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The CAMP effect of Actinobacillus pleuropneumoniae is caused by Apx toxins
ELSEVIER
FEMS Microbiology Letters 126 (1995) 139-144
The CAMP effect of Actinobacillus pleuropneumoniae by Apx toxins
is caused
Ruud Jansen a, Jan Briaire a, Elbarte M. Kamp b, Amo L.J. Gielkens a, Mari A. Smits a~* a Department b Department
of Molecular Biology, DLO-Institute
of Bacteriology,
DLO-Institute
for Animal Science and Health (ID-DLO), Postbox 65,820O AB, Lelystad, the Netherlands. for Animal Science and Health (ID-DLO), Postbox 65, 82OOAl3, Lelystari, the Netherlands
Received 9 September 1994; revised 2 December 1994; accepted 21 December 1994
Abstract Actinobacillus pleuropnezunoniae shows synergistic haemolysis when cocultured with Staphylococcus aureus on blood agar plates. This CAMP effect has been attributed to a discrete CAMP factor, but also to the A. pleuropnezunoniae-RTXtoxins I, II, and III. We examined the CAMP effect of recombinant Escherichia coli strains that secreted each of these toxins, and of A. pleuropneumoniae mutant strains that were devoid of one or more these toxins. We found that the E. coli strains were CAMP positive, whereas the A. pleuropneumoniae strain devoid of functional toxin genes was CAMP negative. This demonstrated that the CAMP effect of A. pleuropneumoniae is caused by the toxins and that no CAMP factor per se
exists. Keywords: Actinobacillus pleuropneumoniae; CAMP; Apx toxins
1. Introduction Actinobacillus pburopneumoniae causes contagious porcine pleuropneumonia, a disease that has
severe economical impact on the pig farming industry world wide [1,2]. The bacterium secretes three different RTX toxins, ApxI, ApxII, and ApxIII [3,4]. These toxins are important virulence factors of the bacterium [1,5]. In vitro these Apx toxins form pores in the membranes of target cells, leading to cell lysis. The three Apx toxins differ in their haemolytic activ-
Corresponding author. Tel: +31 (3200) 73319; Fax +31 (3200) 73473; e-mail: [email protected] l
037%1097/95/$09.50
ities towards sheep erythrocytes and cytotoxic activities towards porcine neutrophils and lung macrophages. ApxI is strongly haemolytic and strongly cytotoxic, ApxII is weakly haemolytic and moderately cytotoxic, and ApxIII is not haemolytic and strongly cytotoxic [3]. In addition, A. pleuropneumoniae strains show the CAMP effect. The term CAMP is a combination of the initials of the authors who first described this phenomenon [6]. The CAMP effect is an enlargement of the haemolytic zones of A. pleuropneumoniae colonies, when grown in the vicinity of a Ptoxigenic bacterium such asStaphyiococcus aureus [6]. This synergistic haemolysis of sheep erythrocytes is caused by a haemolytic compound of A. pleuropneumoniae and a phospholipase
6 1995 Federation of European Microbiological Societies. All rights reserved
SSDI 0378-1097(94)00541-9
140
R. Jansen et al. / FEMS Microbiology
or sphingomyelinase produced by S. aureus. The latter enzymes hydrolyze phospholipids from the erythrocyte membrane and make these cells more susceptible to haemolysis [7]. The CAMP effect is displayed by several bacterial species and is an important tool for taxonomic classifications. Besides its use in diagnosis, the putative CAMP factor of A. pleuropneumoniae drew attention as a virulence factor. Frey et al. identified a gene from A. pleuropneumoniae that conferred CAMP activity on E. coli [7]. This C$LJgene coded for a protein of 27 kDa, which was supposed to be the CAMP factor protein (CFP) of A. pleuropneumoniae [7]. MacInnes et al. described the hlyX gene of A. pleuropneumoniae that coded for the HlyX protein of 27.1 kDa. [8]. The restriction maps of the hlyX and cfp genes were similar, leading to the suggestion that hlyX and cfp are one and the same gene [8]. The hlyX gene conferred a haemolytic phenotype on E. coli, though the HlyX protein itself was not haemolytic [8,9]. Surprisingly, the HlyX protein was found to be homologous to FNR, a regulatory protein of E. coli, and the hlyX gene complemented a FNR defect in E. coli [8,9]. In addition BTR, a PNR homologue of Bordetella pertussis, also conferred a haemolytic phenotype on E. coli. [lo]. This led to the suggestion that these regulatory proteins activate a latent haemolysin of E. coli. [IO]. Devenish et al. associated the CAMP activity of A. pleuropneumoniae with its RTX toxins [ll]. Antibodies raised against Apx toxins of serotype 1 not only neutralized the haemolytic and cytotoxic activity of this serotype and most other serotypes, but also neutralized the CAMP effect of these serotypes [11,12]. Additional evidence for the association of Apx toxins and CAMP activity was obtained from a chemically-induced mutant strain of A. pleuropneumoniae serotype 5 [13]. The parent strain secreted ApxI and ApxII and was CAMP positive, while the mutant strain did not secrete these Apx toxins and was CAMP negative [14]. These observations associated the Apx toxins with the CAMP effect, although they did not rule out the existence of a CAMP factor protein. In this study we determined the CAMP effect of recombinant E. coli strains that secreted ApxI, ApxII, or ApxIII, and of genetically-defined mutant strains of A. pleuropneumoniae serotype 1 that were devoid
Letters 126 (1995) 139-144
of ApxI, ApxII, or both toxins [15]. We demonstrated unequivocally that the CAMP effect of A. pleuropneumoniae is solely caused by the Apx toxins.
2. Materials 2.1. Bacterial
and methods strains
We used the reference strain S4074 of A. pleuropneumoniae serotype 1, that secretes ApxI and ApxII, as the parent strain. Mutants of this strain were generated by targeted mutagenesis [151. In mutant strain 1 the apxZA gene was inactivated and this knockout mutant secretes only ApxII. In mutant strain 14 the apxIL4 gene was inactivated and this knockout mutant secretes only ApxI. In mutant strain 21 the apxL.4 and apxIL4 genes were inactivated and this knockout mutant secretes no Apx toxins [15]. Transformed Escherichia coli DH5 (Y strains were used as a source of recombinant Apx toxins. These strains carried the apxICA, apxIICA, or apxIZICA genes on plasmid pUC19 and the hlyBD secretion genes of E. coli on plasmid pLG575 [16]. A p-toxigenie strain of Staphylococcus aureus was used in the CAMP assay. 2.2. Culture media A. pleuropneumoniae strains were cultured at 37°C in Luria Bertani (LB) medium [17] supplemented with 0.004% NAD (Boehringer Mannheim GmbH, Germany). E. coli was cultured at 37°C in LB supplemented with chloramphenicol (20 pg . ml-’ ) and/or ampicilin (50 pg . ml-‘). S. aureus was cultured at 37°C in LB medium. 2.3. CAMP and haemolysis
assays
The CAMP assay was done essentially as described by Cowan and Steel [18]. Briefly, CAMP and haemolytic activities were assessed on sheep blood agar plates supplemented with 0.004% NAD. CAMP activity was assessed by growth of the bacterial strains in the vicinity of S. aureus or by growth on blood plates containing crude p-toxin of S. aureus (ID-DLO, Lelystad, The Netherlands). This crude
R. Jansen et al. /FEMS
Microbiology Leiters 126 (1995) 139-144
p-toxin preparation is the diluted supernatant of a S. aUreUSculture that confers clear haemolyis to Streptococcus agalactiae on sheep blood agar plates at a thousand-fold dilution. For the CAMP assay with S. aureus, the A. pleuropneumoniae or E. coli strains were streaked from overnight cultures perpendicular to a streak of S. uureus. For the CAMP assay with crude p-toxin, E. coli strains were cultured on blood agar in 87 mm Petri dishes which were spread with 200 ~1 of the crude p-toxin preparation. These plates contained chloramphenicol (20 pg * ml- ’ > and/or ampicilin (50 pg * ml-‘) to stabilise the plasmids of the recombinant E. coli. CAMP activity was assessed as the enlargement of the haemolytic zones after overnight growth at 37°C.
