Construction and characterization of genetically-marked bivalent anti-Shigella dysenteriae1 and anti-Shigella flexneriY live vaccine candidates

Microbial Pathogenesis 1997; 22: 363–376

MICROBIAL PATHOGENESIS

Construction and characterization of genetically-marked bivalent anti-Shigella dysenteriae 1 and anti-Shigella flexneri Y live vaccine candidates Silke R. Kleea, Barbara D. Tzschaschela, Mahavir Singha, Inger Fa¨ltb, Alf A. Lindbergb, Kenneth N. Timmisa & Carlos A. Guzma´na∗ a

Division of Microbiology, GBF – National Research Centre for Biotechnology, Braunschweig, Germany, bDepartment of Immunology, Microbiology, Pathology and Infectious Diseases, Division of Clinical Bacteriology, Karolinska Institute, Huddinge Hospital, Huddinge, Sweden

(Received September 19, 1996; accepted in revised form December 19, 1996)

Bivalent vaccine candidates were developed against Shigella dysenteriae 1 and Shigella flexneri, which are among the most frequent causative agents of shigellosis in developing countries. The rfp and rfb gene clusters, which code for S. dysenteriae serotype 1 O-antigen biosynthesis, were inserted into an arsenite resistance minitransposon and randomly integrated into the attenuated S. flexneri aroD serotype Y strain SFL124. Nine recombinant clones that efficiently expressed both homologous and heterologous O-antigens were obtained. Southern blot analysis showed that in one clone the S. dysenteriae 1 genes had integrated into the chromosome, whereas in all the others they had integrated into the virulence plasmid. All recombinant clones exhibited normal growth characteristics, were able to invade and survive within eukaryotic cells to the same extent as the parental strain, and expressed efficiently the recombinant lipopolysaccharide within invaded cells. Immunization of mice with two of the recombinant clones resulted in the production of antibodies specific for both homologous and heterologous O-antigens. The recombinant clones constitute promising vaccine candidates which can readily be distinguished from endemic shigellae  1997 Academic Press Limited by their non-antibiotic resistance marker. Key words: LPS; O-antigen; Shigella dysenteriae serotype 1, shigellosis, vaccine.

Introduction ∗Author to whom correspondence should be addressed: Carlos A. Guzma´n, Division of Microbiology, GBF-National Research Centre for Biotechnology, Mascheroder Weg 1, D-38124 Braunschweig, Germany. 0882–4010/97/060363+15 $25.00/0 mi960127

Shigellosis is an acute invasive diarrhoeal disease, caused by members of the genus Shigella, which represents a major public health problem  1997 Academic Press Limited

364

in many developing countries. There are at least 250 million cases of shigellosis per year, of which more than 650 000 have fatal outcomes [1]. The causative agents, which spread via the faecal– oral route, are highly infectious and only a few bacteria are needed to initiate an infection [2]. Shigellae cause disease by invading the large bowel and provoking an acute inflammatory reaction, which results in extensive destruction of the mucosal sheet. They are, however, ordinarily restricted to the superficial layers of the mucosa and spread to deeper tissues is infrequent in healthy individuals. Shigella dysenteriae serotype 1, Shigella sonnei and prevalent serotypes of Shigella flexneri account for most cases [1], although infections caused by S. dysenteriae 1 are generally more severe than those caused by other Shigella species and are often characterized by serious complications such as hemolytic–uremic syndrome, leukaemoid reactions and septicemia [3]. Effective clinical management of shigellosis is compromised by the prevalence of multi-resistant strains in endemic areas [4]. Efficacious vaccines are thus urgently needed. Patients recovering from shigellosis are resistant to subsequent infection with strains of the same serotype, suggesting that (a) protection with live vaccine may be effective, and (b) that the major protective antigen is presumably the O-antigen, which is the major surface antigen of Gram-negative bacteria [5, 6]. Numerous approaches have been taken to develop a vaccine against shigellosis. Early parenteral vaccines proved not be protective [7] and pointed to oral vaccines based on live attenuated strains. Several types of oral vaccine candidates have been developed and assessed, including Escherichia coli–Shigella hybrids expressing Shigella O-antigens, which were either unsafe in humans or failed to confer immunity [8, 9], and attenuated Salmonella typhi strains expressing Shigella O-antigens [10–13], which elicited poor antibody responses in animal models, probably because the O-antigen was not covalently linked to the S. typhi lipopolysaccharide (LPS) core. The S. sonnei O-antigen was expressed in the attenuated Vibrio cholerae strain CVD103-HgR [14]. Interestingly, parenteral immunization of rabbits with heat-killed bacteria resulted in a systemic antibody response against the heterologous O-antigen, even though it was not bound to the V. cholerae LPS core [14]. More recently, an attenuated S. flexneri strain (SFL124) carrying an aroD gene deletion has been shown

S. R. Klee et al.

to be safe and immunogenic for animals and humans [15–19], and protective in different animal models against challenge with virulent S. flexneri [20–22]. In S. dysenteriae 1, the genes for O-antigen biosynthesis are located in two unlinked gene clusters, one, rfp [23], on a 9 kb multicopy plasmid, and the other, rfb, on the chromosome near the his locus [24, 25]. The rfp genes and eight contiguous genes of the rfb-cluster, together with their promoter sequences have been combined as a rfp–rfb cassette in a vector plasmid which, when introduced into E. coli K-12 [26], or attenuated derivatives of Salmonella spp. [27], directs the synthesis of S. dysenteriae 1 O-antigen. The recombinant plasmids are, however, unstable and carry an antibiotic resistance gene as selective marker, which is undesirable in vaccine candidates. We report here the construction and characterization of recombinant, geneticallymarked bivalent S. dysenteriae 1/S. flexneri Y vaccine strains obtained by the stable integration of a miniTn10-transposon carrying the rfp–rfb genes of S. dysenteriae 1 into the attenuated carrier S. flexneri SFL124 strain. The growth pattern, stability of expression of both O-antigens, invasiveness, and immunogenicity of the recombinant strains were analysed.

