INFECTION

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IMMUNITY, Dec. 1991,

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Vol. 59, No. 12

0019-9567/91/124497-08$02.00/0 Copyright © 1991, American Society for Microbiology

Cytolysins of Actinobacillus pleuropneumoniae Serotype 9 MARI A. SMITS,1* JAN BRIAIRE,1 RUUD JANSEN,' HILDE E. SMITH,' ELBARTE M. KAMP 2 AND ARNO L. J. GIELKENS1 Departments of Molecular Biology' and Bacteriology,2 Central Veterinary Institute, Postbox 65, 8200 AB Lelystad, The Netherlands Received 15 July 1991/Accepted 2 October 1991

Cytolysin I (ClyI) and cytolysin H (Clyll), which are present in the culture supernatant of Actinobacilus pleuropneumoniae serotype 9, are thought to play an important role in the pathogenesis of pig pleuropneumonia. The purpose of this study was to clone and characterize the genetic determinants of these cytolysins. Cloning was accomplished by the screening of DNA libraries for the presence of cytolytic activity and for the presence of DNA sequences homologous to leukotoxin DNA of PasteureUa haemolytica. Both genetic determinants were found to be members of the RTX cytotoxin family. The ClyII determinant was characterized in more detail. It appeared that Clyll more closely resembled the leukotoxin of P. haemolytica than the alpha-hemolysin of Escherichia coli. The ClyII amino acid sequence was identical to a hemolysin gene sequence of A. pleuropneumoniae serotype 5; this finding indicates that the latter gene also codes for ClyII and not for Clyl, as has previously been suggested. The genetic organization of the ClyIl determinant differed from the genetic organization of other RTX determinants. Genes responsible for secretion of ClyIl were not contiguous with the toxin gene. Instead, secretion genes were present elsewhere in the genome. These secretion genes, however, belong to the Clyl operon. This indicates that the secretion genes of the Clyl operon are responsible for secretion of Clyl and Clyll.

Actinobacillus pleuropneumoniae is the causative agent for pig pleuropneumonia. Although much has been written about the clinical signs and pathology of porcine pleuropneumonia (reviewed in references 2 and 25), little is known about the pathogenesis of the disease. Because investigators have been able to induce pleuropneumonia by inoculating pigs with sterile culture supematant, extracellular toxins are thought to be involved in the development of pneumonic lesions (2, 21, 30). Several investigators have recently suggested that the toxic activities of the 12 serotypes of A. pleuropneumoniae differ qualitatively or quantitatively or both (2, 4, 10, 11, 21). Heat-stable and heat-labile activities have been detected in all serotypes of this bacterium (10, 15, 21). Frey and Nicolet (10) described two different hemolytic activities, hemolysin I (HlyI) and HlyII, and Kamp and van Leengoed (15) distinguished four antigenically different cytotoxic activities. Whether cytotoxic and hemolytic activities are functions of one or more molecules is not known yet. The culture supernatants of all serotypes of A. pleuropneumoniae contain toxin proteins that are approximately 105 kDa (5, 10) and that are structurally and antigenically related to each other (5). The toxin protein that has been best characterized is the 105-kDa HlyI of serotype 1 (4, 8, 9). Gygi et al. (12) cloned the gene encoding this protein, designated hlyIA. Chang et al. (3) cloned a 105-kDa hemolysin-encoding gene from serotype 5, but this one showed only 42% homology with HlyI (7). Both genes appeared to belong to the RTX cytotoxin gene family (27). In a recent study we identified three different cytolytic proteins-cytolysin I (Clyl), cytolysin II (Clyll), and cytolysin III (ClyIII)-in the culture supernatants of the 12 reference strains of A. pleuropneumoniae (14). ClyI and ClyII had molecular weights of 105,000 and 103,000 and were assumed to be the counterparts of HlyI and HlyII. ClyIII had a molecular weight of 120,000 and was assumed to be the *