3. Results 3.1. Haemolytic and CAMP activity of recombinant E. coli that secrete ApxZ, ApxZZ, or ApxZZZ E. coli strains that secreted ApxI or ApxII showed a haemolytic zone of approximately 2 mm and 0.5 mm, respectively. E. coli strains that secreted ApxIII
141
or no Apx toxin (control) were not haemolytic (Fig. 1). These observations were in accordance with the strong haemolytic activity of the purified ApxI protein, the weak haemolytic activity of the ApxII protein, and the absence of haemolytic activity of the ApxIII protein [3]. The E. coli strains that secreted ApxI, ApxII, or ApxIII were CAMP positive, whereas the control E. coli strain was CAMP negative (Fig. 1). These observations demonstrated that the Apx toxins conferred CAMP activity on E. coli. Therefore, the Apx toxins are likely to contribute to the CAMP effect of A. pleuropneumoniae. 3.2. Haemolytic and CAMP activity of parent and mutant strains of A. pleuropneumoniae serotype 1
To determine the contribution of the Apx toxins to the CAMP activity of A. pleuropneumoniae, we assessed the haemolytic and CAMP activity of the parent strain of A. pleuropneumoniae serotype 1, that secreted ApxI and ApxII, and of various mutant strains. We used mutant strain 1 that only secreted ApxII, mutant strain 14 that only secreted ApxI, and mutant strain 21 that secreted no Apx toxins. As expected, the parent and mutant strain 14 were
E. wli
-
Fig. 1. CAMP effect of recombinant E. coli strains and the A. pleuropneumoniae serotype 1 parent and mutant strains. Left of the central streak of S. aureus are E. coli strains that secrete: no toxins (none), ApxI, ApxII or ApxIII. On the right are streaks of the A. pleuropneumoniue serotype 1 parent strain that secretes ApxI and ApxII, mutant strain 1 that only secretes ApxII, mutant strain 14 that only secretes ApxI, and mutant strain 21 that secretes no Apx toxins.
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R. Jansen et al. / FEMS Microbiology Letters 126 (1995) 139-144
Fig. 2. Haemolysis of an E. coli. strain that secretes ApxIII on sheep blood agar plates. (A) without p-toxin, (B) with P-toxin.
strongly haemolytic, mutant strain 1 was weakly haemolytic, and mutant strain 21 was not haemolytic (Fig. 1). The parent strain and mutant strains 1 and 14 were CAMP positive (Fig. 1). Mutant strain 21 that secreted neither ApxI nor ApxII was CAMP negative (Fig. 1). These observations demonstrated that ApxI and ApxII are the sole components of A. pleuropneumoniae serotype 1 that cause the CAMP effect. 3.3. Haemolytic
and CAMP activity of ApxIII
The finding that ApxIII conferred CAMP activity to E. coli, allowed us to set up a plate test to identify bacterial colonies that secrete ApxIII, but not ApxI and/or ApxII. Therefore, we treated sheep blood agar plates with crude p-toxin and inoculated them with the E. coli strain that secreted ApxIII. After overnight incubation at 37°C clear haemolytic zones were visible on the p-toxin treated plates and not on normal plates (Fig. 2A and B). An E. coli strain that only harbored the hlyBD genes was not haemolytic on plates with or without p-toxin (not shown).
