Oral administration of live Shigella vaccine candidates in rhesus monkeys show no evidence of competition for colonization and immunogenicity between different serotypes.

Live oral monovalent Shigella flexneri 2a vaccine candidates as well as bivalent formulations with Shigella sonnei were evaluated in a rhesus monkey model for colonization and immunogenicity. Freshly harvested suspensions of S. flexneri 2a vaccine candidates WRSf2G12 and WRSf2G15 as well as S. sonnei vaccine candidate WRSs3 were nasogastrically administered to groups of rhesus monkeys, Macaca mulatta, either in a monovalent form or when combined with each other. The animals were monitored daily for physical well-being, stools were subjected to quantitative colony immunoblot assays for bacterial excretion and blood and stools were evaluated for humoral and mucosal immune responses. No clinical symptoms were noted in any group of animals and the vaccine candidates were excreted robustly for 48-72h without significant changes in either the magnitude or duration of excretion when given as a monovalent or as bivalent mixtures. Similarly, immunological interferences were not apparent in the magnitude of humoral and mucosal immune responses observed toward Shigella-specific antigens when monkeys were fed monovalent or bivalent formulations. These results predict that a multivalent live oral vaccine of more than one serotype can have a favorable outcome for protection against shigellosis.

Shigella sonnei vaccine candidates WRSs2 and WRSs3 are as immunogenic as WRSS1, a clinically tested vaccine candidate, in a primate model of infection.

Shigella causes diarrhea and dysentery through contaminated food and water. Shigella sonnei live vaccine candidates WRSs2 and WRSs3 are attenuated principally by the loss of VirG(IcsA) that prevents bacterial spread within the colonic epithelium. In this respect they are similar to the clinically tested vaccine candidate WRSS1. However, WRSs2 and WRSs3 are further attenuated by loss of senA, senB and WRSs3 also lacks msbB2. As previously shown in cell culture assays and in small animal models, these additional gene deletions reduced the levels of enterotoxicity and endotoxicity of WRSs2 and WRSs3, potentially making them safer than WRSS1. However the behavior of these second-generation VirG(IcsA)-based vaccine candidates in eliciting an immune response in a gastrointestinal model of infection has not been evaluated. In this study, WRSs2 and WRSs3 were nasogastrically administered to rhesus monkeys that were evaluated for colonization, as well as for systemic and mucosal immune responses. Both vaccine candidates were safe in rhesus monkeys and behaved comparably to WRSS1 in bacterial excretion rates that demonstrated robust intestinal colonization. Furthermore, humoral and mucosal immune responses elicited against bacterial antigens appeared similar in all categories across all three strains indicating that the additional gene deletions did not compromise the immunogenicity of these vaccine candidates. Based on data from previous clinical trials with WRSS1, it is likely that, WRSs2 and WRSs3 will not only be safer in human volunteers but will generate comparable levels of systemic and mucosal immune responses that were achieved with WRSS1.

Characterization of WRSs2 and WRSs3, new second-generation virG(icsA)-based Shigella sonnei vaccine candidates with the potential for reduced reactogenicity.

Live, attenuated Shigella vaccine candidates, such as Shigella sonnei strain WRSS1, Shigella flexneri 2a strain SC602, and Shigella dysenteriae 1 strain WRSd1, are attenuated principally by the loss of the VirG(IcsA) protein. These candidates have proven to be safe and immunogenic in volunteer trials and in one study, efficacious against shigellosis. One drawback of these candidate vaccines has been the reactogenic symptoms of fever and diarrhea experienced by the volunteers, that increased in a dose-dependent manner. New, second-generation virG(icsA)-based S. sonnei vaccine candidates, WRSs2 and WRSs3, are expected to be less reactogenic while retaining the ability to generate protective levels of immunogenicity seen with WRSS1. Besides the loss of VirG(IcsA), WRSs2 and WRSs3 also lack plasmid-encoded enterotoxin ShET2-1 and its paralog ShET2-2. WRSs3 further lacks MsbB2 that reduces the endotoxicity of the lipid A portion of the bacterial LPS. Studies in cell cultures and in gnotobiotic piglets demonstrate that WRSs2 and WRSs3 have the potential to cause less diarrhea due to loss of ShET2-1 and ShET2-2 as well as alleviate febrile symptoms by loss of MsbB2. In guinea pigs, WRSs2 and WRSs3 were as safe, immunogenic and efficacious as WRSS1.

Molecular epidemiology of Shigella flexneri in a diarrhoea-endemic area of Lima, Peru.

