Molecular Microbiology (1990) 4(12), 2167-2178

Attenuated mutants of the intracellular bacterium Usteria monocytogenes obtained by single amino acid substitutions in listeriolysin O E. Michel,^ K. A. Reich,^ R. Favier/ P. Berche^ and P. Cossart'* ' Unite de Genie Mierobiologique, Departement des Bioteehnologies, institut Pasteur, 25 rue du docteur Roux, Paris 75015, France. ^Laboratoire de Microbiotogie, Hopitat Necker-Enfants h/taiades, 149 rue de Sevres, Paris 75015, France. Summary Ljsteriotysjn O (LLO), a major virulence factor of the jntracellular bacterium Listens monocytogenes, shares with other known 'thiol-activated toxins' a conserved undecapeptide, ECTGLAWEWWR, located in the C-terminal region of the protein and containing the unique cysteine of the molecule. Single amino acid substitutions were created in this region to study the role of cysteine and tryptophan residues in the lytic activity of LLO as well as in the virulence of the bacterium. Transformation of a transposon-induced non-haemolytic mutant with plasmids carrying the mutated genes allowed allele exchange and transfer of mutations on to the chromosome by in vivo recombination. The mutant strains secreted a full-length 59 kilodalton LLO. A decrease of 25% in the haemolytic activity in culture supernatants was observed in the case of mutation Cys-484 to Ala and of 80% for mutation Cys-484 to Ser. Mutations Trp-491 and Trp492 to Ala decreased activity by, respectively, 95% and 99.9%. LLOs produced by the mutants, as the wild type, were active at low pH, inhibited by cholesterol, and able to bind to cell membranes. A close relationship was found between virulence of mutants in the mouse model and haemolytic activity in their culture supematants. These results demonstrate that the thiol group of Cys-484 is not essential for either haemolytic activity in vitro or virulence in vivo. In contrast, Trp-492 appears to be required for both haemolytic activity and virulence. The finding that the nearly non-haemolytic mutant Trp-492-Ala persisted in the spleen for several days after inoculation indicates that mutagenesis of a

Received 30 May, 1990; revised 30 July, 1990. 'For correspondence. Tel. (1) 45 68 88 41; Fax {1) 45 68 88 43.

virulence determinant can attenuate virulence and provides a novel approach to the development of live vaccine strains. Introduction Listeria monocytogenes is a bacterial pathogen responsible for a wide range of ctinicat features in humans, the most important of which are meningitis or meningoencephatitis, septicaemia and, in pregnant women, intrauterine infections of the fetus which may resutt in abortion, stitl-birth, or neo-natal infection. As originalty shown by Mackaness (1962), virutence of L monocytogenes can be essentiatly attributed to its capacity to survive in macrophages. Recent genetic studies (Gaitlard et ai, 1986; Kathariou etai, 1987; Mengaud efa/., 1987; Portnoyefa/., 1988; Cossart et ai, 1989) have estabtished that a secreted protein of 524 amino acid residues named listeriotysin O is a virutence factor essentiat for the survivat of L monocytogenes in its infected host (Geoffroy et ai, 1987; Mengaud etai, 1988; Cossart and Mengaud, 1989). tt has been proposed that after entry of the bacterium in the mammalian ceil, LLO damages the phagolysosomat membrane, atlowing retease of the bacterium into the cytosol of the infected cetl (Gaitlard et ai, 1987). LLO is then probably processed and presented to the immune system, as it has been shown to induce a T-cetl-dependent response in infected mice (Berche ef at.. 1987a; 1987b) and production of specific anti-LLO antibodies, in the course of human infection (Berche ef at., 1990). LLO betongs to a famity of biotogicatty and antigenicatly retated membrane-damaging toxins: the 'thiol-activated' toxins. This group includes at least 18 proteins produced by Gram-positive bacteria betonging to the genera Baciltus. Clostridium, Listeria and Streptoeoeeus (Smyth and Duncan, 1978). These toxins have no known enzymatic activity and exert their cytotytic activity by a mechanism not yet ctearty defined, invotving either pore formation (Bhakdi ef ai.. 1985) or tipid phase transition in the membrane due to chotesterol sequestration (Smyth and Duncan, 1978). Cholesterot, which inhibits the activity of the protein, is assumed to be the toxin-binding site on the surface of the eukaryotic celt, since membranes tacking this compound are insensitive to the action of the toxins.

2168 £ Michel etai. ATG Sau%

TAA EcoRI

HindlU

Hindm

Hindm

Sau96

hlyA

E C T 6 L A W E W W R

Cys484Ser

Trp491Ala Trp492Ala

Fig. 1. Partial restriction map of the hlyA region and location of the mutations.

