analysis of hemolysin operons in actinobacillus pleuropneumoniae

8
Gene, 123 (1993) 51-58 0 1993 Elsevier Science Publishers B.V. All rights reserved. 0378-l 119/93/$06.00 GENE 06886 51 Analysis of hemolysin operons in Actinobacillus pleuropneumoniae (RTX toxins; virulence; serotypes; secretion; deletions) Joachim Frey, Marianne Beck, Urs Stucki and Jacques Nicolet Institutefor Veterinary Bacteriology, University of Berne, CH-3012 Berne, Switzerland Received by M. Bagdasarian: 10 September 1992; Accepted: 20 September 1992; Received at publishers: 16 October 1992 SUMMARY Among the twelve different serotypes of Actinobacillus pleuropneumoniae, the causative agent of swine pleuropneumo- nia, a strongly active hemolysin I (HlyI) is produced by serotypes which are particularly virulent, and less active hemolysin II (HlyII) is produced by all serotypes except type 10. In the serotypes 1, 5a, 5b, 9, 10 and 11, which produce HlyI, the hemolysin (My) operon consists of a structural hlyZA gene, encoding pre-HlyI, an activator gene, hlylC, necessary for the activation of pre-Hly to active Hly, and two genes, hlyZB and hlylD, involved in Hly secretion. These genes are clustered in the order, hlylCABD. This is characteristic to RTX toxin (repeats in the structural toxin) operons. The HlyII operons in all serotypes producing HlyII consist only of the pre-HlyII-encoding gene, uppA, and its activator gene, appC. The serotypes, which produce HlyII, but not HlyI, contain a truncated HlyI operon, with the promoter, hlylB and hlyID, and a small segment of the C terminus of hlylA. This partial HlyI operon might have been formed by deletion of hlyIC and most of hlylA. In serotype 3, which produces HlyII, but no HlyI, and which releases only minute amounts of this Hly into the growth medium, none of the hlyl genes and consequently no Hly secretion genes were found. The above results postulate that HlyII is secreted via the products of hlylB and hlylD, and explain the low amount of HlyII secreted by serotype 3. Cloning and analysis of the structural genes encoding pre-HlyI and pre-HlyII among the different serotypes revealed differences in the hlyIA genes which are highly similar in the serologically related serotypes 1, 9 and 11, and differ from the serotypes, 5a, 5b and 10. The hlyIIA genes, in contrast, seem to be conserved in all serotypes. INTRODUCTION A. pleuropneumoniue, the etiological agent of swine pleuropneumonia (Shope, 1964), secretes hemolysins Hly which seem to play an important role in pathogenesis (Nakai et al., 1984; Frey and Nicolet, 1988b; 1990; Rosen- da1 et al., 1988). These Hly are strongly immunogenic in experimentally and naturally infected pigs (Devenish et al., 1990; Frey and Nicolet, 1991). They were also shown to induce protective immunity against A. pleuro- pneumoniue infection and are important components for subunit vaccines (Bhatia et al., 199 1; Van den Bosch et al., Correspondence to: Dr. J. Frey, Institute for Veterinary Bacteriology, Laenggasstrasse 122, CH-3012 Berne, Switzerland. Tel. (41-31) 27 44 84; Fax (41-31) 24 69 22; e-mail: [email protected]. Abbreviations: A., Actinobacillus; aa, amino acid(s); uppA, gene encoding structural HlyII protein (pre-HlyII); appC, gene encoding activator of HlyII; bp, base pair(s); CTP, cytosine triphosphate; A, deletion; dCTP, deoxyCTP; Hly, hemolysis(s); HlyI, hemolysin type I; HlyII, hemolysin type II; hlylA, gene encoding structural HlyI protein (pre- HlyI); hlylB and MyID, secretion genes on the operon specifying pre- HlyI; hlyIC, gene encoding activator of HlyI; kilobase or 1000 bp; NAD, nicotinamide-adenine dinucleotide; nt, nucleotide(s); oligo, oligo- deoxyribonucleotide; PCR, polymerase chain reaction; R, resistance/re- sistant; RTX, repeats in the structural toxin; SDS, sodium dodecyl sulfate; SSC, 0.15 M NaCl/O.OlS M Na,citrate pH 7.6; Tc, tetracycline; T,, melting temperature of DNA:DNA duplexes.