A year-long community-based study of diarrhoeal diseases was conducted in Canto Grande, a periurban community in Lima, Peru. In 109 (34%) houses out of 323 that were visited, at least one individual was detected with shigellosis. The frequency of the 161 shigella isolates obtained was as follows: 117 S. flexneri (73%), 21 S. boydii (13%), 15 S. dysenteriae (9%), and 8 S. sonnei (5%). Using a non-radioactive ipaH gene probe as a molecular epidemiological tool, a total of 41 S. flexneri strains were shown to be distributed in 25 intra-family comparisons by pairs (icp). Further subdivision, based on a comparison of the serotype, plasmid profile, antibiotic resistances and ipaH hybridization patterns indicated that Group I, with 11 icp (44%), had strains that were identical. Group II with 8 icp (32%), had strains that were different and Group III with 6 icp (24%), had strains with the same serotype and identical ipaH profiles but with differences in other markers. This data indicates that a diversity of shigella clones circulated in this community resulting from both clonal spread and horizontal transfer of genetic elements. Furthermore, ipaH profiling of isolates can be used not only to differentiate between closely related shigella strains but also with other parameters, help to understand the dynamics of the generation of new clones of pathogenic bacteria.

Complete genome sequence and comparative genomics of Shigella flexneri serotype 2a strain 2457T.

We determined the complete genome sequence of Shigella flexneri serotype 2a strain 2457T (4,599,354 bp). Shigella species cause >1 million deaths per year from dysentery and diarrhea and have a lifestyle that is markedly different from those of closely related bacteria, including Escherichia coli. The genome exhibits the backbone and island mosaic structure of E. coli pathogens, albeit with much less horizontally transferred DNA and lacking 357 genes present in E. coli. The strain is distinctive in its large complement of insertion sequences, with several genomic rearrangements mediated by insertion sequences, 12 cryptic prophages, 372 pseudogenes, and 195 S. flexneri-specific genes. The 2457T genome was also compared with that of a recently sequenced S. flexneri 2a strain, 301. Our data are consistent with Shigella being phylogenetically indistinguishable from E. coli. The S. flexneri-specific regions contain many genes that could encode proteins with roles in virulence. Analysis of these will reveal the genetic basis for aspects of this pathogenic organism's distinctive lifestyle that have yet to be explained.

Inactivation of DsbA alters the behaviour of Shigella flexneri towards murine and human-derived macrophage-like cells.

The mutants of Shigella flexneri, Sh4 (dsbA::kan) and Sh42 (dsbA33G), behave differently towards murine and human-derived macrophage-like cells in vitro. Sh4 was trapped in the phagocytic vacuoles of the murine J774 cells as evidenced by its colony forming units plus and minus chloroquine exposure in a gentamicin protection assay, and by light and transmission electron microscopy (TEM). Sh42, similar to the wild-type M90TS, was able to escape from the vacuoles and kill host cells presumably by inducing apoptosis. In U937 cells, unlike M90TS that was free in the cytosol, both Sh4 and Sh42 grew poorly. TEM revealed that Sh4 and Sh42 were trapped within the U937 phagocytic vacuoles. Furthermore, the two mutants induced different patterns of interleukin-1beta and tumour necrosis factor-alpha expression, which might explain why they possess different immunogenic properties in vivo.

Complete DNA sequence and analysis of the large virulence plasmid of Shigella flexneri.

The complete sequence analysis of the 210-kb Shigella flexneri 5a virulence plasmid was determined. Shigella spp. cause dysentery and diarrhea by invasion and spread through the colonic mucosa. Most of the known Shigella virulence determinants are encoded on a large plasmid that is unique to virulent strains of Shigella and enteroinvasive Escherichia coli; these known genes account for approximately 30 to 35% of the virulence plasmid. In the complete sequence of the virulence plasmid, 286 open reading frames (ORFs) were identified. An astonishing 153 (53%) of these were related to known and putative insertion sequence (IS) elements; no known bacterial plasmid has previously been described with such a high proportion of IS elements. Four new IS elements were identified. Fifty putative proteins show no significant homology to proteins of known function; of these, 18 have a G+C content of less than 40%, typical of known virulence genes on the plasmid. These 18 constitute potentially unknown virulence genes. Two alleles of shet2 and five alleles of ipaH were also identified on the plasmid. Thus, the plasmid sequence suggests a remarkable history of IS-mediated acquisition of DNA across bacterial species. The complete sequence will permit targeted characterization of potential new Shigella virulence determinants.