Cys484Ala

As cytolysis is usuaiiy tested on erythrocytes, the alternative name 'haemolysins' has also been given to these toxins (Smyth and Duncan, 1978), The biochemicai properties of these haemolysins have been mostly studied in the case of SLO (Smyth and Duncan, 1978; Alouf, 1980; Alouf and Geoffroy, 1984; Bernheimer and Rudy, 1986). As haemolytic activity was inhibited by thioi alkylation and oxidation and was activated by thiol-reducing agents, thioi groups were assumed to be essentiai for activity, hence the name given to these toxins (Neiil and Fleming, 1927; Smyth and Duncan. 1978). Recently, the genes for five of these toxins, pneumolysin (Walker et al.. 1987), streptoiysin O (SLO) (Kehoe ef a/., 1987), LLO(Mengaudefa/., 1988), perfringolysin (Tweten, 1988) and alveolysin (Geoffroy ef al.. 1990), have been sequenced, A comparison of the deduced amino acid sequences reveais that all five proteins share extensive primary sequence homologies. Moreover, each toxin contains a single cysteine residue in the iongest conserved region of identity, the undecapeptide ECTGLAWEWWR, located at an identical position towards the C-terminus of each protein. This suggested that this peptide may be functionaily important; in order to test such a hypothesis, mutants affecting the cysteine residue of SLO (Pinkney et ai. 1989) and pneumolysin (Saunders etai. 1989) were constructed and analysed. The striking result of these studies was the demonstration that the cysteine residue is not essential for activity. Although activity was only slightly reduced by changing Cys to Ala, it was substantially decreased by changing Cys for Ser. In this study, site-directed mutagenesis was used to construct four isogenic mutant strains differing by a single amino-acid substitution in the conserved undeoapeptide

of LLO. Our goai was to eiucidate the role of this peptide in the virulence of L monocytogenes. The unique cysteine was mutated to Ala or Ser and, because of the unusual grouping of tryptophan residues in this region, we also mutated Trp-491 and Trp-492 to Ala. These mutations affected, to different extents, the haemoiytic activity of culture supernatants. As it has been reported that haemoiysin ieveis vary among strains (Geoffroy etai. 1989) and that Ieveis of L monocytogenes haemolysin are not directly proportional to virulence in experimental infections in mice (Kathariou et ai. 1988), mutants were also tested for virulence in the murine model of listeriosis. We found that virulence directly correlated with haemolytic activity. The attenuated virulence of the mutants offers the possibility of their use as live vaccines.

Results In vitro mutagenesis of the hlyA gene of L. monocytogenes In order to investigate the role of the single cysteine residue of LLO as weil as that of the nearby trytophan residues, four mutations were created in vitro by oligonucleotide-site directed mutagenesis. The gene hlyA encoding LLO and its adjacent regions have been previously cloned and sequenced (Mengaud ef ai, 1988; 1989). The 2436 bp Satj96A fragment containing hlyA (Cossart et ai. 1989) was subcloned in M13mp19 and mutagenized as described in the Experimental procedures (Fig. 1). To faciiitate further DNA manipulations, the hlyA gene, which contains an EcoRi site, was first silently mutated to suppress this restriction site without changing the encoded amino acid sequence. The phage

Attenuated mutants of Listeria monocytogenes

2169

pMK4 carrying hlyA with a punctual mutation

ATG

ATG

TAA •

Tn9!7

ll-fIH

I it V H B

Fig. 2. Schematic drawing of the construction of the hlyA mutant strains by allele replacement. The hiyA gene is indicated by a thick line, transposon Tn9f7byanopen box, and the curved line is part of pfw1K4, Restriction sites are abbreviated: H, H/ndlll; E, fcoRI; B, SamHI, The asterisk refers to the position of the mutations in hlyA. The crosses indicate the recombination events required to introduce the mutations onto the Usteria chromosome by allele replacement.

derivative carrying this mutation, called EcoRls, was used to mutate codon Cys-484, Trp-491 and Trp-492. Codon Cys-484 was mutated to a serine and to an alanine. Codons Trp-491 and Trp-492 were mutated to aianine (see the Experimental procedures). After selection of four mutated phages, the inserts were completely sequenced to verify that only the desired mutations were present. Finally, by use of sites present in the poiyiinker of M13mp19, the EcoRI-Ps/l fragments carrying the mutated genes were cloned in the E. coli-Listeria shuttle vector, pMK4 (Sullivan etai, 1984).

Allele replacement of hlyA gene by the genes carrying the missense mutations We had previously complemented a transposon-induced, non-haemolytic strain with a derivative of pMK4, carrying the hlyA gene {Cossart etai, 1989). We first planned to use the same strategy to evaluate the effect of the point mutations on the haemoiytic activity and virulence of L. monocytogenes. However, to circumvent ambiguities due to gene copy number, we chose to transfer the various mutations on to the chromosome. pMK4 derivatives were

