Vol. 58, No. 4

INFECTION AND IMMUNITY, Apr. 1990, p. 920-929 0019-9567/90/040920-10$02.00/0 Copyright C) 1990, American Society for Microbiology

Nucleotide Sequence of the Leukotoxin Gene from Actinobacillus actinomycetemcomitans: Homology to the AlphaHemolysin/Leukotoxin Gene Family ELLEN KRAIG,' TERRY DAILEY,2 AND DAVID KOLODRUBETZ3* Departments of Cellular and Structural Biology,' Periodontics,2 and Microbiology,3 University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, Texas 78284 Received 5 October 1989/Accepted 4 December 1989

The leukotoxin produced by Actinobacillus actinomycetemcomitans has been implicated in the etiology of localized juvenile periodontitis. To initiate a genetic analysis into the role of this protein in disease, we have cloned its gene, lktA. We now present the complete nucleotide sequence of the IktA gene from A. actinomycetemcomitans. When the deduced amino acid sequence of the leukotoxin protein was compared with those of other proteins, it was found to be homologous to the leukotoxin from Pasteurella haemolytica and to the alpha-hemolysins from Escherichia coli and Actinobacillus pleuropneumoniae. Each alignment showed at least 42% identity. As in the other organisms, the lktA gene of A. actinomycetemcomitans was linked to another gene, IktC, which is thought to be involved in the activation of the leukotoxin. The predicted LktC protein was related to the leukotoxin/hemolysin C proteins from the other bacteria, since they shared a minimum of 49% amino acid identity. Surprisingly, although actinobacillus species are more closely related to pasteurellae than to members of the family Enterobacteriaciae, LktA and LktC from A. actinomycetemcomitans shared significantly greater sequence identity with the E. coli alpha-hemolysin proteins than with the P. haemolytica leukotoxin proteins. Despite the overall homology to the other leukotoxin/hemolysin proteins, the LktA protein from A. actinomycetemcomitans has several unique properties. Most strikingly, it is a very basic protein with a calculated pl of 9.7; the other toxins have estimated pIs around 6.2. The unusual features of the A. actinomycetemcomitans protein are discussed in light of the different species and target-cell specificities of the hemolysins and the leukotoxins.

of active toxin (5, 28, 35). In E. coli, the hemolysin structural gene, hlyA, is preceded by the hlyC gene, whose protein product is required posttranslationally to activate HlyA (17). The hlyA gene is followed in the operon by the hlyB and hlyD genes, whose products are necessary for the secretion of HlyA (17). P. haemolytica has four genes, lktC, lktA, lktB, and IktD, arranged in the same order. Furthermore, the protein products of each of the lkt genes are homologous to the corresponding Hly polypeptides. Finally, in a recent report, Chang et al. showed that a porcine pathogen, Actinobacillus pleuropneumoniae, contains a hemolysin gene, hppA, whose protein product is over 65% identical to the pasteurella LktA protein (2a). In spite of the sequence similarities among these proteins, it is interesting that the leukotoxins and hemolysins have distinctly different target-cell specificities. Whereas the hemolysins can lyse both lymphocytic and hematopoetic cells (17), the leukotoxins show exquisite cell selectivity. For example, the leukotoxin from A. actinomycetemcomitans lyses only human polymorphonuclear leukocytes and monocytes (33, 36), whereas the leukotoxin from P. haemolytica is specific for ruminant leukocytes (24). To determine the extent of homology between the leukotoxin and hemolysin genes, we have ascertained the complete nucleotide sequences of the lktA and lktC genes from A. actinomycetemcomitans. The protein sequences encoded by these genes have been compared with those of other members of the alpha-hemolysin/leukotoxin family. Although the overall homologies are quite striking, there are some unique features of the A. actinomycetemcomitans toxin which will be discussed in light of its target-cell specificity.

Actinobacillus actinomycetemcomitans is a gram-negative, capnophilic bacterium that has been implicated in the etiology of localized juvenile periodontitis (26, 31). Most A. actinomycetemcomitans strains isolated from patients with juvenile periodontitis produce a heat-labile leukotoxin that specifically lyses human polymorphonuclear lymphocytes and monocytes (30, 33, 36). Although the role of the leukotoxin in an A. actinomycetemcomitans infection has not been clearly established, it is presumed to inhibit neutrophil defense mechanisms at the site of infection (25, 32). As an initial step in a genetic analysis of the role of the leukotoxin in disease, we recently cloned the structural gene, WktA, from A. actinomycetemcomitans (11). A plasmid containing the entire region was transformed into Escherichia coli; cellular extracts were then tested and shown to have appropriately specific cytolytic activity. Another group of investigators, using antibodies against the A. actinomycetemcomitans leukotoxin protein, also cloned the A. actinomycetemcomitans leukotoxin gene (14). As the partial DNA sequence of their clone was very similar to ours, both serological and functional evidence confirmed that the lktA gene from A. actinomycetemcomitans had been cloned. From the partial sequence we reported previously (11), it was clear that the lktA gene of A. actinomycetemcomitans was related to a family of toxin genes, including the leukotoxin gene from Pasteurella haemolytica (2, 15, 27) and the hemolysin gene from E. coli (5). In addition to sequence homologies, the genes from E. coli and P. haemolytica are in similarly organized operons; each structural gene is flanked by three other genes required for the synthesis and secretion *

Corresponding author. 920

VOL. 58, 1990

LEUKOTOXIN GENE FROM A. ACTINOMYCETEMCOMITANS

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FIG. 1. Nucleotide sequencing strategy utilized. A partial restriction map of the insert from the A. actinomycetemcomitans genomic clone, pTD28 (11), is shown. The positions of the IktA and IktC coding regions are boxed. The approximate positions of the potential IktB and lktD coding regions are also boxed. Restriction sites: B, BamHI; Bg, BglII; D, DraI; E, EcoRI; H, HpaI; Hi, HindIII; Hp, HpaII; N, NaeI; Nh, NheI; P, PstI; R, RsaI; S, Sau3AI; Sc, ScaI; Sp, SspI; only sites used in subcloning are indicated. Below the map, the sequencing strategy is illustrated. Each arrow represents an independent dideoxy-chain termination reaction; these were derived either from independent subclones ( i-p ) or from specific synthetic oligonucleotide primers ( - ). The direction and length of each arrow specify the strand and distance sequenced.

MATERIALS AND METHODS

Bacterial strains and plasmids. The genomic clone was derived from strain JP2 of A. actinomycetemcomitans (33). For sequencing, DNA fragments were subcloned into pUC or into BlueScript (Stratagene) and transformed into E. coli TB-1 or HB101 (18). All experiments involving recombinant constructs with the entire A. actinomycetemcomitans lktA gene cloned into E. coli were done under BL2 containment. Nucleotide sequence determination. The DNA sequence was determined by the Sanger dideoxy-chain termination method (23) as modified for use with Sequenase (U.S. Biochemicals Corp.). All coding sequences were determined independently from both strands. The double-stranded plasmid DNA templates were prepared by an alkaline lysis procedure (18) with the inclusion of an ammonium acetate precipitation to remove contaminating proteins. Computer analyses. The best alignments between the A. actinomycetemcomitans leukotoxin genes and the related lysin or toxin genes were derived in pairwise combinations by using the ALIGN program (3) of the Protein Identification Resource of the National Biomedical Research Foundation. All other sequence analyses were performed by using the Pustell DNA software (International Biotechnologies, Inc.).

RESULTS Nucleotide sequence of two genes from the leukotoxin operon of A. actinomycetemcomitans. Using the P. haemolytica leukotoxin gene as a hybridization probe, we had previously cloned an A. actinomycetemcomitans gene that expressed a leukotoxic activity (11). Partial nucleotide sequencing of this clone identified a segment that could encode a polypeptide with homology to the leukotoxin from P. haemolytica and to the alpha-hemolysin from E. coli. To determine the extent of similarity among these proteins, a region of 4,261 base pairs (bp) of the A. actinomycetemcomitans clone was sequenced. The sequencing strategy (Fig. 1) and the DNA sequence (Fig. 2) are presented. With the exception of a small segment in the 5' noncoding region, the sequence was determined from both DNA strands. Analysis of the DNA sequence revealed two long open reading frames. The longer of these, from nucleotides 520 to 3684, could encode a protein of 1,055 amino acids. Since the predicted protein was homologous to the P. haemolytica LktA protein (Fig. 3), the gene was designated lktA. The second open reading frame was located just upstream of the lktA gene (nucleotides 1 to 504); it could encode a protein of 168 amino acids. This protein was homologous to the P. haemolytica LktC (Fig. 3), so the second open reading frame was designated lktC.

