scholarly journals Microarray-Based Detection of Salmonella enterica Serovar Typhimurium Transposon Mutants That Cannot Survive in Macrophages and Mice

2005 ◽  
Vol 73 (9) ◽  
pp. 5438-5449 ◽  
Author(s):  
Kaman Chan ◽  
Charles C. Kim ◽  
Stanley Falkow
Keyword(s):  
Salmonella Enterica ◽  
Dna Microarrays ◽  
Salmonella Enterica Serovar Typhimurium ◽  
Cultured Cells ◽  
Pathogenicity Island ◽  
Intracellular Survival ◽  
The Novel ◽  
Serovar Typhimurium ◽  
Transposon Mutants ◽  
Tracking Strategy

ABSTRACTDNA microarrays provide an opportunity to combine the principles of signature-tagged mutagenesis (STM) with microarray technology to identify potentially important bacterial virulence genes. The scope of DNA microarrays allows for less laborious screening on a much larger scale than possible by STM alone. We have adapted a microarray-based transposon tracking strategy for use with aSalmonella entericaserovar Typhimurium cDNA microarray in order to identify genes important for survival and replication in RAW 264.7 mouse macrophage-like cells or in the spleens of BALB/cJ mice. A 50,000-CFU transposon library ofS. entericaserovar Typhimurium strain SL1344 was serially passaged in cultured macrophages or intraperitoneally inoculated into BALB/cJ mice. The bacterial genomic DNA was isolated and processed for analysis on the microarray. The novel application of this approach to identify mutants unable to survive in cultured cells resulted in the identification of components ofSalmonellapathogenicity island 2 (SPI2), which is known to be critical for intracellular survival and replication. In addition, array results indicated that a number of SPI1-associated genes, currently not associated with intracellular survival, are negatively selected. However, of the SPI1-associated mutants individually tested for intracellular survival, only asirAmutant exhibited reduced numbers relative to those of wild-type bacteria. Of the mutants unable to survive in mice, significant proportions are either components of the SPI2 pathogenicity island or involved in lipopolysaccharide synthesis. This observation is in agreement with results obtained in the originalS. entericaserovar Typhimurium STM screen, illustrating the utility of this approach for the high-throughput identification of virulence factors important for survival in the host.

scholarly journals Transcriptional Organization and Function of Invasion Genes within Salmonella enterica Serovar Typhimurium Pathogenicity Island 1, Including the prgH,prgI, prgJ, prgK, orgA,orgB, and orgC Genes

2000 ◽  
Vol 68 (6) ◽  
pp. 3368-3376 ◽  
Author(s):  
Joanna R. Klein ◽  
Thomas F. Fahlen ◽  
Bradley D. Jones
Keyword(s):  
Epithelial Cells ◽  
Salmonella Enterica ◽  
Salmonella Enterica Serovar Typhimurium ◽  
Pathogenicity Island ◽  
Pcr Analysis ◽  
Intestinal Epithelial ◽  
Reading Frame ◽  
Bacterial Proteins ◽  
Serovar Typhimurium ◽  
And Function

ABSTRACT Salmonella enterica serovar Typhimurium initiates infection of a host by inducing its own uptake into specialized M cells which reside within the epithelium overlaying Peyer's patches. Entry of Salmonella into intestinal epithelial cells is dependent upon invasion genes that are clustered together inSalmonella pathogenicity island 1 (SPI-1). Upon contact between serovar Typhimurium and epithelial cells targeted for bacterial internalization, bacterial proteins are injected into the host cell through a type III secretion system that leads to internalization of the bacteria. Previous work has established that the prgH, -I, -J, and -K and orgAgenes reside in SPI-1, and the products of these genes are predicted to be components of the invasion secretion apparatus. We report that an error in the published orgA DNA sequence has been identified so that this region encodes two small genes rather than a single large open reading frame. These genes have been designatedorgA and orgB. Additionally, an opening reading frame downstream of orgB, which we have designatedorgC, has been identified and partially characterized. Previously published work has indicated that the prgH, -I, -J, and -K genes are transcribed from a promoter distinct from that used by the gene immediately downstream, orgA. Here, we present experiments indicating that orgA expression is driven by theprgH promoter. In addition, using reverse transcriptase PCR analysis, we have found that this polycistronic message extends downstream of prgH to include a total of 10 genes. To more fully characterize this invasion operon, we demonstrate that theprgH, prgI, prgJ, prgK,orgA, and orgB genes are each required for invasion and secretion, while orgC is not essential for the invasive phenotype.