Results Integration of the rfp–rfb loci in S. flexneri carrier strain SFL124 and analysis of homologous and heterologous O-antigen expression by recombinant clones Initial studies have shown that the hybrid plasmid pMS26-2 [Fig. 1(a)] containing the rfp– rfb cassette that encodes for S. dysenteriae 1 Oantigen biosynthesis is unstable when maintained in SFL124 without selective pressure (see Table 1). Therefore, the rfp–rfb determinants were inserted into a miniTn10 transposon containing an arsenite resistance [28] and the recombinant transposon delivered into strain SFL124 by means of the suicide delivery plasmid pHB120 [Fig. 1(b)]. Plasmid pHB120 was transferred from the donor strain E. coli S17-1(kpir) to recipient strain S. flexneri SFL124 by filter mating [28]. After counterselection on minimal medium, nine arsenite resistant recombinant clones containing the minitransposon were identified (SFL124::Tn(rfp–

Construction and characterization of Shigella vaccines EcoRI SacI KpnI SmaI BamHI

BamHI HindIII (a)

PstI

rfp r

MER

pMS26-2 18.4 kb HindIII BamHI

rfb ori

HindIII SphI PstI PvuII (b) rfb rfp PvuII

XbaI pHB120 26.5 kb

XbaI

PvuII arsB

I end

arsA

IS10R

EcoRI

paer

ptac laclq

O end

PvuII Ap

mobRP4

ori R6K

EcoRI

Figure 1. Constructs containing the rfp–rfb cassette coding for S. dysenteriae 1 O-antigen synthesis (a) plasmid pMS26-2 is a pUC-derivative in which the mercury resistance genes MERr had been cloned into the single ScaI site, thereby inactivating the ampicillin resistance gene. (b) plasmid pHB120 is based on the suicide transposon vector pLOF/Ars [28] and contains the rfp–rfb determinants cloned into the single XbaI site. Abbreviations: I end and O end, inverted repeats of Tn10; IS10R, transposase gene; lacIq, lac repressor gene; Ap, gene for ampicillinresistance; arsA and arsB, genes encoding arsenite resistance; mobRP4, mobilization functions (oriT) of RP4 plasmid; oriR6K, origin of replication of R6K plasmid; ptac, ptac promoter; paer, aerobactin promoter.

rfb)-1 to -9). Absence of the delivery plasmid was analysed by plating clones onto agar plates supplemented with 100 lg/ml ampicillin. Only one out of nine recombinants, SFL124::Tn(rfp–

365

rfb)-9, was resistant to ampicillin and thus results from a cointegration event instead of transposition. LPS from the E. coli S17-1(kpir) donor harboring pHB120, a wild type S. dysenteriae 1, and Congo red-positive colonies of the parental and the recombinant S. flexneri strains, was isolated and separated by tricine-SDS-PAGE. Development of the gels by silver staining [Fig. 2(a)] indicated that approximately the same amount of LPS was loaded on each lane and that all strains exhibited the smooth ladder pattern in which each successive band represents an additional O-antigen unit linked to the lipid Acore. Differences in the LPS pattern and in the number of O-antigen units were observed between parental and recombinant SFL124 strains containing the rfp–rfb cassette either on pMS262 or stably integrated via the miniTn10. Recombinant strain patterns exhibited weak intermediate bands, presumably O-antigen repeat units of S. dysenteriae 1, although the number of O-antigen units was lower that for S. dysenteriae 1 or E. coli S17-1(kpir) (pHB120). Western blot analysis [Fig. 2(b) and (c)] with antibodies against S. flexneri or S. dysenteriae 1 LPS showed expression of homologous Oantigen in all S. flexneri strains and the synthesis of lower amounts of heterologous S. dysenteriae 1 O-antigen in SFL124 (pMS26-2) and in all 9 recombinants. The yield of S. dysenteriae 1 Oantigen was slightly lower in bacteria containing the rfp–rfb cassette in the minitransposon (monoor oligocopy) than in bacteria carrying the plasmid pMS26-2 (high copy) [Fig. 2(c)]. The amount of S. dysenteriae 1 O-antigen produced varied among the different exconjugants, with the weakest expression by SFL124::Tn(rfp–rfb)-6 and the strongest by SFL124::Tn(rfp–rfb)-9, which harbors a cointegrate plasmid. The expression of heterologous antigen does not seem to influence the quality of the homologous O-antigen synthesized by the carrier strain (Fig. 2). This was further supported by slide agglutination performed with specific polyclonal and monoclonal antibodies (not shown). When SFL124 strains carried the rfp–rfb cassette in a pUC-derivative (pSDM4), the sugar analysis revealed that the lipid A-core molecules synthesized by S. flexneri were equally substituted by S. dysenteriae 1 and S. flexneri Oantigens [29], demonstrating that the S. flexneri enzyme responsible for ligation of O-antigen to the core functions equally well with both homologous and heterologous O-antigens. In

SFL124

SFL124 (pMS26-2) with Hga 96.0 78.9 47.0 96.0 78.9 47.0

97.8 90.0 46.4

1

69.0 23.0 10.0

SFL124 (pMS26-2) without Hgb

99.5 97.2 63.9

99.5 97.2 63.9

2

99.6 97.8 90.8

99.6 97.8 90.8

3

99.9 96.8 98.4

99.9 96.8 98.4

4

b

a

100.0 96.6 82.1

100.0 96.6 82.1

5

99.7 99.2 97.6

100.0 100.0 100.0

6

SFL124:Tn(rfp–rfb)-

Strains were cultured and plated on selective medium containing 10 lg/ml HgCl2. Strains were cultured and plated on non-selective medium. c S. dysenteriae 1 O-antigen production was determined by colony blotting. d Congo red-positive colonies were assessed by plating on TS-agar supplemented with 0.01% Congo red.

S. dysenteriae 1 O-antigen productionc: 7 0.0 100.0 14 0.0 100.0 21 0.0 100.0 Congo red-positive phenotyped: 7 99.9 96.8 14 99.3 74.5 21 97.8 2.0

No. of generations

Percentage of colonies positive for the analysed phenotype

100.0 99.2 98.3

100.0 100.0 100.0

7

99.9 99.5 98.0

99.9 99.5 98.0

8

99.9 99.6 99.4

100.0 100.0 100.0

9

Table 1. Stability of S. dysenteriae 1 O-antigen production and the Congo red phenotype in S. flexneri SFL124 hybrids carrying the rfp–rfb determinants.

