Molecular comparison of virulence plasmids in Shigella and enteroinvasive Escherichia coli

Ann. Microbiol. (lnst. Pasteur) 1983, 134 A, 295-318

MOLECULAR

COMPARISON IN

AND

OF V I R U L E N C E

PLASMIDS

SHIGELLA

ENTEROINVASIVE

ESCHERICHIA

COLI

by P. J. Sansonetti, H. d'HauteviUe, C. ]~cobichon and C. Pourcel (*)

Service des Enl~robacl~ries, Unit~ I N S E R M 199, el (*) Unil~ Recombinaison el Expression gdn~lique, Inslilut Pasteur, 75724 Paris Cedex 15

SUMMARY Virulent isolates of ShigeIla dysenteriae and Shigella boydii harboured a 140 Mdal plasmid which was either absent or deleted in spontaneously avirulent strains. Together with previous data concerning S. sonnei, S. flexneri and enteroinvasive Escherichia coli, the present results established the general role of extrachromosomal elements in the virulence of such enteroinvasive species. Among different species, these virulence plasmids showed unrelated endonuclease cleavage patterns, whereas hybridization experiments showed that homologous sequences were present throughout the molecules. These plasmids may therefore have derived from a common ancestor molecule which overcame evolutionary alterations in restriction sites. Furthermore, intraspecies and intraserotype comparison of these plasmids by endonuclease cleavage demonstrated highly conserved sequences. The consequences of these data for evolution, epidemiology and diagnosis of Shigella and enteroinvasive E. coli are discussed. KEV-WOnDS: Shigella, Escherichia coli, Plasmid, Enteroinvasiveness, Virulence; Restriction, Hybridization.

INTRODUCTION

Shigella species, as well as some serotypes of Escherichia coli, cause a dysenteric syndrome through invasion of the human colonic mucosa. S. sonnei accounts for most shigellosis in industrialized countries, whereas Manuscrit resu le 7 janvier 1983, accept6 le 28 f6vrier 1983.

296

P. J. SANSONETTI AND COLL.

S. dgsenleriae I (Shiga bacillus) and S. flexneri are more prevalent in developing countries. Cases due to S. bogdii and enteroinvasive E. coli are sporadic. We have recently demonstrated that non-self-conjugative 120 to 140 Mdal plasmids are necessary for the expression of virulence in S. sonnei [5], S. flexneri [6], and enteroinvasive E. coli [4]. With the exception of S. sonnei, in which this virulence plasmid is also involved in the expression of the serotype specific form I antigen, S. flexneri and E. coli virulence plasmids do not encode alterations in antigenic specificities and are functionally interchangeable within and between both of these species. Although specific functions have yet to be ascribed to these plasmids, they appear to be necessary for the critical step of host cell penetration. Studies of molecular expression in minicells have shown that they encode outer membrane proteins which might interact with host cell surface receptors, thus triggering endocytosis of the bacterium (Hale, Sansonetti et at., accepted for publication). Despite similarities in molecular size and functional expression, no information has been available concerning molecular relatedness among these virulence plasmids. In addition, their participation in the virulence of all ShigelIa species remains speculative. The present work demonstrates that with no known exception, virulent isolates belonging to S. dgsenteriae and S. bogdii species harbour a 140 Mdal plasmid which is either absent or deleted in avirulent isolates. It also shows that despite species- and even serotype-dependent variations in endonuclease restriction patterns, plasmids obtained from the enteroinvasive isolates of Shigella and E. coli share a considerable homology.

MATERIALS AND METHODS

Bacterial strains. These are listed in table I.

Media and cultural conditions. Bacteria were routinely grown at 37~ C in tryptiease soy broth (TSB), or on trypticase soy (TSA) and Salmonella-Shigella (SS) agar (Institut Pasteur Production).

Serological tests. All strains were verified by slide agglutination in the presence of Shigella and E. coli type-specific rabbit antiserum (Institut Pasteur Production).

SDS = sodium dodecyl sulphate. SS = Salmonella-Shigella.

