Sequences homologous to the listeriolysin O gene region of Listeria monocytogenes are present in virulent and avirulent haemolytic species of the genus Listeria

Res. Microbiol. 1989, 140, 631-643

(~ INSTITUTPASTEUR/~LSEVIER Paris 1989

SEQUFI~,ICES HOMOLOGOUS TO THE LISTERIOLYSIN O GENE REGION OF LISTERL4 MONOCYTOGENES ARE PRESENT IN VIRULENT A N D AVIRULENT HAEMOLYI'IC SPECIES OF THE GENUS LISTERIA E. Gormley, $. Mengaud and P. Cossart (*)

Unitd de Gdnie Microbiologique, Institut Pasteur. 75724 Paris Cedex 15

SUMMARY

Various parts of the hlyA gene region of Listeria monoc'~ogenes which encodes a major virulence factor, listeriolysin O, have been used to detect the presence of homologous sequences in other species of the genus Listeria. Under low-stringency hybridization conditions, sequences homologous to the hlyA gene and its 5' adjacent regions were detected in the haemolytic and pathogenic species L. ivanovii, and in the haemolytic but non-pathogenic species L. seeligeri. In contrast, the region located downstream from hlyA appeared specific to L. monocytogenes. None of the probes spanning the region revealed homologies between L. monocytogenes and the non-pathogenic and non-haemolytic members of the genus, L. innocua, L. murrayi and L. welshimeri. Among various strains of L. monocytogenes tested, the gene hlyA and its 3' adjacent region appeared weU-conserved. In contrast, a restriction length polymorphism was detected in the region located upstream from hlyA with no obvious correlation with the haemolytic phenotype or the serovar of the strains tested. KEY-WORDS: Listeria monocytogenes, hlyA gene, Listeriolysin O, Virulence; SH-activated haemolysin, DNA probe.

Submitted December 6, 1989, accepted December 13, 1989, (*) Corresponding author.

632

E. G O R M L E Y E T AL. INTRODUCTION

Listeria monocytogenes is a Gram-positive organism responsible for infections in man and other animal species (Sc¢liger and J ~ e s , 1986; S¢¢liger, 1988). Its virulence is attributed to its ability to survive and multiply inside macrophages (Mackaness, 1962). All strains of L. monocytogen~ isolated from infections produce a hacmolysis on blood agar mediurn and are virulent in the mouse model (Hof, 1984; Rocourt et al., 1983). There is evidence that the secreted haemolysin, listeriolysin O, is a major virulence factor (for a review, see Cossart and Mengaud, 1989). Non-haemoiytic strains of L. monocytogenes are avirulent (Groves and Welshimer, 1977). In addition, insertion of a transposon into the chromosomal locus, hlyA, abolishes haemolysin production and virulence (GailIard et al.. 1986; Kathariou et al., 1987; Cossart et al., 1989), both of which can be restored by spontaneous loss of the transposon, or complementation of the mutants by the ¢¢ild type hlyA gene cloned on a plasmid (Cossart et al., 1989). The hlyA gene coding for listeriolysin O, as well as its adjacent regions, have been cloned from L. monocytogenes and sequenced (Vicente et al., 1985; Mengaud et al., 1987; 1988 ; 1989). In addition, listeriolysin O has been purified and shown to be a protein of 55.8 kDa whose optimal pH for activity is 5.5. It has been proposed that during phagocytosis of the bacterium, listedolysin O might damage the phagosomal membrane, facilitating the escape of the bacterium into the cytosol (Geoffroy et al., 1987; Gaillard et al., 1987). In the genus Listeria, which comprises six distinct species, only thre-, L. monocytogenes, L. ivanovii and L. seeligeri are haemolytic (S~e,liger and Jones, 1986). L. ivanoviiis pathogenic primarily for animals and L. seeh'gerf is non-pathogenic. This raises the q:,'estion of the relationship between the genes coding for the haemolytic factor in these different species and the possibiUty that the non-haemolytic species might have silent copies of the hlyA gene. To determine whether the Listeria monocytogenes hlyA gene or related sequences were present in other members of the genus Listeria, chromosomal DNA was isolated from the six species of Listeri~ an:t hybridized under conditions of low stringency with DNA probes prepared from cloned L. monocytogenes chromosomal fragments, internal and adjacent to the hlyA gene. Sequences homologous to hlyA were detected only in haemolytic species.