366 S. R. Klee et al.

Construction and characterization of Shigella vaccines 1

2

3

4

5

6

367 7

8

9

10

11

12

13

(a)

(b)

(c)

Figure 2. Synthesis of homologous and heterologous O-antigen in parental and recombinant S. flexneri SFL124 strains. LPS was separated by SDS-PAGE and developed by silver staining (a) or by immunoblotting with polyclonal antibodies against S. flexneri (b) or S. dysenteriae 1 (c) O-antigen. LPS from the wild type S. dysenteriae 1 W30864 (lane 1) and from the donor used in the bacterial mating, E. coli S17-1(k-pir) (pHB120) (lane 2), were included as controls. Lane 3, S. flexneri SFL124 (parental strain); lane 4, SFL124 (pMS26-2); lanes 5 to 13, recombinant strains SFL124::Tn(rfp–rfb)-1 to -9.

this study, we found higher amounts of S. flexneri O-antigen than of S. dysenteriae 1 O-antigen in bacteria in which the rfp–rfb genes had been integrated. The rfp determinant is present on a multicopy plasmid in S. dysenteriae 1 [23],

whereas it is present in only one copy in recombinant S. flexneri hybrids in which the rfp–rfb cassette is integrated either in the chromosome or the virulence plasmid. After introduction of a pUC-derivative containing the rfp-gene into

368

SFL124::Tn(rfp–rfb)-6, expression of S. dysenteriae 1 O-antigen was comparable to that in SFL124 (pMS26-2) (not shown). This suggests that the rfp gene product may be the limiting factor in LPS production and that the lower expression of S. dysenteriae O-antigen in the hybrid strains was due, at least in part, to the low rfp copy number. Western blot analysis cannot reveal whether all or only some recombinant bacteria synthesize the heterologous S. dysenteriae 1 O-antigen. Individual bacteria were thus labelled with differentially-marked polyclonal antibodies against S. dysenteriae 1 and monoclonal antibodies against S. flexneri LPS. Immunofluorescence microscopy showed that for all recombinant clones, all bacteria expressed both homologous and heterologous O-antigen (data not shown). Signals for the homologous antigen were stronger than those for the heterologous LPS, with SFL124::Tn(rfp–rfb)-9 giving the strongest heterologous LPS signal and SFL124::Tn(rfp– rfb)-6 giving the weakest. The synthesis of the heterologous O-antigen by recombinant bacteria growing in vitro does not entail that equally good production will be achieved in vivo after invasion of host cells. Therefore, the production of S. dysenteriae type 1 O-antigen by intracellularly growing bacteria was further investigated. Immunofluoresence analysis of bacteria 5 h after injection of Henle cells showed that the heterologous O-antigen was also synthesized by all hybrid S. flexneri bacteria within invaded cells under these in vivo mimicking conditions (Fig. 3).

Southern blot analysis of recombinant SFL124 strains Total DNA from Congo red-positive colonies of parental and recombinant S. flexneri strains and the control plasmid pHB120 were cleaved with PvuII, and the resulting fragments were separated by agarose gel electrophoresis, transferred to nylon membranes, and hybridized with labelled probes specific for the rfpB gene [Fig. 4(a)], the arsA gene [Fig. 4(b)], and the transposase gene IS10R [Fig. 4(c)]. The transposase gene was only detected in clone SFL124::Tn(rfp– rfb)-9, thus confirming that this clone was generated by a cointegration of the entire delivery plasmid [Fig. 4(c)]. The sizes of the PvuII-fragments that hybridized with probes for the rfpB or the arsA gene were approximately the same

S. R. Klee et al.

for clones 1, 3, 4 and 5, and differed from those of clones 2, 6, 7, 8 and 9. This suggested either a hot-spot for integration or the presence of siblings of one initial clone. Two fragments of clone 9 hybridized with the rfpB and the arsA probes [Fig. 4(a) and (b)], suggesting the presence of two copies of the minitransposon. Shigella strains harbor the virulence-associated plasmid and several small cryptic plasmids, in addition to their chromosomes, all of which could be the site of integration of the rfp–rfbcarrying minitransposon. To assess integration in virulence plasmids, plasmids from Congo red-positive and Congo red-negative colonies from each recombinant clone were subjected to electrophoresis on a 0.7% agarose gel (Fig. 5), transferred to nylon membranes and probed with an oligonucleotide specific for the rfpB gene [Fig. 5(b)]. Deletions were observed in the virulence plasmid of all Congo red-negative colonies [Fig. 5(a)]. The rfp-probe hybridized with the plasmids of all Congo red-positive colonies, except that of clone 6, and also with the deletion plasmids of Congo red-negative colonies of clones 7 and 9. This suggests that the rfp–rfb genes are integrated into the chromosome of only SFL124::Tn(rfp–rfb)-6, and that in all other recombinants the genes are integrated into the virulence plasmid. Interestingly, the deletions responsible for the Congo red-negative phenotype of Congo red derivatives of clones 7 and 9 do not encompass the rfp–rfb genes, whereas in the other clones the deletions do. These results were confirmed by Southern blot analysis of total DNA, where only DNA of Congo red-negative colonies of clones 6, 7 and 9 hybridized with a probe for the rfpB gene (data not shown).

Growth curves and stability of the Oantigen expression in the recombinant strains In order to analyse whether integration of the minitransposon inactivated genes encoding functions important for growth and robustness of the host organism, growth curves of parental and recombinant strains were compared. Overnight cultures of each strain were diluted to an OD600 of approximately 0.1, and growth to the stationary phase was monitored. The results obtained failed to reveal any impairment in growth characteristics of recombinant strains resulting from integration of the rfp–rfb cassette,

Construction and characterization of Shigella vaccines

369

Figure 3. Intracellular expression of homologous and heterologous LPS, and actin polymerization by recombinant strains. Five hours after infection, the intracellular expression of LPS by S. flexneri SFL124 (a to c) and SFL124::Tn(rfp–rfb)-8 (d to f) was assessed using antibodies against S. dysenteriae 1 (a and d) and S. flexneri (b and e) LPS, as described in Materials and methods. The ability of SFL124::Tn(rfp–rfb)-8 to direct actin polymerization was analysed 5 h after infection by staining invaded cells for F-actin with rhodaminephalloidin (g), and for bacterial LPS with fluorescein-labelled antibodies against S. flexneri LPS (h). Actin tails are indicated by arrowheads (g). Phase-contrast micrographs of the corresponding fields are also shown (c, f and i). Similar results were observed for all the other recombinant strains. The magnification is the same for all panels; the bar represents 10 lm.

similar results were obtained using either rich or minimal medium (data not shown). Recombinant strains were subcultured for 3 days to assess the stability of the virulence plasmid and rfp–rfb expression. Approximately 100% of the SFL124 (pMS26-2) bacteria synthesized S. dysenteriae 1LPS after 21 generations of growth in medium with HgCl2 (see Table 1),

whereas 90% of them lost the plasmid when cultured without selective pressure. In contrast, almost 100% of the Congo red-positive bacteria from the hybrid clones expressed S. dysenteriae 1 LPS after 21 generations. Congo red-negative colonies of clones 1, 2, 3, 4, 5 and 8 were segregated at different frequencies, and these failed to synthesize S. dysenteriae 1 LPS, whereas

370

S. R. Klee et al. 1

(a)

2

3

4

5

6

7

8

9

10 11

kb 12.0

1

2

3

4

5

6

7

8

9

10

11

12

(a)

7.9 6.0 4.8 4.3 (b)

2.3 2.0 (b) 14.0 8.7 7.0

4.2 3.9

(c) 5.6

Figure 4. Southern blot analysis of parental and recombinant S. flexneri SFL124 strains. DNAs were digested with PvuII and the fragments thereby generated separated on agarose gels, transferred to nylon membranes, and hybridized with labelled oligonucleotides complementary to either the rfpB (a), or the arsA (b), or the transposase (c) genes. Lane 1, PvuII-digested pHB120 control, lane 2, total DNA from SFL124 strain, lanes 3 to 11, total DNA from recombinants SFL124::Tn(rfp–rfb)-1 to -9. The sizes of the fragments are indicated in kb on the right side by arrowheads.