4. Discussion In this paper we present conclusive evidence that the CAMP effect of A. pleuropneumoniae serotype
1 is solely caused by its RTX toxins ApxI and ApxII. This implicates that no CAMP factor per se exists. This conclusion is mainly based on the observation that mutant strain 20, which is devoid of ApxI and ApxII, did not show the CAMP effect. The CAMP effect of the parent strain is caused by both ApxI and ApxII, since the mutant strains 1 and 14, each secreting one of the toxins, still showed the CAMP effect. We demonstrated that, besides ApxI and ApxII, ApxIII also confers the CAMP effect on E. coli. This implicates that also ApxIII may contibute to the CAMP effect of the A. pleuropneumoniae serotypes that secrete this toxin, i.e. serotypes 2, 3, 4, 6, and 8. There is strong evidence from the work of Devenish et al. [ll] that supports this hypothesis. These authors assessed the neutralization of CAMP activity of the twelve serotypes of A. pleuropneumoniae with an antiserum that was raised against the Apx toxins of serotype 1. They found that this serum completely neutralized the CAMP effect of the serotypes that secrete ApxI and/or ApxII, i.e. serotypes 1, 5, 7, 9, 10, 11, and 12. They found partial neutralization for the serotypes that secrete a mixture of ApxII and ApxIII, i.e. serotypes 3, 4, and 8. This is apparently due to the absence of ApxIII neutralizing antibodies in the serum. The data of Devenish et al. with respect to the serotypes 2 and 6 do not support our hypothesis. However, the cytotoxic and haemolytic activities of the serotypes 2 and 6 strains that were used differed from the activities reported by others for these serotypes [3,7,11]. The finding that CAMP activity of serotype 1 is solely caused by ApxI and ApxII demonstrates unequivocally that the cloned cfp gene cannot be responsible for the CAMP effect of A. pleuropneumoniae serotype 1[7]. This is in support of the suggestions of MacInnes et al. and Green et al. [8,9] that the cfi gene codes for a regulatory protein that activates a latent haemolysin in E. coli. M$ller et al. described A. pleuropneumoniae strains that were non-haemolytic, but CAMP positive [19]. Such a phenotype would be expected from strains that only secrete ApxIII and no other RTX toxins [20]. Such strains have, however, not been recognized thus far by others. A possible explanation for this could be that haemolytic activity is one of the most important traits that are used for classification. The CAMP activity of ApxIII on p-toxin treated
R. Jansen et al. / FEMS Microbiology
blood agar plates might be an useful laboratory tool for the identification of such A. pleuropneumoniae strains.
References [l] Bertram, T.A. (1990) Actinobacillus pleuropneumoniae: Molecular aspects of virulence and pulmonary injury. Can. J. Vet. Res. 54, S53-S56. [2] Nicolet, J. (1986) Haemophilus infections. In: A.D. Lemon, B. Straw, R.D. Glock, W.1. Mengeling, R.H.C. Penny and E. Scholl (eds.), Diseases of Swine, 6th ed. Iowa State University Press. Ames. 131 Kamp, E.M., Popma, J.K., Anakotta, J. and Smits, M.A. (1991) Identification of hemolytic and cytotoxic proteins of Actinobacillus pleuropneumoniae by using monoclonal antibodies. Infect. Immun. 59, 3079-3085. .I Frey, J., Bosse, J.T., Chang, Y.-F. et al. (1993) Actinobacillus pleuropneumoniae RTX-toxins: uniform designation of haemolysins, cytolysins, pleurotoxin and their genes. J. Gen. Genet. 139, 1723-1728. ;I Inzana, T.J. (1991) Virulence properties of Acfinobacillus pleuropneumoniae. Microbial Pathogen. 11, 305-316. 161 Christie, R., Atkins N.E. and Munch-Petersen, E. (1944) A note on a lytic phenomenon shown by ~-streptococci. Austral. J. Exp. Biol. Med. Sci. 22, 197. [7] Frey, J. Penin, J. and Nicolet, J. (1989) Cloning and expression of a cohemolysin, the CAMP factor of Actinobacillus pleuropneumoniae. Infect. Immun. 57, 2050-2056. [8] MacInnes, J.I., Kim. J.E., Lian, C.-J. and Soltes, G.A. (1990) Acfinobacillus pleuropneumoniae hlyX gene homology with the fir gene of Escherichia coli. J. Bacterial. 172, 4587-4592. [9] Green, J., Sharrocks, A.D., Machutes, J.I. and Guest, J.R. (1992) Purification of HlyX, a potential regulator of haemolysin synthesis, and properties of HlyXFNR hybrids. Proc. R. Sot. Land. B 248,79-84. [lo] Bannan, J.D., Moran, M.J., Machines, J.I., Soltes, GA. and Friedman, R.L. (1993) Cloning and characterization of brr, a Bordetella pertussis gene encoding an FNR-like transcriptional regulator. J. Bacterial. 175, 7228-7235.