first introduced by electroporation in a non-haemolytic derivative of strain LO28. This mutant strain, LO28(htyA.:Tn917), carries a single copy of transposon Jr\917 inserted in the hlyA gene and has been fully characterized (Cossart ef a!., 1989). In this mutant strain, Tn977 has inserted after codon 502 and the mutant is non-haemolytic. In order to get recombination between plasmid and chromosome, as represented in Fig. 2, cells were grown without selection and screened as described in the Experimental procedures. Clones where putative allele exchange had occurred were analysed further. We first verified that allele exchange and loss of plasmid had been successfully obtained without chromosomal rearrangements by performing Southern blot analysis of chromosomal DNA isolated from the various candidates. DNAs were digested with EcoR\. which restricts the gene once, with H/ndlll which restricts the gene WyA twice, and SamHI, which has no site within the haemolysin gene (Fig. 2). Restriction digests were probed with the 41 Obp H/ndlll fragment containing the 3' end of hlyA (Fig. 3). Controls included DNAs from wild-type strain LO28, from LO28{hlyA::Tr\917) and from pLis4, a plasmid harbouring the 2436bp Sau96A fragment containing hlyA (Cossart et ai.

E. Michel et al

T

1

in

TO
10^

a. The LDw of LO28 is slightly different from the reported value (Cossart et al.. 1989). This is due to growth conditions of the culture before inoculation. Bacteria grown under shaking were reproducibly more virulent (LDjo = l O ' ' " versus 10^^),

2172 £ Michel etai

10 20 TtME (hri)

10 20 TIME (hri)

Fig. 5. In vitro growth curves and haemoiytic activity of culture supernatants of wild-type and mutanl strains. The ODeoo is represented by the open squares and the haemoiytic activity, by the closed diamonds.

various mutant culture supernatants. Haemolysin titrations were performed in the presence of various concentrations of cholesterol to evaluate the concentration which inhibited 50% of the haemoiytic activity. Inhibition occurred at similar cholesterol concentrations for all of the mutants tested: 1.2 x 10"^Mfor the wild-type strain LO28 and for mutant Cys-484-Ala, and 3 x 10"^M for mutants Cys-484-Ser and Trp-491-Ala. We were unable to measure the cholesterol inhibition for mutant Trp-492-Ala due to its low haemoiytic activity. As cholesterol is believed to be the binding site of the toxin on the eukaryotic cell membrane, it was of interest to test whether the various mutant proteins were affected in their capacity to insert in cholesterol-containing membranes. Culture supernatants were incubated with sheep red blood cells. LLO bound to cell membrane proteins was detected with rabbit a-LLO antibody, after sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDSPAGE) electrophoresis {see the Exper/menfa/procedures). All four mutant proteins retained their capacity to bind red blood cells, even mutant Trp-492-Ala, which is nearly non-haemolytic. This was also the case for the protein produced by LO28(Wy/A::Tn9)7). Finally, as LLO is maximally active at low pH (5.5) and nearly inactive at pH 7 (Geoffroy etat.. 1987), we measured the haemoiytic activity of the various mutants at various pHs, but no significant differences could be detected among the various mutants.

Virulence of the mutants: LD50 and growth oun/es of L. monocytogenes in the liver and spleen of infected mice Virulence of the four mutant strains was evaluated by their LD50 (50% lethal dose) values after intravenous (i.v.) inoculation in mice. Controls included the wild-type strain LO28 and strain LO28(h/y^::Tn977). The LD50 values of the various mutants decreased in parallel with the decrease in the haemoiytic titres of culture supernatants (Table 1). Mutant strains had LD50 values that ranged between that of wild-type LO28 (LD50 = 10" ^^) and that of the non-haemolytic strain LO2Q{hlyA:-r[n917), (LD50 > 10^. Mutant Cys-484-Ala had an LD50 of 10"^°, mutant Cys-484-Ser of 10^^^ mutant Trp-491 -Ala of 10^^^ while mutant Trp-492-Ala was the least virulent strain with an LD5oOf10'^^ The infectious process was further analysed by measuring the growth curves of bacteria in the spleen and liver of mice infected i.v. with 10^ bacteria and sacrified 1, 2 and 3 days post inoculation (Fig. 6). In the wild-type strain LO28, bacteria unrestrictedly replicated in the spleen and liver, reaching 10^-10^ counts per organ by day three of infection. Mutants behaved differently. Mutant Cys-484-Ala replicated rapidly in the liver and spleen during the first days of infection, then bacterial counts decreased. For mutant Cys-484-Ser, replication in the organs was also observed, but elimination of bacteria occurred more rapidly. For mutant Trp-491-Ala, rep-

Attenuated mutants of Listeria monocytogenes

LO2e Cys484Ala Cys484Ser Trp49IAIa Ttp492Ala LO28(hlyA::Tn917)

lication was mainly observed in the spleen. No growth of mutant Trp-494-Ala occurred in the spleen or liver although bacteria persisted in the spleen for several days. In contrast, LO28ihiyA::Tn917) was rapidly destroyed in host tissues, as described by Cossart et ai. (1989).

