Identification of a virulence locus encoding a second type III secretion system in Salmonella typhimurium.

Mapping the insertion points of 16 signature-tagged transposon mutants on the Salmonella typhimurium chromosome led to the identification of a 40-kb virulence gene cluster at minute 30.7. This locus is conserved among all other Salmonella species examined but is not present in a variety of other pathogenic bacteria or in Escherichia coli K-12. Nucleotide sequencing of a portion of this locus revealed 11 open reading frames whose predicted proteins encode components of a type III secretion system. To distinguish between this and the type III secretion system encoded by the inv/spa invasion locus known to reside on a pathogenicity island, we refer to the inv/spa locus as Salmonella pathogenicity island (SPI) 1 and the new locus as SPI2. SPI2 has a lower G+C content than that of the remainder of the Salmonella genome and is flanked by genes whose products share greater than 90% identity with those of the E. coli ydhE and pykF genes. Thus SPI2 was probably acquired horizontally by insertion into a region corresponding to that between the ydhE and pykF genes of E. coli. Virulence studies of SPI2 mutants have shown them to be attenuated by at least five orders of magnitude compared with the wild-type strain after oral or intraperitoneal inoculation of mice.

[1]  J. Devereux,et al.  A comprehensive set of sequence analysis programs for the VAX , 1984, Nucleic Acids Res..

[2]  J. Galán,et al.  Cloning and molecular characterization of genes whose products allow Salmonella typhimurium to penetrate tissue culture cells. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[3]  H. Sambrook Molecular cloning : a laboratory manual. Cold Spring Harbor, NY , 1989 .

[4]  D. Botstein,et al.  Advanced Bacterial Genetics: A Manual for Genetic Engineering , 1980 .

[5]  C. Sasakawa,et al.  Eight genes in region 5 that form an operon are essential for invasion of epithelial cells by Shigella flexneri 2a , 1993, Journal of bacteriology.

[6]  C. Ginocchio,et al.  Identification and molecular characterization of a Salmonella typhimurium gene involved in triggering the internalization of salmonellae into cultured epithelial cells. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[7]  H. Ochman,et al.  Molecular, functional, and evolutionary analysis of sequences specific to Salmonella. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[8]  D. Botstein,et al.  Advanced bacterial genetics , 1980 .

[9]  Frederick M. Ausubel,et al.  Conserved domains in bacterial regulatory proteins that respond to environmental stimuli , 1987, Cell.

[10]  Philip J. Reeves,et al.  Membrance traffic wardens and protein secretion in Gram-negative bacteria , 1993 .

[11]  D. Botstein,et al.  Functional interchangeability of DNA replication genes in Salmonella typhimurium and Escherichia coli demonstrated by a general complementation procedure. , 1984, Genetics.

[12]  J Hacker,et al.  Large, unstable inserts in the chromosome affect virulence properties of uropathogenic Escherichia coli O6 strain 536 , 1986, Journal of bacteriology.

[13]  P. Youderian,et al.  Packaging specific segments of the Salmonella chromosome with locked-in Mud-P22 prophages. , 1988, Genetics.

[14]  G. Cornelis,et al.  Analysis of virC, an operon involved in the secretion of Yop proteins by Yersinia enterocolitica , 1991, Journal of bacteriology.

[15]  F. Sanger,et al.  DNA sequencing with chain-terminating inhibitors. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[16]  H. Ochman,et al.  Relationship between evolutionary rate and cellular location among the Inv/Spa invasion proteins of Salmonella enterica. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[17]  C. Ginocchio,et al.  Contact with epithelial cells induces the formation of surface appendages on Salmonella typhimurium , 1994, Cell.

[18]  M. Starnbach,et al.  Morphological and cytoskeletal changes in epithelial cells occur immediately upon interaction with Salmonella typhimurium grown under low‐oxygen conditions , 1992, Molecular microbiology.

[19]  J. Kronstad,et al.  Mutation in a heat‐regulated hsp70 gene of Ustilago maydis. , 1989, The EMBO journal.

[20]  B. Bachmann,et al.  Linkage map of Escherichia coli K-12, edition 8 , 1990, Microbiological reviews.

[21]  H. Ochman,et al.  How to become a pathogen. , 1994, Trends in microbiology.

[22]  F. Collins,et al.  THE ROUTE OF ENTERIC INFECTION IN NORMAL MICE , 1974, The Journal of experimental medicine.

[23]  Catherine A. Lee,et al.  A 40 kb chromosomal fragment encoding Salmonella typhimurium invasion genes is absent from the corresponding region of the Escherichia coli K‐12 chromosome , 1995, Molecular microbiology.