ORF

=

open reading frame.

hlyA G E N E H O M O L O G Y

STUDIES IN LISTERIA

633

MATERIALS AND METHODS B a c t e r i a strains and m e d i a .

Lister~a strains used in this study are listed in table 1. Listeria were routinely grown in brain-heart infusion broth (BHI, Difco) at 37°C; 5 °Tosheep blood BHI agar plates were used to detect haemolytic activity. C h e m i c a l s and e n z y m e s .

Restriction enzymes were purchased from Amersham, Bochringer or C~nof~! ap.d u~ed a~ ~r~¢:ib~ b7 the manufa~urer. ~32p-dCTP (3000 Ci/mmol¢~ was p u r c h a s e d from Amersbam.

TABLE I. - - Strains tested in D N A hybridization studies. Strain

Serovar

Haemolytic phenotype

Origin or references

L, monoeytogenes LO28 EGD (*) ATCC43251 CLIP11289 LM32 LM44 LM37 CIP82110T ATCC43250 CLIP11~88

i/2c 1/2a l/2a I/2a l/2b 4a 4b 1/2a 1/2a 1/2a

q+ +

Ramon y Cajal collection Gaillard et al., 1986 Pine et al., 1987 E. Espaze I . e . Piffaretti

-

Pine et aL, 1987

-

E. Espaze

L. ivanovii CIP7842 T

5

+

J. Rocourt

L. seeh'ged 8LCC3503

l/2a

+

L. innocua CIP8011T

6a

L. welshimeri CIPSI49T

6a

+ +

+

77

L. murrayi CIP76124 T ATCC---American "IXype Culture Collection, Rockville, MD; C(L)lP=Collcction (Listeria) de rInstitut Pasteur; SLCC = Special Listeria Culture Collection, H.P.R. Seoliger, University of WtlrzbuTg, FRG, and lnstitut Pan,cur; LM = J.C, Piffaretti C;olleotion,Is0tuto Cantanale Battcriologico, 6904 Lugano, SwRzerhnd. (*) Trudeau Institute. Type strain is indicated by raised T.

634

E. GORMLEY E T AL.

DNA techniques. Listeria chromosomal DNA was prepared by a method described previously (Mengaud et al., 1988). For the preparation and purification of DNA probes, p!asmid DNA was purified by ultracentrifugation in caesium chloride gradients (Mantatis et al., 1982). After digestion with appropriate restriction enzymes, the DNA was dectrophore~ed on thin polyacrylamide (0.35 ram) gels. Generally, 100 [tg of plasmid digest were layered in a 10-em well. Electrophoresis was carried out m Tris-bor~te buffer (9× 10-2 M Tris base, 2× 10-3 M EDTA pH 8.4, 9× 10-2 M H3BO3). DNA fragments were purified by electroelutiolt from gel slices in dialysis bags or diffusion into elution buffer (0.3 M sodium acetate, 1 mM EDTA, 0.1% SDS, pFI 7.5) as previously described (Mengaud et al., 1988). Southern blot hybridizations. For DNA hybridizations, purified fragments were labelled by the multiprime labelling system (Kit RPN. 160 1, Amersham). Listeria DNA was digested by HindllI or EcoRI, and elec~ophoresis was carried out in Tris-borate buffer in horizontal 0.7 % agarose gels for 18 h at 25V. DNA was transferred from the agarose ~o "Hybond-N" nylon membranes (Amersham). For low-stringency hybridization, pre-hyobridization (1 h) and hybridization with labelled probes (16 h) were carried out at 37 C in (35 070 formaraide, 5 × SSPE, l0 × Denhardt) buffer containing 200 ttg/ml sonicated salmon' DNA, in sealed plastic bags. The fiIters were then washed twice in 5 × SSC, 0.1 070 SDS for 1 h and exposed overnight or longer with an intensifying screen at - 80°C. For high-stringency hybridization, the buffer contained 50 % formamide, and prehybridization and hybridizations were carried out at 42°C. The filters were then washed twice in 1 × SSC, 0.1 070SDS, fox 30 rain at room temperature and exposed to film at - 80°C. To remove bound probe, the filters were washed in 0.4 M NaOH for 45 rain at 42°C and rinsed in 0.1 ×SSC, 0.1 eta SDS, 0.2 M Tris pH 7.5, for 1 h at 42°C. The filters were then exposed to film overnight to check efficiency of removal.