Discussion This paper describes the first allele replacement experiments in L. monocytogenes designed to obtain isogenic strains differing by a single amino acid in the sequence of the secreted toxin, LLO. Haemolytic titration in culture supernatants allowed us to evaluate the role of residues conserved among thiol-activated toxins, particularly the unique cysteine residue of the protein. Our results demonstrate that the thiol group of the cysteine is not absolutely required for activity, in agreement with results obtained with pneumolysin and SLO. However, the results obtained by mutating the neighbouring residues Trp-491 and Trp492 support the hypothesis that the single cystoine in LLO, Oys-484, is located in a functionally important region. The virulence of the mutant strains was attenuated when compared to that of the parental strain. Effect of mutations at Cys-484-Ala. Cys-484-Ser, Trp491-Aia and Trp-492-Aia on haemolytic activity Comparison of the amino acid sequences of five thiol-activated toxins reveals the presence of a conserved motif (ECTGLAWEWWR) located in the C-terminal part of these proteins. This peptide contains the unique cysteine present in each of these proteins. Since thiol-activated toxins are strongly inhibited by thiol alkylating or oxidizing agents, it was tempting to hypothesize that the conserved motif played a crucial role in the mechanism of action of these toxins. Two groups of investigators have recently reported the effects of mutations at the cysteine residue. In the case of pneumolysin, the mutation Cys-428 to Ala did not affect the haemolytic activity of the protein. In

2173

Fig. 6. Growth curves of strain LO28 and mutant derivatives in spleen and liver of infected mice inoculated i.v. with 10^ bactena. Each time point corresponds to the mean value for a group of five mice. Mice were inoculated with LO28 ([J), LO28(hlyA:.Tn917) (U), mutant Cys-484-Ser (O), mutant Cys-484-Ala (+), mutant Trp-491 -Ala (O). mutant Trp-492-Ala (•) and sacrificed on days 1, 2 and 3 after inoculation.

contrast, mutations Cys-428 to Ser or Gly caused a reduction of activity of 85% and 99%, respectively (Saunders etai., 1989). Similarly, for SLO, the mutation Cys-530 to Ala did not significantly alter the haemolytic activity; a 12% reduction was observed while the mutation Cys-530 to Ser caused a decrease of 75% (Pinkney et al., 1989). In the present study, we mutated Cys-484 of LLO to Ser and to Ala and observed a reduction in activity of 85% and 28%, respectively. These results are similar to those obtained with pneumolysin and SLO and again demonstrate that the thiol group of the unique cysteine residue of these toxins is not essential for haemolytic activity. These data may seem difficult to reconcile with the inactivation of these proteins by alkylation or oxidation, but it is to be recalled that inactivation by oxidation has been observed in culture supernatants and not with purified toxins {Alouf, 1980). Therefore, these results may favour the hypothesis that a component, e.g. a protein present in culture supernatants, could react with and inactivate these toxins under oxidizing conditions, through formation of a disulphide bond. Although the cysteine residue is not essential for activity, our results obtained with the tryptophan mutants highlight the fact that the cysteine-containing region is involved in the membrane-damaging activity of the toxins. Both Trp-491 and Trp-492 are within the conserved peptide and both residues also appear to be important for haemolytic activity since mutation Trp-491-Ala reduces the activity by 95% and mutation Trp-492-Ala nearly abolishes haemolytic activity. The reason(s) for the lower activity of the mutant proteins remains unclear. The concentration of cholesterol required to inhibit haemolytic activity of the four mutants and of the wild type was similar. The point mutants described for pneumolysin and SLO behave similarly. In addition, the mutated LLO proteins retained their capacity to bind to red blood cell membranes. It can be argued that the test used for this assay was not quantitative but a two-fold difference in cell membrane binding capacity

2174

E. M/cA?e/etal.

would have been detected. Since no differences between the mutant haemolysins and the wild-type protein were observed, further experiments are required to clarify this point. It is nevertheless highly probable that cholesterol binding is not mediated by the C-terminal portion of the protein. This is in agreement with a recent publication reporting that a truncated fragment of LLO, lacking the C-terminal part of the protein and the cysteine region, could be complexed with and precipitated by cholesterol (Vazquez-Boland et ai, 1989). The conclusion that the cholesterol-binding site is not affected by the mutations is in agreement with the description of haemolysis as a two-step process, as first described by Neill and Fleming (1927). The first step is temperature-independent and would involve binding to cholesterol. This was not affected by the mutations constructed here. The second step, which is temperaturedependent, involves toxin oligomer formation and lysis of cell membranes. In the cases of SLO and pneumolysin, it has been shown that mutations at the cysteine residue do not prevent oligomer formation. This is probably also the case for LLO. Therefore, oligomer formation in the membrane, thought to be the crucial step for membrane disruption {Bahkdi etai, 1985), may be necessary but not sufficient to lyse cells. Alternatively, it is possible that the oligomers obtained with the mutant proteins are not in a conformation appropriate to efficiently mediate membrane lysis.