Letters 126 (1995) 139-144 [ll]
143
Devenish, J., Brown, J.E. and Rosendal, S. (1992) Association of the RTX proteins of Actinobacillus pleuropneumoniae with hemolytic, CAMP, and neutrophil-cytotoxic activities. Infect. Immun. 60, 2139-2142. [12] Devenish, J., Rosendal, S., Johnson, R. and Hubler, S. (1989) Immunoserological comparison of 104kilodalton proteins associated with hemolysis and cytolysis in Actinobacillus pleuropneumoniae, Actinobacillus suis, Pasteurella haemolytica, and Escherichia coli. Infect. Immun. 57, 32103213. [13] Inxana, T.J., Todd, J., Ma, J. and Veit, H. (1991) Cbaracterization of a non-hemolytic mutant of Actinobacillus pleuropneumoniae serotype 5: role of the 110 kilodalton hemolysin in virulence and immunoprotection. Microb. Pathogen. 10, 281-296. [14] Remer, D.J. Frey, R. Jansen, R. and Inxana, T.J. (1993) Molecular investigations of the role of ApxI and ApxII in the virulence of Actinobacillus pleuropneumoniae serotype 5., abstr. 225, p. 44. 74th Annu. Meet. Conference of Res. Workers Animal Disease. [15] Jansen, R., Briaire, J., Smith, H.E., Dom, P., Haesebrouck, F., Kamp, E.M., Gielkens, A.L.J. and Smits, M.A. (1995) Knock-out mutants of Actinobacillus pleuropneumoniue serotype 1 that are devoid of RTX toxins, do not activate or kill porcine neutrophils. Infect. Immun. (in press). 1161 Gygi, D., Nicolet, J., Frey, J., Cross, M.K., Koronakis, V. and Hughes, C. (1990) Isolation of the Actinobacillus pleuropneumoniae hemolysin gene and the activation and secretion of the prohemolysin by the HlyC, HlyB and HlyD proteins of Escherichia coli. Mol. Microbial. 4, 123-128. [17] Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular cloning. A laboratory manual. Second edition. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, New York. [18] Cowan, S.T. and Steel, K.J. (1974) Cowan and Steel’s manual for the identification of medical bacteria. 2nd edn. Cambridge, Cambridge University Press. [19] Mbller, K. and Kilian, M. (1990) V-factor dependent members of the family Pasteurellaceae io the porcine upper respiratory tract. J. Clin. Microbial. 28, 2711-2716. 1201 Jansen, R., Briaire, J., van Geel, A.B.M., Kamp, E.M., Gielkens, A.L.J. and Smits, M.A. (1994) Genetic Map of the Actinobacillus pleuropneumoniae RTX-Toxin (Apx) Oper011s: Characterization of the ApxIII Gperons. Infect. Immun. 62, 4411-4418.
FEMS Microbiology Letters 126 (1995) 139-144
The CAMP effect of Actinobacillus pleuropneumoniae by Apx toxins
is caused
Ruud Jansen a, Jan Briaire a, Elbarte M. Kamp b, Amo L.J. Gielkens a, Mari A. Smits a~* a Department b Department
of Molecular Biology, DLO-Institute
of Bacteriology,
DLO-Institute
for Animal Science and Health (ID-DLO), Postbox 65,820O AB, Lelystad, the Netherlands. for Animal Science and Health (ID-DLO), Postbox 65, 82OOAl3, Lelystari, the Netherlands
Received 9 September 1994; revised 2 December 1994; accepted 21 December 1994
Abstract Actinobacillus pleuropnezunoniae shows synergistic haemolysis when cocultured with Staphylococcus aureus on blood agar plates. This CAMP effect has been attributed to a discrete CAMP factor, but also to the A. pleuropnezunoniae-RTXtoxins I, II, and III. We examined the CAMP effect of recombinant Escherichia coli strains that secreted each of these toxins, and of A. pleuropneumoniae mutant strains that were devoid of one or more these toxins. We found that the E. coli strains were CAMP positive, whereas the A. pleuropneumoniae strain devoid of functional toxin genes was CAMP negative. This demonstrated that the CAMP effect of A. pleuropneumoniae is caused by the toxins and that no CAMP factor per se
exists. Keywords: Actinobacillus pleuropneumoniae; CAMP; Apx toxins
1. Introduction Actinobacillus pburopneumoniae causes contagious porcine pleuropneumonia, a disease that has