RESULTS Sequences homologous to the L. monocytogenes hlyA gene and its 5' adjacent region are present in L. ivanovii and L. seeligeri, but not in the other species. Chromosomal D N A prepared from L. monocytogenes (LO28), L. ivanovii (CIP7842x), L. seeligeri (SLCC3503), L. innocua (CIP8011T), L. murrayi (CIP76124 w) and L. welshimeri (CIP8149 T) were digested with HindllI and used in Southern blot hybridization experiments (table I). The D N A probes used for hybridization originated from L. monocytoge~ies LO28 (serovar l/2c). As shown in figure IA, probes ! and 2 are HindIII fragments of 0.7 kb and 9.4 kb, respectively, corresponding to an internal fragment and to the 3' end o f the hlyA gene. Probe 3 and probe 4 are 0.4-kb HindIII and 0.5-kb Sau961-BamHI fragments which correspond to regions downstream from hlyA. Probe 5 is a 0.8-kb HindIII-Hpal fragment located immediately upstream from hlyA. Probe 6 and probe 7 are 0.5-kb EcoRI-HindIiI and 0.9-kb HindIII fragments respectively, which are located upstream o f probe 5.

hlyA G E N E H O M O L O G Y

S T U D I E S I N LISTERIA

635

A L,

monoeylogenes

UO~$

" - ' ~ r i' E

'

XI

0.9

3.5

--

OaF U" : n? 1.$

' E

l .g

I

M~A orv 'a .I 0 . 7 x 0. ~,o.~ ,.+_/~.. ' E Pr~nhe I Probe

Probe :$ Probe 6 Probe "/

2

Probe

3 Probe

L

t~anouJi

H I

C~t~Tg42T L,

0+9

seeli~erJ H

23

SLfCJ$O3

H i

H 0.75 ,

HI

4

H

1.75 ,

I

II I

2.7

B [,. monoeytoge~es EGD

CL1PllZIl$ CLIPIt2D LM:I2

LM3?

Ii i

H g'~l H

~

S E

H l

I~

2~5

2~$$

3.b

1

~,

....

i1~

It ,0"75 I E

t~.~ 0.75 H ~ 0,8

H ClPg2tlFt" ~TC¢,3150

[+S

!

.$

tlo 0 . 7

" ~ ' ~ ) E

2:6 ~

iii

2.251.8

1,8~|'$

~-

H H H 10.410.4t

1.3~+,1

H

E

H

E LM44

0.9 '

.,~*:~

,, 0 . , .0.+,

H

.~,._.

A tt

! E

II

iH 0.,? +0.4,0.4j

|,$

, ': 0+ ?0,r°,,

t,O

E

1+$ I.~

i..7

H

H

H

H

1.3

., ,/__" H

' | 0,? I fl'41 0"41 E

1.3/~_

El

FIG. 1. -- Restriction map of the hlyA locus. A) Restriction map of hlyA gene and adjacent regions of L. monocytogenes, L. ivanovii and L seeligeri. H =HindlIl, E =EcoRI. Numbers refer to size of DNA fragments (kb). probe 1 : 0.7-kb HindIIl fragment. Probe 2: 0.4-kb HindlI1 fragment. Probe 3: 0.4-kb Hindlll fragment. Probe 4: 0.5-kb Sau961-BamHI fragment, probe 5: 0.8-kb HindlIl-t/pal fragment. Probe 6: 0.5-kb EcoRI-HindIII fragment. Probe 7:0.9 kb H/ndIII fragment. 13) H/DDIII CA)and EcoRI OE)restriction map of the hlyA locus in different L. monocy2ogenes strains.

