An important virulence factor of Salmonella spp. is their ability to gain access to host cells. A type III secretion system encoded in the inv and spa loci of these organisms is essential for this phenotype. We have identified two proteins, SipA and SipD, whose secretion from the bacterial cells is dependent on this system. The genes encoding these proteins are located at centisome 63 on the S. typhimurium chromosome, immediately downstream of the previously identified sipB and sipC genes (K. Kaniga, S. Tucker, D. Trollinger, and J. E. Galán, J. Bacteriol. 177:3965-3971, 1995). Nucleotide sequence analysis of the genes encoding these proteins indicated that SipA and SipD have significant sequence similarity to the Shigella IpaA and IpaD proteins. A nonpolar null mutation in sipD rendered S. typhimurium severely deficient for entry into cultured epithelial cells. In addition, this mutant strain exhibited increased secretion of a selected group of proteins whose export is controlled by the inv-and spa-encoded translocon. In contrast, a nonpolar mutation in sipA did not result in an invasion defect or in a significant decreased in virulence in a mouse model of infection. In addition, we have found an open reading frame immediately downstream of SipA that encodes a predicted protein with significant similarity to a family of acyl carrier proteins.The ability of Salmonella typhimurium to gain access to host cells is largely encoded by a contiguous region of the chromosome located at centisome 63 (4, 9, 13, 18, 20, 22, 25, 29, 38-40, 43, 56). At least two loci of this region, inv (13,18,22,25,39) and spa (29), encode components of a sec-independent protein secretion system. This protein secretion apparatus, termed type III, is also present in other mammalian pathogens such as Shigella spp. (1,2,6,7,60,65), Yersinia spp. (3,10,52,53,70), and enteropathogenic Escherichia coli (37) as well as in a variety of plant-pathogenic bacteria from the erwiniae and the families Pseudomonaceae and Xhanthomonaceae (19,23,27,28,35,44,64,68). These secretion systems are required for the export of proteins thought to, directly or indirectly, elicit responses in infected host cells. These responses include the induction of membrane ruffling and bacterial internalization (Salmonella and Shigella spp.), cytotoxicity and other cellular responses (Yersinia spp.), intestinal epithelial cell damage (enteropathogenic E. coli), and induction of pathogenicity or local defense reactions in susceptible or resistant plant hosts (plant pathogens). Identification of proteins that exit the bacterial cells via these systems is of great interest since such proteins presumably include effectors of the various responses elicited by the corresponding pathogens. Protein targets of these type III secretion systems have been identified in Shigella spp. (Ipas) (reviewed in reference 59), Yersinia spp. (Yops) (reviewed in reference 63), enteropathogenic E. coli (EaeB) (17), Pseudomonas syringae and Erwinia amylovora (harpins) (33, 69), and Pseudomonas solanace...

Processes as diverse as receptor binding and signaling, cytoskeletal dynamics, and programmed cell death are manipulated by mimics of host proteins encoded by pathogenic bacteria. We show here that the Salmonella virulence factor SspH2 belongs to a growing class of bacterial effector proteins that harness and subvert the eukaryotic ubiquitination pathway. This virulence protein possesses ubiquitination activity that depends on a conserved cysteine residue. A crystal structure of SspH2 reveals a canonical leucine-rich repeat (LRR) domain that interacts with a unique E3 ligase [which we have termed NEL for Novel E3 Ligase] C-terminal fold unrelated to previously observed HECT or RING-finger E3 ligases. Moreover, the LRR domain sequesters the catalytic cysteine residue contained in the NEL domain, and we suggest a mechanism for activation of the ligase requiring a substantial conformational change to release the catalytic domain for function. We also show that the N-terminal domain targets SspH2 to the apical plasma membrane of polarized epithelial cells and propose a model whereby binding of the LRR to proteins at the target site releases the ligase domain for site-specific function.microbial pathogenesis ͉ type III secretion ͉ crystallography ͉ SspH2

Invasion of intestinal epithelial cells is an essential virulence factor of salmonellae. A group of genes, invABC and invD, that allow SalmoneUa typhimurium to penetrate cultured epithelial cells have previously been characterized (J. E. Galain and R. Curtiss III, Proc. Natl. Acad. Sci. USA 86:6383-6387, 1989). The distribution of these genes among Salmonella isolates belonging to 37 different species or serovars was investigated by Southern and colony blot hybridization analyses. Regions of high sequence similarity to the invABC genes were present in all SalmoneUa isolates examined, while regions of sequence similarity to the invD gene were present in all but one (S. arizonae) of the isolates tested, with little restriction fragment length polymorphism. Sequences similar to these genes were not detected in strains of Escherichia coli, Yersinia spp., or Shigella spp. invA mutants (unable to express the invABC genes) of several SalmoneUa species or serovars, including S. typhi, were constructed and examined for their ability to penetrate Henle-407 cells. All mutants were deficient for entry into cultured epithelial cells, indicating that the invABC genes were not only present in these strains but also functional.