Two other genes, designated B and D, have been identified in the toxin operons from E. coli (35) and P. haemolytica (28). The proteins encoded by the E. coli hlyB and hlyD genes are involved in the transport of the alpha-hemolysin from the cell (17). Preliminary sequence analysis of two regions downstream of the A. actinomycetemcomitans lktA gene (designated a and b in Fig. 1) revealed sequences homologous to both the hlyB and hlyD genes. Specifically, translation of the 230 bases sequenced just downstream of lktA revealed a 73-amino-acid open reading frame that shared 67% identity with the amino-terminal sequence of the E. coli HlyB protein. Similarly, a deduced amino acid sequence from the 200 bases near the end of clone pTD28 had 72% identity with a region near the carboxy terminus of the E. coli HlyD. Therefore, A. actinomycetemcomitans appears to have a leukotoxin operon structure similar to those of E. coli and P. haemolytica, with lktB and lktD downstream of lktA and 1ktC. Similarities among the toxins produced by A. actinomycetemcomitans and other pathogenic microorganisms. The alignments between the deduced amino acid sequences of the actinobacillus LktA protein and other toxin proteins of the leukotoxin/hemolysin family are presented (Fig. 3). The extent of identity between all pairwise combinations of these proteins was at least 42% (Table 1). The overall homology among the toxin proteins from the four organisms was reflected in their very similar hydropathy plots (Fig. 4A) (27, 34). As has been found before, the amino halves of the protein had several strong hydrophobic domains, whereas the carboxy portions were essentially all hydrophilic (27, 34). In the alignment shown (Fig. 3), 24% of the amino acid residues matched in all four proteins. These highly conserved amino acids seemed to cluster in three regions. The first of these was a strongly hydrophobic region encompassing residues 200 to 500 (Fig. 4A). Mutational analyses of the E. coli hemolysin have demonstrated that this hydrophobic domain is required for lytic function (16). The second conserved region, from amino acids 754 to 864, is a series of 12 repeats with the consensus sequence X-G-G-X-G-N/ D-D-X-I/L (27, 34). These glycine-rich repeats have been shown to be necessary for lytic activity in E. coli (6). Finally, a block of conserved amino acids was found around position 970; of the 15 residues, 5 were identical in all four toxin proteins and 9 others were shared by three of the four polypeptides. A role for the third conserved region in leukotoxin activity has not been demonstrated. Differences between the A. actinomycetemcomitans LktA and the other leukotoxin/hemolysin proteins. Although the four toxin proteins compared in Fig. 3 had a number of

922

INFECT. IMMUN.

KRAIG ET AL. -493 -400

-300 -200

-100 1

91

161 271 361

451

GATC AAAACCTGAT AACAGTATTT TCTCAGTCTA ATTTTTGCGT ATTAATACAA TACGGGATTG CGTAGATAAA TGTATTATCA AAAAACTAAT MATTTTATGA AATTAAATAA TTTTTTCTAT_TGACTATTAA AGAATCCGGA GTAAATTAGT CTCCAAAATT AACCAAAACT AGGTAATTTA TCCGGTCMAA GGTTATCTTA AGTATTAACC CTAAGAAAAA GGAAAACGAG TATGTCCAGT ACAGAATATG CTCCATTTTA TCTCCGTTTT ATTCAGTTCC CAA6TAATGA A6TTTTACTC TATGAATACT GGAAACTTGT TCAGMATTTT GTACAAAAGG TTAGTAAAAT AACGGTAAGA TTAGCACAAA TC6TTGGCAT TCTCGGCGAA AAAACTATTT GGAATACCA MAGTACTTTT MATGATGGCA TGCTGGATAT TGTGGTTTGG TTATCTTATT CAAMTAMAT TATTAACAM..MTTTAAT ATG GAG AAA MAT AAT MAT TT GMA GTG TTA GGG TAT GTG GCT TGG TTA TGG GCA MAT TCT CCT TTG CAT AGA MAT TGG TCT TTA TCA Not Giu Lys Asn As. As. Pho Glu Val Lou Gly Tyr Val Ala Trp Lou Trp Ala Asn Sor Pro Lou His Arg Asn Tip Sor Lou Sor CTT GCT ATT MAT GTA CTG CCT GCA ATT CMA TAT GGG CMA TAT ACA TTA TTA ATG AGA GAT GGG GTT CCT ATT GCT TTT TGT A6C TGG Lou Ala Ile Asn Val Lou Pro Ala Ioe Gin Tyr Gly Gin Tyr Thr Lou Lou Not Arg Asp Gly Vol Pro Ile Ala Pho Cys Sor Tip MAT TTA AGC CTT GAG MAT GAG ATA MAA TAT CTT GMA GAT GTA TCA TCT CTT GTT TAT GAC GAC TGG MAT TCC GGT GAT CGT AMA TGG As. Lou Sor Lou Glu Asn Glu RIoe Lys Tyr Lou Glu Asp Vol Sor Sor Lou Val Tyr Asp Asp Trp Asn Sor Gly Asp Arg Lys Tip ATT GAT TGG AlT GCG CCA TTT GGT CAC MAT TAT GTG CTT TAT MAA CAT ATG CGT MAA TCA TTT CCC TAT GAT TTA TTC AGA TCA ATT Rio Asp Tip RIoe Ala Pro Pho Gly His Asn Tyr Vol Lou Tyr Lys His Hot Arg Lys Sor Pho Pro Tyr Asp Lou Pho Arg Sor RIle GTT TAT MAA GGT TCG TCA GAG GGC MAA ATT ACT GMA TTC CAT GGA GGT AMA GTC GAT AMA CMA TTA GCT MAT AMA ATC TTT CMA CMA Vol Tyr Lys Gly Sor Sor Giu Gly Lys RIoe Thr Glu Pho His Gly Gly Lys Vol Asp Lys Gin Lou Ala Asn Lys RIoe Pho Gin Gin CAT TTT GAG TTA ATC MAT GMA TTA MAA MAC AAA TCT GMA GTT ATA TCT ATT MAT TA.A.&W..M TAG ATT ATG GCA ACT ACT ACA CTG

Phw Glu Lou RIoe Asn Glu Lou Lys Asn Lys Sor Glu Val RIoI Sor RIoe Asn Torim

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GAT AGA GCT Asp Arg Ala TAT ATC CCT Tyr RIoe Pro MAA GAC GGC

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CCA Thr Lou Pro TG CMA AM Lou Gin Lys GCG CTT ATA Ala Lou 666 AAA MAA Gly Lys Lys MAT TCT 6GT

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Sor Asn Sor Vol

GCA TTT TC6 Ala Phe Sor MAC CTG ATT As. Lou Rio CTA GGA CMA Ala Lou Gly Gin TTA TCC GGT GTA Lou Sor Gly Vol TTG ACA MAT AAA Lou Thr As. Lys CCT GTC GCA 666 Pro Vol Ala Gly

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Vol TTG Lou GTG

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GGT ATT GCG MAA CMA TTT GAT CGT GCG AGA ATG CTT 6AM GMA TAC Gy RIoe Ala Lys Gin P1w Asp Arg Ala Ar, Not Lou Glu Blew Tyr

TTA CTT GGT CMA TTC TAC MAA MAT ACA GGG ATC GCA GAT GCT 6C6 ATT ACA Lou Lou Gly Gin P1w Tyr Lys Asn Thr Gly RIoe Ala Asp Ala Ala RIoe Thr

GGT GCA GCC TCC GCC GGT TCT TTA GTT GGT GCG CCA ATC 6GT TIB TTA BT6 Gly Ala Ala Sot Ala Gly Sot Lou Vol Gly Ala Pro RIoe Gly Lou Lou Vol

1711 MGT GCG ATT ACC MGC TTA ATT TCA GGA ATT CTT GAT GCT TCT AAA CMA GCC GTT TTT GMA CAT ATC GCG MAT CMG CTC GCC GAT AAA ATT Sot Ala RIoe Thr Sot Lou RIoe Sot Gly RIoe Lou Asp Ala Sot Lys Gin Ala Vol P1w Giu His RIoe Ala Asn Gin Lou Ala Asp Lys RIol 1801 AAA GCA Lys Ala 1891 TTT P1w Asn

MAT

TGG GAG MAT MG6 TAC GGT MG Trp Glu Asn Lys Tyr Gly Lys GAG TTA CGT GMA MAA TAT AAA Giu Lou Arg Glu Lys Tyt Lys

MAT TAC TTT Asn Tyr P1w ACC GMA MAT Tht Giu Asn

GMA MAT GGC TAT GAT GCC CGT CAT TCC Glu Asn Gly Tyt Asp Ala Arg His Sot ATA TTA TCT ATC ACT CMA CMA GGT TGG RIoe Lou Sot RIoe Tht Gin Gin Gly Ttp