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Gene, 123 (1993) 51-58 0 1993 Elsevier Science Publishers B.V. All rights reserved. 0378-l 119/93/$06.00

GENE 06886

51

Analysis of hemolysin operons in Actinobacillus pleuropneumoniae

(RTX toxins; virulence; serotypes; secretion; deletions)

Joachim Frey, Marianne Beck, Urs Stucki and Jacques Nicolet

Institutefor Veterinary Bacteriology, University of Berne, CH-3012 Berne, Switzerland

Received by M. Bagdasarian: 10 September 1992; Accepted: 20 September 1992; Received at publishers: 16 October 1992

SUMMARY

Among the twelve different serotypes of Actinobacillus pleuropneumoniae, the causative agent of swine pleuropneumo-

nia, a strongly active hemolysin I (HlyI) is produced by serotypes which are particularly virulent, and less active

hemolysin II (HlyII) is produced by all serotypes except type 10. In the serotypes 1, 5a, 5b, 9, 10 and 11, which produce

HlyI, the hemolysin (My) operon consists of a structural hlyZA gene, encoding pre-HlyI, an activator gene, hlylC,

necessary for the activation of pre-Hly to active Hly, and two genes, hlyZB and hlylD, involved in Hly secretion. These

genes are clustered in the order, hlylCABD. This is characteristic to RTX toxin (repeats in the structural toxin) operons.

The HlyII operons in all serotypes producing HlyII consist only of the pre-HlyII-encoding gene, uppA, and its activator

gene, appC. The serotypes, which produce HlyII, but not HlyI, contain a truncated HlyI operon, with the promoter,

hlylB and hlyID, and a small segment of the C terminus of hlylA. This partial HlyI operon might have been formed by

deletion of hlyIC and most of hlylA. In serotype 3, which produces HlyII, but no HlyI, and which releases only minute

amounts of this Hly into the growth medium, none of the hlyl genes and consequently no Hly secretion genes were

found. The above results postulate that HlyII is secreted via the products of hlylB and hlylD, and explain the low

amount of HlyII secreted by serotype 3. Cloning and analysis of the structural genes encoding pre-HlyI and pre-HlyII

among the different serotypes revealed differences in the hlyIA genes which are highly similar in the serologically related

serotypes 1, 9 and 11, and differ from the serotypes, 5a, 5b and 10. The hlyIIA genes, in contrast, seem to be conserved

in all serotypes.

INTRODUCTION

A. pleuropneumoniue, the etiological agent of swine

pleuropneumonia (Shope, 1964), secretes hemolysins Hly

which seem to play an important role in pathogenesis

(Nakai et al., 1984; Frey and Nicolet, 1988b; 1990; Rosen-

da1 et al., 1988). These Hly are strongly immunogenic in

experimentally and naturally infected pigs (Devenish

et al., 1990; Frey and Nicolet, 1991). They were also

shown to induce protective immunity against A. pleuro- pneumoniue infection and are important components for

subunit vaccines (Bhatia et al., 199 1; Van den Bosch et al.,

Correspondence to: Dr. J. Frey, Institute for Veterinary Bacteriology,

Laenggasstrasse 122, CH-3012 Berne, Switzerland. Tel. (41-31) 27 44

84; Fax (41-31) 24 69 22; e-mail: [email protected].

Abbreviations: A., Actinobacillus; aa, amino acid(s); uppA, gene encoding

structural HlyII protein (pre-HlyII); appC, gene encoding activator of

HlyII; bp, base pair(s); CTP, cytosine triphosphate; A, deletion;

dCTP, deoxyCTP; Hly, hemolysis(s); HlyI, hemolysin type I; HlyII,

hemolysin type II; hlylA, gene encoding structural HlyI protein (pre-

HlyI); hlylB and MyID, secretion genes on the operon specifying pre-

HlyI; hlyIC, gene encoding activator of HlyI; kilobase or 1000 bp;

NAD, nicotinamide-adenine dinucleotide; nt, nucleotide(s); oligo, oligo-

deoxyribonucleotide; PCR, polymerase chain reaction; R, resistance/re-

sistant; RTX, repeats in the structural toxin; SDS, sodium dodecyl

sulfate; SSC, 0.15 M NaCl/O.OlS M Na,citrate pH 7.6; Tc, tetracycline;

T,, melting temperature of DNA:DNA duplexes.