[

l

TSA ~ trypticase soy agar. TSB = trypticase soy broth.

VIRULENCE

PLASMIDS

TABLE I. - -

Species

IN

SHIGELLA

AND

E. C O L I

B a c t e r i a l strains.

Designation

Scrotype

Virulence

Source

M25-8 177-81 330-81 02-81 07-81 43-81 J17-B 08-80 109-80 291-81 351-81 M76-39 MOOT 6154-61 138-80 2924-71

1 1 1 2 2 2 3 3 3 4 4 4 5 5 6 6

+ + + + + + + + + + + + + + + +

WRAIR IP IP IP IP IP WRAIR IP IP IP IP WRAIR WRAIR CDC IP CDC

31-81 47-80 02-82 20-82 D989 1007-74 01-82 13-80 36-81 16-81 41-06-65

1 1 1 1 1 1 2

IP IP IP IP IP CDC IP

2 2 2

+ + + + --+ + + + --

IP IP CDC

S. boydii

31-81 33-81 33-81 2854-67 871-74 1-81 Foy 74 17-79 3073-50

2 2 2 2 4 4 5 7 8

+ + + + + ----

IP IP IP CDC CDC 1P IP IP CDC

S. sonnei

482-79 01-82 07-82

form I ,, ,,

+ + +

IP IP IP

0143

+

WRAIR

S. flexneri

S. dys~nteriae

E. coli

4608-58

297

2

IP

W R A I R = Walter Reed Army Institute of Research. IP = Institut Pasteur (Centre National de R6f6rence des Shigella). CDC = Center for Diseases Control.

Virulence assays. Virulence was always determined by tissue culture infection and the Sereny t e s t . N o n - c o n f l u e n t m o n o l a y e r s of H e L a cells w e r e i n o c u l a t e d w i t h b a c t e r i a a n d i n c u b a t e d f o r 90 m i n a t 370 C. T h e p r o c e d u r e u s e d f o r m a i n t e n a n c e , c u l t u r e a n d i n f e c t i o n of cells h a s a l r e a d y b e e n d e s c r i b e d b y H a l e a n d F o r m a l [2]. T h e S e r e n y t e s t [711 w a s u s e d as d e s c r i b e d in a p r e v i o u s p a p e r [5].

298

P. J. SANSONETTI AND COLL.

Plasmid D N A isolalion and agarose gel eleclrophoresis. Rapid plasmid detection was performed according to the procedure of Casse

el al. [1]: 20-~1 samples of DNA were electrophoresed for 2 h at 100 V in a 0.7 % agarose gel prepared with TE buffer (40 mM tris-hydroxymethylaminomethane, 2 mM ethylenediaminetetraacetic acid, adjusted to pH 7.9 with acetic acid); gels were stained and photographed as previously described [4]. Large quantities of plasmid DNA were isolated as follows. A 250-ml overnight culture in TSB was pelleted and lysed by incubation for 30 rain at 65o C in 60 ml of 50 mM tris-hydrochloride buffer with 1 % SDS (pH 12.6). The lysate was adjusted to pH 7.0 with 2.0 M tris-hydrochloride buffer and centrifuged at 15,000 g for 30 rain at 4o C. The supernatant was collected and mixed with CsC1~ and ethidium bromide, ultracentrifuged for 40 h at 100,000 g and the plasmid DNA band was collected. Ethidium bromide was extracted with isopropanol, and the DNA solution was dialyzed against T E buffer. This dialyzed solution was mixed with 0.3 M sodium acetate, ethanol-precipitated, frozen in dry ice, and centrifuged at 15,000 g for 30 rain at 40 C. The resulting DNA pellet was resuspended in TE buffer and stored at 4 ~ C.

Reslriclion endonuclease analgsis. Two ~g of purified plasmid DNA were digested by EeoR1, BamH1 or HindlII restriction endonucleases under conditions recommended by the supplier (Boehringer). This mixture was heated to 70 ~ C for 5 min and cooled to 4 ~ C. Stop mix (0.07 % bromophenol blue, 7 % SDS and 2 ~o Ficoll) was added and the final mixture was applied to a vertical 0.7 % agarose gel in Tris-borate buffer (89 mM Tris 89 mM boric acid, 2.5 mM Na2 EDTA, pH 8). Gels were then stained and photographed.