Shigella flexneri IpaH(7.8) facilitates escape of virulent bacteria from the endocytic vacuoles of mouse and human macrophages.

The behavior of Shigella flexneri ipaH mutants was studied in human monocyte-derived macrophages (HMDM), in 1-day-old human monocytes, and in J774 mouse macrophage cell line. In HMDM, strain pWR700, an ipaH(7.8) deletion mutant of S. flexneri 2a strain 2457T, behaved like the wild-type strain 2457T. This strain caused rapid host cell death by oncosis, and few bacterial CFU were recovered after incubation in the presence of gentamicin as previously described for 2457T-infected HMDM. However, analysis of bacterial compartmentalization within endocytic vacuoles with gentamicin and chloroquine indicated that more pWR700 than 2457T was present within the endocytic vacuoles of HMDM, suggesting that ipaH(7.8) deletion mutant transited more slowly from the vacuoles to the cytoplasm. In contrast to findings with HMDM, CFU recovered from pWR700-infected mouse J774 cells were 2 to 3 logs higher than CFU from 2457T-infected J774 cells. These values exceeded CFU recovered after infection of J774 cells with plasmid-cured avirulent strain M4243A1. Incubation with gentamicin and chloroquine clearly showed that pWR700 within J774 cells was mostly present within the endocytic vacuoles. This distribution pattern was similar to that seen with M4243A1 and contrasted with the pattern seen with 2457T. Complementation of pWR700 with a recombinant clone expressing ipaH(7. 8) restored the intracellular distribution of bacteria to that seen with the wild-type strain. Strains with deletions in ipaH(4.5) or ipaH(9.8), however, behaved like 2457T in both HMDM and J774 cells. The distribution profile of pWR700 in 1-day-old monocytes was similar to that seen in J774 cells. Like infected J774 cells, 1-day-old human monocytes demonstrated apoptosis upon infection with virulent Shigella. These results suggest that a role of the ipaH(7. 8) gene product is to facilitate the escape of the virulent bacteria from the phagocytic vacuole of monocytes and macrophages.

Native and mutant forms of cholera toxin and heat-labile enterotoxin effectively enhance protective efficacy of live attenuated and heat-killed Shigella vaccines.

Both native and mutant forms of cholera toxin (CT) and heat-labile enterotoxin (LT) are effective adjuvants for antigens and killed whole-cell preparations. To determine whether these toxin molecules could also boost the immunogenicity and efficacy of live attenuated vaccines directed against shigellosis, the guinea pig keratoconjunctivitis model was used to evaluate the adjuvant effect of these toxin molecules on EcSf2a-3, a DeltavirG DeltaaroD Escherichia coli-Shigella flexneri 2a hybrid vaccine strain that was previously found to be less protective than its parent strain in the guinea pig model. Experiments using native and mutant toxin molecules showed that both CT and LT and mutant derivatives were effective as an adjuvant for EcSf2a-3 and that the mutant toxin molecules, which were developed to retain adjuvanticity without the toxicity associated with the native molecules, were as effective as the native toxin molecules as adjuvants. Protective efficacy was enhanced for both the oral and intranasal routes of immunization. Serum antibody response to the S. flexneri 2a O antigen, the primary antigen for protective immunity, was not dependent on the addition of an adjuvant. However, enumeration of the O-antigen-specific immunoglobulin G (IgG) and IgA antibody-secreting cells in the spleen and draining lymph nodes following intranasal immunization suggested that enhancement of the local immune response by the toxin molecules may contribute to the observed increase in protective efficacy. The efficacy of heat-killed S. flexneri 2a was enhanced only by mutant LT molecules. These results suggest that the best candidates for enhancing the efficacy of both live attenuated and heat-killed Shigella vaccines with minimal reactogenicity are the mutant toxin molecules.

Vaccination against shigellosis with attenuated Shigella flexneri 2a strain SC602.