InvA, which is essential for Salmonella spp. to enter cultured epithelial cells, is a member of a family of proteins involved in either flagellar biosynthesis or the secretion of virulence determinants by a number of plant and mammalian pathogens. The predicted overall secondary structures of these proteins show significant similarities and indicate a modular construction with a hydrophobic amino-terminal half, consisting of six to eight potential transmembrane domains, and a hydrophilic carboxy terminus which is predicted to reside in the cytoplasm. These proteins can be aligned over the entire length of their polypeptide sequences, with the highest degree of homology found in the amino terminus and clusters of conserved residues in the carboxy terminus. We examined the functional conservation among members of this protein family by assessing the ability of MxiA of Shigella flexneri and LcrD of Yersinia pseudotuberculosis to restore invasiveness to an invA mutant of Salmonella typhimurium. We found that MxiA was able to complement the entry defect of the invA mutant strain of S. typhimurium. In contrast, LcrD failed to complement the same strain. However, a plasmid carrying a gene encoding a chimeric protein consisting of the amino terminus of LcrD and the carboxy terminus of InvA complemented the defect of the Salmonella invA mutant. These results indicate that the secretory systems in which these proteins participate are functionally similar and that the Salmonella and Shigella systems are very closely related. These data also suggest that determinants of specificity may be located at the carboxy termini of these proteins.

Mucosal nasopharyngeal immunoglobulin A (IgA) and IgG responses to proteins of Streptococcus equi were studied in horses after the experimental production of strangles. S. equi-specific IgA and IgG titers in nasopharyngeal mucus were much higher in samples from animals 1 to 2 weeks after challenge than in samples from control animals. Although IgA was the major immunoglobulin in nasal mucus, there was more antibody activity associated with IgG as measured by radioimmunoassay. Great differences between the specificities of antibodies in nasal mucus and in serum were detected. IgA and IgG of mucus origin recognized only two major proteins with molecular weights of about 41,000 and 46,000 in acid extracts of S. equi and gave no detectable reaction with culture supernatant proteins. Only one protein of about 62,000 molecular weight was recognized in acid extracts of an equine strain of S. zooepidemicus. In contrast, immunoglobulins in serum recognized a great variety of proteins in culture supernatants and acid extracts of S. equi and S. zooepidemicus which did not include those proteins recognized by immunoglobulins in mucus. These findings provide good evidence for the independence of the local and systemic immune responses of the horse to S. equi. Horses rechallenged shortly after recovery from the first infection were resistant to challenge with an inoculum of S. equi 10 times greater than that to which they were originally susceptible. This resistance appeared to be independent of the levels of bactericidal antibody in serum. We therefore suggest that immunity to S. equi infection is mediated by locally produced nasopharyngeal antibodies.

A series of isolates of Streptococcus equi from the United States and Europe were compared by the bactericidal test, immunoblotting, DNA restrictions, and Southern hybridization analysis. All isolates tested were sensitive to the same bactericidal serum. In addition, immunoblotting revealed no differences in M proteins prepared by acid or mutanolysin extraction. Immunoblotting of acid extracts of the isolates with mucosal nasopharyngeal mucus from a convalescent horse revealed the presence of the 41,000and 46,000-Mr polypeptide fragments of the M protein of S. èqui known to be important in stimulating mucosal nasopharyngeal immune responses. DNA restriction analysis of total cell DNA digests, as well as Southern hybridizations using an S. equi M protein gene probe, did not detect any differences among these isolates. Our results, therefore, confirm the antigenic homogeneity of the M proteins of S. equi isolates and suggest that variation in this antigen is not a reason for the failure of commercial vaccines in the field. Interestingly, the protoplast M proteins of all isolates showed remarkable size homogeneity, in contrast to the size variation reported in M proteins of group A streptococci.

Erysipelothrix rhusiopathiae is a primary pathogen of swine and turkeys and sporadic cause of disease in a variety of other hosts, including humans. A genomic library of the highly virulent strain of E. rhusiopathiae E1-6P was constructed in the expression-cloning vector Xgtll and screened with serum from a pig convalescent from an E. rhusiopathiae experimental infection. Immunoreactive clones were screened for their ability to protectively immunized mice. Two clones, AgtlllersA and Agtll/ersB, were obtained that protected mice against challenge with E. rhusiopathiae E1-6P. Antisera against the recombinant clones reacted with polypeptides of molecular weights 66,000, 64,000, and 43,000 in detergent-solubilized surface antigen preparations and whole-cell lysates of E. rhusiopathiae. These polypeptides were also the major antigens recognized by convalescent pig serum when reacted with the same preparations. Western immunoblot and Southern blot analysis revealed that the cloned genes and gene products were present in all of the E. rhusiopathiae strains tested.

A Streptococcus equi gene bank was constructed in the bacteriophage Agtll cloning vector, and hybrid phage plaques were screened with S. equi M protein antiserum. A hybrid phage expressing the S. equi M protein (Xgtll/SEM7) was identified and lysogenized into Escherichia coli Y1089. The cloned M protein appeared in immunoblots as three polypeptides with relative molecular weights of 58,000, 53,000, and 50,000. When reacted with S. equi M protein antiserum in an agar double-diffusion assay, the cloned M protein formed a line of identity with a protein in an acid extract of S. equi. Furthermore, Agtl 1/SEM7 protein inhibited opsonization of S. equi by antiserum to S. equi M protein. In addition, the recombinant protein expressed determinants of the antigen in the immune complexes of purpura hemorrhagica. Native M protein obtained from S. equi and recombinant M protein showed very similar molecular weight distributions on immunoblots, appearing as multiple closely spaced bands with molecular weights ranging from 52,000 to 60,000. Antisera prepared separately against each of the acid-extracted polypeptides shown to be important in serum bactericidal responses (molecular weight, 29,000) and nasopharyngeal local antibody responses (molecular weights, 41,000 and 46,000) of the horse each reacted with all three polypeptides in an acid extract. Moreover, antisera against protoplasts and against recombinant M protein of S. equi also reacted with these polypeptides. These results suggest that the entire M protein molecule of S. equi is present in these preparations and that the fragments in acid extracts carry overlapping segments.