Preparation o[ lhe radioaclive probe bg nick lranslalion. Plasmid DNA was labelled to high-specific activity according to Bigby el el. [3]. Specific activity of ~ P d C T P and ~ P d T T P (Amersham) was 3,000 Ci/mole; 100 ~Ci of each of the nucleotides was used for 500-rig of plasmid DNA.

Nitrocellulose filler hgbridizalion. This was performed under stringent conditions according to Southern's procedure with modifications [8]. EeoRl-digested DNA was depurinated by immersing 0.7 % agarose gel in 0.25 M HCI for 10 min, denatured by two 15 min immersions in 1.5 M NaC1, 0.5 N NaOH, and neutralized by two 30-min immersions in 3 M NaC1, 0.5 M Tris HC1, pH 7.0. The DNA was then transferred overnight onto a nitrocellulose filter along a 20X-2X SSC gradient (1X SSC: 0.15 M NaC1 0.015 M sodium citrate). The filter was rinsed in 2 • SSC for 20 rain, air-dried and baked for 3 h at 80 ~ C. Plasmid DNA from different enteroinvasive strains was radiolabelled by nick translation using 82p deoxyribonucleotides achieving a specific activity of 2.5 x 107 cpm/~g DNA. The nitrocellulose filter was prehybridized by immersion in the following solution for at least 4 h: formamide 40 %, 5 • SSC, 5 • Denhardt solution, 50 mM sodium phosphate pH 6.5, heat-denatured calf thymus DNA 100 ~g/ml. Hybridization was carried out in a sealed plastic bag at 420 C for 17 h in 10 ml of the following solution: formamide 50 %, 5 x SSC, 1 • Denhardt solution, 20 mM sodium phosphate pH 6.5, heat-denatured calf thymus DNA 100/~g/ml and radioactive probe (5 • 106 cpm). The filter was then washed 3 times with 250 ml of 2 • SSC, 0 . 1 % SDS for 5 min, and twice with 250 ml of 0.1 • SSC, 0 . 1 % SDS at 50 ~ C for 30 rain, air-dried and exposed overnight to a ,r Kodak ~ XAR5 film at --80 ~ C.

VIRULENCE PLASMIDS IN SHIGELLA AND E. COLI

299

RESULTS

Presence of a 140 Mdal plasmid in the virulent isolates of S. dysenteriae and S. boydii. Isolates belonging to the different serotypes of S. dysenleriae and S. boydii were found to be invasive in HeLa cell monolayers and in the Sereny test. The keratoconjunctival exudate was streaked onto SaImonella-Shigella (SS) agar. After overnight incubation at 370 C, isolated virulent colonies were checked for proper serotype and used for plasmid DNA isolation. Agarose gel electrophoretic profiles showed that they all harboured a 140-Mdal plasmid as well as smaller plasmids of various sizes. Additional strains belonging to both species were verified for avirulence in HeLa cell monolayers. Avirulence was consistently confirmed by a negative Sereny test. These strains were originally either virulent isolates which had been kept at room temperature in nutrient agar after isolation from human faeces, or virulent isolates which had been subcultured for several generations until they produced avirulent variants. They were smooth, and expressed their original antigenic specificity. Agarose gel electrophoretic profiles showed that these avirulent variants fell into two sets. In one set, a large plasmid was still present, although consistently smaller than the plasmid observed in the virulent isolates (i. e. 100 Mdal or less), suggesting that the large plasmid was either lost or deleted from sequences involved in the invasive process. In other set, no large plasmid was observed. Selected virulent and avirulent isolates belonging to several S. dysenleriae and S. boydii serotypes are shown in figure 1. These results indicate that a plasmid is also necessary in order for S. dysenteriae and S. boydii to penetrate into cells. They support the common observation that virulence in S. dysenteriae is highly unstable and irreversibly lost upon subculture. Inlerspecies comparison of Shigella and enteroinvasive E. coli virulence plasmids. The observation that all enteroinvasive isolates harbour virulence plasmids of similar molecular size does not imply that these plasmids are related. An extensive screening of virulent strains belonging to the five enteroinvasive species provided isolates harbouring only one large virulence plasmid, thus facilitating interpretation of endonuclease restriction patterns. EcoR1 restriction patterns of virulence plasmids belonging to selected isolates of S. dgsenleria, S. flexneri, S. bogdii, S. sonnei form I and enteroinvasive E. coli are shown in figure 2. Two comigrating bands (arrows) were observed among an average of 20 restriction fragments. BamH1 and HindIII produced no comigrating fragments (data not shown). These results suggested a rather limited (if any) relation between virulence plasmids belonging to different enteroinvasive species.