severe economical impact on the pig farming industry world wide [1,2]. The bacterium secretes three different RTX toxins, ApxI, ApxII, and ApxIII [3,4]. These toxins are important virulence factors of the bacterium [1,5]. In vitro these Apx toxins form pores in the membranes of target cells, leading to cell lysis. The three Apx toxins differ in their haemolytic activ-
Corresponding author. Tel: +31 (3200) 73319; Fax +31 (3200) 73473; e-mail: [email protected] l
037%1097/95/$09.50
ities towards sheep erythrocytes and cytotoxic activities towards porcine neutrophils and lung macrophages. ApxI is strongly haemolytic and strongly cytotoxic, ApxII is weakly haemolytic and moderately cytotoxic, and ApxIII is not haemolytic and strongly cytotoxic [3]. In addition, A. pleuropneumoniae strains show the CAMP effect. The term CAMP is a combination of the initials of the authors who first described this phenomenon [6]. The CAMP effect is an enlargement of the haemolytic zones of A. pleuropneumoniae colonies, when grown in the vicinity of a Ptoxigenic bacterium such asStaphyiococcus aureus [6]. This synergistic haemolysis of sheep erythrocytes is caused by a haemolytic compound of A. pleuropneumoniae and a phospholipase
6 1995 Federation of European Microbiological Societies. All rights reserved
SSDI 0378-1097(94)00541-9
140
R. Jansen et al. / FEMS Microbiology
or sphingomyelinase produced by S. aureus. The latter enzymes hydrolyze phospholipids from the erythrocyte membrane and make these cells more susceptible to haemolysis [7]. The CAMP effect is displayed by several bacterial species and is an important tool for taxonomic classifications. Besides its use in diagnosis, the putative CAMP factor of A. pleuropneumoniae drew attention as a virulence factor. Frey et al. identified a gene from A. pleuropneumoniae that conferred CAMP activity on E. coli [7]. This C$LJgene coded for a protein of 27 kDa, which was supposed to be the CAMP factor protein (CFP) of A. pleuropneumoniae [7]. MacInnes et al. described the hlyX gene of A. pleuropneumoniae that coded for the HlyX protein of 27.1 kDa. [8]. The restriction maps of the hlyX and cfp genes were similar, leading to the suggestion that hlyX and cfp are one and the same gene [8]. The hlyX gene conferred a haemolytic phenotype on E. coli, though the HlyX protein itself was not haemolytic [8,9]. Surprisingly, the HlyX protein was found to be homologous to FNR, a regulatory protein of E. coli, and the hlyX gene complemented a FNR defect in E. coli [8,9]. In addition BTR, a PNR homologue of Bordetella pertussis, also conferred a haemolytic phenotype on E. coli. [lo]. This led to the suggestion that these regulatory proteins activate a latent haemolysin of E. coli. [IO]. Devenish et al. associated the CAMP activity of A. pleuropneumoniae with its RTX toxins [ll]. Antibodies raised against Apx toxins of serotype 1 not only neutralized the haemolytic and cytotoxic activity of this serotype and most other serotypes, but also neutralized the CAMP effect of these serotypes [11,12]. Additional evidence for the association of Apx toxins and CAMP activity was obtained from a chemically-induced mutant strain of A. pleuropneumoniae serotype 5 [13]. The parent strain secreted ApxI and ApxII and was CAMP positive, while the mutant strain did not secrete these Apx toxins and was CAMP negative [14]. These observations associated the Apx toxins with the CAMP effect, although they did not rule out the existence of a CAMP factor protein. In this study we determined the CAMP effect of recombinant E. coli strains that secreted ApxI, ApxII, or ApxIII, and of genetically-defined mutant strains of A. pleuropneumoniae serotype 1 that were devoid
Letters 126 (1995) 139-144
of ApxI, ApxII, or both toxins [15]. We demonstrated unequivocally that the CAMP effect of A. pleuropneumoniae is solely caused by the Apx toxins.