GCC TTC TTG GMA Ala P1w Lou Glu GAT CAG CGC ATT Asp Gin Arg RIoe

GAT TCA CTA Asp Sor Lou GGT GM TTA Gly Glu Lou

AAA TTA Lys Lou GCA GGT Ala Gly

FIG. 2. DNA sequence of the A. actinomycetemcomitans lktA and lktC genes. The complete nucleotide sequence determined is presented. The deduced positions and amino acid sequences of the actinobacillus lktA and lktC gene products are shown. Sequences that are presumed to play a role in transcription (-) and translation (=) are indicated. The asterisk designates the C not found in the sequence presented elsewhere (13). Sequence data will appear in the EMBL/GenBankIDDBJ nucleotide sequence data bases under accession number X16829.

similarities, some interesting differences were also noted between the LktA of A. actinomycetemcomitans and the other three proteins. First was the fact that the A. actinomycetemcomitans LktA protein was 32 amino acids longer than the HlyA from E. coli and 100 amino acids longer than the other two proteins. When the proteins were aligned (Fig. 3), essentially all of the extra amino acids were found in the carboxy 200 amino acids, which was the least conserved region among these proteins. The second difference was in the number of glycine-rich repeats present in the proteins. There were 12 of these repeats in the A. actinomycetemcomitans LktA protein (Fig. 2, positions 754 to 864) and 11 in the HlyA protein of E. coli

but only 6 in the other two proteins. The significance of the extra repeats is unclear; however, it was recently reported that increasing the number of repeats in the pasteurella LktA protein did not affect its activity (R. Y. C. Lo, B. J. Cooney, and P. E. Shewen, Abstr. Annu. Meet. Am. Soc. Microbiol. 1989, B269, p. 75). The final major difference was found when the amino acid compositions of the four proteins were compared (Table 2). For the most part, the compositions were very similar, including the absence of any cysteine residues. However, the A. actinomycetemcomitans LktA had 20 extra basic amino acids (Table 2). Because of this, the calculated pl of the LktA of A. actinomycetemcomitans was 9.7 as com-

LEUKOTOXIN GENE FROM A. ACTINOMYCETEMCOMITANS

VOL. 58, 1990

923

1961 ATC ACT CGT AAT GGA GAT CGT ATT CAA AGT GGT AAA GCT TAT GTG GAT TAT TTG AAA AAG GGT GAG GAG CTT GCA AAG CAT AGC GAT AAA Ile Thr Arg Asn Gly Asp Arg le Gln Ser Gly Lys Ala Tyr Val Asp Tyr Leu Lys Lys Gly Glu Glu Leu Ala Lys His Ser Asp Lys 2071 TTC ACT AAA CAG ATT TTA GAT CCA ATC MA GGT AAT ATT GAT CTT TCG GGT ATA AAA GGT TCT ACC Phe Thr Lys Gln Ile Leu Asp Pro Ile Lys Gly Asn lie Asp Leu Ser Gly lie Lys Gly Ser Thr 2161 TTA ACC GCA GGT AAG GAA GAA CGG AAA ACA CGT CAG TCA GGT AAA TAT GM TTT ATT ACT GAA TTA Liu Thr Ala Gly Lys Glu Glu Arg Lys Thr Arg Gln Ser Gly Lys Tyr Glu Phe lie Thr Glu Leu 2251 MB BTA AAA GGT 6TT CCT AAT TCT AAT GGT GTA TAT GAT TTT TCT AAC TTA ATT CM CAT GCC GTT Lys Val Lys Bly Val Pro Asn Ser Asn Gly Val Tyr Asp Phe Ser Asn Leu Ile Gln His Ala Val 2341 BCA MA TTA ATT GCT AAT TTG GGT GCT AAA GAT GAT TAT GTT TTT GTC GGA TCC GGT TCA ACA ATA Ala Arg Leu Ile Ala Asn Leu Gly Ala Lys Asp Asp Tyr Val Phe Val Gly Ser Gly Ser Thr Ile 2431 6T6 GTG BAC TATAGT AAA GGT CGC ACC GGT GCA TTA ACA ATC GAC GGT CGT AAT GCT ACT AAA GCC Vai Vai Asp Tyr Ser Lys Gly Arg Thr Gly Ala Leu Thr Ile Asp Gly Arg Asn Ala Thr Lys Ala 2521 CTT ABC BBT ACT CM GTC TTG CAG GAA ACC GTA TCA MG CAA GAA ACT AAA CGA GGG AAG GTT ACC Leu Ser Bly Thr Gin Vai Leu Gln Glu Thr Val Ser Lys Gln Glu Thr Lys Arg Gly Lys Val Thr 2611 AA TTA BAT TAC TAT TAT ACG AAT AAG GGC TTT Lye Liu Asp Tyr Tyr Tyr Thr Asn Lys Gly Phe 2701 AM TTT TAT GGT TCT AAA TTT AAT GAT GTT TTC Lys Phe Tyr Gly Ser Lys Phe Asn Asp Vai Phe

AAA GCT CAT GAT GM TTA AAC Lys Ala His Asp Glu Leu Asn CAT GGT CAC GAT GGC GAT GAT His

Gly

His

Asp Gly Asp Asp

2791 GBC BAT MT BBB AAT GAC GAA ATT CAT GGC GGC CAA GGT AAT GAT AAG CTC TAT 61y Asp Asn 61y Asn Asp Glu Ile His Gly Gly Gln Gly Asn Asp Lys Leu Tyr 281661 C AC AAC TAT CTT GAC GGT GGA GAA GGC GAC GAC CAC TTA GAG GGA GGC MT 61y Asn Asn Tyr Leu Asp Gly Gly Glu Gly Asp Asp His Leu Glu Gly Gly Asn

66A MC CAA GGA Lys Liu Phe Bly Asn Gln Gly 3061 TAC CBT A GM TAT G66 CAC Tyr Arg Lye 6Bu Tyr Gly His 3151 TCA TTT BAB CGT AAC GGC AAT Ser Phe 6Bu Arg Asn Bly Asn

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3241

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3421 3511

3601 3691

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ACA CGT GAT AAT AAA GTT CTA GAA Thr Arg Asp Asn Lys Val Leu Glu

GTT AAT GCT GGA GAC GGT Vai Asn Ala Gly Asp 61y GGA CAA TATAAG GTT GM Gly Gln Tyr Lys Vai Glu

TAT GAT Tyr Asp MA GAT Arg Asp

BAT CTA CTT GMA TAT CBT AMC TAT Asp Leu Leu Blu Tyr Arg Asn Tyr TCA GTA GAG GAA ATT ATC GGC AGC ACA CTA CBT BAT Ser Val Glu G6u Ile Ile Gly Ser Thr Liu Arg Asp TTG ATT TAT GGT TAT GAT GGC BAT GAT CGT TT6 TAT Leu Ile Tyr Gly Tyr Asp Gly Asp Asp Arg Lou Tyr GGT GGT GCC GGT AAC GAT AGG CTC TTT GBT 6M TAT Gly Gly Ala Gly Asn Asp Arg Leu Phi Bly Biu Tyr G6T TCC GAT ATT CTA AGA G6T 6GA AMT GBC MT GAT Gly Ser Asp Ile Leu Arg Gly Bly Ser Bly Asn Asp