Congo red-negative colonies of clones 6, 7 and 9 retained their ability to synthesize S. dysenteriae 1LPS, confirming the data of the Southern blot analysis (Table 1). Since S. flexneri strain SFL124 has been attenuated through deletion of the aroD gene and the integration of foreign genes might influence the auxotrophic properties of this strain, dependence of recombinant clones on aromatic amino acids was tested by plating on minimal medium. No change in the auxotrophic phenotype in any of the recombinant clones was detected (data not shown).

Figure 5. Preferential integration of the rfp–rfb minitransposon into the invasion plasmid of S. flexneri. The invasion plasmids were isolated, subjected to electrophoresis on a 0.7% agarose gel and stained with ethidium-bromide (a) or, after transfer to a nylon membrane by Southern blotting, hybridized to a probe homologous to the rfpB gene (b). Lane 1, S. flexneri SFL124 Congo red-positive (cr+) colony; lane 2, SFL124 Congo red-negatie (cr−) colony; lane 3 and 4, cr+ and cr− colonies of SFL124::Tn(rfp–rfb)-4; lane 5 and 6, cr+ and cr− colonies of SFL124::Tn(rfp–rfb)6; lane 7 and 8, cr+ and cr− colonies of SFL124:: Tn(rfp–rfb)-7; lane 9 and 10, cr+ and cr− colonies of SFL124::Tn(rfp–rfb)-8; lane 11 and 12, cr+ and cr− colonies of SFL124::Tn(rfp–rfb)-9. The intact and deleted virulence plasmids of SFL124::Tn(rfp–rfb)-1, 2, -3, and -5 gave the same result as those of SFL124:: Tn(rfp–rfb)-4 and -8 (data not shown).

Invasiveness of recombinant strains Since invasiveness is a critical feature of the SFL124 vaccine strain, and many virulenceassociated proteins required for invasion are encoded on the 220 kb virulence plasmid [30–32], the preferential insertion of the rfp–rfb minitransposon in the large plasmid might have inactivated functions necessary or important for invasion and hence for vaccine efficacy. That the plasmid is retained in the recombinant clones was ascertained by checking for the Congo redpositive phenotype and by PCR amplification of a specific 320 bp fragment of the invasion plasmid (data not shown). Invasion assays using the eukaryotic cell line Henle 407 were subsequently performed to compare the invasiveness of the parental SFL124 strain with that of the recombinants. All recombinant clones retained their invasive and survival capabilities or survive even better than the parental strain

% cfu of control SFL 124 strain

Construction and characterization of Shigella vaccines 400 * 300

200

*

*

* *

**

*

Immunogenicity of recombinant SFL124 strains

* 1

be seen in Fig. 3, recombinant strains retained the ability to direct actin polymerization in a polar fashion and thereby spread within the invaded cell.

*

100

0

371

2

3

4

5

6

7

8

*** * * 9 NC SFL1 S.d.

Figure 6. Invasiveness and intracellular survival of recombinant S. flexneri strains. Henle cells were infected with the parental strain SFL124, the recombinant S. flexneri strains (1 to 9 refers to SFL124:: Tn(rfp–rfb)-1 to 9), a Congo red-negative non-invasive derivative of SFL124 (NC), the wild type S. flexneri (SFL1), and S. dysenteriae 1 (S.d.), and intracellular bacteria were harvested 2 h, 5 h, and 24 h after infection. The cfu recovered per well were compared with the number of viable bacteria harvested from cells infected with the strain SFL124. The number of cfu recovered per well for strain SFL124 was 1.2×105 (0.4% of the initial inoculum), 4.0×105, and 2.4×104 after 2, 5, and 24 h, respectively. The results reported are mean values of three independent assays, standard errors are represented by vertical lines. Results which differ significantly (PΖ0.05) from the control strain SFL124 are indicated by an asterisk (∗). Φ: 2 h; ∆: 5 h; Ε: 24 h.

(Fig. 6). The recovery of the wild type S. flexneri Y (SFL1) and S. dysenteriae 1 control bacteria was much lower 24 h after infection due to lysis of the invaded cells [33]. The underlying mechanism involved in the improved survival has not been elucidated. However, we can speculate that a modification in surface hydropathy may lead to an improved bacterial attachment and invasion. Alternatively, the specific structure of the Oantigenic polysaccharide may contribute by itself to the virulence, as has been previously hypothesized [34]. However, it seems unlikely that this may have any impact in the safety of the vaccines prototypes since the auxotrophic phenotype is maintained and eukaryotic cells infected with the recombinant clones were intact 24 h after infection in contrast to those infected with virulent Shigellae. Then, we analysed whether bacterial interaction cytoskeletal proteins, which mediates intracellular and cell-to-cell spread [35], was also maintained intact. This interaction was examined by simultaneous labelling of cellular Factin and bacterial LPS in invaded cells. As can

Mice were injected intraperitoneally with heatkilled recombinant strains SFL124::Tn(rfp–rfb)-8, and -9 (highest expression of heterologous Oantigen), the parental strain SFL124, and the wild type S. dysenteriae 1 strain, and IgG and IgM antibodies elicited against S. flexneri and S. dysenteriae LPS were measured. Mice immunized with S. flexneri SFL124 and S. dysenteriae 1 exhibited elevated titers of antibodies against the homologous LPS and marginally elevated crossreacting antibodies against the heterologous Oantigens (Table 2). Antibody titers against the homologous LPS elicited by immunization with recombinant clones were similar to those of the control group immunized with the SFL124 strain (P>0.05). This demonstrates that the immunogenic properties of the carrier strain are retained in recombinants. Titers against the heterologous O-antigen induced in mice immunized with the recombinant vaccine candidates were also significantly elevated (PΖ0.05) over those of the group immunized with the parental strain, particularly in mice immunized with SFL124::Tn(rfp–rfb)-9 bacteria, which harbors a cointegrate and seems to synthesize the highest amounts of heterologous O-antigen [see Fig. 2(c)]. These data demonstrate that enough heterologous LPS molecules are expressed on the surface of the recombinant bacteria to induce a specific immune response, a prerequisite for a bivalent vaccine strain.