Virulence of haemolysin mutant strains The availability of a good animal model for listeriosis allowed us to test the effect of the mutations on virulence. LLO has been shown to be a virulence factor involved in the escape of the internalized bacterium from the acidic compartment of the phagosome and in its release to the cytosoi, a hypothesis reinforced by the finding that LLO is maximally active at low pH (Gaillard etai, 1987; Geoffroy et ai, 1987). Therefore, it was to be expected that altering the haemolytic activity of LLO would alter the virulence of L. monocytogenes. Non-haemolytic mutants have been isolated by use of theconiugativetransposonsTnT545(Gaillard etai, 1986; Porinoy ef a/., 1988), Tr\916 (Kathariou et ai, 1987; Portnoy et al.. 1988) or by use of the non-conjugative transposon Tn9r7 (Cossart ef a/., 1989), In all of these mutants, a dramatic reduction in virulence was observed relative to the haemolytic parent strains. In addition, spontaneous non-haemolytic mutants have been Isolated after subculture of haemolytic strains. The first example is the type strain ATCC 15313"^, which is non-haemolytic on blood agar plates but does contain the gene hlyA (Mengaud etai, 1988) and produces detectable activity (Geoffroy ef ai, 1989). Spontaneous loss of

haemolytic phenotype has also been described for strain ATCC 35152 (Pine ef ai, 1987). We and others (Gormley ef ai. 1989; Leimeister-Wachter and Chakraborty, 1989) have recently detected, in these strains, a deletion in the region located upstream from the haemolysin structural gene, but evidence for the effect of this deletion on the phenotype still awaits further genetic evidence. Ali these spontaneous non-haemolytic strains, like the transposoninduced mutants, are avirulent. Finally, it has been reported that haemolysin levels vary among strains (Geoffroy ef ai. 1989). However, other studies failed to correlate the haemolytic titres of various strains of L. monocytogenes with their virulence (Kathariou ef a/., 1988). In this paper, we studied four missense mutants obtained after in vitro mutagenesis of gene hlyA followed by allele replacement, in the same L monocytogenes strain. As previously mentioned, the haemolytic titres of the mutants ranged between those of the wild-type (1025 HU ml"') and of the Tn9^7-induced non-haemolytic mutant (0 HU ml"'). The LD50 values of the mutant strains, when tested after i.v, inoculation of mice, also ranged between LD50 values of the wild type and that of the transposon-induced non-haemolytic mutant. These results indicated that for a given strain, virulence and haemolysin production are correlated. It is to be noted that virulence of mutant Trp-491 -Ala was still high although the haemolytic activity had dropped by a factor of 20. This implies that even when low levels of haemolytic activity are detected in culture supernatants, the corresponding strains can still be virulent. It is of interest that although mutant Trp-492-Ala had a haemolytic activity barely detectable in culture supernatants, its LD50 was high (10^-^^) but still lower than that of the Tn977-induced mutant (>10^ which is non-haemolytic and avirulent. This difference was even more apparent when bacterial counts in the liver and spleen of infected animals were monitored. At day three post infection, in contrast to the Tn97 7"non-haemolytic mutant, which was totally eliminated from these organs, mutant Trp-492-Ala was still present in the spleen and to a lesser extent in the liver. These results demonstrate that even a low titre of haemolytic activity can mediate survival in the infected cell. Nevertheless, one could argue that measure of haemolytic activity in culture supernatants, as performed here, may not be an exact measure of haemolysin production in vivo. However, we recently obtained evidence that restoration of haemolysin production in vitro correlates with restoration of virulence (Cossart ef ai, 1989), which is in agreement with results presented here. The observation that mutant Trp-492-Ala, which has been obtained by a double mutation (TG to GC), had an attenuated virulence and yet persisted in the spleen for several days post infection, may offer a new approach for

Attenuated mutants of Listeria monocytogenes the development of live vaccine strains. Other attenuated intracetlutar bacteria, such as aro~ mutants of Salmonella typhimurium, have been used to express heterologous antigens and to study the immune response to these antigens (Hoiseth and Stoker, 1981). Although it has been reported that S. typhimurium can trigger the cetlutar immune system {Brov^rn ef a/.,1987), it is to be expected that the Gram-positive bacterium L. monocytogenes, which is able to secrete proteins into the external medium and to induce a bacteriat replication-dependent protective T-cetl response {Berche et ai, 1987a), may be a more efficient 'antigen-presenting system'. Recent advances in introducing and expressing genes and ptasmids in L. monocytogenes should facititate this approach (Cossati and Mengaud, 1989).

2175

electric discharge of 2.3 t^V at 25^lf and 200 Ohms. The sample was then immediatety removed from the cuvette, transferred to 5mt of BHt broth and incubated at 37°C with agitation for 1-2h. Atiquots of 200|i.t were ptated on antibiotic-containing BHt agar plates. After 2 days, transformants appeared at a frequency of 10^-10^ per n,g DNA. The presence of ptasmid was demonstrated after rapid ptasmid isolation from the transformants (Birnboim and Doly. 1979), retransformation into E. co//with this preparation and ptasmid isolation followed by restriction enzyme anatysis.