Under conditions o f low stringency, probe I hybridized with the corresponding 0.7-kb fragment in L. monocytogenes LO28 (fig. 2A). In L. ivanovii and L. seeligeri, hybridization was observed with a 1.75-kb and a 2.7-kb fragment, respectively, demonstrating that sequences h o m o l o g o u s to hlyA were present in these species. N o signal w a s detected in the Other species. After washing, the filter was rehybridized with probe 1 under conditions o f high stringency, and hybridization was only observed with L. monocytogenes,

636

E. G O R M L E Y E T A L .

A 1 2 3 4 5 6 7 8

B 1 2 3 4 5 6 7 8

23 9,4 6.5 4.3

2.3

6

o

0.5

FIG. 2. -- Hybridization of probe I to chromosomal DNA of Listeria sp. digested with Hindlll under conditions of low (A) and high stringency (B). Lane I: MW size standards; 2: L. monocytogenes (LO2_8); 3: L. ivanovii (CIP7842T); 4: L. seeligeri (SLCC3503)_; 5: L. innoc~a (cIPg011~); 6: L. murrayi (CIP76124T), 7: L. welshimeri (CIP~I49'); 8: probe 1 (0.7 kb).

demonstrating the* the probe was species-specific under high-strix,gency conditions, in agreement with our previous results 0Vlengaud et al., 1988) (fig. 2[3). Probe 2, which confabs the 3' end of hlyA, hybridized under low stringency with the corresponding 0.4-kb fragment in L. monocytogenes and, as with probe !, with a 1.75-kb fragment in L. ivanovii and with a 2.7 kb-fragment in L. seeligeri (fig. 3A). No homologous sequences were detected in the nonhaemolytic species. Thus, DNA sequences homologous to hlyA of L. monocytogenes were detected only in the haemolytic species of the genus. However, divergence occurred between these species, as shown by changes in the positions of HindIII sites, and in the strengh of hybridization signals. Probe 5, located immediately 5' to hlyA, hybridized with a 1.5-kb HindIII fragment in L. monocytogenes and with a 0.75-kb fragment in L. ivanovii, revealing the presence of a new HindIII site in this species (fig, 4A). In L. seeligeri, the 2.7-kb fragment, previously detected with probes 1 and 2, was again detected. Hybridization with probe 6 revealed a 0.9-kb band both in L. monocytogenes and L. ivanovii (fig. 4B). In L. seeligeri, a 2.3-kb fragment was detected. No homologous sequences were observed in the other species with either probe 5 or probe 6.

hlyA GX,~:E HOMOLOGY STUDIES IN LISTERIA A 1

2

3

637

B 4

5

6

1

2

3

4

5

6

9.4 6.5 4.3

2.3 o

0.5

O

JO

FIG. 3. - - Hybridization o f probes 2 (A) and 3 (B) to chromosomal DNA o f Listeria sp. digested with Hindlll under conditior, s o f low stringency. Lane I : L. monocytogenes ~LO28); 2: L. lean,vii (CIP7842T); 3: L. seeligeri (SLCC3503); 4: L. innocua (CIPS011'); 5: L. murrayi (CIP76124t); 6: L. welshimeri (CIP8149t).

Thus, sequences homologous to the region located upstream from hlyA were detectable in L. ivanovii and in L. seeligeri, but not in the non-haemolytic species. The strength of the signals obtained with L. ivanovii and L. seeligeri was weaker with upstream probes than with the probes containing hlyA sequences. Although there are no experimental data to strictly demonstrate [hat the HindIII fragt~ents detected in L. i~anovii or L. seeligeri were adjacent, their sizes suggest that the HindIII restriction maps of the hlyA loci of L. ivanovii and L. seeligeri were as presented in figure 1A. The region located downstream from hlyA is specific for L. monocytogenes. Probe 3, which covers sequences immediately downstream from hlyA, detected the corresponding fragment of 0.4 kb in L. monocytogenes (fig. 3B). In contrast to the results obtained with *~heprobes containing the hlyA gene sequences or upstream sequences, no homologous sequences were detected

638

E. G O R ~ L E Y

ET AL.

A 1

5.1

2

S

3.456

1

2 3 4

!~i~i: ~ i : i

.

5

6

~

3.5 •

.

.

.

1.9 1.3 0.8

e

,b.

q

0.5

FIG. 4. -- Hybridization o[flrobes 5 (,4) and 6 (B) to chromosomal DATA of Listeria sp.

digested with Hindlll under conditions of low st.~.ngency. Lane I : L. monocytogenes ~LO28);2: L. ivanovff (CIP7_842T);3: L. seeligeri (SLCC35_03); 4: L. ~nnocua (CIF8011J); 5: L. murreyi (CIP76124~); 6: L. welshimeri (CIP8149~).