GAT BAT TTA CTT GAC GGT GGA GAA GGC GAT GAC CAA CTT GCC GGT GGA GAA GGA MAT AT ATT TAT 6TT

Asp Asp Leu Leu Asp Gly Gly Glu Gly Asp Asp Gln Leu Ala Gly Gly Glu Bly Asn Asp Ile Tyr Vai CAC ACT ATT ACG GAA CAT ABC GGT GAT AAA GAT A TTA TCA TTA GCA AAT ATC AAT CTC AM 6AT BT6 His Thr Ile Thr Glu His Ser Gly Asp Lys Asp Lys Leu Ser Leu Ala Asn Ile Asn Lou Lys Asp Vai BAT CTA CTA TTG AAA ACA AAT AAT AGA ACA GCA GTA ACA TTT AAA GGA TGG TTT AGT AM CCT MT TCA Asp Leu Liu Liu Lys Thr Asn Asn Arg Thr Ala Vai Thr Phi Lys Gly Trp Phe Ser Lye Pro Asn Ser TTA BAT BAG TAT BCA 6BA CAA AGA AAA CTT CTT GAA TAC GCA CCT GAA AAG GAT CGT GCA CGA CTT AAG AGA CAA TTT BAB TTA CAG TC6 Ser Ala 61y Lou Asp Glu Tyr Gln Arg Lys Leu Leu Glu Tyr Ala Pro Glu Lys Asp Arg Ala Arg Leu Lys Arg 61n Phe Blu Liu 61n CBA 6BT AM GTC BAC AAA TCA CTC AAT AAT AM GTT GM GAA ATT ATC GGT MA GAT GGG GAG CGG ATT ACT TCG CAA GAC ATT BAT MT Arg 61y Lys Vii Asp Lys Ser Leu Asn Asn Lys Vii Glu Glu Ile Ile Gly Lys Asp Gly Glu Arg Ile Thr Ser Bin Asp Ile Asp Asn CTT TTT GAT MG AGT 6G6 MC MA MG ACA ATT TCA CCT CAA GAG CTT GCC GGA CTT ATT MG MT AM GGT MG TCA AGT AGC CTT AT6 Leu Phe Asp Lye Ser Bly Asn Lys Lys Thr Ile Ser Pro Gln Glu Leu Ala Gly Leu Ile Lys Asn Lys Gly Lye Ser Sir Ser Leu it TCT TCT TCT CGT TCB TCA AGT ATG CTT ACA CAA AAG TCC GGT TTG TCA AAT GAT ATT AGT CGT ATT ATT TCA GCA ACC AGT BBT TTT BBT Ser Ser Ser Arg Ser Ser Ser Mit Leu Thr Gin Lys Ser Gly Leu Ser Asn Asp Ile Ser Arg Ile Ile Ser Ala Thr Ser 61y Phi 61y TCA TCC BGT AM GCG TTA TCC GCT TCG CCA TTG CAG ACC AAT AAT AAC TTT AAC TCT TAC GCA AAT TCG TTA GCA ACT ACT BCT TM T6A Ser Ser Bly Lys Ala Leu Ser Ala Ser Pro Leu Gln Thr Asn Asn Asn Phe Asn Ser Tyr Ala Asn Ser Leu Ala Thr Thr Ala Term GTTATTGGC TTTTTAGTGA TTCCGCATAT TTGTGTGTGC GGAATCACTG TTTTTCAGGA TATGTAATGG ACTCACABA

FIG. 2-Continued

pared with 6.0 to 6.2 for the other three proteins. Half of the extra basic amino acids were found near the carboxy terminus of the A. actinomycetemcomitans LktA in its unique sequence area. The remainder were distributed fairly evenly throughout the LktA molecule. Comparison of A. actinomycetemcomitans LktC protein to other hemolysin/leukotoxin C proteins. The LktC protein of A. actinomycetemcomitans was over 70% identical to the HlyC protein of E. coli, whereas the alignments with the C proteins from the other organisms ranged from 52 to 55% identity (Table 3). The strong homology among all of these C proteins is emphasized by the fact that 35% of the residues matched in all four proteins (Fig. 3). As would be expected from these sequence homologies, the hydropathy plots for the C proteins from these four organisms were very similar (data not shown); each had a rather hydrophobic amino half and a very hydrophilic carboxy region. Despite these similarities, there was one rather incongruous result: the calculated pl of the LktC protein from A. actinomycetemcomitans was over 1 pH unit more acidic (8.7) than the calculated pIs for the C proteins from the other organisms, which range from 9.8 to 10.0. The difference in pl was not due to an increase of acidic residues in a specific region of the A. actinomycetemcomitans C protein. Rather, additional basic residues were scattered throughout the C proteins from the

other organisms. The significance of the difference in pl, if any, is unclear. Comparison of the noncoding regions of the leukotoxin/ hemolysin gene clusters from four organisms. Although nothing is known about promoter sequences in A. actinomycetemcomitans, there are likely to be sequences resembling those commonly found in E. coli (8), especially since the A. actinomycetemcomitans lktA gene is expressed in E. coli (11). When the nucleotide sequence was examined for the presence of the E. coli -10 consensus (TATAAT) and -35 consensus (TTGACA) elements, a number of sequences that matched these sequences at five out of six positions were found. However, none of the pairs of elements had the 16- to 18-bp spacing typically found in most E. coli promoter elements. The sequences that most closely resembled a functional promoter were located 344 bp 5' to the lktC gene (Fig. 2). This region contained a -35 consensus sequence followed 16 bp downstream by a sequence that matched the -10 consensus at its four most highly conserved nucleotides. None of the other potential -35 sequences had a correctly spaced -10 sequence that matched at more than three of six nucleotides. Whether these sequence elements promote transcription in either A. actinomycetemcomitans or E. coli remains to be determined. Both lktA and lktC contained potential ribosome-binding

924

KRAIG ET AL. A.

a.

LKTC

Z.C. HLYC

INFECT. IMMUN. M M M

5 10 15 20 25 30 35 E X N N N F E VIL G Y V A WLWAN SPLHR N W S L S LLA I N V L N R N N P L E VIL G H V S W L W ass P L H R N W P V S LIFA I N V L

40

45

50

P A IQ Y G Q Y T L L M R D IR A N Q Y A L L T R D

A. p. HPPC P. h. LKTC

P A L X - N F N VIL G Q I A W L W A N S P M H R N W S V L LMK N V I P A IE N D P I F V T V D D H N Q 8 Y - F N LIL G N I T W L H H S S L H K E W S C E LA R N V I P A I E N E Q Y H L L I D N

A.a. LRTC

C V _ I W F C S W A N LS L E N E I K

Z.C. HLYC

A.p. HPPC P.h. LXTC

a.a. LKTC C.C. HLYC A.p. HPPC P.h. LKTC

A.a. LKTC E.C. HLYC A.p. HPPC

P.h. LKTC

a. a. LITA E.ec. HLYA A.p..HPPA P.h. LXTA

a.a. LKTA 1.C. HLYA a.p. NPPA

P.h. LITA

s

55

G

I

P

60

A

Y

A.a. LITA l.C. KLYA A.p. HPPA P.h. LKTa A.a. LITA Z.c. HLYA A.p. HPPA P.h. LKTA

a.a. LITA Z.c. HLYA A.p. HPPA

P.h. LITA

A.a. LKTA C.c. HLYA A.p. HPPA P.h. LITA A.a. LITA

l.c. HLYA

A.p. NPPA

P.h.

LTA

70

75

85

80

95

90

W

A

S

105

110

Y VIL Y K H R K S F

115

Y D L FTS I

120

RIV Y K G

125 -

S S

AIL Y KIYIMIR K K FIPID E L FIRIAII RIV D P K - T H S L|L Y KXHIM|R Q R F|PIY D I GIRIA I R|I Y P S K K D Q LIL Y K KHC Q K YPD H I V RS I RIF Y P K Q K E G

155 H F I I EII QIE I Q E LA

YIH H

N T T T

E E A A

EIG VIG T|G LIG

130

KII T KIV S K|I I KIIA

E E Y Y

135

F HIG G F HIG G L KG G F KIG G

KIV KII KII KIL

140

D

100

GIH N GID N GID S

GIH S

145

150

Q L N I TQ Q

LANLIIKIIIFIK DIKIQ TIKIKVIAIEIKITIFIL D JK T AXK KR FD

Q

Q

T

160 165 170 L K N K S E V - I S I N V K N K S D F N F S L T G L Q L K N E F N F I K K -

45 5 10 15 20 25 30 35 40 50 H A T T T L P N T K Q Q A A Q F A N S V A D R A K E N I D AAKEQmQKAL D K L G K T G K K L T H P T I T a A Q I K S T L Q S A K Q S A A N K L H S A G Q S T K D A|L|K K A A E Q T R NA G N R L I H s K I T L S S L K S S L Q Q G L K N G K N K L N Q A G T T L K N G|L|T Q T GH S L Q NG A K K L I - - - - - H G T R L T T L S N G L K N T L T A T K S G L H K A G Q S LT Q A GS S L K TG A K K I I

55

fLY[I P|K NI1K|L|LI P|X D|Y|X LIYI PIQGIYI- Y I PIQ N Y Q Y L

65 70 75 80 85 - KmN G L T AR1I K AlXQ KIL G I E VIYH E G K G QIGIS S L N D|L|V R T|A|D E|L G I E VIQ Y D E K D S G QIGIN G V Q DILIV K AI IN DL G I E V|W R E E R D T E QlGJN G L Q DL V K A A E EIL G I E VIQ R E E R