52

1990). To date, two different Hly, both proteins with an

apparent molecular weight of 105 kDa, have been iso-

lated (Frey and Nicolet, 1988a; 1988b: 1992). Both the

strongly hemolytic Hlyl which is very similar to the Esch-

erichia coli x-Hly and the weakly hemolytic Hlyll which

resembles more the Pasteurella haemolytica leukotoxin,

belong to the group of RTX toxins as determined from

trarzs-complementation experiments with E. coli x-Hly ac-

tivator and secretion genes and from their DNA derived

aa sequence (Chang et al., 1989; Gygi et al., 1990; Frey

et al., 1992; Smits et al., 199 1). In addition, a third secreted

protein toxin of 120-125 kDa, named pleurotoxin or cy-

tolysin III, was identified among certain serotypes and

was shown to have cytotoxic activity (Rycroft et al., 1991;

Kamp et al., 1991). Among the twelve different serotype

reference strains, the serotypes 1, 5a, 5b, 9, 10 and 1 I,

known generally as virulent strains, have been shown to

secrete Hlyl. All serotypes except serotype-10 secrete

Hlyll (Kamp et al., 1991; Frey et al., 1992), but serotype-

3 seems to secrete substantially lower amounts of Hlyll

than most other strains (Frey and Nicolet, 1991). The

genetic determinant encoding HlyI in A. pleuropneumon- iae serotype-1 is a single operon comprising four contigu-

ous genes, the posttranscriptional activator gene hlylC,

the gene for the structural 105kDa protein hlylA, and

the secretion genes hlyIB and hlylD, arranged in the order

hlylCABD (Gygi et al., 1992). An intragenic Rho-

independent transcription termination signal is located

between hlylA and hlylB (Frey et al., 1991). The genes

encoding the weakly hemolytic Hlyll, also named App,

were postulated to be located on two different operons,

one containing the activator and structural genes appC and appA and a second operon containing appB and

appD (Chang et al., 1991; Smits et al., 1991). Comparison

of the secretion genes appB and appD with the secretion

genes hlylB and hlylD from the hlyl operon shows that

the genes are the same, or at least highly similar and seem

to belong to the hlyl operon (Gygi et al,. 1992). Due to

the fact that certain serotypes of A. pleuropneumoniae

contain two different Hly operons, which were detected

subsequently and by different laboratories, various desig-

nations for these genes and their products have been used,

which are listed in Table I for a better understanding.

In order to clarify the role of the different genes in-

volved in Hlyl and Hlyll production, activation and

secretion in the different serotypes, we have analyzed the

genetic organisation of the hlyl and hlyll determinants

in the serotype reference strains. In addition, the degree

of conservation of the genes encoding the structural pro-

teins of these Hly among the different serotypes was esti-

mated by gene cloning or amplification and subsequent

restriction enzyme fragment analysis.

RESULTS AND DISCUSSION

(a) Analysis of the h&Z operon in serotypes 1-12

In order to get a complete picture of all genes belonging

to the hlyl operon in the different A. pleuropneumoniae serotype reference strains (Table II), Southern blot analy-

sis was performed using total genomic DNA digested

with Pstl and EclXI and probes for the individual hly genes and for a segment upstream from hlylC, containing

the promoter sequences, The location of the different

probes on the hlyl operon is shown in Fig. I. Table II

summarizes the results obtained from the hybridization

experiments. For the serotypes 1, 9, 10 and 11 all probes

for the Hly genes hlylC, hlylA(both N-and C-terminal),

hlylB and hlylD and the segment upstream from hlylC hybridized with a 9.2-kb Pstl-EclXI fragment, indicating

that these genes are closely linked like the genes of the

hlyl operon of serotype 1 (Fig. 2). This genetic arrange-

ment is common to most RTX operons found in many

different pathogenic bacteria (Welch, 1991). In serotypes

5a and 5b, the probes for hlylC, hlylA(N-and C-terminal),

hlylB and hlylD and the probe for the segment upstream

from hlylC hybridized to a 20-kb PstI-EclXI fragment.

When EcoRI + Pstl-digested genomic DNA of serotypes

1, 5a, 5b, 9, 10 and 11 was used: hlylC and hlylA probes

hybridized to a 4.8 kb-fragment in all these serotypes

(Fig. 3 B). Further hybridizations indicated that in sero-

types 5a and 5b the structure of the hlyl operon is the

same as in serotype 1 and that the segment downstream

from the hlyl operon must be different (Fig. 2). The hy-

bridization results with serotypes 2, 4, 6, 7, 8 and 12

showed no signal for h/y16 and no signal for the N-termi-

nal part of the hlylA gene, but a weak reaction with a

6-kb Pstl-EclXI fragment when the C-terminal hlylA probe was used. This 6-kb fragment also hybridized to

the probe for the sequences upstream from hlylC and to

the hlylB and hlylD probes (Table II). When

EcoRI + Pstl-digested genomic DNA was used, a weak

signal of a 1.3-kb fragment, hybridizing to the C-terminal

hlylA probe, could be seen in these serotypes (Fig. 3 B).