The Shigella flexneri 2a SC602 vaccine candidate carries deletions of the plasmid-borne virulence gene icsA (mediating intra- and intercellular spread) and the chromosomal locus iuc (encoding aerobactin) (S. Barzu, A. Fontaine, P. J. Sansonetti, and A. Phalipon, Infect. Immun. 64:1190-1196, 1996). Dose selection studies showed that SC602 causes shigellosis in a majority of volunteers when 3 x 10(8) or 2 x 10(6) CFU are ingested. In contrast, a dose of 10(4) CFU was associated with transient fever or mild diarrhea in 2 of 15 volunteers. All volunteers receiving single doses of >/=10(4) CFU excreted S. flexneri 2a, and this colonization induced significant antibody-secreting cell and enzyme-linked immunosorbent assay responses against S. flexneri 2a lipopolysaccharide in two-thirds of the vaccinees. Seven volunteers who had been vaccinated 8 weeks earlier with a single dose of 10(4) CFU and 7 control subjects were challenged with 2 x 10(3) CFU of virulent S. flexneri 2a organisms. Six of the control volunteers developed shigellosis with fever and severe diarrhea or dysentery, while none of the vaccinees had fever, dysentery, or severe symptoms (P = 0. 005). Three vaccinees experienced mild diarrhea, and these subjects had lower antibody titers than did the fully protected volunteers. Although the apparent window of safety is narrow, SC602 is the first example of an attenuated S. flexneri 2a candidate vaccine that provides protection against shigellosis in a stringent, human challenge model.

Genetic analysis of Shigella sonnei form I antigen: identification of a novel IS630 as an essential element for the form I antigen expression.

The form I coding region of Shigella sonnei was cloned and shown to have an operon-like rfb organization. It was found that the 11.0 kb HindIII-XbaI fragment of pHH201 encoding the form I antigen contains 10 contiguous open reading frames (ORF), ORF1 to ORF10. Deletions from either end of pHH201, within ORF1 or ORF10, eliminated form I expression. ORF1 and ORF2 share significant nucleic and amino acids homologies to two ORF's of the Salmonella typhi Vi antigen genes. ORF5 in pHH201 is identical to IS630. pHH2064, derived from pHH201, lacks the IS630 element and can stably express the form I antigen inE. coli HB101. However, pHH2064 is structurally unstable in a S. sonnei form II host. This indicates that the presence of the IS630 gene within the S. sonnei rfb operon may be necessary for the stability of form I expression in S. sonnei. This finding is substantiated by the observation that all virulent S. sonnei isolates examined in this study retained the IS630 element within their rfb operon.

Construction of a stable attenuated Shigella sonnei DeltavirG vaccine strain, WRSS1, and protective efficacy and immunogenicity in the guinea pig keratoconjunctivitis model.

Construction of a stable Shigella sonnei vaccine has been complicated by the instability of the virulence phenotype caused by the spontaneous loss of the invasion plasmid. To select a suitable candidate for vaccine construction, 16 S. sonnei strains were screened for stability of the virulence phenotype. A stable strain, S. sonnei Mosely, was selected for further work. pDeltavirG2, a deletion derivative of the virG gene in the sacB suicide vector pCVD442, was used to generate an S. sonnei virG deletion strain, WRSS1, which was invasive in HeLa cells but negative in the Sereny test. WRSS1 was found to be both immunogenic and protective in the guinea pig keratoconjunctivitis model.

Hemolysin-positive enteroaggregative and cell-detaching Escherichia coli strains cause oncosis of human monocyte-derived macrophages and apoptosis of murine J774 cells.

Infection of human monocyte-derived macrophages (HMDM) and J774 cells (murine macrophage cell line) with several enteroaggregative and cytodetaching Escherichia coli (EAggEC and CDEC, respectively) strains demonstrated that some strains could induce macrophage cell death accompanied by release of lactate dehydrogenase activity and interleukin 1beta (IL-1beta) into culture supernatants. The mode of cell death differed in the two types of macrophages. Damage to macrophage plasma membrane integrity without changes in nuclear morphology resulted in cytolysis of HMDM. This mechanism of cell death has been previously described for virulent Shigella infection of HMDM and is termed oncosis. In contrast, infection of J774 cells by EAggEC and CDEC strains resulted in apoptosis. The presence of alpha-hemolysin (Hly) in EAggEC and CDEC strains appears to be critical for both oncosis in HMDM and apoptosis in J774 cells. Bacteria lacking Hly, including Hly- EAggEC strains as well as enterotoxigenic, enteropathogenic, and enterohemorrhagic E. coli strains, behaved like avirulent Shigella flexneri in that the macrophage monolayers were intact, with no release of lactate dehydrogenase activity or IL-1beta into the culture supernatants.

Characterization of Shigella type 1 fimbriae: expression, FimA sequence, and phase variation.