60 -

-

-

-

D N S N

G G N N

90 95 100 PA L T N GI L NfG K TA I T K QV F GIT|A E LD I A K TS F DITIT Q IA T A Q TS L G TI Q

120 125 130 135 115 140 145 150 T L F|A PE L[K W I Q G N K H L S N S VmS T G - N L T K A I DV Q SVG T I F AA IAGSIVLIS PIQ LIDX L L Q K Y Q K A G N K LIGG S A E N I G D N V L FIA PIQ LID|N L L K K N P K I G N T LIGIS A S - S I S QN GGKIA I N TIV LG G I V L SIA PIQ I D K L L Q K T - K A G Q A L G S A E - S I V Q N A N KA K TIV LIS IGSLTER 105

A.a. LITA .C. HLYA A.P. HPPA P.h. LITA

65

L E V S|S LIV Y D D N S|G D R K F D I A P F c s A N L S L E N E I K YL N|DIV TIS L|V A E DIWIT SIG D R|KIW|F|I|V|W|I|A P F Y c s R K L T L E S E A R YIV KIDIT NIS LIK I DDIWIN AIG D RIIIWIIIIIDIWIIIA P F I a Y c s w A D L N L E T E V K Y I K DJI NIS LIT P E E Q SIG D R RWII DWV A P F

N Y P V GFI P I

110

L LGLfERL CL LIICGLIT|E|R IV KI LIGIFITID|R GII K

T a

165

160

155 T LmA Fm1N T T T FIQIN FL

170

a F S G H D L D A L I A L S S H K I D E L I IIQIS IILIG S V L S G V N L N E L L G I Q S I JGS V L A G M D L D E A L G

210 205 I T N N V D TF1S KrL L - N N V N S|F|S Q Q L S V Q T V D A|F|A EIQ|I S V K T L D ELFG EIQJI 255 v L S A I S rA IIL S A I SIAIS L|L SIG A S|A|G

215

220

N K LArE N K L|G|S S K

175 180 K A R Q N G K N V T D V Q IL K K Q K S G G N V S S S EAL Q N - - - - K D P N Q L EAL Q N - - - - - N S N Q H AIL 225

G

S KL Q N I

K K K K

195

200

245

250

AIS L NIIIE 1;I G T I S S I L IIINIQILIV D T A a S AIS E AIG L EILITINIEILIV G N I a S AIG L EWL T NJS LI E N I a N

235

240

L P KrT1G N L GKXI LIaG Lr cl N|K LIQINIL P NILID N I AGLD T VIS L G G L S N|K LQ|NIL P D|LIG K A S L LD I IS GI G L G T L G DIK L K N I G G L D K A G LIG LID V IIS G

G S F G DIK A1G QV[IH F LSKFr

V|L|S N T K H LG|S H|L|Q N V|K|G K

S Q F

230

190

185

A A A A

L N G V G

G

270 275 280 285 290 295 300 L L r 1AN KD RDmfATTA A 1A A -EL T N K V L rG N I IG A I T Q L I 1A Q R IA F IILIS N A D|A|D|TIG TIKIA AIAIG VIE|L T T K V LIG NIV GIKIG I S QIYII IIA Q R AII

260

L

IIL|A

265

D K

EIA|SIT|E KIK|A

AIAIG VIEIFA N Q I

L L SIG a TAA L VLA D K NASTA KKV GAG FEL A

H

305

310

315

320

325

330

355

360

365

370

375

380

I|G N|V T|K|A

V S

S|Y|I

LA Q RI VIA

Q V VIG N.II TA V S SY I LIA Q RVA 335

340

345

350

390

395

400

440

445

450

alc L SIT T G P V[A GWL I A SIVmS L AI S P L S F L G I T1K Q mFD RW1R N L E EmYS KIR FIKm FW QIG L SIT S A A AIAIGIL ASIVIVITIL AIIIS P LISIFIL S IIA|D KIFIK RIAIN K I E EIYIS QIR FIKIK LIG SIG L S|S T G PVIA|AIL I A S|TIVIA|L A|V|S P LSIF|L N V|AID KIFIK QIAID L I K SY|S E|R F|Q|KI L|G I A SATS ALG L SIS T G P VAAAL IIS P L AFA G I AD KFN HK S L E SYA ERFKK L mN[G IYIDIG IYIDIG YDG

DISIL LIG Q F Y K N GI A|D A|A ITmNVTS A I S V DIS|L LA A F H K EIT GIA IID AIS LITIRIIISITIVILIAA DIRL L|A D F H R EIT GIT IID A|S VITIT|I|NITIA|LLA A I DINI LIA E Y Q R GIT GIT IID AIS VTAINT.ALA A I 405

410

415

420

425

430

385

A

A GTL V G AI G L AIA T TISIL V G AIPIV S A AIS A GISIL V G A|PIV a L AIA A GWV I a S Pa L

AWV GIA AIS

S SIGIISI S GIGIV GIA A GGV SIA 435

IVSA I MTS L mS GII LD A K IIVF N Q L A E Km K A WI- N K Y G riN Y F EN Y VIT|G I111S GII LIE ASK QA FEVA|S K A D VII|A EW E-- KK K HH G NN YY F EINYD EIQYD E K NK HPPA V|A V|TG L|IIT TI LIE Y1YS K QAIFEIVIAIN KK VI HH ND RV KV EKW N N H GK N Y F EINICYD LITA cI G VWS T LIQ KQ NLI E VLN FIG. 3. Protein similarities among related toxin genes. The proteins encoded by the toxin genes from A. actinomycetemcomitans [A.a], A. pleuropneumoniae [A.p] (2a), E. coli [E.c] (5), and P. haemolytica [P.h] (27) have been aligned to achieve maximum homology. The residues showing identity among all four polypeptides have been boxed. A.a. C.c. A.p. P.h.

LKTA HLYA

V

L VG A IL L GL

sites that matched the consensus E. coli Shine-Dalgarno sequence (7) at five of six positions (Fig. 2). The two actinobacillus ribosome-binding sites were identical and, as expected, resided 6 or 7 bp in front of their respective translation initiation codons. Interestingly, although the Shine-Dalgarno sequences found 5' to the hlyC, IktC, and hppC (C genes) differed among the four bacteria, the ribosome-binding sites between the A and C genes were identical in A. actinomycetemcomitans, A. pleuropneumoniae, and P. hemolytica: AGGAGA. To look for transcriptional or translational regulatory sequences common to the toxin genes from various organisms, the DNA sequences of the three noncoding regions around the lktCA, hlyCA, and hppCA gene clusters were

N

F

examined for homologies. First, the 5' noncoding sequences (260 to 400 bp in length) were aligned in register with the initiator codon for the C gene. Between 27 and 30% sequence identity was observed for each pairwise combination (data not shown). This was significantly lower than the 67% nucleotide identity found in the coding region immediately downstream. The sequences were also compared in different alignments to identify any highly conserved regions that may have different spacing relative to the coding region. Although numerous matches of 7 to 10 nucleotides were found, none of these occurred in more than two of the DNAs being compared. Many of the identities included stretches rich in A and T, a common feature of 5' regions. We concluded that there were no highly conserved primary sequence homolo-

LEUKOTOXIN GENE FROM A. ACTINOMYCETEMCOMITANS

VOL. 58, 1990 LKTA E. c. HLYA A. p. HPPA P. h. LKTA

A.a.

455

460

ED S L E D N L Q D N AR Y L a N Q D N A

R H

S a

H A A F SR H L A D

505

A.a.

LXTA

E.c. HLYA A.p. HPPA P.h. LKTA

A.a.

LKTA

E.c. HLYA A.p. HPPA P.h. LKTA

I Q

555

LITA

E.c. HLYA A.p. HPPA P.h. LKTA

E.c. HLYA HPPA P.h. LKTA

A.p.

470 E

L R

E

R

NL I

H

Y

480 L

S

L

H

K F L L N

LNKEL

K

T

V

N

I I

Q

T

Q

Q

525

565

570

KY

E E N S KY E

K T RS S E R V T

610

575

Y

W D QRI G E L W D T LI G EL R W D NQ I G D L Q W D N N I G D G

A

G I

T

H

A

G

V

A

a

T

LA

HNIV V|QIR

I

V.R

I

JV

E.c. HLYA A.p. HPPA P.h. LKTA

G R T D K G E

E.c. HLYA A.p. HPPA P.h. LKTA

GK V

E.c. HLYA A.p. HPPA P.h.