in L. ivanovii and L. seeligeri, or in the other species, even in low stringency

conditions. With probe 4, which contgns sequences downstream from probe 3, a 1.3-kb fragment was detected in L. monocytogenes, and as with probe 3, no homology was detected in any of the other species under low stringency conditions (data not shown). Thus, the region lying downstream from hlyA appeared specific to L. monocytogenes. Idel~lifieation of a polymorphic locus upstream of hlyA ~ L . m o n o e ~ o g ~ e s . Since it is known that in vitro haemolytic activity production varies considerably between different strains of L. monocytogenes (Geoffroy et ai., 1989), we were interested in determining whether these differences might be correlated with changes in restriction patterns either within, or in regions ad-. jaccnt to, hlyA. A series of L. monocytogenes strains chosen on the basis of their different serotypes or haemolytie phenotype were analysed. Strains LO28 (serovar 1/2c), EGD (serovar 1/2a), ATCC43251 (serovar 1/2a), CLIPlI289 (serovar l/2a), LM32 (serovar 1/2b), LM44 (serovax 4a) and LM37 (serovar 41)) are haemolytic and virulent. Strains CIP821 l0 T (serovar 1/2a),

hlyA GENE HOMOLOGY STUDIES I N LISTERIA

639

CLIPl1288 (serovar l/2a) and ATCC43250 (serovar l/2a) are 3 nonhaemolytic, avirulent strains (table I). Chromosoma! DNA digested with EcoRl and HindIIl were initially probed with probe 7. This probe is longer than probe 6, and was therefore more appropriate for this study. With EcoRl digests, different hybridization patterns were observed. Two fragments of 3.5 kb and 1.8 kb were observed in haemolytic strains LO28, EGD, ATCCA3251, CLIP11289, LM32 and LM37 and also m the non-~emolytie strain CLIP11288. In the non-haemolytic strains CIP82110 T and ATCCA3250, the 3.5-kb fragment was replaced by a fragment of 3.1 kb, indicating a 0.4-kb deletion or a new EcoRI site. In LM44, the 1.8-kb fragment was replaced by a fragment of 0.8 kb, again suggesting either a deletion or a new EeoRI site. A restriction map constructed from the Southern blot analysis is presented in figure tB. With HindIll digests, using the same probe (probe 7}, a single fragment of 0.9 kb was observed with haemolytic strains LO28, EGD, ATCCA3251, CLIPII289 and the non-haemolytic strain CLIPl~288. In the other nonhaemolytic strains CIP82110 x and ATCCA3250, the 0.9-kb fragment was absent and was replaced by a 2.6-kb fragment. This was consistent with the EcoRI restriction pattern and indicated a 400-bp deletion which removed a HindIIl site, resulting in fusion of adjacent HindIII fragments. In LM32, LM37 and LM44, two fragments were detected in each strain, 2.5 kb and 2.25 kb for LM32, 2.5 kb and 0.75 kb for LM37 and 0.75 kb and 0.25 kb for LM44. These results indicated differences in the positions of the HindIII sites in the region covered by the probe (fig. IB). It therefore appeared that the region covered by this probe had diverged between the different grains tested, but no particular restriction pattern was strictly associated with either a specific serovar or a haemolytic phenotype. The different L. monocytogenes DNA were then rehybridized with probe 5, which is adjacent to p.~obe 7 and covers the region immediately upstream, from hlyA. In the EcoRI digests, an identical 1.8-kb EcoRl fragment was detected in all strains except LM44, where two fragments of 1.0 kb and 0.8 kb were detected, identifying a new EcoRI site in this strain (fig. 113). In the case of the HindIII digests, all patterns were identical except that of LM32, where a fragment of 2.25 kb in place of a 1.5-kb fragment was detected due to loss of a ttindIII site. Thus, it appeared that this region was much less polymorphic than the adjacent upstream sequences. With probe 1, intern~ to hlyA, an identical 0.7-kb fragment was detected in all L. monocytogenes strains tested, in agreement with our previous results (Mengaud et al., 1988). Using probe 3, which covers a region located immediately downstream from hlyA, a 0.4-kb HindIII fragment was found in all strains with the exception of LM32 where a 1.7-kb fragment wa~ detected. Finally, using probe 4, which covers a region further downstream from hlyA, a 1.3-kb HindIIl fragment was found in all strains except LM32, where the 1.7-kb fragment was again detected. With probe 4, EcoRI restriction patterns were identical in all L. monoc),togenes strains tested.