300

P. J. SANSONETTI AND COLL.

We therefore studied the amount and distribution of homologous sequences among these plasmids by nitrocellulose filter blot-hybridization (fig. 3). The virulence plasmid of enteroinvasive E. coli 4608-58 was selected for ~P labelling because this strain carries no other plasmid. It was used as a probe for hybridization with the EcoR~ restriction fragments of the Shigella virulence plasmids shown in figure 2. Unlike what is observed in endonuclease cleavage patterns, hybridization experiments showed t h a t these virulence plasmids shared mainly homologous sequences which were distributed throughout the DNA molecule. These results were confirmed when virulence plasmids form S. flexneri and S. sonnei were used as probes (data not shown).

Intraspecies comparison of Shigella virulence plasmids. S. flexneri. Virulent isolates representing the six serotypes of Shigella flexneri and harbouring only one large virulence plasmid along with a varying number of small plasmids were selected for further study by endonuclease cleavage. EcoR1 restriction patterns are shown in figure 4. Small plasmids had no EcoR~ restriction site. Four comigrating bands were observed among an average of 20 bands. However, when the virulence plasmid of S. flexneri 6 was left over, eight comigrating bands were detected (arrows). Their molecular weights were 17, 12, 11.5, 8, 6.2, 4.6, 3.2 and 3 Mdal, respectively, thus representing a total of 65.5 Mdal (i. e. 47 % of the plasmid). Similar results were obtained with BamH1, and HindIII restriction (data not shown). When the ~2P-labelled virulence plasmids of S. flexneri 5 M90T and enteroinvasive E. coli 4608-58 were hybridized with the nitrocelluloseblotted restriction fragments of S. flexneri virulence plasmids shown in figure 4, homology was detected with all fragments (data not shown). These results indicate that, in addition to a general background of homology, about 50 % of the plasmid sequences are highly conserved among S. flexneri virulence plasmids, with the exception of S. flexneri serotype 6.

F I 6 . 1. - A B C D E F G H I J K L

~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~

0,7 % agarose gel electrophoresis of p l a s m i d D N A .

S . d g s e n t e r i a e l, 47-80, virulent (vir+); S. d y s e n t e r i a e 1, 20-82, vir+; S. dgsenteriae 1, D989, avirulent (vir-); S. dgsenteriae 1, CDC 1007-74, vir-; S. dysenteriae 2, 16-81, vir+; S. dgsenteriae 2, CDC 4106-65, vir-; S. bogdii 2, CDC 2854-67, vir+; S. boydii 4, CDC 871-74, vir+; S. bogdii 4, 1-81, vir ; S. bogdii 5, F o y 74, vir-; S. boydii 7, 19-79, vir-; S. bogdii 8, CDC 3073-50, vir-.