counterpart of what has been described as pleurotoxin (22). Clyl and ClyllI were strong cytolysins, whereas ClyII was weaker. Most serotypes of A. pleuropneumoniae produced more than one cytolysin; serotype 9 produced Clyl and ClyII. To study the role of Clyl, ClyII, and ClyIII in the pathogenesis of pig pleuropneumonia, the most profitable way forward would be the cloning of their genes. Therefore, in the present study we cloned the genes encoding Clyl and ClylI of A. pleuropneumoniae serotype 9. These genes and the operons to which they belong were analyzed, and we determined the primary amino acid sequence of Clyll. MATERIALS AND METHODS Bacterial strains, plasmids, and cloning vectors. The reference strain CVI 13261 of A. pleuropneumoniae serotype 9 was used as a source of DNA. The gene library was made in bacteriophage lambda Gemll (Promega Biotec) and propagated in Escherichia coli LE 392 (23). Specific DNA fragments were (sub)cloned in pKUN plasmid vectors (16) and transformed into E. coli JM101 (23). Plasmid pLKT52, containing the RTX determinant of Pasteurella haemolytica, was kindly provided by R. Lo (27). Plasmid pLG575, a pACYC184-based plasmid containing the hlyB and hlyD secretion genes of E. coli, was kindly provided by C. Hughes (12). Construction and screening of the DNA library. Highmolecular-weight DNA was isolated by sodium dodecyl sulfate (SDS)-proteinase K lysis, phenol and phenol-chloroform extractions, and precipitation with ethanol (23). This DNA was partially digested with the restriction enzyme Sau3A, and a library was constructed in lambda Gemll according to the methods recommended by the supplier of the cloning vector (Promega). The plaques from this library were hybridized with a 3.7-kbp PvuI-SalI DNA fragment, derived from the leukotoxin determinant of P. haemolytica (27), which had been labelled with [32P]dATP (Amersham

Corresponding author. 4497

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Corp.) by a nick translation kit (Boehringer Mannheim Biochemicals). Plaques that hybridized were visualized by exposure to X-ray film (Eastman Kodak Co.). The DNA library was also screened for the presence of hemolytic plaques. Plaques were grown at 37°C on a Luria broth agar plate and were overlaid with 0.8% agarose that contained 5% sheep erythrocytes, 10% Serum Plus (Hazelton Research Products, Lexena, Kans.), and 50% Eagle minimal essential medium (Flow Laboratories, Irvin, United Kingdom) diluted in phosphate-buffered saline. The plates were incubated at 37°C for 6 to 12 h. Selected plaques were purified to homogeneity by at least two cycles of plating and screening. DNA manipulation and sequence analysis. DNA was digested to completion with restriction enzymes according to the specifications of the enzyme supplier (Pharmacia LKB, Uppsala, Sweden). The resulting fragments were separated by electrophoresis on 0.8% agarose gels. Desired fragments were electrophoretically eluted from gel slices and further purified by extractions with phenol and chloroform and precipitation with ethanol (23). Fragments were (sub)cloned in pKUN plasmid vectors (16) by standard molecular biological techniques (23). Progressive unidirectional deletions were made with the Erase-a-base system from Promega. Nucleotide sequences were determined by the dideoxy chain termination method (24). The sequences were analyzed by using the PCGENE (Intelligenetics Corp., Mountain View, Calif.) and University of Wisconsin Genetics Computer Group analysis software packages. GeneScreen Plus nylon membranes (du Pont de Nemours & Co., New England Nuclear Corp., Boston, Mass.) were used for Southern blot analysis. The blots were hybridized with DNA probes labelled as described above, according to the instructions of the membrane supplier. Before exposure the blots were washed a final time with a solution of 0.1 x SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate) and 0.1% SDS for 30 min at 65°C for homologous probes and with a solution of lx SSC and 0.1% SDS for 30 min at 50°C for heterologous probes. All other DNA manipulations were performed with standard molecular biological techniques (23). Immunoblotting, MAbs, and toxin bioassays. Proteins present in recombinant plaques, cells, or supernatants of logarithmic-growing or stationary-phase cultures were separated by electrophoresis on reducing and denaturing 6% polyacrylamide gels (19). The separated proteins were stained with silver or blotted onto nitrocellulose with a semidry blotting apparatus (Bio-Rad Laboratories, Richmond, Calif.). The blots were incubated according to the method of Towbin et al. (29) with convalescent swine serum derived from an A. pleuropneumoniae serotype 9-infected pig or with monoclonal antibodies (MAbs) specific for Clyl and/or ClyII. MAbs CVI-ApCly 9.1 and 9.2 recognize Clyl. MAb CVI-ApCly 9.3 recognizes ClyII. MAb CVI-ApCly 9.4 recognizes ClyI and ClyIl (14). Bound antibodies were detected with an anti-mouse or anti-swine serum conjugated with alkaline phosphatase (Zymed Laboratories Inc.). The substrates nitroblue tetrazolium (E. Merck AG, Darmstadt, Germany) and 5-bromo-4-chloro-3-indolyl phosphate (Boehringer Mannheim Biochemicals) were used for color development.