640

E. GORMLEY E T AL.

All these results (fig. 1B) showed that the region located upstream from hlyA ~.nL. monocytogenes was polymorphic, as indicated by loss or addition of restriction sites and even, in some strains, a deletion event. In the latter cases, which corresponded to two non-haemolytic strains, a strict correlation between the non-haemolytic phenotype and the deletion was not established. In contrast, hlyA and its adjacent downstream region appeared strongly conserved.

DISCUSSION

In previous experiments, performed under conditions of high stringency, hlyA was detected in all L. monocytogenes strains tested, even in the nonhaemo|ytic type strain, but was not detected in the other species (Mengaud et al., 1988). In this study, by using lower stringency conditions, sequences homologous to the hlyA gene of L. monocytogenes and its upstream adjacent region were detected in the haemolytic species L. ivanovii and L. seeligeri, but not in the non-haemolytic species L. innocua, L. welshimeri and L. murrayi. The use of various gent probes allowed us to show that nucleotide sequence divergence occnlrred among the haemolytic species of the genus Listeria. Probes containing parts of the ~lyA gene hybridized with L. ivanovii and L. seeligeri to fragments of sizes different from those of L, monocytogenes, thus indicating different restriction site positions; this was in contrast with a high conservation of restriction sites in L. monocytogenes strains tested. In addition, the strength of the signal produced in L. ivanovii and L. seeligeri was weaker than that obtained with L. monocytogenes, but varied with the probe used: the strongest signals were obtained with probe 2, which covers the 3' end of the gvne. This region encodes the C.terminal part of the protein which contains a peptide of 11 amino acids, strictly conserved among SH-activated cytolysins and thought to be essential for activity (Alouf and •Geoffrey, 1984; Kehoe et al., 1987; Twcten, 1988). Thus, there might be a stronger selection for conservation of DNA sequence in this regicm. Hybridization with probes corresponding to regions located upstream from hlyA revealed that homologous sequences were present in L. ivanovii and L. seeligeri. However, on the basis of different restriction site positions and strength of hybridization signals, it appears that these sequences have also diverged. For L. monocytogenes, the rc~ion immediatelyupstream from hlyA, detected by probe 5, appeared well conserved in all of the strains tested, with a single restriction site change observed in only two strains, LM44 and LM32. Interestingly, an open reading frame (ORF-U) has been identified in this region, but its function has not been determined (Mengaud et aL, 1989). The region located upstream from ORF-U was found to be polymorphic in L. monocytogenes, as shown by changes in the restriction patterns among the haemolytic strains and a deletion crept in two of the three non-haemolytic strains. However, this polymorphism could not be correlated with a specific serovar or haemolytic phcnotypc.

hlyA GENE HOMOLOGY STUDIES IN LISTERIA

641

No homology was detected between the region located downstream from the hlyA gene in L. monocytogenes and the correspor~ding regions in L. ivanovii and L. seeligeri. It is possible that the DNA sequences have diverged in such a way that relatedness cannot be detected even underlow-stringency hybridization conditions. Alternatively, one can speculate that the region is absent in these species. The presence in this region of an open reading frame (ORF-D) (Mengaud et al., 1989) and of only two types of restriction patterns among the L. monocytogenes strains tested suggests that this downstream region may be functional and play a role in the specific properties of L. monocytogenes. The DNA-DNA hybridization results presented here are in agreement with the results of Geoffroy et al. (1989) who, using a sensitive immunoassay detected cross-reactivity between the haemolysins of L. monocytoge,ws, L. ivanovii and L. seefigeri, and demonstrated that all L. monocytogenes strains, even the phenotypicaUy non-haemolytic type strain, produced listeriolysin O, albeit at very different levels. Our study is also in agreement with the work of Leimeister-Wachter and Chakraborty (1989) who detected sequences homologous to the L. monocytogenes hlyA ,~-eneregion in the other haemolytic species. Their study involved different sets of probes and strains. They reported that polymorphism in the hlyA gene region could be correlated with specific serovars. However, this conclusion was reached from a study of only three different serovars. In the present study, five serovars were tested, and in some cases (serovars l/2a and 1/2c), the restriction: digest profiles were identical. In addition, our set of probes demonstrated that the region specific to L. monocytogenes begins immediately ~ownstream from hiyA. Finally, Leimeister-Wachter and Chakraborty (1989) found that homology between L. monocytogenes and L. ivanovii or L. seeligeri could be detected under highstringency conditions. In our work, as already published (Mengaud et al., 198~), under high-stringency conditions of hybridization, probe I detects only the hlyA gene of L. monocytogenes. Nevertheless, all of these data clearly establish that haemolysins and haemolysin genes of the different Listeria spe~es are related and emphasize that other virulence factors are necessary for the pathogenicity of L. monocytogenes. RI~SUMI~ PRI~SENCE DE S]~QUENCES HOMOLOGUES ~t LA R~-GION DU GI~NE DE LA LIST~RIOLYSINE O DE LISTERIA MONOCFTOGENESCHEZ DES ESP~CES H~MOLYTIQUES VIRULENTES El" AVIRULENTES DU GENRE I.ISTERIA