ABCDE

F

GH

FIG, I

I J KL

FIG. 2. - - E c o R 1 restriction patterns o/ virulence p l a s m i d s belonging to dil~erent enteroinvasive species. A = S. dgsenteriae 1, 47-80; B = S. dysenteriae 2, 1-82; C ~ S. flexneri 5, M9OT; D = S. bogdii 2, 33-~t; E = S. s o n n e t l , 482-79; F = E . r 0143, 4608~

AB

E3 E3

D,-

Fro. 2

F

Fia. 3. - - Hybridization of the 82P-labelled virulence p l a s m i d o[ E. coli 4608-58 with nitrocellulose blotted restriction ]ragments of the virulence plasmids shown in figure 2. L e g e n d s are similar to figure 2. EcoRl-restricted ~ p h a g e is p r e s e n t as a n e g a t i v e control.

FIG. 3

Ann. Microblol. (Inst. Pasteur), 134 A, nO 3, },983.

21

F~G. 4. - - EcoR1 restriction patterns o[ virulence p l a s m i d s belonging to lhe s i x serotgpes o[ S. flexneri. A ~ S . flexneri 1: M25-8; B = S. flexneri 2: 0%81; C = S. flexneri 3: 109-80; D = S. flexneri 4, 291-81; E ~ Si flexneri 5: M9OT; F = S. flexlTeri 6, CDC 2924-71.

B

C

lip

FiG. 4

D

L

Fro. 5. - - Intraserotype comparison o[ E c o R ~ restriction patterns of S. f l e x n e r i virulence plasmids. S. S. S. S. S. S.

flexneri 1: A = M 2 5 - 8 ; B = 1 7 7 - 8 1 ; C = 3 3 0 - 8 1 . flexneri 2: D = 0 2 - 8 1 ; E = 0 7 - 8 1 ; F = 4 3 - 8 1 . flexneri 3: G = J 1 7 - B ; H = 0 8 - 8 0 ; I = 1 0 9 - 8 0 . flexneri 4: J = 2 9 1 - 8 1 ; K = 3 5 1 - 8 1 ; L = M 7 6 - 3 9 flexneri 5: M = M 9 O T ; N = C D C 6 1 5 4 - 6 1 . flexneri 6: O = 1 3 8 - 8 0 ; P = C D G 2 9 2 4 - 7 1 .

~2

Fro. 6. - - Intraserotgpe comparison of E c o R ~ restriction patterns o[ S. d y s e n t e r i a e l and 2, S. b o y d i i 2 and S. s o n n e i l virtzlence plasmids. S. S. S. S.

dgsenleriae dgsenteriae boydii 2: G sonnei 1: J

1: 2: : :

A : 31-81; B = 47-80; C : 02-82. D : 01-82; E = 13-80; F = 36-81. 31-81; H = 33-81; I ~ 38-81. 482-79; K : 01-82; L : 07-82.

C:

o

0 .Q

1:1

'1~

Fie. 7. - - Hybridization of Ihe ~2P-tabelted virulence plasmid oI E. colt 4608-58 with the nitrocellulose blotled plasmids ol S. d y s e n t e r i a e and S. boydii shown in figure 1. L e g e n d s are similar to figure 1. H y b r i d i z a t i o n of t h e probe w i t h linear D N A over t h e average b a c k g r o u n d c o r r e s p o n d s to linearized h o m o l o g o u s p l a s m i d s m i x e d w i t h c h r o m o s o m a l D N A .

J K L !i i

m

J

FIG. 7

VIRULENCE PLASMIDS IN SHIGELLA AND E. COLI

315

Intraserotype homology among virulence plasmids of S. flexneri was also studied. EcoR~ restriction patterns are shown in figure 5. Striking intraserotype homology, suggesting an average of 80 % conserved sequences was observed among plasmids belonging to serotypes 1, 2, 3 and 4. Serotypes 5 and 6 appeared to be much more heterogeneous.