LXTA

K

A

T

Q H

I

L

595

635

640

645

650

625

630

665

670

675

680

TWAmTIDG R N AmK AmQ KVE D TIGIYILITIIDIG T K AISIE AIGINIY TVITIRV Y|G|A|L|V|I D|A T A EIT|E K|G| S|YIV|K|R|Y

DIA T

K EJE

QLGSYT

720

715

710

D L L

T

E[Y RIN Y K L D Y Y Y

QIY RIS Y E F T H I RR E D D R F R E E K I

GK R

T E

GN R E E

K T

E|Y X I EIY RIH

DV F H

S NN Q H

D

-

-

T N K

VJNRF 730

725

A H G F K E5ER N

700 690 685 695 LS G T Q V-Q 1TT V S X Q r T x It L G G D V K VILIQIEIV V X E Q E V S V V G D S - K I aT H Q T1 V V T S TH T ALV V E TG - K A L H -

A|L|H|E|T

735

-

-

770

DmoLmf VYfYD G

Dm

R

Y T

775 D N

NGIVIDIT|I|D|G|N|D G|DID|LIFIGIG

L D G G E G D D H L E G G N H N Y GA G N N Y L N G G D G D D E L Q V Q G

E Y G -

-

-

-

-

-

-

-

K

rF Y1[1T

Q|

830

r TISIX F a RAIDIK F Q F NID I F xGISIQ F D r 1N S H

T

N|!2JI Fri

-

- - -

E

-

- - - -

G

N [1

Y

V

Y R

K E

IGINDL L KIG GIY G NID IIY R Y L S G

GN|D

I I S G GI K D JNWL L HIG G K G GH

NID IIY DID I F

Y A G N D R L F G D Q L Y G G D G 1 D X LI

N D K L -

-

-

-

-

-

-

-

-

-

- - - - - - - - - - - - - - - - -

845

840

835

D I LR G G S G N D X L FG N Q GD D L LD N V L S GG K G N D X L Y G S E a D L L D

Y

- - - - - - - - - - - - - - - - - - -

875

T A V T N V L S E K V R E K V T

880

H H Tm T E H S

YIG|H

G D

III|D D D GIGIK D SI1 D S GIG Q H

T V H K T G D GIN V H R K G D0 GN D IWIT D

915

910

L L K T N N R N D G N D LI I M Y K A E G D S S L E I I N Q K G K H N LWV I T N S K K

G

-

870

865

860

G G

-

925 920 F K G W F S K P N S S A - - - - - - - - I I G N W F L E D - - - I Q N W F R E A - - - -

S D

J N

885

K D X L|S DID -D -

D

930 G L D E Y Q

L

A

X LIS L X LI A F

A

890 N I N L

D I D F

S D V N L LI S F S D S N L

935

R X

L L E

Y

A

X

P G

1010

L A G S N N L S K L S K

L I K N K G K K A S Y V Y G V V N D - - V V D N - - -

L S A A A A RN N R N V

S P L Q S L L Q L G T Y L V A P

r1

V

RID|V X XK

S

A A

Tr

L

T

-

LFE

A

- -

R X

900 N

V V

950

L F

X R x N

- - - - 1000

995 T

Li

AIPIR R

DlL T|F|X

X X

E

I S P Q Z L E Y Q Q I S A E A IT Q D

1050 1040 0145 1035 A I S R I [TS1 A T S G1G N Fl V K ZE IR I S x III SI A A - - - - - - - - - - Y N T S K D R Q N V S|N|S L A K L|I S|S V G SIFIT S S S D SIS V S A T S S N D S - - - - - - - - - - Y E L L K H S K N V T N S L D X L 1020 1015 S S S R S S S S S S L N D A L A Y G S Q G N L

1060 N N N L S G N V P S S T S H L

T

895

E X D R AR H X N C I T - T N D T x D - - -

T|P

D|11R

N

945

940

- - - - - - - - - - - - - - - D L A S T V A N Y K - - - D F A K E V P N Y X

850 '

GCI.E GICITX

PL V D D F I D G

990 985 980 975 970 965 960 955 N E R T|S Q D I D N L F D K S I G K D F E L Q R G K V D K S L N N K V E E V D S L - - - - - - - - - X F E K E S G D I S - - - N H Q I E Q I|F D K N N Q - - - - - - - - - - - - - - R K I E E I1I G K GIG E R IITIS E Q V D X L I K E I G Q NLJE RI IS K Q V D D L I a K G N G X - - - - - - - - - - - - - - E K I EEE

Q W

800

795

790

785

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - N

- - - - - - - - - - - -

855 G D[5] Q L A

G S N S L A -

780 E I HG 1

N

KIG|N|D|T

825

820

815

810

E|I1I G|S EIIII GST

H a G Y Y T KDJTLK AIV E

-

GIT

E EILII E

750

745

740

S|V E E II GI S TL R[

E T|DEINILY SIV V T|D|S|L|K S|V

N G K N L T HT G Y T

765

760

H

1055

A.a. LKTA

G

K V K G R T D K V K G V P N S N G V Y|B1F S L V K G V D K T V G V Q D X S V Y|D|Y S H I Q R V D S T V T D G D A S S S V -|D|F T I N R V D S K I T D G A a s s T F -L T TKN

620

DR VJH YS R G N Y GA L T I

1005

A.&. LKTA

S

E

615

660

VVmD mS K IDIVIVIYIYID ID|RIV|H|Y|S R

905

A.a. LKTA

VL JN

A V T R D N K V L E A R L I A N [L G|A K D1Y iV F|V G S G S T I V N A1D Y R E I R I E S GI D GIDIKIV FIL S A G S A N I Y aGIXICIH A S V G N N Q Y HEL N| V F V G S S T T V I D CICIDICIN A V K F D D A G N I I E S K D T K I I A N L G|A GNID G G I E L D N A G N V T K T K E T K I I A KIL GIE DG DNV FIV G S G TT E I D GSj ElJ Y

805

E.c. HLYA A.p. HPPA P.h. LKTA

-

- - - - -

755

A . a . LKTA

-

- -

600

L S|G GNGI D IDI F NGIA D|D|D|H|I|E|GENID GIN|D||L|Y LG|D AIDIDV I DIG GI NiG|D|V F H|G|G KG D DI L DIG Gj NGD D GNLD GNLD R LF G G V D T I DAFN G

P.h. LKTA

-

590

LKTA E.c. HLYA A.p. HPPA P.h. LKTA

A. a. LKTA E.c. HLYA A.p. HPPA

550 T

IS|D S K S S S L L X SIN T N R KK T Q S V L

L

585

|CN

E.c. HLYA A.p. HPPA P.h. LKTA

E

G

NIL IIQIH - - - - - -

705

A.a. LKTA

L

540

580

A.a. LKTA E.c. HLYA A.p. HPPA P.h. LKTA

A.a.

S

I

C D R G D X R TD X X

N

N

R

S R

I

G

500

R

545 mNmD LmG I K G S T T L

535

530

TE

F Y YI Y

495

490

485

T Q

V VA I TQ Q Q A E R V I A I T Q

520 515 E E L A K H

560

-

475

K

I

NIP L L TA K E T|P T|P|G|E|N TIP L L IrP T H

655

A.&. LKTA

N

IFIV TIP L L TIPIGIEIEIIIE R R L L FIR E R IE

605 A.a.

F

Y L K K T S D K F T KQ I L D P I K K R L E K K P D E F Q K Q V F D P L Y Y E E GKEG|N|I|D A F E E G Q H Q S Y D S S V - - - - Q L D N K NIGIIIII A F E E G K H I K A D K L V Q L D S A NWG I I D

TL S G K S Y I D ISS S G K A Y VD VL S G K A Y V D

L

L

F K I L S Q Y N K EY S V E R S K F L I N L N K E L Q A E R N

510

YD V

S Kj G a

465

K

F

AR

925

1065

1030 1025 L T Q K S G L S

-

1070

F N S Y A N - - - - - S A S D F S Y G R N S I T I D V S N N- - - - I Q D Q S L S S - - - - L Q

- - - - N P L I

iOD

NIE

1075

L A T Tm

L T A SIAI L A R AIAI F A R A A

FIG. 3-Continued

regions of these genes. In addition, there regions of potential secondary structure conserved among the four 5' regions, even in the areas identified previously for the 5' region of the E. coli hlyC gene (5). The second DNA region examined was the intercistronic region between the A and C genes. The sizes of these regions in the various organisms ranged from 11 to 38 bp. With the exception of the Shine-Dalgarno sequence, there were no significant identities among the sequences in this region. The region 3' to the A gene is another intercistronic region in E. coli and P. haemolytica. Both organisms make two lktA-containing transcripts; one transcript contains all four genes in the leukotoxin/hemolysin gene cluster, and the second terminates between the hly and lkt A and B genes (29,

TABLE 1. Percent sequence similarities among LktA/HlyA proteinsa

among the 5' were no obvious

gies

% Sequence similarity source Protein surce Protln

A. A. E. P.

actinomycetemcomitans pleuropneumoniae coli haemolytica

A. actino- A. pleuromycetem- pneumo- E. coli niae HlyA comitans HppA LktA

100 42 51 43

100 44 64

100 42

P.haeLktA

molytica

100

The proteins being compared were adjusted by the ALIGN program to optimize sequence matches. The percent sequence similarity was then calculated as 100 x (the number of amino acid identities/number of matches possible) between two proteins. I

926

INFECT. IMMUN.