The results indicate that the serotypes 2,4, 6, 7, 8 and 12,

which do not produce Hlyl (Frey and Nicolet, 1990; Frey

et al., 1992) contain part of the hlyl operon including the

promoter sequences, the genes hlyIB and hlylD and

aproximately 300-500 bp of the 3’ end of the hlylA gene,

but are lacking hlylC and most of hlyIA (Fig. 2). These

hybridization results and the comparison of the restric-

tion map of the hlyl operon (Gygi et al., 1992) with that

of the genes appB and appD (Chang et al., 1991) show

that the truncated hlyl operon in serotypes 2,4,6,7,8 and

12, which contains a hlylC-hlylA deletion (Fig. 2), is iden-

tical or very similar to the appB and appD gene segment.

Further evidence for our finding that appB and appD

53

TABLE I

Synonyms used for the hemolysin proteins and genes among different Actinobacillus pleuropneumoniae strains

Original designation Other names used

Name Abbreviation Name Abbreviation

toxin protein toxin protein

genes genes

Hemolysin I HlyI”’ Cytolysin I Cly1’5’

hlylC’=’ clylC’6’

hlylA’2J) clylA’6’

hlylB’=’ clylB’“’

hlyZP3’ clyLD’6’

Name

toxin

Abbreviation

protein

genes

.ppBb”’

,pplF)

Name

toxin

Abbreviation

protein

genes

Hemolysin II HlyII’”

hlyZIC”‘4’

hlyllA”‘4’

Cytolysin II ClyI1’5’

clyllC’6’

cly1’1A’~’

Hemolysin App”’ Cytolysin Cyt’9’

appC”’ cyu?

appA’7’ cytA@

The hemolysin of A. pleuropneumoniae were initially named hemolysin I”’ (HlyI), hemolysin II”’ (HlyII). Various groups have later on used different

designations for the proteins and for their genes which are compiled in this table.

a Names for hemolysin II genes in analogy to the protein HlyII.

b Initially believed to belong to the app determinant “) but later found to be hlylB and hlylDc3’ References: (1) Frey and Nicolet, 1988b; (2) Gygi et al., , 1990; (3) Gygi et al., 1992; (4) Frey et al., 1992; (5) Kamp et al., 1991; (6) Jansen et al., 1992; (7) Chang et al., 1989; (8) Chang et al., 1991; (9) Anderson

et al., 1991)

TABLE II

Hybridization of hlyl probes to genomic DNA of Actinobacillus pleuropneumoniae serotypes reference strains digested with PstI and EclXI

Serotype

Probe

1 2 3

Fragment size (kb)”

4 5a 5b 6 7 8 9 10 11 12

Upstream 9.2 6 1 6 20 20 6 6 6 9.2 9.2 9.2 6

hlyIC

hlylC 9.2 20 20 9.2 9.2 9.2 hlyZA N-term 9.2 20 20 - - 9.2 9.2 9.2

hlyIA C-term 9.2 6 6 20 20 6 6 6 9.2 9.2 9.2 6

hlyIB 9.2 6 6 20 20 6 6 6 9.2 9.2 9.2 6 hlylD 9.2 6 - 6 20 20 6 6 6 9.2 9.2 9.2 6

a Numbers indicate the band size in kb. -, indicates no hybridization signal was detected. Probes for Southern blots are described in Fig. 1 and the

conditions for hybridizations are given in Fig. 3 B. The A. pleuropneumoniae serotype reference strains used were: serotype 1, 4074; serotype 2, S1536;

serotype 3, S1421; serotype 4, M62, serotype 5a, K17; serotype 5b, L20; serotype 6, ferna; serotype 7, WF83; serotype 8, 405; serotype 9, CVI 13261;

serotype 10, 13039; serotype 11, 56153; serotype 12, 8329; as described previously (Frey et al., 1992). A. pleuropneumoniae strains were grown in

Columbia broth supplemented with 1% IsoVitaleX (BBL Microbiology Systems, Cockeysville, MD. USA) and 10 pg/ml NAD (Sigma Chemicals Co.,