This study documents the presence of type 1 fimbriae on Shigella and confirms these mannose-sensitive adherence structures to be bona fide components of the Shigella surface. While laboratory-passaged Shigella strains and lyophilized clinical isolates failed to express type 1 fimbriae, 6 of 20 recent clinical isolates, including 4 Shigella flexneri strains, 1 Shigella boydii strain, and 1 Shigella dysenteriae strain, produced type 1 fimbriae as detected by mannose-sensitive hemagglutination (MSHA) and electron microscopy. Optimal production of a predominantly Fim+ population required serial passage every 48 to 72 h in unshaken brain heart infusion broth at 37 degrees C. Fim+ Shigella cultures were capable of reversibly switching to a non-MSHA, afimbriated phase during serial aerobic cultivation on tryptic soy agar plates. The amino acid sequence of S. flexneri type 1 FimA contained 18 substitutions compared to that of Escherichia coli fimbrillin. Indirect immunoelectron microscopy suggested the presence of both shared and unique epitopes on E. coli and S. flexneri type 1 fimbriae. Random phase variation between fimbriated and afimbriated states in Shigella was accompanied by the genomic rearrangement associated with phase variation in E. coli.

Human monocyte-derived macrophages infected with virulent Shigella flexneri in vitro undergo a rapid cytolytic event similar to oncosis but not apoptosis.

Infection of human monocyte-derived macrophages in vitro with virulent Shigella flexneri resulted in cell death which involved rupture of the plasma membrane, cell swelling, disintegration of ultrastructure, and generalized karyolysis. These features bore resemblance to oncosis and are in striking contrast to previously described observations of mouse macrophages, where a similar infection by virulent Shigella resulted in cell death by apoptosis. Cell death by oncosis in human macrophages was confirmed by lactate dehydrogenase release, light microscopy, electron microscopy, terminal deoxynucleotidyltransferase end labeling of DNA ends, DNA fragmentation assays, and fluorescence-activated cell sorter analysis of propidium-labeled nuclei. Thus, the phenomena of cell death induced by virulent Shigella in human and mouse macrophages reflect different biochemical pathways. Interleukin-1beta (IL-1beta) was released in culture supernatants of human macrophages infected with virulent bacteria. Inhibition with IL-1beta-converting enzyme inhibitors indicated, however, that this release occurred as a passive event of cell lysis. The patterns of intracellular survival of Shigella strains within human and mouse macrophages reflect differences that exist not only between Shigella serotypes but also between the two different macrophage cell types.

Identification and molecular characterization of a 27 kDa Shigella flexneri invasion plasmid antigen, IpaJ.

Shigella species and enteroinvasive Escherichia coli contain a core set of virulence genes whose coordinated expression results in the invasion of host colonic epithelial cells and the dysenteric syndrome. A number of virulence determinants are carried by the 230 kb invasion plasmid found in all virulent strains of Shigellae. Many of these invasion plasmid genes encode immunogens that are recognized by convalescent serum, including proteins that mediate the invasion (IpaB, IpaC, IpaD) and cell spreading (VirG or IcsA and IcsB) phenotypes. In this report, we describe the molecular characterization of a novel invasion plasmid antigen from Shigella flexneri, designated IpaJ. The ipaJ gene encodes a 780 bp open reading frame (ORF), separated from the ipaR (virB) stop codon by 944 bp. The predicted amino acid sequence for IpaJ revealed a consensus signal peptide for protein export. TnphoA mutagenesis of the ipaJ ORF confirmed the presence of export signal sequences in IpaJ. Unlike ipaBCDA genes, transcription analysis of ipaJ indicated that the gene is not expressed in a temperature-dependent fashion. The IpaJ protein was expressed and purified as a His6-tagged fusion protein that reacted with convalescent sera in Western blot analyses, confirming its identification as a Shigella immunogen. Construction and phenotypic characterization of ipaJ mutants in two serotypes of S. flexneri showed that the mutants were not compromised in their ability to invade cultured epithelial cells or to form plaques on BHK cell monolayers. In addition, the ipaJ mutants were Sereny positive indicating a capacity for intercellular dissemination; however, in the limited number of guinea-pigs tested, the keratoconjunctivitis reaction appeared attenuated.

A Shigella flexneri invasion plasmid gene, ipgH, with homology to IS629 and sequences encoding bacterial sugar phosphate transport proteins.

Sequences representing the 2684 bp flanking the ipaH4.5 gene on the invasion plasmid of Shigella flexneri 5 indicate an unusual fusion gene, designated ipgH, in which the first 27 amino acids (aa) are identical to ORF2 of IS629. The aa sequence encoded by the remainder of ipgH bears significant homology to Escherichia coli and to Salmonella typhimurium GlpT and UhpT proteins and to the S. typhimurium PgtP protein, which are involved in the uptake of high-energy sugar phosphates from an external source.