Diff6rents fragments du locus hlyA codant pour la iist~riolysineO, un f~cteur essentiel/tla virulencede Listeria monocytogenes, om ~ ntilis6scomme sonde dans des exp£'riencesd'hybfidation de type <
642

E. GORMLEY ET AL.

l'csp~c¢ h~molytique et avirulente L. seeligeri. Par contre, la r~gion situ6e en aval de hlyA est Sl~Cifique de L. monocytogenes. Aucune des sondes utilis~es n'a r~v*l~ d'homologies entre L. monocytogenes et les esp~ccs non h~molytiques et non pathog~nes relies L. innocua, L. murrayi ct L. welshimeri. De plus, parmi les diff6rentes souches de L. monocytogenes test~es, le g~ne hlyA et la r~gion adjacente situ~e en aval de ce # n e se sont av, r~es conserv~es. Par contre, dam la r6gion situ~e en amont de hlyA, un polymorphisme des sites de restriction a ~t* d~tect6, sans correlation ~,idente avec le ph~notype h~molytique ou Iv s~rovar des diff~rentes souches test~s. MOTS-CL~S: Listeria monocytogenes, G~ne hly..~, Listeriolysine O, Virulence; H6molysine SH-d6pendante, Sondes ADN.

ACKNOWLEDGEMENTS We thank J.E. Davies for constant support, E. Espaze, J.C. Piffaretti and J. Rocourt for their generous gifts of strains, and K. Reich for critical reading of the manuscript. This work was supported by a "Contrat de Jumelage" from the EEC (STJ20319-C) and a "Contrat de Recherche Externe" (eRE 891003) from INSERM. E. G. is a recipient of an EEC Biotechnology Action Progrmn fellowship. J. M. was a recipient of a short-term "Acad~mie d'Agriculture de France" fellowship.

REFERENCES ALOUF, J.E. & GEOFFREY,C. (1984), Structure-activity relationships in sulfhydrylactivated toxins, in "Bacterial protein toxins" (J.E. Alouf, F.J. Fehrenbach, LH. Freer & J. Jeljaszewicz) (pp. 165-171). Academic Press, London, New ~rork. COSSART. P. & M~N~AUD. J. (1989L Listeria monocyto~enes: a model for the molecular study of intracellular parasitism. MoL Biol. Meal. (in press). CosseT, P., VICENT~,M.F., ME~AUD, J., BAQUERO,F., P~d~z-Dmz, J.C. & BERCHE, P. (1989), Listeriolysin O is essential for the virulence of Listeria raonocytogenes: direct evidence by genetic complementation. Infect. Immun., 57, 3629-3636. GAtLL~a~n, J.L., BFRCla~, P. & SANSO~Tt, P. 0956), Transposon mutagenesis as a tool to study the ro~e of hemolysin in the virulence of Listeria monocytogenes. Infect. lmmun., 52, 50-55. GAILLARD,J.L., BERCHE,P., Motmle_~, P.J., RICHARD,S. & SANSONETTt,P. (1987), In vitro model of penetration and intracell~flar growth of L. monocytogenes in the human ~mterocytvfike cell fine Caco-2. Infect. Immun., 55, 2822-2829. (~EOFFROY, C., GAILLARD,J.L., ALOUV, J.E. & BERCI-Ie, P. (1987), Purification, characterization, and toxiei,y of the sulfhydryl-activated hemolysin listeriolysin O from Listeria monoeytogenes. Infect. Iramun.. 55, 1641-1646. GEo~oY, C., GAtLLmU~,J.L., ALOUF, J.E. & BERCnE, P. (1989), Production of. thiol-dependent hemolysins by Listeria monocytoge~es and related species. J. gen. MicrobioL, 135, 481-487. GnovEs, R.D. & WBLSmMBR, H.J. (1977), Separation of pathogenic from apathogenic LL~teria monocytogenes by three in vitro reactions, j. clin. MicrobioL, 5, 539-563. HoF, H. (1984), Virulence of different strains of Listeria monocytogenes serovar |/2a. Med. Microbiol. Immunol., 173, 207-218.