Bacterial growth media E. coli strains hartjouring ptasmids were routinely grown in L-brothandL-agar(Mitter, 1972) incorporating ampicitiin at 50 fig ml ', when appropriate. For propagating Ml 3 phages, 2YT media and H-base agar were used, as previously described (Mengaud ef at., 1988). Listeria strains were routinely grown in BHI, supplemented with erythromycin (5ji.g ml ^) and/or chloramphenicol (7 ^g ml ') or plated on sheep blood agar plates (bioMerieux).

Experimental procedures Bacteriai strains and plasmids

DNA maniputations The bacteriat strains used in this study are tisted in Table 2 and the ptasmids in Tabte 3.

DNA-modifying or restriction enzymes were purchased from Cetus, Bioiabs, BRL or Boehringer and were used according to the manufacturers' instructions. Oligonucleotides were synthesized on a Milligen oligonucleotide synthesizer (Miliipore).

Transfer of plasmids Plasmids were transformed into E. coli strains made competent by CaClj treatment (Maniatis et ai. 1982) and into L monocytogenes by eiectroporation as fotlows: an overnight culture of L monocytogenes 6\\uied 1/100 in brain-heart infusion (BHI, Difco) was incubated at 3 7 X untit the ODeoo reached 0.5-0.8. One hundred millilitres of the culture was then centrifuged at 4 X for 15 min at 5000 r.p.m, and washed three times in 30mt of cold eiectroporation buffer (816 mM sucrose, 3 mM MgCt2, pH 4.5) and resuspended in 4mt of the same buffer, tn an etectroporation cuvette, 200 fif of bacteria was mixed with 1 |i.g of DNA in solution in lOmM Tris-HCt, 1 mM ethytenediamine tetraacetic acid (EDTA), pH 8.0 and incubated for 10 min at 0°C, then submitted to an

Table 2. List of bacteriai strains.

DAM preparations Plasmid DNA was purified by ultracentrifugation in caesium chloride gradients. L monocytogenes chromosomal DNA was prepared by a rapid protocol, derived from Ausubel ef ai. (1990), and modified by J. Mengaud (personal communication). Single-stranded DNA was prepared from phages grown on agar plates as follows: 5ml of LB was inoculated with 200^1 of a TGI overnight culture and a single plaque. Phage adsorption was allowed to proceed for 10 min at 37"O without agitation. Four millilitres of H agar was then added to the culture and the mixture

Strain

Genotype/Relevant properties

Source/Reterence

TGI

Gibson (Medical Research Council, Cambridge, UK) Kramer era/. (1984)

LO28 Bot415 Bug 288 Bug 290 Bug 335 Bug 337

£ co/( K12, A{l3C-pro). supE. thi. hsdD5/ F' traD36.proA'B'. iacP. iacZ\M15 E. coli K^2, Miac^pro). supE. thi. mutL: JniO/FproA'B'. LacP. iacZ\M15 L. monocytogenes. serotype 1/2c LO2a{hlyA\.lr,917) LO28(Cys-484-Ser) LO28(Cys-4a4-Aia) LO28n"rp-491-Ala) LO28n'rp-494-Ala)

Plasmid/Phage

Host

Markers

pMK4 pLis4 phi 27

E. ooli/Usteria E. colilListeria

Amp, Cm Amp, Cm

BMH71-18mtJfi.

Vicente efa'. (1965) Cossart e( a/. (1989) This study This study This study This study

Table 3. Rasmids and phages. Relevant propertie:i

Source/Reterence

pMK4 derivative M13 carrying hlyA

Sullivan er a/, (1984) Cossart etai. (1989) This study

2176

E. Michel et a\.

was immediately poured on LB plates. After 7-8h of incubation al 37X, 4-5ml of SM buffer (lOOmM NaCI, 8mM MgS04, 50mM Tris-HCt, 0.01 % gelatine, pH 7.5) was added to the plates placed for 12-16h at 4''C under gentle agitation. Four milliiitres of solution was removed, centrifuged and 1 ml of 20% PEG 6000, 2.5 M NaCI was added to the supernatant. After inverting the tube, the solution was left for 30 min at room temperature and supernatant carefully removed after centrifugation. To the visible phage pellet, 460hLl of lOmM NaCI, 0.1 mM EDTA were added; 60|i.l of this solution was kept as phage stock at 4°C. To the remaining 400 M.1, 200 M.1 of neutralized phenol was added. After mixing, 400 M^I of the upper phase was transferred to a new tube and again extracted with 200 M^' of phenol, and then with 1 ml of ether. The single-stranded DNA was precipitated by the addition of 40^.1 of 3M ACONa, pH 5.5, and 1 ml of cold ethanol. After centrifugation, the pellet was dried and resuspended in 160p.l of 10mM Tris-HCI, 1 mM EDTA and stored at 4 X ; 7 M-I of the solution was used for sequence analysis.

