, J. J. Sninsky, and T. J. White (ed.). 1990. PCR protocols: a guide to methods and applications. Academic Press, Inc., San Diego, Calif. 16. Kamp, E. M., J. K. Popma, J. Anakota, and M. A. Smits. 1991. Identification of hemolytic and cytotoxic proteins of Actinobacillus pleuropneumoniae by use of monoclonal antibodies. Infect. Immun. 59:3079-3085. 17. Kamp, E. M., J. K. Popma, and L. A. M. G. von Leengoed. 1987. Serotyping of Haemophilus pleuropneumoniae in the Netherlands: with emphasis on heterogeneity within serotype 1 and

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Identification of a second hemolysin (HlyII) in Actinobacillus pleuropneumoniae serotype 1 and expression of the gene in Escherichia coli.

Hemolysin genes of the reference strains of Actinobacillus pleuropneumoniae serotypes 1 and 2 were identified, cloned, and expressed in Escherichia co...
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