hlyA G E N E H O M O L O G Y STUDIES I N LISTERIA

643

KATHAR]OU,S., MBTZ,P., Her, H. & GORBEL,W. (1987), Tn916-induced mutations in the hemolysin determinant affecting virulence of Listeria monocytogene$. J. Bact., 169, 129-137. IOmoE, M.A., M[LL~n, J.A., WALKEX,J.A. & BOULNOIS,G.J. (1987), Nucleotide sequence of the strcptolysin O ~SLO) gene: structural homologies between SLO and other membrane-damaging, thiol-activated toxins. Infect. lmmun., $5, 3228-3232. LEKMEISTEX-WACHT~R,M. & CH~XAUORTY,T. (1989), Detection of listeriolysin, the thiol-dependent hemolysin in Listeria monocytosen~, Listeria ivanovii, and Listeria seeligeri. Infect. Immun., 57, 2350-2357. MAc~, G.B. (1962), Cellular resistance to infection. J. exp. Med., 116, 381-406. M~L~TIS, T., FRITSCH, E.F. & SAMSnOOK, J. (1982), Molecular cloning - - A laboratory manu~!. Cold Spring Harbor Laboratory, New York. M~NGAUD,J,, CH~NEVERT,J., GEOF~'RO~,C., GAILLARD,J.L. & COSSART,P, (1987), Identification of the structu_-al't,,"gone encoding the SH-activated hemolysin of Listeria monocytogenez: listeriolysin O is homologous to streptolysin O and pneumolysin. Infect. Immun., $S, 3225-3227. ME~C~UD, J., VICEN~, M.F., CH~N~V~T, J., MONIZP~1P.A, J., G E e z e r , C., GICQUeL-S~zEY, B., BAQUFRO,F., PF~Z-DL~Z, J.C. & COSSET, P. (1988), Expression in E. coil and sequence analysis of the listeriolysin O determinant of L~steria monocytogenes. Infect. lmmun., 56, 766-772. Me~c~uD, J., Vxc~wr~, M.F. & COSSAltT, P. (1989), Transcriptional mapping and nucleotid© sequence of the Listeria monocytogenes hlYA regmn reveal structural features that may be involved in regulation. Infect. =rmmun., 57, 3695-3701. PINE, L., WEAVER,R.E., CAXLON~,G.M., Pl~mA, P.A., ROCOUgT,J., GO,EL, W., KATHAXIOU, S., BIss, W.F. & MALCOLM, G.B. (1987), Listeria monocytogenes ATCC35152 and NCTC 7973 contain a nonhemolytic, nonvirulent varient. J. ciin. Microbiol., 25, 2247-2251. ROCOURT,J., ALOe~So,J.M. & S~L[G~, H.P.R. (1983), Virulence compar~e des cinq groupes g~nomiques de Listeria monocytogenes (sensu late). Ann. Microbiol. (Inst. Pasteur), 1MA, 359-364. SI~I~LI~n, H.P.R. & J o ~ s , D. (1986), Genus Listeria P|rie i940, in "Bergey's manual of systematic bacteriology" (P.H.A. Sneath, N.S. Malt, N.E. Sharpe & J.G. Holt, ed.) eel. 2 (pp. 1235-1245). The Williams & Wilkins Co., Baltimore. S~U~eR, H.P.R. (1988), Listeriosis -- history and actual developments. Infection, TW~TE~,~.~I~'."~'l~88~,~tucleotidesequence of the gone for perfringolysin O (thetatoxin) from Clostridium perfringens: significant homology with the genes for streptolysin O and pneumolysin. Infect. Immun., $6, 3235.3240. V]c~rr~, M.F., BAQUI~RO,F. & PI~REzoDIAZ,J.C. (1985), Cloning and expression of the Listeria monocytogenes haemolys~n in E. coil FEMS Microbiol. Letters, 30, 7%79.