USA) at 37°C with shaking (Frey et al., 1988a). Genomic DNA was isolated by the guanidinium thiocyanate method (Pitcher et al., 1989).

segment is identical to the hlyIB and hlyID genes of the

truncated hlyl operon is provided by the fact that the ‘hly

pseudo-gene’ upstream from appl? and appD (Chang

et al., 1991) corresponds to the 3’ end of the partially

deleted hlyIA gene. In serotype 3 the probe for the se-

quences upstream from hlylC hybridized to a 1-kb PstI-

EclXI fragment indicating that all genes of the hly1 op-

eron are deleted in this serotype (Fig. 2).

The above results indicate that the presence of the

hlyIA gene encoding the structural pre-HlyI protein can

be used as indicator to show whether a strain belongs to

the highly virulent HlyI producing group of A. pleuro-

pneumoniae. PCR amplification with total DNA of sero-

types 1-12 as template and oligo primers HLYIA-L and

HLYIA-R corresponding to the N-terminal and C-termi-

nal coding sequences of the hlyZA gene from serotype-1

strain 4074 (Frey et al., 1991) was used for the detection

of hlyZA. As demonstrated in Fig. 3 A, PCR amplification

produced a 3.1 -kb fragment in serotypes I,9 and I 1 corre-

sponding to the full length DNA of the hlyZA gene. Diges-

tion of this DNA with frequently cutting restriction

enzymes AluI, D&I, Dpd, Hid, HphI, MaeII and RsaI

showed identical restriction fragment profiles indicating

that these genes are highly similar or identical (results not

shown). However, no PCR amplification of a hlylA gene

could be obtained with the above named primers in sero-

types 5a, Sb and 10, even when the annealing temperature

was reduced stepwise from 54°C to 44°C. This was in

I I I I I I I I II I I, II I I III I \II II 1 I I

ml WA II hlylB II hiylD 1

upstream h/y/C

h/y/C

h/y/A

h/y/A N-terminal h/y/A C-terminal

h/v/D

Fig. 1. Location of the different probes for hly genes used for the hybridization experiments. The upper part represents a physical and genetic map

of the h/y1 operon as described (Gygi et al., 1992). The scale is given in kb. The DNA probes for the A. pleuropneumoniae hlyl genes shown in the

lower part were isolated from plasmid pJFF750, containing the full hIyICABD operon from serotype 1 strain 4074 (Gygi et al., 1992). The probe for

the regions upstream from the hlylC gene was a I.l-kb PstI-XhoI fragment. Probe hlylC was a 300-bp SspI-XhoI fragment, hlylB was a 2.1-kb BglII-

BglII fragment and MyID a 800-bp EcoRI-EcoRV fragment. Methods: The hlylA probe was generated by PCR amplification of the 3.1.kb coding

sequence of hlylA with the primers HLYIA-L (5’-TGGCTAACTCTCAGCTCG-3’) and HLYIA-R (5’-ATAGACTAACGGTCCGCC-3’). Cutting

the hlyIA PCR amplified fragment with the restriction enzyme EcoRV gave a 2.3-kb N-terminal hlyIA probe and a 800-bp C-terminal hly1A probe.

Probes for the hly11 genes (not shown in the figure) were produced by PCR amplification. Probe appA was a 2%kb fragment amplified from

chromosomal serotype 1 DNA with the primers APPSA-LT (5’-CCCATATGGATCCGTCAAAAATCACTTTGTCATCATT-3’) and APPSA-RT (5’-

TCCGGAATTCAAGCGGCTCTAGCTAATTGA-3’) corresponding to the beginning and end of the appA sequence (Chang et al., 1989). Probe appC was produced by first amplifying a 3.3-kb appCA fragment with the primers APPSC-LT (5’-CGCGGATCCGTTGCCTTGTTTTCCTTCAC-3’)

corresponding to the beginning of the appC gene (Chang et al., 1989) and oligo APPSA-RT, and subsequent isolation of a 500-bp appC fragment

obtained by digestion with &/I. PCR amplifications were performed in a thermal cycler (Perkin Elmer Cetus, Norwalk, Conn. USA) with the following

parameters: denaturation at 94°C for 1 min, annealing at 54°C for 1 min for the primers HLYIA-L and HLYIA-R or at 52°C for 1 min for the primers

APPSC-LT, APPSA-LT and APPSA-RT, elongation at 74°C for 2.5 min, total of 35 cycles, by using the protocol as described (Innis et al., 1990). The

oligos were produced by Microsynth, Windisch, Switzerland.

contrast to biochemical and serological evidence for the

production of a HlyI protein by serotypes 1, 5a, 5b, 9, 10

and 11 (Kamp et al., 1991; Frey et al., 1992) and to the

results of Southern hybridization using the hlyIA gene as

a probe (Fig. 3 B).