KRAIG ET AL.

A

Glycine Repeats

Hydrophobic Domains

3 2 1

LI IIa AlhhI i dII 11.I .1

A

-1 -2 m Cu

t0. 10 0

I

-3

3 2 1

-1 -2 -3

I

0

400

200

600

800

1000

B m

3

-

2

-

A

1

,-S

-P1

0

-2

A.a. LKTA, Lally et al.(13)

-

-3

-

6

200

400

600

800

1000

Sequence Number FIG. 4. Hydropathy plots of the hemolysin/leukotoxin proteins. The method of Kyte and Doolittle was used to calculate the values (12). The positive values are hydrophobic and the negative values are hydrophilic. (A) The plots for the E. coli HlyA (5) and A. actinomycetemcomitans (A.a.) LktA (this paper) are shown. (B) Plot for the A. actinomycetemcomitans LktA from Lally et al. (13).

35). Consistent with this, the DNA between the A and B genes in both E. coli and P. haemolytica have a potential rho-independent transcription termination signal, consisting of an inverted repeat followed by a run of T residues (21). The A. actinomycetemcomitans intercistronic region also has the potential to form such a structure from positions 3705 to 3738 (Fig. 2). Although each of these intercistronic regions has a sequence capable of forming a stem-loop structure (ranging from 9 to 16 bp for the stem and from 5 to 15 bp in the loop), the primary sequences in this region were not conserved. It remains to be determined whether A. actinomycetemcomitans also uses this transcription terminator. Comparison of the coding regions reported for the leukotoxin from A. actinomycetemcomitans. Lally et al. (13, 14) have also recently reported the cloning and characterization of the lktA and lktC genes from A. actinomycetemcomitans. Since the restriction map of their clone matched ours (11), it appeared that both groups had identified the same gene. Therefore, it was surprising that a comparison of the two predicted amino acid sequences for LktA indicated significant divergence over the last 124 amino acids. This was particularly evident when the hydropathy plots of the two predicted toxins were compared (Fig. 4). When the DNA

sequences obtained by the two laboratories were aligned, several differences were noted (Table 4). Although these changes caused some minor amino acid switches in both LktA and LktC, the major difference at the carboxy terminus was due to an additional C residue at position 3298 in our sequence (Fig. 2). The lack of this C caused a shift in the reading frame which yielded a protein of approximately the same length but with a very different sequence. The C residue was confirmed in our laboratory on two sequencing gels of the noncoding strand and seven gels of the coding strand; the appropriate region from one of these is shown in Fig. 5. We concluded that our sequence in this region is correct.

DISCUSSION We determined the entire nucleotide sequence of the lktA and lktC genes from A. actinomycetemcomitans. The deduced amino acid sequence of LktA was over 42% identical to the sequences of the hemolysins from E. coli and A. pleuropneumoniae and the leukotoxin from P. haemolytica. The sequence of the A. actinomycetemcomitans LktC protein was over 52% identical to the sequences of the C

VOL. 58, 1990

LEUKOTOXIN GENE FROM A. ACTINOMYCETEMCOMITANS

TABLE 2. Comparison of the amino acid compositions of the leukotoxin/hemolysin proteins from four bacteriaa

927

TABLE 4. Nucleotide and amino acid differences found between two sequences for the lktA and lktC genes of A. actinomycetemcomitans

No. of each amino acid in:

Amino acid

LktAb

Nonpolar Ala Val Leu Ile Pro Met Phe Trp Polar Gly

HlyAc

HppAd

LktAe

Nucleotide position"

5' region _59

89

88

73

88

42

54

65

54

118 60 15 5 34 5

100 64 12 5 30 4

88 69 11 3 29 5

92 69 9 5 26

119 78

96 88 21 71 45

97 70 67 0 23 68 41

4

Asn Gln

37 74 40

113 88 56 0 40 65 43

Acidic Asp Glu

74 50

76 56

73 46

64 53

Basic Lys Arg His

99 37 15

83 29 17

67 32 17

77 26 20

Ser Thr Cys Tyr

64 0

57 0

a Calculated pIs were 9.65 for A. actinomycetemcomitans LktA, 6.06 for E. coli HlyA, 6.01 for A. pleuropneumoniae, and 6.27 for P. haemolytica. b A. actinomycetemcomitans (this paper). c E. coli (5). d A. pleuropneumoniae (2a). P haemolytica (27). p.

proteins from the other three bacteria. Surprisingly, the A. actinomycetemcomitans LktA and LktC proteins 'were more identical to the E. coli A and C proteins than to the analogous' proteins from P. haemolytica or A. pleuropneumoniae (Tables 1 and 3). This result was unexpected, since both phenotypic features and overall DNA hybridization kinetics have placed A. actinomycetemcomitans, A. pleuropneumoniae, and P. haemolytica in a family of organisms distinct from the Enterobacteriaceae family containing E. coli (19,''22). We concluded that there may h'ave been a relatively recent transfer of the leukotoxin genes between A. actinomycetemcomitans and E. coli. Although this could TABLE 3. Percent sequence similarities among

LktC/HlyC proteinsa % Sequence similarity

Protein source

A. actino- A. pleuro-

mycetemcomitans LktC

pneumoniae HppC

E. coli HlyC

lktC gene 25 lktA gene 1238 1297 1526 1766 1835 2013 2690 3298

Nucleotide(s) found by: Kraig et al.b Lally et al.c

Amino acid found by: Kraig et al.b Lally et al.c

_d

GCATG

Noncoding

G

A

Val

Met

TA G A T T T CA C

AT

Leu Asp Glu Phe Phe Gly Thr Arg

Tyr His Ala Ser Ser Gly Asn Frameshift

C C C C C AC -

aThe positions given are from this paper. b The sequence for lktCA reported in this paper. C The sequence for lktCA reported by Lally et al. (13). d No nucleotide at the corresponding position.

occur by any number of mechanisms, the fact that some strains of E. coli have a plasmid-borne copy of the hemolysin genes (9, 20) suggests plasmid transmission as a possible route. If so, one might expect'that the A. actinomycetemcomitans LktA protein would resemble more closely the plasmid-encoded HlyA protein than the chromosomally encoded HlyA protein. Apparently, this is not the case. The two forms of the E. coli toxin differ at 30 residues (9); at these positions, the A. actinomycetemcomitans LktA shares 6 residues with the chromosomally encoded toxin but only 4 residues with the plasmid-encoded peptide. It is interesting to note that the highest sequence similarity for the C proteins was found between E. coli and A. actinomycetemcomitans (70%), whereas the highest homology observed for the A proteins was between A. pleuropneumoniae and P. haemolytica (64%). The biological relevance of this finding is unclear but suggests that the A and C genes may be under different evolutionary constraints in the various bacteria. Despite the sequence similarities among these proteins, the leukotoxins and hemolysins demonstrate different target cell specificities. The E. coli and A. pleuropneumoniae hemolysins lyse both lymphocytic and hematopoietic cells (17), whereas the leukotoxins from A. actinomycetemcomitans and P. haemolytica demonstrate lytic activity only toward certain cell subsets. When the protein sequences were analyzed for regions of similarity unique either to the leukotoxins or to the hemolysins, no significant homologies that could account for the target-cell specificities were

P

LktC

T

A C G T 3300

T

A. A. E. P.

actinomycetemcomitans pleuropneumoniae coli haemolytica

100 55 70 52

100 49 56

100 50

100

a The proteins being compared were adjusted by the ALIGN program to optimize sequence matches. The percent sequence similarity was then calculated as 100 x (number of amino acid identities/number of matches possible) between two proteins.

FIG. 5. Sequencing gel of the region around nucleotide 3298. The numbers indicate the nucleotide positions as assigned in Fig. 2. The asterisk designates the C not found in the sequence presented elsewhere (13).

928

INFECT. IMMUN.