scholarly journals HilE Interacts with HilD and Negatively Regulates hilA Transcription and Expression of the Salmonella enterica Serovar Typhimurium Invasive Phenotype

2003 ◽  
Vol 71 (3) ◽  
pp. 1295-1305 ◽  
Author(s):  
M. Aaron Baxter ◽  
Thomas F. Fahlen ◽  
Rebecca L. Wilson ◽  
Bradley D. Jones
Keyword(s):  
Gene Expression ◽  
Salmonella Enterica ◽  
Salmonella Enterica Serovar Typhimurium ◽  
Gastrointestinal Disease ◽  
Pathogenicity Island ◽  
Specific Gene ◽  
Negative Effects ◽  
Invasive Phenotype ◽  
Serovar Typhimurium ◽  
Two Hybrid

ABSTRACT The ability of Salmonella enterica serovar Typhimurium to traverse the intestinal mucosa of a host is an important step in its ability to initiate gastrointestinal disease. The majority of the genes required for this invasive characteristic are encoded on Salmonella pathogenicity island 1 (SPI1), and their expression is controlled by the transcriptional activator HilA, a member of the OmpR/ToxR family of proteins. A variety of genes (hilC, hilD, fis, sirA/barA, csrAB, phoB, fadD, envZ/ompR, fliZ, hilE, ams, lon, pag, and hha) have been identified that exert positive or negative effects on hilA expression, although the mechanisms by which these gene products function remain relatively unclear. Recent work indicates that the small DNA-binding protein, Hha, has a significant role in repressing hilA transcription and the invasive phenotype, particularly in response to osmolarity signals. We have characterized the Salmonella-specific gene, hilE, and found that it plays an important regulatory role in hilA transcription and invasion gene expression. Mutation of hilE causes derepression of hilA transcription, and overexpression of hilE superrepresses hilA expression and the invasive phenotype. Bacterial two-hybrid experiments indicate that the HilE protein interacts with HilD, suggesting a possible mechanism for HilE negative regulation of hilA gene expression and the Salmonella invasive phenotype. Finally, we have found that the hilE gene resides on a region of the serovar Typhimurium chromosome that has many characteristics of a pathogenicity island.

scholarly journals Role of Nod1 in Mucosal Dendritic Cells during Salmonella Pathogenicity Island 1-Independent Salmonella enterica Serovar Typhimurium Infection

2009 ◽  
Vol 77 (11) ◽  
pp. 5203-5203 ◽  
Author(s):  
Lionel Le Bourhis ◽  
Joao Gamelas Magalhaes ◽  
Thirumahal Selvanantham ◽  
Leonardo H. Travassos ◽  
Kaoru Geddes ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Salmonella Enterica ◽  
Salmonella Enterica Serovar Typhimurium ◽  
Pathogenicity Island ◽  
Salmonella Pathogenicity Island ◽  
Serovar Typhimurium

scholarly journals Secretion of the orgC Gene Product by Salmonella enterica Serovar Typhimurium

2003 ◽  
Vol 71 (11) ◽  
pp. 6680-6685 ◽  
Author(s):  
James B. Day ◽  
Catherine A. Lee
Keyword(s):  
Gene Product ◽  
Type Iii Secretion ◽  
Salmonella Enterica ◽  
Virulence Genes ◽  
Salmonella Enterica Serovar Typhimurium ◽  
Type Iii Secretion System ◽  
Pathogenicity Island ◽  
Secretion System ◽  
Serovar Typhimurium ◽  
Transposon Insertions

ABSTRACT HilA activates the transcription of genes on Salmonella pathogenicity island 1 (SPI1), which encodes a type III secretion system (TTSS). Previous studies showed that transposon insertions in orgC, a gene located on SPI1, increase hilA expression. We characterize the orgC gene product and show that it is secreted via the SPI1 TTSS. We propose a model whereby OrgC functions as a secreted repressor of the SPI1 virulence genes.

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