[24]  C. Ginocchio,et al.  Molecular and functional characterization of the Salmonella typhimurium invasion genes invB and invC: homology of InvC to the F0F1 ATPase family of proteins , 1994, Journal of bacteriology.

[25]  S. Barve,et al.  LcrD, a membrane-bound regulator of the Yersinia pestis low-calcium response , 1991, Journal of bacteriology.

[26]  T. McDaniel,et al.  A genetic locus of enterocyte effacement conserved among diverse enterobacterial pathogens. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[27]  C. Boucher,et al.  Conservation of secretion pathways for pathogenicity determinants of plant and animal bacteria. , 1993, Trends in microbiology.

[28]  H. Ochman,et al.  Cognate gene clusters govern invasion of host epithelial cells by Salmonella typhimurium and Shigella flexneri. , 1993, The EMBO journal.

[29]  A. Takeuchi Electron microscope studies of experimental Salmonella infection. I. Penetration into the intestinal epithelium by Salmonella typhimurium. , 1967, The American journal of pathology.

[30]  K. Rudd,et al.  Genetic map of Salmonella typhimurium, edition VIII. , 1995, Microbiological reviews.

[31]  T. Bergman,et al.  The lcrB (yscN/U) gene cluster of Yersinia pseudotuberculosis is involved in Yop secretion and shows high homology to the spa gene clusters of Shigella flexneri and Salmonella typhimurium , 1994, Journal of bacteriology.

[32]  K. Sanderson,et al.  The XbaI-BlnI-CeuI genomic cleavage map of Salmonella typhimurium LT2 determined by double digestion, end labelling, and pulsed-field gel electrophoresis , 1993, Journal of bacteriology.

[33]  B. Finlay Molecular and cellular mechanisms of Salmonella pathogenesis. , 1994, Current topics in microbiology and immunology.

[34]  B. Finlay,et al.  Cloning and molecular characterization of a gene involved in Salmonella adherence and invasion of cultured epithelial cells , 1993, Molecular microbiology.

[35]  P. Cossart,et al.  The virulence gene cluster of Listeria monocytogenes is also present in Listeria ivanovii, an animal pathogen, and Listeria seeligeri, a nonpathogenic species , 1994, Infection and immunity.

[36]  R. Perry,et al.  Loss of the pigmentation phenotype in Yersinia pestis is due to the spontaneous deletion of 102 kb of chromosomal DNA which is flanked by a repetitive element , 1992, Molecular microbiology.

[37]  V. de Lorenzo,et al.  Analysis and construction of stable phenotypes in gram-negative bacteria with Tn5- and Tn10-derived minitransposons. , 1994, Methods in enzymology.

[38]  M. Venkatesan,et al.  Surface presentation of Shigella flexneri invasion plasmid antigens requires the products of the spa locus , 1992, Journal of bacteriology.

[39]  J. Hacker,et al.  Excision of large DNA regions termed pathogenicity islands from tRNA-specific loci in the chromosome of an Escherichia coli wild-type pathogen , 1994, Infection and immunity.

[40]  C. Collazo,et al.  Functional analysis of the Salmonella typhimurium invasion genes invl and invJ and identification of a target of the protein secretion apparatus encoded in the inv locus , 1995, Molecular microbiology.

[41]  J. Galán,et al.  The Salmonella typhimurium invasion genes invF and invG encode homologues of the AraC and PulD family of proteins , 1994, Molecular microbiology.

[42]  B. Goldman,et al.  Rapid mapping in Salmonella typhimurium with Mud-P22 prophages , 1992, Journal of bacteriology.

[43]  B. Finlay,et al.  Cytoskeletal rearrangements accompanying salmonella entry into epithelial cells. , 1991, Journal of cell science.

[44]  J. Shea,et al.  Simultaneous identification of bacterial virulence genes by negative selection. , 1995, Science.

[45]  J Hacker,et al.  Deletions of chromosomal regions coding for fimbriae and hemolysins occur in vitro and in vivo in various extraintestinal Escherichia coli isolates. , 1990, Microbial pathogenesis.

[46]  A. Maurelli,et al.  mxiA of Shigella flexneri 2a, which facilitates export of invasion plasmid antigens, encodes a homolog of the low-calcium-response protein, LcrD, of Yersinia pestis , 1992, Infection and immunity.

[47]  P. Sansonetti,et al.  MxiD, an outer membrane protein necessary for the secretion of the Shigella flexneri Ipa invasins , 1993, Molecular microbiology.

[48]  W. Gilbert,et al.  Direct genomic sequencing of bacterial DNA: the pyruvate kinase I gene of Escherichia coli. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[49]  C. Ginocchio,et al.  Molecular and functional characterization of the Salmonella invasion gene invA: homology of InvA to members of a new protein family , 1992, Journal of bacteriology.