Serial twofold dilutions of recombinant toxin, isolated from logarithmic-growing cells, was tested for hemolytic and cytotoxic activity as described earlier (15). Hemolysis was detected by eye, and the titers were expressed as the reciprocal of the highest dilution showing at least 50% lysis of the erythrocytes. Cytotoxin was detected by nigrosin

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FIG. 1. Southern blot of HindIlI-restricted DNA from A. pleuropneumoniae serotype 9 (lane 1), from two hemolytic recombinants (lanes 3 and 4), and from six recombinants selected with the IktCAB probe (lanes 2 and 5 to 9). The blot was hybridized with a 3.7-kb PvuI-SaII fragment of plasmid pLKT52 (27) containing the IktA gene and the 3' end of IktC and the 5' end of IktB (probe lktCAB). After exposure of the blot and removal of the probe, the blot was rehybridized with DNA fragments of 1.2 and 0.7 kb containing parts of the lktB and 1ktD genes (probe lktBD). Since this probe did not hybridize with the blot except in lane 1, only the part of the blot corresponding to lanes 1 to 3 is shown (lanes 10 to 12). The positions of molecular mass markers are shown on the left in kilobase pairs.

staining and determination of the percentage of stained macrophages by using an inverted microscope. The cytotoxin titer was expressed as the reciprocal of the highest dilution showing at least 50% stained macrophages. The ClyI and ClyIl protein contents were estimated by SDS-polyacrylamide gel electrophoresis followed by Coomassie brilliant Blue or silver staining. Nucleotide sequence accession numbers. The GenBankEMBL accession numbers for primary nucleotide sequence data of the presented sequences are X61111 (clyIICA) and X61112 (clyIBD). RESULTS Gene cloning. To determine whether A. pleuropneumoniae serotype 9 encoded toxins related to the RTX cytotoxin family, we used a 3.7 kilobase pairs (kb) PvuI-SalI DNA fragment that was derived from the leukotoxin (Ikt) determinant of P. haemolytica (27) and that contained the lktA gene, the 3' end of the lktC gene, and the 5' end of the lktB gene (lktCAB). This fragment was hybridized to genomic DNA of A. pleuropneumoniae serotype 9. Three specific DNA fragments were homologous to the probe (Fig. 1, lane 1). The lktCAB probe was then used to screen a library of A. pleuropneumoniae serotype 9 DNA, which was prepared in the vector lambda Gemll; 40 recombinants had strongly positive reactions. To determine whether there existed recombinants with cytolytic activity but with no detectable homology to lkt DNA, we also screened the library for recombinants that hemolyzed sheep erythrocytes; three recombinant plaques showed clear hemolytic activity. These hemolytic clones, however, hybridized with the lktCAB probe, indicating that they shared identical sequences with the clones that reacted positively with the Ikt DNA probe (Fig. 1, compare lanes 3 and 4 with lanes 2 and 5 to 9). The hemolytic clones expressed a 103-kDa protein that was