2. Materials 2.1. Bacterial
and methods strains
We used the reference strain S4074 of A. pleuropneumoniae serotype 1, that secretes ApxI and ApxII, as the parent strain. Mutants of this strain were generated by targeted mutagenesis [151. In mutant strain 1 the apxZA gene was inactivated and this knockout mutant secretes only ApxII. In mutant strain 14 the apxIL4 gene was inactivated and this knockout mutant secretes only ApxI. In mutant strain 21 the apxL.4 and apxIL4 genes were inactivated and this knockout mutant secretes no Apx toxins [15]. Transformed Escherichia coli DH5 (Y strains were used as a source of recombinant Apx toxins. These strains carried the apxICA, apxIICA, or apxIZICA genes on plasmid pUC19 and the hlyBD secretion genes of E. coli on plasmid pLG575 [16]. A p-toxigenie strain of Staphylococcus aureus was used in the CAMP assay. 2.2. Culture media A. pleuropneumoniae strains were cultured at 37°C in Luria Bertani (LB) medium [17] supplemented with 0.004% NAD (Boehringer Mannheim GmbH, Germany). E. coli was cultured at 37°C in LB supplemented with chloramphenicol (20 pg . ml-’ ) and/or ampicilin (50 pg . ml-‘). S. aureus was cultured at 37°C in LB medium. 2.3. CAMP and haemolysis
assays
The CAMP assay was done essentially as described by Cowan and Steel [18]. Briefly, CAMP and haemolytic activities were assessed on sheep blood agar plates supplemented with 0.004% NAD. CAMP activity was assessed by growth of the bacterial strains in the vicinity of S. aureus or by growth on blood plates containing crude p-toxin of S. aureus (ID-DLO, Lelystad, The Netherlands). This crude
R. Jansen et al. /FEMS
Microbiology Leiters 126 (1995) 139-144
p-toxin preparation is the diluted supernatant of a S. aUreUSculture that confers clear haemolyis to Streptococcus agalactiae on sheep blood agar plates at a thousand-fold dilution. For the CAMP assay with S. aureus, the A. pleuropneumoniae or E. coli strains were streaked from overnight cultures perpendicular to a streak of S. uureus. For the CAMP assay with crude p-toxin, E. coli strains were cultured on blood agar in 87 mm Petri dishes which were spread with 200 ~1 of the crude p-toxin preparation. These plates contained chloramphenicol (20 pg * ml- ’ > and/or ampicilin (50 pg * ml-‘) to stabilise the plasmids of the recombinant E. coli. CAMP activity was assessed as the enlargement of the haemolytic zones after overnight growth at 37°C.
3. Results 3.1. Haemolytic and CAMP activity of recombinant E. coli that secrete ApxZ, ApxZZ, or ApxZZZ E. coli strains that secreted ApxI or ApxII showed a haemolytic zone of approximately 2 mm and 0.5 mm, respectively. E. coli strains that secreted ApxIII
141
or no Apx toxin (control) were not haemolytic (Fig. 1). These observations were in accordance with the strong haemolytic activity of the purified ApxI protein, the weak haemolytic activity of the ApxII protein, and the absence of haemolytic activity of the ApxIII protein [3]. The E. coli strains that secreted ApxI, ApxII, or ApxIII were CAMP positive, whereas the control E. coli strain was CAMP negative (Fig. 1). These observations demonstrated that the Apx toxins conferred CAMP activity on E. coli. Therefore, the Apx toxins are likely to contribute to the CAMP effect of A. pleuropneumoniae. 3.2. Haemolytic and CAMP activity of parent and mutant strains of A. pleuropneumoniae serotype 1
To determine the contribution of the Apx toxins to the CAMP activity of A. pleuropneumoniae, we assessed the haemolytic and CAMP activity of the parent strain of A. pleuropneumoniae serotype 1, that secreted ApxI and ApxII, and of various mutant strains. We used mutant strain 1 that only secreted ApxII, mutant strain 14 that only secreted ApxI, and mutant strain 21 that secreted no Apx toxins. As expected, the parent and mutant strain 14 were
E. wli
-
Fig. 1. CAMP effect of recombinant E. coli strains and the A. pleuropneumoniae serotype 1 parent and mutant strains. Left of the central streak of S. aureus are E. coli strains that secrete: no toxins (none), ApxI, ApxII or ApxIII. On the right are streaks of the A. pleuropneumoniue serotype 1 parent strain that secretes ApxI and ApxII, mutant strain 1 that only secretes ApxII, mutant strain 14 that only secretes ApxI, and mutant strain 21 that secretes no Apx toxins.