Discussion The high mortality rates of infections caused by Shigella spp. and the detection of isolates which exhibit multiple antibiotic resistance [4] prompted the WHO to assign a high priority to the development of efficacious vaccines [36]. The aromatic amino acid-dependent S. flexneri aroD serotype Y strain SFL124 is a promising oral vaccine candidate for shigellosis [16–19, 21]. We

372

S. R. Klee et al.

Table 2. Serum antibodies elicited after mice immunization with S. flexneri SFL124, S. dysenteriae 1, or recombinant S. flexneri SFL124:: Tn(rfp–rfb) strains. Titersa of antibody against

Immunizing strain (n=5)

S. flexneri LPS

Non-immunized SFL124 S. dysenteriae 1 SFL124::Tn(rfp–rfb)-8 SFL124::Tn(rfp–rfb)-9

S. dysenteriae 1 LPS

IgG

IgM

IgG

IgM

44∗ 4850 606∗ 3200 5572

400∗ 29407 1600∗ 44572 19401

87∗ 1600 7352∗ 2111 7352∗

303∗ 1393 12232∗ 3200∗ 3676∗

a Geometric mean of the maximal reciprocal dilution which elicited an optical density greater than 0.1 after 1 h of incubation with substrate. ∗Titer differs significantly from titer of control group immunized with SFL124 (PΖ0.05).

describe here the development of hybrid bivalent vaccine candidates against S. flexneri serotype Y and S. dysenteriae serotype 1 based on a hybrid SFL124 derivative which expresses both homologous and S. dysenteriae 1 O-antigens. The rfp–rfb determinants encoding biosynthesis of the O-antigen of S. dysenteriae 1 were stably integrated into either the chromosome or the invasion plasmid of the carrier strain by means of a minitransposon containing a non-antibiotic arsenite resistance selection marker. Furthermore, the absence of a transposase gene in the minitransposon ensures stability of the integrated genes and eliminates the possibility of further transposition. The S. dysenteriae 1 LPS molecules produced by the recombinant S. flexneri strains somewhat were shorter than those produced by S. dysenteriae 1 itself (Fig. 2), presumably due either to a weaker synthesis of S. dysenteriae 1 Oantigen units compared to that of S. flexneri, or to a lack of the S. dysenteriae 1-specific rol gene (which determines chain length [37]) in the rfbcluster of pHB120, and reliance of S. dysenteriae 1 O-antigen formation on the S. flexneri rol gene [38]. Comparison of the parental and recombinant strains in terms of growth characteristics and dependence upon aromatic amino acids failed to detect any differences, indicating that important genes were not affected by integration of rfp–rfb cassette. Expression of the heterologous O-antigen by SFL124::Tn(rfp–rfb)-6 was stable with no loss after 21 generations in the absence of

selective pressure, whereas in the other recombinants heterologous O-antigen expression was linked to the stability of the virulence plasmid. Since this plasmid is essential for the efficacy of the vaccine strain and the stability of the Congo red-positive phenotype was unaffected in the recombinant strains, it is in principle immaterial whether the rfp–rfb determinants are integrated into the chromosome or the virulence plasmid. In fact, integration into the virulence plasmid seems to provide increased expression yields. Concerns that integration of the minitransposon into the invasion plasmid might result in inactivation of critical virulence functions proved to be unwarranted since immunofluorescence and infection studies showed that the recombinant strains interact with the host cell in the same way as the parental strain. Furthermore, both the homologous and heterologous O-antigens were efficiently expressed within invaded eukaryotic cells. Immunization of mice with heat-killed recombinant strains induced specific antibody responses against both homologous and heterologous LPS. This demonstrates that at least as assessed in the mice model the recombinant O-antigen is delivered by the bacteria in an immunogenic conformation which does not interfere with the expression of the homologous LPS, suggesting that in principle protection against both pathogens can be achieved. Finally, the presence of genes encoding arsenite resistance in the recombinant strains will allow ready differentiation between recombinant and

Construction and characterization of Shigella vaccines

wild type strains and detection of any virulent revertants of a vaccine. This will be important in field trials of vaccine strains in countries where shigellosis is endemic, where vaccines may shed both the vaccine strain and endemic shigellae.

Materials and methods Bacterial strains, plasmids and media The aroD auxotrophic mutant of S. flexneri serotype Y strain SFL124 was used as carrier strain [16]. The S. flexneri Y SFL1 [15] and the S. dysenteriae 1 W30864 [23] wild type strains were used as controls. E. coli S17-1(kpir) [39] was used to mobilize hybrid plasmid pHB120 into strain SFL124. Plasmid pMS26-2 [Fig. 1(a)] contains the rfp–rfb cassette in a pUC19-derivative in which the ampicillin resistance gene had been replaced by the mercury resistance genes from plasmid pHP45X-Hg [40]. Plasmid pHB120 [Fig. 1(b)]; H. N. Brahmbhatt, unpubl. data) contains the rfp–rfb cassette from plasmic pSS37 [26] cloned into the XbaI-site of the pLOF/Ars vector [28]. Shigella strains were grown in trypticase soy (TS) broth (Difco Laboratories, Augsburg, Germany) or TS agar supplemented with 0.01% Congo red (Sigma Chemie GmbH, Deisenhofen, Germany) to detect the presence of the virulence plasmid. 121-salt minimal medium [41] supplemented with 200 lm K2HPO4, 0.2% glucose, 10 lg/ml nicotinic acid, aromatic compounds (40 lg/ml of typtophane, tyrosine, and phenylalanine, 10 lg/ml of p-aminobenzoic acid and 2,3-dihydroxybenzoic acid) and 1.5 mm NaAsO2 was used to counterselect donor bacteria in matings. Aromatic amino acid dependence of transconjugants was checked by streaking parental and recombinant shigellae on 121-salt minimal medium lacking aromatic compounds. E. coli S17-1(kpir) with pHB120 was grown on Luria Bertani medium [42]. Where required, ampicillin (100 lg/ml) and HgCl2 (10 lg/ml) were used for selection, and 100 lm isopropyl-b-D-thiogalactopyranoside (IPTG) was used for induction.