5'-TACTAATTCCGAGTTCGC-3' for mutation EcoRls, 5'CCAGTGCTrTCTTTAGC-3' for mutation Cys-484-Ser, 5'CCAGTGGCTTCTTTAGCG-3' for mutation Cys-484-Ala, 5'TCTCCACGCTTCCCAAG-3' for mutation Trp-491-Ala, 5'CCGTTCTCGCCCATTCC-3' for mutation Trp-492-Ala (changes from the wild-type sequence are underlined). After annealing of the kinased mutagenic primer to the Ml 3 derivative containing the 2436bp htyA fragment, elongation in the presence of dNTPs, and ligation, the newly synthesized double-stranded DNA was used to transform strain BMH 71-18 mutL and plated in the presence of TGI cells. Plaques were then streaked on L-agar plates and putative mutants screened by colony hybridization with the labelled mutagenio oligonucleotide. Selected colonies were then inoculated in 1 ml of L-broth and an aliquot of the mixture was streaked on to a plate subsequently covered with H-agar containing TGI cells. Single-stranded DNA from isolated plaques was then prepared for subsequent sequence analysis. Oligonucleotides, derived from the published sequence (Mengaud ef a/., 1988) were used to completely sequence the inserts of the mutated phages.

DAW sequencing The dideoxy chain-terminator sequencing technique was used on M13 single-stranded DNA (Sanger et ai. 1977) with the modification of Biggin etai (1983) using p^S]-dATP (Amersham, 800Ci mmol ') and T7 DNA poiymerase (Sequenase Kit no. 2, USB). Alternatively, sequencing was performed on poiymerase chain reaction (PCR)-amplified fragments. PCR amplification was performed on chromosomal DNA using two oligonucleotides: 5'-CATCGATCACTCTGGAGGATAC-3' and 5'-CGTGTGTGTTAAGCGGTnTATTC-3'. which are located at positions 1311-1332 and 1665-1688, respectively, on the sequence of hlyA (Mengaud et ai. 1988). The amplified 378bp fragment was purified on a 6% poiyacrylamide gel, eluted overnight in 1ml of 0.3 M ACONa, I m M EDTA, 0.1% SDS, pH 7.5, ethanol precipitated and resuspended in water. To approximately 1 M-g of purified fragment, 2^^.\ of Sequenase buffer (Sequenase Kit no. 2, USB) and 5pmol of one of the primers used for the amplification were added. Hybridization was performed at 100°C for 5 min. One microlitre of single-stranded DNA-binding protein (1 mg ml ') (SSB, USB) was then added. The mixture was incubated at 42°C for 30 min and immediately put on ice. The sequencing was performed with the Sequenase kit no. 2 (USB) as described by the manufacturer but the labelling reaction was performed for 30s at 42X and the termination reaction for 5 min at 42''C. One microlitre of proteinase K (0.2 mg ml ') was then added. The mixture was incubated for 20 min at 65°C. Finally, 5^^! of stop solution was added before loading on a 6% sequencing gel.

Hybridizations Southern blots were performed using the rapid-hybridization system (Amersham) with probes labelled with pP]-dCTP at 3000CimM '(Amersham).

In vitro mutagenesis Oligonucleotide site-directed mutagenesis in Ml 3 was performed using the protocol of Carter (Carter ef ai, 1985). Oligonucleotides used for the mutagenesis were:

Construction of isogenic strains carrying mutations in hlyA Allelic exchange between plasmid and chromosome was obtained after transformation of LO28{hlyA::Jr\917) with plasmids carrying the mutated hlyA genes. Strains carrying the various plasmids were first grown in the presence of erythromycin and chioramphenicol, the two antibiotics corresponding to the resistance genes carried by transposon Jn917, and plasmid pMK4. After a 1000-fold dilution of saturated cultures into fresh medium, cells were grown without selection, for c. 100 generations until stationary phase was attained. This procedure was repeated 10-15 times. Bacteria were then plated on BHI agar plates and screened for loss of both antibiotic resistance markers by replica-plating on medium containing either erythromycin or chloramphenicol. For each mutation, four to six erythromycinand chloramphenicol-sensitive clones were obtained after screening of 1000-2000 colonies. All clones in which putative allele exchange had occurred were analysed.