142
R. Jansen et al. / FEMS Microbiology Letters 126 (1995) 139-144
Fig. 2. Haemolysis of an E. coli. strain that secretes ApxIII on sheep blood agar plates. (A) without p-toxin, (B) with P-toxin.
strongly haemolytic, mutant strain 1 was weakly haemolytic, and mutant strain 21 was not haemolytic (Fig. 1). The parent strain and mutant strains 1 and 14 were CAMP positive (Fig. 1). Mutant strain 21 that secreted neither ApxI nor ApxII was CAMP negative (Fig. 1). These observations demonstrated that ApxI and ApxII are the sole components of A. pleuropneumoniae serotype 1 that cause the CAMP effect. 3.3. Haemolytic
and CAMP activity of ApxIII
The finding that ApxIII conferred CAMP activity to E. coli, allowed us to set up a plate test to identify bacterial colonies that secrete ApxIII, but not ApxI and/or ApxII. Therefore, we treated sheep blood agar plates with crude p-toxin and inoculated them with the E. coli strain that secreted ApxIII. After overnight incubation at 37°C clear haemolytic zones were visible on the p-toxin treated plates and not on normal plates (Fig. 2A and B). An E. coli strain that only harbored the hlyBD genes was not haemolytic on plates with or without p-toxin (not shown).
4. Discussion In this paper we present conclusive evidence that the CAMP effect of A. pleuropneumoniae serotype
1 is solely caused by its RTX toxins ApxI and ApxII. This implicates that no CAMP factor per se exists. This conclusion is mainly based on the observation that mutant strain 20, which is devoid of ApxI and ApxII, did not show the CAMP effect. The CAMP effect of the parent strain is caused by both ApxI and ApxII, since the mutant strains 1 and 14, each secreting one of the toxins, still showed the CAMP effect. We demonstrated that, besides ApxI and ApxII, ApxIII also confers the CAMP effect on E. coli. This implicates that also ApxIII may contibute to the CAMP effect of the A. pleuropneumoniae serotypes that secrete this toxin, i.e. serotypes 2, 3, 4, 6, and 8. There is strong evidence from the work of Devenish et al. [ll] that supports this hypothesis. These authors assessed the neutralization of CAMP activity of the twelve serotypes of A. pleuropneumoniae with an antiserum that was raised against the Apx toxins of serotype 1. They found that this serum completely neutralized the CAMP effect of the serotypes that secrete ApxI and/or ApxII, i.e. serotypes 1, 5, 7, 9, 10, 11, and 12. They found partial neutralization for the serotypes that secrete a mixture of ApxII and ApxIII, i.e. serotypes 3, 4, and 8. This is apparently due to the absence of ApxIII neutralizing antibodies in the serum. The data of Devenish et al. with respect to the serotypes 2 and 6 do not support our hypothesis. However, the cytotoxic and haemolytic activities of the serotypes 2 and 6 strains that were used differed from the activities reported by others for these serotypes [3,7,11]. The finding that CAMP activity of serotype 1 is solely caused by ApxI and ApxII demonstrates unequivocally that the cloned cfp gene cannot be responsible for the CAMP effect of A. pleuropneumoniae serotype 1[7]. This is in support of the suggestions of MacInnes et al. and Green et al. [8,9] that the cfi gene codes for a regulatory protein that activates a latent haemolysin in E. coli. M$ller et al. described A. pleuropneumoniae strains that were non-haemolytic, but CAMP positive [19]. Such a phenotype would be expected from strains that only secrete ApxIII and no other RTX toxins [20]. Such strains have, however, not been recognized thus far by others. A possible explanation for this could be that haemolytic activity is one of the most important traits that are used for classification. The CAMP activity of ApxIII on p-toxin treated
R. Jansen et al. / FEMS Microbiology
blood agar plates might be an useful laboratory tool for the identification of such A. pleuropneumoniae strains.
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