Transfer of plasmids to Shigella spp. Plasmid pMS26-2 was introduced into strain SFL124 by electroporation [43], whereas plasmid

373

pHB120 was transferred from the donor strain E. coli S17-1(kpir) into strain SFL124 by mobilization using a filter mating technique [28].

Lipopolysaccharide (LPS) isolation and immunological analysis LPS from whole cell lysates was prepared as described by Hitchcock and Brown [44], fractionated by tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) [45], and gels were developed either by silver staining [46] or Western blotting. For Western blotting LPS was transferred as previously described [29] to Immobilon polyvinylidene difluoride membranes (Millipore, Eschborn, Germany). The membranes were then blocked, incubated with rabbit polyclonal antiserum against O-antigen of either S. dysenteriae type 1 or S. flexneri (Behringwerke, Marburg, Germany), washed, and then incubated with horseradish-peroxidase-conjugated goat–anti-rabbit IgG (Jackson ImmunoResearch Laboratories, Inc., PA, USA). LPS bands were visualized by chemiluminescence using the Amersham ECL system (Amersham, Braunschweig, Germany). Colony immunoblotting was carried out by transferring the colonies from agar plates to nylon membranes (Biodyne A, Pall, Dreieich, Germany), baking the filters under vacuum in a gel drier (BioRad Laboratories, Mu¨nchen, Germany) at 60°C for 20 min, and then performing the immunodetection as described for Western blots.

Detection of the virulence-associated plasmid by polymerase chain reaction (PCR) To confirm the presence of the virulence plasmid, one Congo red-positive colony of each clone was suspended in 200 ll of distilled H2O and heated for 12 min at 95°C. 10 ll of this suspension was then subjected to PCR with the PCR reagent kit from Perkin Elmer (Vaterstetten, Germany), in accordance with manufacturer’s instructions, using two primers that flank a 320 bp region of the virulence plasmid [47], followed by analysis of the PCR-products by electrophoresis on a 1.5% agarose gel.

374

S. R. Klee et al.

Southern analysis of chromosomal and virulence plasmid DNA

Analysis by immunofluorescence microscopy

Chromosomal DNA or virulence plasmids of Congo red-positive and -negative colonies obtained as previously described [48, 49] were separated on 0.7% agarose gels and transferred to Biodyne B nylon membranes (Pall). The DNA was probed with digoxigenin-labelled oligonucleotides (DIG Oligonucleotide 3′-End Labelling Kit; Boehringer, Mannheim, Germany) that were complementary to sequences of the Tn10transposase (5′-CCAGGCATTACTTGACTGTA AAACTCT-3′) [50], the rfpB (5′-GGGATT TCAGTCTATGCTTTTGCTACT-3′) [51, 52], and the arsA (5′-GCTGGATTATCAATAACAGCC TTTCCA-3′) [53] genes. After hybridization using the DIG Luminescent Detection Kit for Nucleic Acids (Boehringer), the membranes were washed and the light emission of bound probes was recorded on Kodak X-ray film.

To examine bacterial synthesis of homologous and heterologous LPS within invaded eukaryotic cells, the cells were infected as described above and, after incubation for 5 h, were fixed with 3.7% formaldehyde in PBS, permeabilized with 0.2% Triton X-100 in PBS, washed with PBS, and immunofluorescence was performed using polyclonal rabbit antibodies against S. dysenteriae 1 O-antigen and the monoclonal antibody MASF Y-5 [55] against S. flexneri Y as first antibodies, and fluorescein-labelled goat–antirabbit IgG and rhodamine-labelled goat–antimouse IgG (Jackson ImmunoResearch Laboratories) as secondary antibodies. Samples examined under phase contrast or epifluorescence using a Zeiss axiophot microscope (Carl Zeiss, Oberkochen, Germany). The interaction of bacteria with host cell actin after infection [35] was visualized by the staining of F-actin with rhodamine-labelled phalloidin used at a 1:100 dilution in 5% FCS in PBS (Medac, Hamburg, Germany). Bacteria were stained with rabbit polyclonal antibodies against S. flexneri LPS diluted 1:10 in 5% FCS in PBS and fluorescein-labelled goat–anti-rabbit IgG (1:50) as secondary antibodies.

Stability of the virulence plasmid and rfp–rfb expression Congo red-positive colonies of SFL124 (pMS262) were inoculated into TS broth with or without HgCl2, and Congo red-positive colonies of the hybrid SFL124::Tn(rfp–rfb)-1 to -9 clones were inoculated into TS broth without arsenite. The cultures were diluted 1:100 in fresh medium every day (24 h of growth corresponds to approximately seven generations). Aliquots were removed after 24, 48 and 72 h, approximately diluted, and plated on TS-agar containing 0.01% Congo red to assess the Congo red-positive phenotype, whereas the synthesis of S. dysenteriae 1 LPS was determined by colony blotting.

Immunization by mice Groups of five 6-week-old female BALB/c mice were immunized on days 0, 14 and 28 by injecting intraperitoneally 0.2–1.0×108 heat-killed (60°C, 1 h) bacteria suspended in PBS. Two weeks after the last immunization the mice were sacrificed, blood samples were collected and antibody titers against LPS of S. flexneri SFL124 and S. dysenteriae 1 were determined by enzymelinked immunosorbent assay (ELISA).

Invasion assays

ELISA

Invasion assays were performed using Henle 407 cells as previously described [54]. The results reported are expressed as percentages of the cfu recovered per well from cells infected with strain SFL124 and are mean values of three independent assays±standard errors. They were analysed for significance by Student’s t test; differences were considered significant at P<0.05.

LPS was prepared by the method of Westphal and Jann [56], further purified by treatment with RNase, DNase, and proteinase K, and used in a 10 lg/ml solution in 0.1 m NaHCO3 (pH 9.6) to coat 96-well microtiter plates (Nunc MaxiSorp; Inter Med Nunc, Wiesbaden, Germany) (100 ll per well). After overnight incubation at 4°C, plates were washed, blocked, and serially diluted mice sera (100 ll/well) were added and

Construction and characterization of Shigella vaccines

incubated for 2 h at 37°C. Then, plates were washed and horseradish peroxidase-conjugated goat anti-mouse IgG (Fc fragment specific) or IgM (Mu chain specific) (Jackson ImmunoResearch Laboratories) were added for 1 h at 37°C. After three washes, 200 ll of peroxidase substrate solution (o-phenylenediamine dihydrochloride; Sigma Chemie GmbH) were added per well, incubation continued for 1 h. The antibody titer noted is expressed as the geometric mean of the maximal reciprocal dilution which elicited an optical density (490/ 595 nm) equal or above the cutoff value of 0.1. Statistical analysis was conducted using the Statgraphics Plus for Windows 1.11 Program (Statistical Graphics Corp.). Differences between the immunization groups were calculated using the Mann–Whitney (Wilcoxon) test for two-sample comparison and were considered statistically significant at PΖ0.05.