KRAIG ET AL.

found. Therefore, it will be necessary to undertake a directed mutagenesis approach to ascertain the molecular basis of target-cell specificity. Despite the overall homology to the other leukotoxin/ hemolysin proteins, the LktA protein from A. actinomycetemcomitans has several unique features. Most strikingly, the leukotoxin from A. actinomycetemcomitans had a calculated pI that was significantly more basic than those of the other toxin proteins. The basic pl may play a role in determining the target-cell specificity of the LktA or in its capability to invade the periodontal pocket. The latter possibility is supported by the finding that the site of A. actinomycetemcomitans infection, the gingival crevice, usually has a pH above 8.0 (1, 10). Alternatively, the difference in isoelectric points between the A proteins may be important in the differential cell distribution of the toxins. It has been shown that the E. coli hemolysin is secreted from the cell (17), whereas the A. actinomycetemcomitans leukotoxin is localized in the periplasm (4). The secretion of HlyA requires functional HlyB and HlyD. We have found that A. actinomycetemcomitans contains at least part of lktB and part of lktD. However, 90% of each of these genes has yet to be sequenced, so they may not be functional. Nonetheless, since the carboxy domain is required for secretion of HlyA (6), it is interesting to speculate that the addition of basic residues in this region of LktA might, in fact, affect its secretion from the actinobacillus cell. Since the carboxy terminus may play a crucial role in the function and/or target-cell specificity of the toxin, the report of an A. actinomycetemcomitans leukotoxin A gene with an entirely different sequence for the last 124 amino acid residues is particularly interesting (13). This divergence is clearly seen on the hydropathy comparison of the two reported lktA sequences (Fig. 4). The critical nucleotide difference between these two sequences is the C present at position 3298 in the clone described here (Fig. 2). Lally et al. (13) did not report a residue at this position; this would cause a shift in reading frame, which would yield a protein of approximately the same length but of a very different sequence. In fact, their predicted protein would have four cysteine residues, all in this carboxy-terminal region. This is noteworthy, since none of the other members of the toxin family contains any cysteines (Table 2). The recombinant clones reported by both laboratories have been shown to produce functional toxin when expressed in E. coli (11, 13). Therefore, it is important to determine whether one or both of these sequences are correct. There are several possible explanations for the apparent conflict in the sequences reported for lktA. First, there could be two genes in the A. actinomycetemcomitans genome that are capable of encoding functional toxins. However, Southern blot analysis of A. actinomycetemcomitans DNA showed that there was only a single gene (11). Second, there could be genetic polymorphisms between the A. actinomycetemcomitans strains used. Although this possibility cannot be formally excluded, both groups used strain JP2, so again this is an unlikely explanation. Third, the addition or deletion of the C residue may have been introduced during cloning of the gene. This is unlikely for our clones, since the sequence reported here was derived from two independently isolated genomic clones. Last, the difference may have been due to an error in the DNA sequencing itself. Again, we feel that there is no mistake in our sequence; in nine sequencing gel runs, the C at position 3298 was unambiguously present in the coding strand and/or the

complementary G was seen in the noncoding strand. We conclude that it is unlikely that there are two different leukotoxins produced by A. actinomycetemcomitans, and we argue that the sequence presented in this paper is the correct one. In summary, we have shown that although the LktA and LktC proteins of A. actinomycetemcomitans are highly homologous to the equivalent leukotoxin/hemolysin proteins from E. coli, P. haemolytica, and A. pleuropneumoniae, there are some potentially interesting differences. The major difference would seem to be that the isoelectric points of the A. actinomycetemcomitans LktA and LktC proteins are quite different from those of the homologous proteins from the other organisms. The possible role of these differences in the host and tissue specificity of the A. actinomycetemcomitans leukotoxin is being investigated. ACKNOWLEDGMENTS

We thank Ryland Young and Doug Struck (Texas A&M University) for providing us with the DNA sequence from the A. pleuropneumoniae leukotoxin gene before its publication. We are also grateful to Jeff Ebersole for many helpful discussions and for introducing us to this very interesting microorganism. We thank Jack Spitznagel for his assistance with the graphics and Rick George, Jimmy Buchanan, and Michael Kolodrubetz for help with photography. We also acknowledge D. Dailey, J. Ebersole, J. Spitznagel, and J. Guthmiller for their critical reading of the manuscript. This work was supported by Public Health Service grant DE08521 from the National Institutes of Health. LITERATURE CITED 1. Bickel, M., and G. Cimasoni. 1985. The pH of human crevicular fluid measured by a new microanalytical technique. J. Periodontal Res. 20:35-40. 2. Chang, Y. F., R. Y. Young, D. Post, and D. K. Struck. 1987. Identification and characterization of the Pasteurella haemolytica leukotoxin. Infect. Immun. 55:2348-2354. 2a.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. 3. Dayhoff, M. O., W. C. Barker, and L. T. Hunt. 1983. Establishing homologies in protein sequences. Methods Enzymol. 91: 524-545. 4. DiRienzo, J. M. 1986. Application of monoclonal antibodies to the study of oral bacteria and their virulence factors, p. 250-293. In A. J. L. Macario and E. C. de Macario (ed.), Monoclonal antibodies against bacteria, vol. 3. Academic Press, Inc., New York. 5. Felmlee, T., S. Pellett, and R. A. Welch. 1985. Nucleotide sequence of an Escherichia coli chromosomal hemolysin. J. Bacteriol. 163:94-105. 6. Felmlee, T., and R. A. Welch. 1988. Alterations of amino acid repeats in the Escherichia coli hemolysin affect cytolytic activity and secretion. Proc. Natl. Acad. Sci. USA 85:5269-5273. 7. Gold, L., D. Pribnow, T. Schneider, S. Shinedling, B. S. Singer, and G. Stormo. 1981. Translational initiation in prokaryotes. Annu. Rev. Microbiol. 35:365-403. 8. Hawley, D. K., and W. R. McClure. 1983. Compilation and analysis of Escherichia coli promoter DNA sequences. Nucleic Acids Res. 11:2237-2255. 9. Hess, J., W. Wels, M. Vogel, and W. Goebel. 1986. Nucleotide sequence of a plasmid-encoded hemolysin determinant and its comparison with a corresponding chromosomal hemolysin sequence. FEMS Microbiol. Lett. 34:1-11. 10. Kleinberg, I., and G. Hall. 1968. pH and depth of gingival crevices in different areas of the mouths of fasting humans. J. Periodontal Res. 3:109-117. 11. Kolodrubetz, D., T. Dailey, J. Ebersole, and E. Kraig. 1989. Cloning and expression of the leukotoxin gene from Actinoba-

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cillus actinomycetemcomitans. Infect. Immun. 57:1465-1469. 12. Kyte, J., and R. F. Doolittle. 1982. A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157:105-132. 13. Lally, E. T., E. E. Golub, I. R. Kieba, N. S. Taichman, J. Rosenbloom, J. C. Rosenbloom, C. W. Gibson, and D. R. Demuth. 1989. Analysis of the Actinobacillus actinomycetemcomitans leukotoxin gene. J. Biol. Chem. 264:15451-15456. 14. Lally, E. T., I. R. Kieba, D. R. Demuth, J. Rosenbloom, E. E. Golub, N. S. Taichman, and C. W. Gibson. 1989. Identification and expression of the Actinobacillus actinomycetemcomitans leukotoxin gene. Biochem. Biophys. Res. Commun. 159:256262. 15. Lo, R. Y. C., C. A. Strathdee, and P. E. Shewen. 1987. Nucleotide sequence of the leukotoxin genes of Pasteurella haemolytica Al. Infect. Immun. 55:1987-1996. 16. Ludwig, A., M. Vogel, and W. Goebel. 1987. Mutations affecting activity and transport of haemolysin in Escherichia coli. Mol. Gen. Genet. 206:238-245. 17. Mackman, N., J.-M. Nicaud, L. Gray, and I. B. Holland. 1986. Secretion of haemolysin by Escherichia coli. Curr. Top. Microbiol. Immunol. 125:159-181. 18. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 19. Mannheim, W., S. Pohl, and R. Hollander. 1980. On the taxonomy of Actinobacillus, Haemophilus, and Pasteurella: DNA base composition, respiratory quinones, and biochemical reactions of representative collection cultures. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. Reihe A 246: 512-540. 20. Noegel, A., U. Rdest, W. Springer, and W. Goebel. 1979. Plasmid cistrons controlling synthesis and excretion of the exotoxin a-haemolysin of Escherichia coli. Mol. Gen. Genet. 175:343-350. 21. Platt, T. 1986. Transcription termination and the regulation of gene expression. Annu. Rev. Biochem. 55:339-372. 22. Pohl, S. 1981. DNA relatedness among members of Haemophilus, Pasteurella and Actinobacillus, p. 245-253. In M. Kilian, W. Frederiksen, and E. L. Biberstein (ed.), Haemophilus, Pasteurella and Actinobacillus. Academic Press, Inc., London. 23. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463-5467. 24. Shewen, P. E., and B. N. Wilkie. 1982. Cytotoxin of Pasteurella haemolytica acting on bovine leukocytes. Infect. Immun. 35: 91-94.

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