For further analysis of the hly1A genes in serotypes 1,

5a, 5b, 9, 10 and 11 we have cloned the hlyICA genes as

4%kb &I-EcoRI fragments from genomic DNA of these

strains onto vector pBluescriptIISK (Stratagene, La

Jolla, CA, USA). The clones from all these serotypes pro-

duced in E. coli XLl-blue strain (supE44, hsdR17, recA1, endA1, gyrA96, thi, relA1 A[proAB-LX], [F’proAB laclq

lacZAM15, TnZO (TcR)]) (Bullock et al., 1978) a narrow

hemolytic zone without induction of the vector borne lac

promoter. The 105-kDa HlyI protein could be identified

in these clones with monoclonal anti-HlyI antibodies on

immunoblots. These results showed that the recombinant

clones expressed the hlyZCA genes. No HlyI was detected

in culture supernatants of cultures from these clones re-

flecting the absence of the Hly secretory genes hlyZB and

hlyZD. The hlyIA genes from the clones were subcloned

as XhoI-EcoRI fragments onto vector pBluescriptIISK -.

Restriction analysis of the resulting plasmids using the

enzymes AluI, DdeI, DpnI, HinfI, HphI, Mae11 and RsaI

showed two different types of restriction fragment profiles

indicating two classes of genes (Fig. 3 C). The restriction

fragment patterns of the cloned genes from serotypes 1,

9 and 11 were identical (Fig. 3 C) and corresponded to

the DNA sequence as determined (Frey et al., 1991). The

patterns obtained from the clones of serotypes 5a, 5b and

10 were identical among each other but showed clear

differences to hlylA from serotypes 1, 9 and 11 (Fig. 2 C).

In this respect, it is important to note that the strains of

serotypes 1,9 and 11 are serologically closely related

(Mittal, 1990; Nicolet, 1988). The degree of divergence

between the hlylA genes from the group of serotypes 1,

9 and 11 compared to the group of serotypes 5a, 5b and

10 is estimated from the number of different restriction

fragments in the patterns obtained with the frequently

cutting restriction enzymes to be approximately 5%. This

explained the failure of PCR amplification of the hlyZA genes from the serotypes 5a, 5b and 10 with the probes

corresponding to the sequence of hly1A from serotype 1.

The HlyI proteins from both groups of serotypes seem to

be biochemically very similar. So far, no monoclonal anti-

bodies have been found which are able to distinguish

between HlyI from different serotypes. However, the

above described differences in the hlyIA genes might be

useful for taxonomic purposes and as epidemiological

markers.

55

0 1 2 3 4 5 5 7 8 9

I I I I I I I I I I

Serotypes

1

2

3

4

5a

5b

6

7

8

9

10

11

12

h/YlC h/y/A h/y/B h/y/D r-- ._ I :x- . I I I I P

.---._ I E E L ._

.._ .._ !

P ‘.,E‘. E I_ - . . _._- .

_-. L _.. - -.: -__

--G---l P L

7-- ----- h/y/B h/y/D

r I I I P E E I.

h/y/C h,ylA h/y/B hlylD I I I I

II ,

P x E E L

/lryrc h/ylA /IlylE hlylD

I I I I I P x E E I_

i Ii ~-__

h/y/B htyylD I I I P E E L

h/y/B h/y/D

I 3 II I

I

P E E L

1 II h/y/B hlyl0 I I 1 P E E I.

h/y/C My/A hly/B h/y/D

,‘-- 1

I I I x E E L

h/y/C h/y/A hlylt3 hly/D

I I I I I P x E E L

h/y/C h/y/A h/y/B i&ID I I I I I P x E E L

1 II h/y/B h/y/D I I I P E E L

Fig. 2. Genetic organisation of the hlyl operons. Open boxes represent the location of the various hlyl genes. Broken boxes represent the remaining

part of hlyIA in A. pleuropneumoniae serotypes 2, 4, 6, 7, 8 and 12. Broken lines show the possible deletion formations. P, PSI, E, EcoRI, L; EclXI.