375

8

9 10

11

12

13

Acknowledgements We thank H. N. Brahmbhatt for providing plasmid pHB120 and H. Watanabe for providing the S. dysenteriae 1 strain W30864. K. N. T. gratefully acknowledges generous support from the Fonds der Chemischen Industrie.

14

15

16

References 17 1 Katz SL. The prospects for immunizing against Shigella spp., In: Institute of Medicine. New Vaccine Development: Establishing Priorities. Vol. 2. Diseases of Importance in Developing Countries. Washington DC: National Academy Press, 1986: 329–37. 2 DuPont HL, Levine MM, Hornick RB, Formal SB. Inoculum size in shigellosis and implications for expected mode of transmission. J Infect Dis 1989; 159: 1126–8. 3 Levine MM. Bacillary dysentery. Mechanisms and treatment. Med Clin North Amer 1982; 66: 623–38. 4 Casalino M, Nicoletti M, Salvia A, Colonna B, Pazzani C, Calconi A, Mohamud KA, Maimone F. Characterization of endemic Shigella flexneri strains in Somalia: antimicrobial resistance, plasmid profiles, and serotype correlation. J Clin Microbiol 1994; 32: 1179–83. 5 Levine M, Gangarosa EJ, Werner M, Morris GK. Shigellosis in custodial institutions. J Pedia 1974; 84: 803–6. 6 Robbins JB, Chu C, Schneerson R. Hypothesis for vaccine development: protective immunity to enteric diseases caused by nontyphoideal Salmonella and Shigella may be conferred by serum IgG antibodies to the Ospecific polysaccharide of their lipopolysaccharides. Clin Infect Dis 1992; 15: 346–61. 7 Formal SB, Maenza RM, Austin S, LaBrec EH. Failure

18

19

20

21

22

of parenteral vaccines to protect monkeys against experimental shigellosis. Proc Soc Exp Biol Med 1967; 25: 347–9. Hale TL, Keren DF. Pathogenesis and immunology in shigellosis: applications for vaccine development. In: Sansonetti PJ, Ed. Current Topics in Microbiology and Immunology, Vol. 180. Berlin: Springer Verlag, 1992: 117– 37. Lindberg AA, Pa´l T. Strategies for development of potential candidate Shigella vaccines. Vaccine 1993; 11: 168–79. Baron LS, Kopecko DJ, Formal SB, Seid R, Guerry P, Powell C. Introduction of Shigella flexneri 2a type and group antigen genes into oral typhoid vaccines strain Salmonella typhi Ty21a. Infect Immun 1987; 55: 2797–801. Formal SB, Baron LS, Kopecko DJ, Washington O, Powell C, Life CA. Construction of a potential bivalent vaccine strain: introduction of Shigella sonnei form I antigen genes into the galE Salmonella typhi Ty21a typhoid vaccine strain. Infect Immun 1981; 34: 746–50. Seid RC, Kopecko DJ, Sadoff JC, Schneider H, Baron LS, Formal SB. Unusual lipopolysaccharide antigens of a Salmonella typhi oral vaccine strain expressing the Shigella sonnei form I antigen. J Biol Chem 1984; 259: 9028–34. Tramont EC, Chung R, Berman S, Keren D, Kapfer C, Formal SB. Safety and antigenicity of typhoid-Shigella sonnei vaccine (strain 5076-1C). J Infect Dis 1984; 149: 133–6. Viret J-F, Cryz SJ Jr, Lang AB, Favre D. Molecular cloning and characterization of the genetic determinants that express the complete Shigella serotype D (Shigella sonnei) lipopolysaccharide in heterologous live attenuated vaccine strains. Mol Microbiol 1993; 7: 239–52. Lindberg AA, Ka¨rnell A, Pa´l T, Sweiha H, Hultenby K, Stocker BAD. Construction of an auxotrophic Shigella flexneri strain for use as a live vaccine. Microb Pathog 1990; 8: 433–40. Ka¨rnell A, Stocker BAD, Katakura S, Reinholt FP, Lindberg AA. Live oral auxotrophic Shigella flexneri SFL124 vaccine with a deleted aroD gene: characterization and monkey protection studies. Vaccine 1992; 10: 389–94. Li A, Pa´l T, Forsum U, Lindberg AA. Safety and immunogenicity of the live oral auxotrophic Shigella flexneri SFL124 in volunteers. Vaccine 1992; 10: 395–404. Li A, Ka¨rnell A, Huan PT, Cam PD, Minh NB, Tram LN, Quy NP, Trach DD, Karlsson K, Lindberg G, Lindberg AA. Safety and immunogenicity of the live oral auxotrophic Shigella flexneri SFL124 in adult Vietnamese volunteers. Vaccine 1993; 11: 180–9. Li A, Cam PD, Islam D, Minh NB, Huan PT, Rong ZC, Karlsson K, Lindberg G, Lindberg AA. Immune responses in Vietnamese children after a single dose of the auxotrophic, live Shigella flexneri Y vaccine strain SFL124. J Infection 1994; 28: 11–23. Hartman AB, Powell CJ, Schultz CL, Oaks EV, Eckels KH. Small-animal model to measure efficacy and immunogenicity of Shigella vaccine strains. Infect Immun 1991; 59: 4075–83. Ka¨rnell A, Sweiha H, Lindberg AA. Auxotrophic live oral Shigella flexneri vaccine protects monkeys against challenge with S. flexneri of different serotypes. Vaccine 1992; 10: 167–74. Mallett CP, VanDeVerg L, Collins HH, Hale TL. Evaluation of Shigella vaccine safety and efficacy in an intranasally challenged mouse model. Vaccine 1993; 11: 190–6.