CYTOLYSINS OF A. PLEUROPNEUMONIAE

VOL. 59, 1991

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absent in nonhemolytic clones (data not shown). Moreover, the 103-kDa protein reacted with MAbs specific for ClyII but not with MAbs specific for ClyI (see below). From these data we concluded that we had cloned the gene for Clyll. To locate the ClyII gene in the 9- to 21-kb-long inserts of the selected recombinants, we digested the DNA of 23 positive clones, including the hemolytic ones, with the restriction enzyme HindlIl. The resulting fragments were separated by electrophoresis, blotted onto nylon membranes, and hybridized with the lktCAB probe. All recombinants contained a 2.4-kb fragment homologous to the probe (Fig. 1, lanes 2 to 9). Several recombinants also contained a 4.4-kb fragment that hybridized. Some also contained hybridizing fragments of variable lengths; apparently these clones contain only a part of the 4.4-kb HindlIl fragment which has been ligated to one of the vector arms (Fig. 2A). These findings were used to locate the gene for ClyII (clyIIA). To determine whether the genome of A. pleuropneumoniae serotype 9 also contained sequences related to the RTX secretion genes B and D, we hybridized a 1.2- and a 0.7-kb EcoRV DNA fragment, covering the 3' end of the lktB gene and the 5' end of the lktD gene of P. haemolytica (lktBD [27]), with Southern-blotted genomic DNA of A. pleuropneumoniae serotype 9. A 4.3-kb HindIII fragment hybridized (Fig. 1, lane 10). None of the 9- to 21-kb-long inserts of the selected clones contained this 4.3-kb HindIlI fragment or a part of it (Fig. 1, lanes 11 and 12). Thus, the genome of A. pleuropneumoniae appears to contain sequences related to the RTX secretion genes B and D, but these sequences are not contiguous to the clyIIA gene. To clone the DNA of genes B and D, we used genomic DNA of strain CVI 13261 that was digested with HindIII and size fractionated. The fraction that hybridized with the lktBD probe was ligated into a pKUN19 plasmid that had been digested with HindIII. The ligation mixture was used to transform E. coli cells, and the transformed cells were

hybridized with the lktBD probe. A clone that contained the 4.3-kb HindIII fragment was then isolated. Using this fragment, we also isolated a 7.1-kb BglII-EcoRV fragment that overlapped the 4.3-kb HindIII fragment at the 5' end and a 4.2-kb BamHI fragment that overlapped the 4.3-kb HindlIl fragment at the 3' end (Fig. 2B). By restriction enzyme analysis of the 4.3-kb HindIII, the 7.1-kb BglII-EcoRV, and the 4.2-kb BamHI fragment and by Southern hybridization with the lktBD probe, we were able to locate the putative secretion genes clyB and clyD as indicated in Fig. 2B. Nucleotide sequence analysis. The nucleotide sequences of the clylHCA and clyBD loci were analyzed. The established sequences and the amino acid sequences of the major open reading frames are shown in Fig. 3. Both loci contained two major open reading frames: clyIIC and clyIIA for the first locus and clyB and clyD for the second (see also Fig. 2). Maps of the restriction sites that were deduced from the sequences correlated well with the maps of restriction sites deduced from the cloned DNA and the genomic DNA (data not shown). Thus, the sequenced areas were not detectably rearranged during the cloning procedure. The sequences were numbered starting at -231 (clylICA locus) and -592 (clyBD locus) to correspond to the orientation and location of the major open reading frames. In locus clylICA the open reading frame from 1 to 477 (clylIC) codes for a polypeptide of 159 amino acids (18.5 kDa), and the frame from 519 to 3386 (clyIIA) codes for a polypeptide of 956 amino acids (102.5 kDa). The latter protein is the ClylI toxin and like other RTX toxins contains glycine-rich repeats near the carboxyl terminus. In locus clyBD the frame from 1 to 2133 (clyB) codes for a polypeptide of 711 amino acids (80.2 kDa), and the frame from 2142 to 3575 (clyD) codes for a polypeptide of 478 amino acids (54.9 kDa). These protein sequences closely resemble the protein sequences of the RTX determinants of E. coli, P. haemolytica, and A. pleuropneumoniae serotype 5 (Table 1). Their mutual hydropathy profiles (18) were also very similar (data

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Cytolysins of Actinobacillus pleuropneumoniae serotype 9.

Cytolysin I (ClyI) and cytolysin II (ClyII), which are present in the culture supernatant of Actinobacillus pleuropneumoniae serotype 9, are thought t...
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