The scale on the top is given in kb.

(b) Analysis of the hZyZZ operon tion on Southern blots using genomic DNA from the

The hlyII operon contains the genes appA encoding different serotypes digested with PstI and probes for the

pre-HlyII also named AppA protein (see Table I) and its appA and appC genes confirmed these results and re-

activator gene appC (Chang et al., 1989; Frey et al., 1992). vealed small differences upstream from appC and down-

Downstream of appA only a small segment resembling stream from appA (results not shown). Initially it was

the 5’ beginning of a hlyB-like protein was found (Chang believed that the genes appB and appD were the corre-

et al., 1991; Jansen et al., 1992). PCR amplification using sponding secretion genes belonging to this Hly determi-

total genomic DNA of each serotype as template and the nant (Chang et al., 1991). However, we have shown that

primers APPSC-LT and APPSA-RT, corresponding to appB and appD are indeed hlyIB and hlyID and belong

the beginning of the appC and the end of the appA gene to the HlyI operon as demonstrated in section a. This

(Chang et al., 1989), produced a 3.4-kb fragment in all explains the fact that these secretion genes were initially

serotypes except 10, showing the presence of the linked found to be unlinked to the structural and activator

appIICA genes in these serotypes. DNA:DNA hybridiza- genes. We have found no minor bands on the autoradio-

56

Hlyl Hlyll

M, 1 2 3 4 5a5b 6 7 8 9 1011 12

I ii 1 2 3 4 5a 5b 6 7 8 9 ICI II 12 1 Ii I 2 3 4 5a5b 6 7 8 9 10 Ii 12

M, 1 2 3 4 5a5b 6 7 8 9 1011 12

c

M, i 2 3 4 5a5b 6 7 8 9 1011 12

Fig. 3. Analysis of the structural genes encoding HlyI and HlyII. Left part: hlyI.4 genes encoding the structural protein, HlyI. Right part: app.4 genes encoding the structural protein HlyII. (Panel A) PCR amplified hfq’fA, and upp.4 genes respectively, from serotypes I-12 separated on 0.8% agarose

gels. M, is the molecular size standard, phage 1. DNA digested with Bind111 (23.1, 9,4,6.6,4,4,2.3,2.0,0.56 kb). (Panel B) Southern blot hybridizations

of P~~I~~c~RI-digested total DNA with probes hty1A N-te~inal (left) or appA (right). The open arrowheads give the position of the i. Hind111

markers 4.4 kb, 2.3 kb and 2.0 kb. Black arrowheads indicate the position of the 1.3-kb fragments in serotypes 2,4,6,7,8 and 12. The DNA fragments

were separated on 0.8% agarose gels, depurinated for 10 min in 0.2 M HCL, denatured for 60 min in 0.5 M NaGH/I.S M NaCl, neutralized for 30

min in 1 M TrisHCl pH 7.5/1.5 M NaCI, and transferred to nitrocellulose sheets by passive transfer as described (Ausubel et al., 1990). The DNA

probes were labelled with [a-3ZP]dCTP (3000 Ci/mmol, Amersham plc, UK) using random priming (Feinberg and Vogelstein, 1983). Hybridizations

were carried out for 25 h at 37°C in 5 x SSCjSO% formamide~S% polyethylene glycol 6000~0.5a~ SDS and 100 nglml denatured and sonicated salmon

sperm DNA. Filters were washed in 0.1 x SSC/O.l% SDS at 25°C corresponding to the melting temperature (T,) of DNA:DNA duplexes with

approximately 75% sequence identity. Autoradiography was carried out for 24 h on Fuji RX films using an intensifying screen. (Panel C) left:

Restriction enzyme profile of cloned hl,ylA genes from serotypes 1; 5a, 5b, 9, 10 and 11 digested with D&I. Growth of recombinant E. coli strains,

purificatjon of plasmids and DNA fragments from agarose gels, ligation and selection for recombinant clones are described (Ausubel et al., 1990: Sambrook et al., 1989). (C) right: Restriction enzyme profile of PCR amplified appA genes from the different serotypes digested with Hid. Mb is the molecular size standard, plasmid pBR322 digested with Hi& (1631. 517, 506, 396, 344, 298, 221/220, 154 and 75 bp).

graphs of the Southern hybridizations when hEylB and

I+lD probes were hybridized to chromosomal DNA of

each serotype at low stringency conditions, which pre-

viously allowed the detection of the hEylB gene of A. pleurqneumoniae with an E. coli hlyB probe (Gygi et al.,

1990). Since the genes encoding the HlyB secretion pro-

teins are generally the most conserved genes in the RTX

operons of various bacterial species, we expect that the

strains which we analyzed do not contain secretion genes

specific to the HlyII operon, analogue to h~~~~ and hEyfD.