376 23 Watanabe H, Timmis KN. A small plasmid in Shigella dysenteriae 1 specifies one or more functions essential for O antigen production and bacterial virulence. Infect Immun 1984; 43: 391–6. 24 Hale TL, Guerry P, Seid RC, Kapfer C, Wingfield ME, Reaves CB, Baron LS, Formal SB. Expression of lipopolysaccharide O antigen in Escherichia coli K-12 hybrids containing plasmid and chromosomal genes from Shigella dysenteriae 1. Infect Immun 1984; 46: 470–5. 25 Sturm S, Jann B, Jann K, Fortnagel P, Timmis KN. Genetic and biochemical analysis of Shigella dysenteriae 1 O antigen polysaccharide rfb gene cluster. Microb Pathog 1986; 1: 307–24. 26 Sturm S, Timmis KN. Cloning of the rfb gene region of Shigella dysenteriae 1 and construction of an rfp–rfb gene cassette for the development of lipopolysaccharidebased live anti-dysentery vaccines. Microb Pathog 1986; 1: 289–97. 27 Mills SD, Sekizaki T, Gonzalez-Carrero MI, Timmis KN. Analysis and genetic manipulation of Shigella virulence determinants for vaccine development. Vaccine 1988; 6: 116–22. 28 Herrero M, de Lorenzo V, Timmis KN. Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in gram-negative bacteria. J Bacteriol 1990; 172: 6557–67. 29 Fa¨lt IC, Schweda EKH, Klee S, Singh M, Floderus E, Timmis KN, Lindberg AA. Expression of Shigella dysenteriae serotype 1 O-antigenic polysaccharide by Shigella flexneri aroD vaccine candidates and different S. flexneri serotypes. J Bacteriol 1995; 177: 5301–5. 30 Sansonetti PJ, Kopecko DJ, Formal SB. Involvement of a plasmid in the invasive ability of Shigella flexneri. Infect Immun 1982; 35: 852–60. 31 Hale TL. Genetic bases of virulence in Shigella species. Microbiol Rev 1991; 55: 206–24. 32 Sasakawa C, Buysse JM, Watanabe H. 1992. The large virulence plasmid of Shigella. In: Sansonetti PJ, Ed. Current Topics in Microbiology and Immunology, Vol. 180. Berlin: Springer Verlag, 1992; 21–44. 33 Sansonetti PJ, Mounier J. Metabolic events mediating early killing of host cells infected by Shigella flexneri. Microb Pathog 1987; 3: 53–61. 34 Lindberg AA, Ka¨rnell A, Weintraub A. The lipopolysaccharide of Shigella bacteria as a virulence factor. Rev Infect Dis 1991; 13(suppl 4): S279–84. 35 Goldberg MB, Sansonetti PJ. Shigella subversion of the cellular cytoskeleton: a strategy for epithelial colonization. Infect Immun 1993; 61: 4941–6. 36 World Health Organization. Programme for control of diarrhoeal diseases, seventh programme report, 1988– 1989. Document WHO/CDD/90.34. Geneva: World Health Organization, 1990. 37 Batchelor RA, Alifano P, Biffali E, Hull SI, Hull RA. Nucleotide sequences of the genes regulating O-polysaccharide antigen chain length (rol) from Escherichia coli and Salmonella typhimurium: protein homology and functional complementation. J Bacteriol 1992; 174: 5228– 36. 38 Klena JD, Schnaitman CA. Function of the rfb gene cluster and the rfe gene in the synthesis of O antigen by Shigella dysenteriae 1. Mol Microbiol 1993; 9: 393–402. 39 De Lorenzo V, Eltis L, Kessler B, Timmis KN. Analysis of Pseudomonas gene products using laclq/Ptrp-lac plasmids and transposons that confer conditional phenotypes. Gene 1993; 123: 17–24.

S. R. Klee et al. 40 Fellay R, Frey J, Krisch H. Interposon mutagenesis of soil and water bacteria: a family of DNA fragments designed for in vitro insertional mutagenesis of Gramnegative bacteria. Gene 1987; 52: 147–54. 41 Kruezer K, Pratt C, Torriani A. Genetic analysis of regulatory mutants of alkaline phosphatase of E. coli. Genetics 1975; 81: 459–68. 42 Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1989. 43 O’Callaghan D, Charbit A. High efficiency transformation of Salmonella typhimurium and Salmonella typhi by electroporation. Mol Gen Genet 1990; 223: 156–8. 44 Hitchcock PJ, Brown TM. Morphological heterogeneity among Salmonella lipopolysaccharide chemotypes in silver-stained polyacrylamide gels. J Bacteriol 1983; 154: 269–77. 45 Lesse AJ, Campagnari AA, Bittner WE, Apicella MA. Increased resolution of lipopolysaccharides and lipooligosaccharides utilizing tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis. J Immunol Meth 1990; 126: 109–17. 46 Tsai C-M, Frasch CE. A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels. Anal Biochem 1982; 119: 115–9. 47 Islam D, Lindberg AA. Detection of Shigella dysenteriae type 1 and Shigella flexneri in feces by immunomagnetic isolation and polymerase chain reaction. J Clin Microbiol 1992; 30: 2801–6. 48 Richards E. Preparation of genomic DNA from bacteria. Miniprep of bacterial genomic DNA, unit 2.4.1. In: Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K, Eds. Current Protocols in Molecular Biology. New York: John Wiley & Sons, 1987: 1–2. 49 Kado CI, Liu S-T. Rapid procedure for detection and isolation of large and small plasmids. J Bacteriol 1981; 145: 1365–73. 50 Halling SM, Simons RW, Way JC, Walsh RB, Kleckner N. DNA sequence organization of IS10-right of Tn10 and comparison with IS10-left. Proc Natl Acad Sci USA 1982; 79: 2608–12. 51 Klena JD, Ashford RS II, Schnaitman CA. Role of Escherichia coli K-12 rfa genes and the rfp gene of Shigella dysenteriae 1 in generation of lipopolysaccharide core heterogeneity and attachment of O antigen. J Bacteriol 1992; 174: 7297–307. 52 Go¨hmann S, Manning PA, Alpert C-A, Walker MJ, Timmis KN. Lipopolysaccharide O-antigen biosynthesis in Shigella dysenteriae serotype 1: analysis of the plasmid-carried rfp determinant. Microb pathog 1994; 16: 53–64. 53 Chen C-M, Misra TK, Silver S, Rosen BP. Nucleotide sequence of the structural genes for an anion pump. The plasmid-encoded arsenical resistance operon. J Biol Chem 1986; 261: 15030–8. 54 Tzschaschel BD, Klee SR, de Lorenzo V, Timmis KN, Guzma´n CA. Towards a vaccine candidate against Shigella dysenteriae 1: expression of the Shiga toxin Bsubunit in an attenuated Shigella flexneri aroD carrier strain. Microb Pathog 1996; 21: 277–88. 55 Carlin NIA, Bundle DR, Lindberg AA. Characterization of five Shigella flexneri variant Y-specific monoclonal antibodies using defined saccharides and glycoconjugate antigens. J Immunol 1987; 138: 4419–27. 56 Westphal O, Jann K. Bacterial lipopolysaccharides. Extraction with phenol-water and further applications of the procedure. Methods Carbohydr Chem 1965; 5: 83–91.