Construction and characterization of virG (icsA)-deleted Escherichia coli K12-Shigella flexneri hybrid vaccine strains.

Human challenge studies with EcSf2a-2, an aroD deletion-attenuated Escherichia coli K12-Shigella flexneri hybrid vaccine expressing S. flexneri 2a somatic antigen and the invasive phenotype indicated that, at doses of 2 x 10(9) bacteria, EcSf2a-2 was immunogenic but also reactogenic and therefore not sufficiently attenuated. Two factors that may contribute to the residual reactogenicity are the spontaneous appearance of plaque-positive variants in the E. coli K12 recipient and the presence of the arg locus encoding enterotoxin or cytotoxin, transferred from S. flexneri 2a into the E. coli recipient. EcSf2a-3 was derived from EcSf2a-2 by introducing a deletion in the virG gene, whose expression is required for plaque formation and keratoconjunctivitis in guinea pigs. EcSf2a-5 contains the same deletion in the E. coli-S. flexneri hybrid strain, 7921, but does not contain the arg locus. Lack of virG expression in these hybrid strains did not affect the immune response to LPS or the development of protective immunity in the guinea pig model.

Evaluation of alkaline phosphatase-labelled ipaH probe for diagnosis of Shigella infections.

The presence of many enteropathogens which are not easily detectable by routine stool culture has led to the development of alternative diagnostic methods. One of these techniques, nucleic acid probe hybridization, has been used to identify Shigella spp. and enteroinvasive Escherichia coli (EIEC) in stool specimens through the detection of genetic material encoded by a specific large approximately 200-kbp virulence-related plasmid. In the present study, an alkaline phosphatase-labelled oligonucleotide probe developed to detect the gene for ipaH, a repetitive genetic sequence thought to be present on both the virulence-related plasmid and the chromosomes of all strains of Shigella and EIEC, was tested in a developing-country setting through a prospective clinical trial. In a group of 219 Peruvian adults and children with acute gastroenteritis, the ipaH probe detected 85% of cases of shigellosis and demonstrated a specificity of 95% when compared with simultaneous detection by several stool culture techniques. Additionally, three cases of EIEC infection which could not be diagnosed by culture methods alone were detected with the ipaH probe and were confirmed by plasmid analysis and Sereny testing. These preliminary results suggest that, with further research, the ipaH probe should prove to be a useful and rapid adjunct in the diagnosis of acute gastroenteritis in developing countries.

Surface presentation of Shigella flexneri invasion plasmid antigens requires the products of the spa locus.

An avirulent, invasion plasmid insertion mutant of Shigella flexneri 5 (pHS1059) was restored to the virulence phenotype by transformation with a partial HindIII library of the wild-type invasion plasmid constructed in pBR322. Western immunoblot analysis of pHS1059 whole-cell lysates revealed that the synthesis of the invasion plasmid antigens VirG, IpaA, IpaB, IpaC, and IpaD was similar to that seen in the corresponding isogenic S. flexneri 5 virulent strain, M90T. IpaB and IpaC, however, were not present on the surface of pHS1059 as was found in M90T, suggesting that the transport or presentation of the IpaB and IpaC proteins onto the bacterial surface was defective in the mutant. pHS1059 was complemented by pWR266, which carried contiguous 1.2- and 4.1-kb HindIII fragments of the invasion plasmid. pHS1059(pWR266) cells were positive in the HeLa cell invasion assay as well as colony immunoblot and enzyme-linked immunosorbent assays, using monoclonal antibodies to IpaB and IpaC. These studies established that the antigens were expressed on the surface of the transformed bacteria. In addition, water extraction of pHS1059 and pHS1059(pWR266) whole cells, which can be used to remove IpaB and IpaC antigens from the surface of wild-type M90T bacteria, yielded significant amounts of these antigens from pHS1059(pWR266) but not from pHS1059. Minicell and DNA sequence analysis indicated that several proteins were encoded by pWR266, comprising the spa loci, which were mapped to a region approximately 18 kb upstream of the ipaBCDAR gene cluster. Subcloning and deletion analysis revealed that more than one protein was involved in complementing the Spa- phenotype in pHS1059. One of these proteins, Spa47, showed striking homology to ORF4 of the Bacillus subtilis flaA locus and the fliI gene sequence of Salmonella typhimurium, both of which bear strong resemblance to the alpha and beta subunits of bacterial, mitochondrial, and chloroplast proton-translocating F0F1 ATPases.