These results agree with the findings that downstream

from the nppCA genes no genes for Hly secretion were

found (Chang et al., 1989; Smits et al., 1991). Secretion of

the the HlyIf protein seems therefore to occur by ~t”nns-

complementation with the help of the hE_ylB and hlylD gene products from the Hoyt operon. This view is sup-

57

by the fact that in serotypes 2, 4, 6, 7, 8 and 12,

which actively secrete HlyII (Frey and Nicolet, 1988b),

the truncated hlyl operon contains the whole hlyZB and

hlyID genes. Further evidence is given by the serotype 3

strain, which is devoid of hlyIB and hlyZD (Fig. 2), and

which releases only minute amounts of HlyII into the

growth medium (Frey and Nicolet, 1991).

In order to analyze whether the appA genes show heter-

ologies among the various serotypes we have amplified,

by PCR, the uppA genes from chromosomal DNA

(Fig. 3 C right). PCR amplification using oligo APPSA-

LT and APPSA-RT (see legend to Fig. 1) corresponding

to the beginning and end of the appA sequence (Chang

et al., 1989) produced a 2.8-kb fragment in all serotypes

except 10 (Fig. 3 A). Restriction fragment analysis of these

PCR fragments with AM, DdeI, DpnI, Hi&, HphI, Mae11

and RsaI showed no differences indicating that all appA genes in these strains are very similar. In contrast to re-

sults published by Jansen et al. (1992), our experiments

exclude a large insertion in the appA gene (corresponding

to hlyZ1A and to clyllA) in serotype 6. A small difference

among the appA genes was found with EcoRI in serotypes

6, 8, and 12 where appA contained an additional site.

Southern blot hybridization of PstI-EcoRI digested geno-

mic DNA from the different serotypes using the appA gene as a probe showed the internal 2-kb PstI fragment

which contains most of appA in all serotypes except 10

(Fig. 3 B). The second fragment seen on the blot in sero-

types 1, 2, 4, 5a, Sb, 7, 9 and 11 corresponds to a 3.5-kb

PstI fragment containing the N-terminal part of the appA gene and sequences upstream since the same fragment is

obtained with PstI-digested genomic DNA (data not

shown). In serotypes 6, 8 and 12 this fragment is lacking,

but a 500-bp fragment that hybridized with appA is pre-

sent due to the additional EcoRI site which is estimated

to be located about 200 bp downstream from the begin-

ning of uppA. Such EcoRI site could have been created

by a single G + A transition at nt 711 nt in the coding

sequence of appA (Chang et al., 1989) changing the aa

valine to the similar aa isoleucine which is expected not

to change significantly the structure or immunogenic epi-

topes of the HlyII protein. In serotype 3, a 5.5-kb frag-

ment is seen instead of the 3.5-kb fragment, indicating

that sequences outside the appA gene are different

(Fig. 3 B).

(c) Conclusions

(I) All serotypes of A. pleuropneumoniae contain two

different hly operons (hlyl and hly1Z), although frequently

truncated. Our data indicate that the two different Hly

operons did undergo rearrangements in A. pleuropneu- moniue by deletion formation.

(2) During evolution, the hlyll operon has probably

lost its genes encoding the secretion proteins B and D,

possibly due to high similarity to the hlylB and hlylD

genes.

(3) Subsequently, certain serotypes might have un-

dergone further deletions and lost the activator and struc-

tural genes hlyIC and hlyIA from the hlyl operon.

However, they retained the hlyZB and hlyID genes as well

as the segment containing the promoter of this operon

that allows the expression of hlyIB and hlylD. (4) Serotype 3 seems to have lost all genes of the hlyl

operon retaining only some sequences upstream the

hlyIC gene.

(5) One can therefore speculate that ancestors of A. pleuropneumoniae have contained two full hly operons

with all four genes (CABD).

ACKNOWLEDGMENTS

We are grateful to Han van den Bosch and Ruud

Segers, Boxmeer, Netherlands, for valuable help and

stimulating discussions. This work was supported by

grant 31l28401.90 from the Swiss National Science

Foundation.

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