Shigella flexneri, the causative agent of shigellosis, is a gram-negative bacterial pathogen that initiates infection by invading cells within the colonic epithelium. Contact with host cell surfaces induces a rapid burst of protein secretion via the Shigella type III secretion system (TTSS). The first proteins secreted are IpaD, IpaB, and IpaC, with IpaB and IpaC being inserted into the host cell membrane to form a pore for translocating late effectors into the target cell cytoplasm. The resulting pathogen-host cross talk results in localized actin polymerization, membrane ruffling, and, ultimately, pathogen entry. IpaD is essential for host cell invasion, but its role in this process is just now coming to light. IpaD is a multifunctional protein that controls the secretion and presentation of IpaB and IpaC at the pathogen-host interface. We show here that antibodies recognizing the surface-exposed N terminus of IpaD neutralize Shigella's ability to promote pore formation in erythrocyte membranes. We further show that MxiH and IpaD colocalize on the bacterial surface. When TTSS needles were sheared from the Shigella surface, IpaD was found at only the needle tips. Consistent with this, IpaD localized to the exposed tips of needles that were still attached to the bacterium. Molecular analyses then showed that the IpaD C terminus is required for this surface localization and function. Furthermore, mutations that prevent IpaD surface localization also eliminate all IpaD-related functions. Thus, this study demonstrates that IpaD localizes to the TTSA needle tip, where it functions to control the secretion and proper insertion of translocators into host cell membranes.

An avirulent, invasion plasmid insertion mutant of Shigea flexneri 5 (pHS1059) was restored to the virulence phenotype by transformation with a partial HindUI 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. feneri 5 virulent strain, M9OT. IpaB and IpaC, however, were not present on the surface of pHS1059 as was found in M9OT, 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 HinduI 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. Miniceli 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 Bacilus subtUisflaA locus and the fliI gene sequence of SalmoneUa typhimurium, both of which bear strong resemblance to the a and 13 subunits of bacterial, mitochondrial, and chloroplast proton-translocating FoF, ATPases.

An in vitro tissue culture plaque assay was developed to investigate the intracellular replication and intercellular spread of virulent shigellae. Shigella plaques were formed in HeLa cell monolayers in the presence of an agarose overlay containing tissue culture medium and gentamicin, which eliminated extracellular bacterial growth. Microscopically, the plaques were characterized by a central area of dead host cells surrounded by cells infected with shigellae. Cells further away from the plaque center were uninfected. Inclusion of chloramphenicol or nalidixic acid in the overlay completely abolished plaque formation. Plaque formation was completely inhibited when infected monolayers were shifted from 37 to 30°C. Shifting infected monolayers from 30°C, where plaques do not form, to 37°C resulted in the formation of plaques. Cultures of Shigella boydii, Shigella sonnei (form I), and all six serotypes of Shigellaflexneri produced plaques. Shigellae isolated from plaques were Sereny test positive, contained a 140-megadalton plasmid, and were gentamicin sensitive. Noninvasive shigellae did not form plaques.

The serum antibody response to proteins encoded by the virulence-associated plasmid of Shigellaflexneri was determined in monkeys challenged with virulent S. flexneri serotype 2a. With water-extractable antigen in an enzyme-linked immunosorbent assay, a significant increase in antibody titer against proteins from a plasmid-carrying, virulent strain of S. flexneri serotype 5 could be demonstrated in convalescent sera. There were minimal antibody titers against proteins from an avirulent (plasmid-free) organism. Previously identified plasmid-coded polypeptides a, b, c, and d were predominant antigens recognized by a majority of the convalescent sera in immunoblots. An additional 140-megadalton plasmid-coded polypeptide was also recognized by half of the sera. Convalescent serum from an infected monkey recognized antigens on the bacterial surface in several different plasmid-containing Shigella species and in an enteroinvasive Escherichia coli strain. A survey of sera obtained from children 5 to 10 years of age who had been infected with S. flexneri or S. sonnei revealed high enzyme-linked immunosorbent assay titers in both acute and convalescent sera against a water extract from a virulent Shigella strain. In contrast, children under 3 years of age had no antibody titer in either acute or convalescent sera against the virulence-associated shigella proteins, while 3to 4-year-old children mounted an immune response against these proteins only in convalescence.

Tn5-tagged invasion plasmid DNA (pWRllO) from Shigeglaflexneri serotype 5 (strain M9OT) was cloned into the expression vector Agtll. Recombinant phage (XgtllSfl) expressing pWR11O-encoded polypeptide antigens were identified by using rabbit antisera directed against S. flexneri M9OT invasion plasmid antigens. Antigens encoded by AgtllSfl recombinant phage were characterized by reacting affinity-purified antibodies, eluted from nitroceilulose-bound plaques of AgtllSfl recombinants, with virulent, wild-type S. flexneri M9OT polypeptides in Western blot analyses. AgtllSfl clones directing the synthesis of complete, truncated, and B-galactosidase fusion versions of three previously identified outer membrane polypeptides antigens) were isolated. A fourth polypeptide, similar in size to the 57-kDa antigen (ca. 58 kDa) but unrelated as determined by DNA homology and serological measurements, was also identified. Southern blot analysis of S. flexneri M9OT invasion plasmid DNA hybridized with AgtllSfl insert DNA probes was used to construct a map of invasion plasmid antigen genes (ipa) corresponding to the 57-kDa (ipaB), , and 39-kDa (ipaD) polypeptides. Genes ipaB, ipaC and ipaD mapped to contiguous 4.6-kilobase (kb) and 1.0-kb Hindm fragments contained within a larger (23-kb) Bam-H fragment. The ipall gene, which encodes the synthesis of the 58-kDa polypeptide, did not map in or near the ipaBCD gene cluster, suggesting a distinct location of ipaH on the invasion plasmid.The complex pathology of the dysenteric syndrome, caused by Shigella spp. and enteroinvasive Escherichia coli, is reflected in the diversity of genetic components controlling the virulence of these organisms. Both chromosomal and extrachromosomal loci that are essential for the expression of the virulent phenotype have been identified (21, 22; reviewed in reference 10). One aspect of this phenotype, the invasion of colonic epithelial cells, has its genetic components located on a large 120-to 140-megadalton (MDa) nonconjugative plasmid found in all Shigella and enteroinvasive E. coli strains (11,23,24). Loss of the plasmid is accompanied by loss of the invasive phenotype, as measured by in vitro infection of cultured mammalian cells, and by the inability of spontaneously cured shigellae to elicit keratoconjunctivitis (i.e., the Sereny reaction) in guinea pigs (21,23,24,27). Reintroduction of the invasion plasmid into a plasmid-free avirulent Shigella strain restores the invasive phenotype (23,24,30).A 37-kilobase (kb) region of the S. flexneri serotype 5 invasion plasmid cloned into the cosmid vector pJB8 restores the HeLa cell invasiveness of plasmid-cured Shigella spp. but does not restore the ability to cause a positive Sereny reaction (13). At least eight polypeptides, ranging in size from 12 to 140 kDa, have been identified as unique products of the invasion plasmid (7,8 temperature for the invasive phenotype (13, 14). These polypeptides, plus an additional 140-kDa outer membrane protein, are also important immunogens, since convalescentstage sera...