S. sonnei. S. sonnei comprises a single serotype (i. e. form 1 antigen) which is encoded by the virulence plasmid. EcoR1 restriction patterns (fig. 6 J, K and L) showed considerable homology among these plasmids, despite the worldwide origin of the strains. S. dysenleriae I (Shiga bacillus). This is the most prevalent and harmful enteroinvasive species in developing countries. As shown in figure 6 A, B and C, EcoR1 cleavage of virulence plasmids from the strain of worldwide origin produced heterogeneous patterns as compared ot S. dysenteriae 2 isolates, which displayed highly conserved patterns, (fig. 6 D, E and F). Similarly, isolates of S. boydii 2 harboured virulence plasmids which appeared totally identical after EcoR1 digestion (fig. 6 G, H and I). Figure 3 demonstrates homology among S. dysenteriae 1 and 2, S. boydii 2 and enteroinvasive E. coli virulence plasmids. Taking advantage of this observation, S. dysenteriae and S. boydii plasmids shown in figure 1 were blotted onto a nitrocellulose filter and hybridized with the 3*P-labelled virulence plasmid of E. coli 4608-58. As shown in figure 7, this probe hybridized with all the 140-Mdal plasmids of virulent strains, but, unexpectedly, also hybridized with smaller plasmids either in virulent or in avirulent strains. These results could simply indicate that common sequences like IS or transposons were present on both the probe and on smaller plasmids. However, spontaneous deletions were obtained with such facility in the virulence plasmids that hybridization studies suggest that on the contrary, our cultures were highly heterogeneous, containing a mixed population of virulent strains with the intact 140-Mdal plasmid and avirulent strains with plasmid deletions of various sizes comprising sequences necessary for virulence. In certain cases, dissociation of these plasmids in two replicons may have occurred. DISCUSSION Tile involvement of plasmids in the virulence of S. sonnei [5], S. flexneri [16] and enteroinvasive serotypes of E. coli [4] is well established; for the S. dysenteriae and S. boydii species, however, it has been purely speculative up to now. In addition, except for similarities in molecular size, no data existed on homology among these virulence plasmids.

316

P.J.

SANSONETTI AND COLL.

The present work demonstrates that a 140-Mdal plasmid is indeed necessary for S. dgsenteriae and S. bogdii invasiveness. Strains harbouring this plasmid invaded HeLa cell monolayers and evoked a positive Sereny test, whereas strains lacking this plasmid didnot. Aceording to serology avirulent variants displayed intact LPS suggesting that, like S. flexneri, virulence plasmids, these plasmids might encode outer membrane proteins involved in surface interactions during the key step of host cell penetration. These virulence plasmids appeared to be structurally unstable. Over several generations, they overcame deletions of various size which contained virulence sequences. This was in agreement with the common observation that the virulence of isolates is highly unstable, especially in S. dgsenteriae strains, and is irreversibly lost upon subculture. The results definitively establish that extrachromosomal elements are consistently necessary for enteroinvasiveness of ShigeUa and E. coli species. Although endonuclease cleavage of plasmids representative of each of these enteroinvasive species suggested very limited - - if any - relatedness, blotting hybridization experiments using different virulence plasmids as 32P-labelled probes showed that all these virulence plasmids shared primarily homologous sequences distributed throughout the molecule. These observations indicate that all the virulence plasmids were derived from a common ancestor molecule, which followed the evolution of its host strain into the different ShigeUa and E. coli species. Microevolution led to modifications in restriction cleavage sites, although the overall homology was conserved. This interpretation is strengthened by the observation that none of the virulence plasmids thus far studied in Shigella spp. are self-conjugative. Unlike numerous R factors which are easily interchangeable among enterobacteria, these virulence plasmids are associated with enteroinvasive species, and were not found in other species when colony hybridization screening was performed (Sansonetti el al., unpublished data). Moreover, restriction cleavage usually showed a striking homology among plasmids belonging to the same serotype. Although these data may have been biased because some of the strains were isolated in the same area (i. e. all S. bogdii serotype 2 were isolated in France), the clonality of some serotypes was obvious even when strains were isolated on a worldwide basis (i. e. S. sonnei). In addition to the usual epidemiological markers such as serotypes, biotypes, lysotypes and antibiotypes, the epidemiology of Shigella infections might now rely upon plasmid fingerprinting and, in particular, upon the restriction pattern comparison of virulence plasmids. Moreover, given the absence of an effective enrichment medium for Shigellae, a high-homology background among all virulence plasmids would make any one of them suitable for use as a diagnostic 32P-labelled probe for enteroinvasive strains present in faeces, contaminated food and water. Our current research is continuing along these two lines.