SDS-PAGE and Western biotting To 100|il of culture supernatants, 25|j.l of 5x sample buffer (Laemmli, 1970) was added. Samples were heated for 5 min at 10OX in a dry heating block. Twenty-five microlitres of the sample was loaded on an 11.25% polyacrylamide mini-gel (Hoeffer) run at 25mA until the dye front had reached the bottom of the gel (about 1 h). The gel was electrophoretically transferred to a membrane of Immobilon P (Millipore), (in 192mM glycine, 25mM Tris, 0.1 % SDS, 20% MeOH) for 30 min at 1 A in a 'Little Genie' apparatus (Idea Scientific). Electrophoresis and transfer were monitored by visual inspection of pre-stained molecular weight standards (BRL). The membrane was probed overnight with a rabbit polyclonal anti-LLO antiserum, diluted 1/1000 in buffer A (lOmM Tris-HCI, pH 8, 2mM EDTA, 50mM NaCI, 1 % haemoglobin) with gentle shaking. The blot was subsequently washed three times in buffer B (100 mM Tris-HCI, 200 mM NaCI, 1 % NP40, pH 8.0) and incubated for 2h In50mlof buffer A containing 1 jj.Ci [i25|].protein A (30M^Ci m g " ' , Amersham). The membrane was

Attenuated mutants of Listeria monocytogenes washed three times with buffer B and autoradiographed ovamight with an intensifying screen (Cronex, Dupont).

Mierotitre plate haemolysin titration Sheep red blood cells (RBCs), less than three weeks old, were washed at 4 X three times in phosphate-buffered saline, pi-l 7.4 (PBS pH 7.4). They were diluted just before use into PBS pH 6.7, The dilution factor was calculated such that the ODs^, of hypotonically lysed diluted RBC would be 1,85. Microtitre plates were prepared as follows: non-sterile, untreated, V-bottom microtitre plates (Costar) were filled (200HLl/well) with 2% bovine serum albumin (BSA) in PBS pH 7.4. After incubation for 1 hour at 37''C, solution was removed and wells of the first column were filled with 100J.LI of 80mM dithiothreitol, 240mM NaCI, in PBS pH 6.0, 0.2% BSA. Routinely, lOOji' of culture supernatant was assayed and added to the wells of the first column. The other wells were filled with lOOixl of PBS pH 6.7, 0.2% BSA. One hundred microiitres from wells of the first column were then diluted 1:1 into the 100 jil of the wells of the second column and mixed. This operation was then repeated, i.e. the second column was diluted into the third, for the 12 columns of the microtitre plate, the final lOOfxl from column 12 being discarded. One hundred microiitres of washed, diluted RBCs was added. The plate was incubated for 30 min at 37X and centrifuged at 2000 r.p.m. for 5 min to pellet unlysed RBCs. Visual reading of the microtitre plate was used to estimate the well number (n) that reveals 50% lysis: the haemolytic titre in the supernatant is calculated to be 2 " ' HU ml"''. One haemolytic unit corresponds to the complete lysis of 1.5 x 10^ erythrocytes.

Cholesterol inhibition Solutions of cholesterol ranging between 3 x 10"^ M and 3 x 10 ' M were prepared in ethanol. One microlitre of these solutions was added to 99 ^,1 of culture supernatants and incubated at room temperature for 20 min. Assay for haemolytic activity was as described above.

Binding to membranes Seventy microiitres of RBCs prepared as described above was mixed with 90\x\ of culture supernatant. The mixture was incubated at room temperature for 40 min. Cells were then hypotonically lysed by addition of 160 hil of water and centrifuged for 30 min at 13 000 r.p.m. The pellet was washed three times in 1 ml of PBS pH 7.4, resuspended in 90hi.l of sample buffer (Laemmli, 1970), and heated for 5 min at 100°C. Twenty-five microiitres of the sample was then loaded on to an SDS/polyacrylamide gel and Western blotted as described above.

Virulence assays: LD^Q and growth curves Pathogen-free, ICR female Swiss mice (Charles River), 6-8 weeks old, were used in this study. Bacteria were grown in BHI broth at 37°C for lOh under agitation and harvested in log phase ( 2 x 1 0 ^ bacteria/ml) and stored at -80°C. For each experiment, aliquots were rapidly thawed and appropriately diluted inO,15M NaCI for intravenous (i.v.) inoculations (0.5ml per injection in the tail). Virulence was estimated by determining the LD50 by the log-pro-

2177

bit method (probit of % of death versus log of inoculated bacteria) on groups of five mice using five inocula for each strain (lO'', 10^, 10^, 10^ and 10^ and by monitoring bacterial growth in the liver and spleen. The aseptically removed spleens and livers from mice, killed by cervical dislocation, were separately homogenized and samples (0.1 ml) of serial dilutions of whole organ homogenates were plated on BHI agar. Colonies were counted after 24 h of incubation at S/^C, and results were expressed as the log,o bacterial counts per organ. The detection limit was 100 bacteria per organ.

Acknowledgements This investigation received financial support from the World Health Organization, as part of its programme for 'Transdisease Vaccinology', from the EEC (action de stimulation, contrat de jumelage no, STJ2-0319-C) from INSERM (CRE no. 891003 and CRE no. 873004) and from Universite Paris V (DES 3009). K.A.R. was a recipient of a Fogarty NIH-CNRS fellowship. We thank Julian Davies who enthusiastically encouraged the development of this project. We acknowledge C. Geoffroy, J. Mengaud and J. L. Gaillard for helpful discussions during this work. The technical assistance of I. Razafimanantzoa is gratefully acknowledged.

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