VIRULENCE PLASMIDS IN SHIGELLA AND E. COLI

317

RI~SUMt~ COMPARAISON MOLt~CULAIRE DES PLASMIDES DE V I R U L E N C E CHEZ LES (( SHIGI-IELLA )) ET (, E S C H E R I C H I A COLI )) ENTI~ROINVASIF

Toutes les souches virulentes appartenant aux esp~ces Shigella dysenleriae et S. boydii h6bergent un plasmide de 140 Mdal qui est perdu ou d616t6 dans les souches spontan6ment avirulentes. Ces donn6es 6tablissent d6finitivement l'intervention d'un 616ment extrachromosomique dans la virulence de routes les souches ent6roinvasives appartenant au genre Shigella et ~ Escherichia coll. D'une esp~ce fi l'autre, les profils de coupure par les endonucl6ases de restriction ne montrent pas de parent6 6vidente. Par contre, les exp6riences d'hybridation montrent des s6quences homologues r6parties sur ]'ensemble des plasmides qui d6rivent probablement d'un an@tre commun ayant subi au cours de l'6volution des modifications darts les sites de restriction. La comparaison de ces plasmides d'espbce esp~ce et de s6rotype fi s6rotype par restriction montre des s6quences h a u t e m e n t conserv6es. Les cons6quences de ces r6sultats pour l'6volution, l'6pid6miologie et le diagnostic des shigelles et des E. coli ent6roinvasifs est discutde. MOTS-CLI~S : Shigella, Escherichia coli, Enteroinvasion, Virulence, Plasmide ; Restriction, Hybridation. ACKNOWLEDGMENTS

We wish to thank M. Toucas, M. Popoff and L. Le Minor for their constant interest in this study, P. A. D. Grimont for his careful reading and D. de Champs for typing this manuscrit. This work was supported by grant 82/1130 from DRET.

REFERENCES F., BOUCHER, C., JUILLIOT, J. S., MICHEL, M. & DENARIE, J., Identification and characterization of large plasmids in Rhizobium meliloti using agarose gel electrophoresis. J. gen. Microbiol., 1979, 113, 229-242. [2] HALE, T. L. & FORMAL, S. B., Protein synthesis in HeLa or Henle 407 cells infected with Shigella dysenleriae 1, S. flexneri 2a or Salmonella typhimurium Wl18. Infect. Immun., 1981, 32, 137-144. [3] RIGBY, P. W. J., DIECKMAN, M., RHODES, C. BERG, P., Labelling DNA to high specificity activity in vitro by nick-translation with DNA polymerase I. J. tool. Biol., 1977, 113, 237-251. [4] SANSONETTI,P. J., d'HAuTEVlLLE, H., FORMAL,S. B. & TOUCAS,M. Plasmid mediated invasiveness of (( Shigella-like )) Eseherichia coll. Ann. Microbiol. (lEst. Pasteur), 1982, 133 A, 351-355. [1] CASSE,

318

P. J. SANSONETTI AND COLL.

[5] SANSONETTI,P. J., KOPECKO, D. J. & FORMAL,S. B., Shigella sonnei plasmids: evidence that a large plasmid is necessary for virulence. In/ect. Immun., 1981, 34, 75-83. [6] SANSONETTI, P. J., KOPECKO, D. J. & FORMAL,S. B., Demonstration of the involvement of a plasmid in the invasive ability of Shigella flexneri. In/ect. Irnmun., 1982, 35, 852-860. [7] SERENY, B., Experimental Shigella conjunctivitis. Acta microbiol. Acad. Sci. Hung., 1955, 2, 293-296. [8] SOUTHZRN, E., Gel electrophoresis of restriction fragments, in (( Methods in enzymology, recombinant DNA )), (R. Wu), 68, (pp. 152-176), Academic Press, London, New-York, 1979.