Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Transcriptional profiling of 14 regulators required for Salmonella virulene

ABSTRACT: To cause a systemic infection, Salmonella must respond to many environmental cues during mouse infection and express specific subsets of genes in a temporal and spatial manner, but the regulatory pathways are poorly established. To unravel how micro-environmental signals are processed and integrated into coordinated action, we constructed in-frame non-polar deletions of 83 regulators inferred to play a role in Salmonella enteriditis Typhimurium (STM) virulence and tested them in three virulence assays (intraperitoneal [i.p.], and intragastric [i.g.] infection in BALB/c mice, and persistence in 129X1/SvJ mice). Overall, 35 regulators were identified whose absence attenuated virulence in at least one assay, and of those, 14 regulators were required for systemic mouse infection, the most stringent virulence assay. As a first step towards understanding the interplay between a pathogen and its host from a systems biology standpoint, we focused on these 14 genes. Transcriptional profiles were obtained for deletions of each of these 14 regulators grown under four different environmental conditions. These results, as well as publicly available transcriptional profiles, were analyzed using both network inference and cluster analysis algorithms. The analysis predicts a regulatory network in which all 14 regulators control the same set of genes necessary for Salmonella to cause systemic infection. We tested the regulatory model by expressing a subset of the regulators in trans and monitoring transcription of 7 known virulence factors located within Salmonella pathogenicity island 2 (SPI-2). These experiments validated the regulatory model and showed that the response regulator SsrB and the MarR type regulator, SlyA, are the terminal regulators in a cascade that integrates multiple signals. Furthermore, experiments to demonstrate epistatic relationships showed that SsrB can replace SlyA and, in some cases, SlyA can replace SsrB for expression of SPI-2 encoded virulence factors. A total of 55 RNA pools from 14 strains in 4 conditions were prepared. An additional 12 RNA samples were prepared from the parental strain. All were subjected to microarray analysis according to the procedures described (Porwollik S et al. 2003 Nucleic Acids Res 31: 1869M-bM-^@M-^S1876). Genomic DNA from 14028s was isolated and used as reference for hybridization.

ORGANISM(S): Salmonella enterica subsp. enterica serovar Typhimurium str. 14028S

SUBMITTER: Bryan Frank

PROVIDER: E-GEOD-38378 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Coordinated regulation of virulence during systemic infection of Salmonella enterica serovar Typhimurium.

Yoon Hyunjin H McDermott Jason E JE Porwollik Steffen S McClelland Michael M Heffron Fred F

PLoS pathogens 20090220 2

To cause a systemic infection, Salmonella must respond to many environmental cues during mouse infection and express specific subsets of genes in a temporal and spatial manner, but the regulatory pathways are poorly established. To unravel how micro-environmental signals are processed and integrated into coordinated action, we constructed in-frame non-polar deletions of 83 regulators inferred to play a role in Salmonella enteriditis Typhimurium (STM) virulence and tested them in three virulence ...[more]

PMID: 19229334

Publication: 1/2

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Project description:To cause a systemic infection, Salmonella must respond to many environmental cues during mouse infection and express specific subsets of genes in a temporal and spatial manner, but the regulatory pathways are poorly established. To unravel how micro-environmental signals are processed and integrated into coordinated action, we constructed in-frame non-polar deletions of 83 regulators inferred to play a role in Salmonella enteriditis Typhimurium (STM) virulence and tested them in three virulence assays (intraperitoneal [i.p.], and intragastric [i.g.] infection in BALB/c mice, and persistence in 129X1/SvJ mice). Overall, 35 regulators were identified whose absence attenuated virulence in at least one assay, and of those, 14 regulators were required for systemic mouse infection, the most stringent virulence assay. As a first step towards understanding the interplay between a pathogen and its host from a systems biology standpoint, we focused on these 14 genes. Transcriptional profiles were obtained for deletions of each of these 14 regulators grown under four different environmental conditions. These results, as well as publicly available transcriptional profiles, were analyzed using both network inference and cluster analysis algorithms. The analysis predicts a regulatory network in which all 14 regulators control the same set of genes necessary for Salmonella to cause systemic infection. We tested the regulatory model by expressing a subset of the regulators in trans and monitoring transcription of 7 known virulence factors located within Salmonella pathogenicity island 2 (SPI-2). These experiments validated the regulatory model and showed that the response regulator SsrB and the MarR type regulator, SlyA, are the terminal regulators in a cascade that integrates multiple signals. Furthermore, experiments to demonstrate epistatic relationships showed that SsrB can replace SlyA and, in some cases, SlyA can replace SsrB for expression of SPI-2 encoded virulence factors.

Project description:Salmonella Typhimurium is an enteric food-borne pathogen responsible for causing salmonellosis associated with acute diarrhea. The bacterial invasion initially requires flagellar motility to reach the intestinal lumen and to cross the mucus layer of the intestinal epithelia and subsequent adhesion to the host cell, mainly mediated by fimbriae .The key genes involved in the pathogenic process are encoded within highly conserved regions of the bacterial genome called Salmonella Pathogenicity Islands (SPIs). The key regulator HilA, which is positively regulated by HilC and HilD, is located in SPI-1;however, other virulence-related regulators, such as RtsA, are encoded outside this island . During the invasion, proteins encoded by the prgHIJK, spaMNOPQRS, and invABCEFGH SPI-1 operons constitute a Type-3 Secretion System. Effector proteins,such as SipA and SipC (encoded in SPI-1) and SigD/SopB (encoded in SPI-5), translocate to the host cytosol through this system causing intracellular changes and inducing the immune response . The survival and replication of intracellular Salmonella inside Salmonella-containing vacuoles (SCVs) is mainly mediated by the genes located in SPI-2. The immune response is activated through the recognition of pathogen-associated molecular patterns (PAMPs) by specific receptors expressed on the surface and/or inside different cell types. The most important PAMPs are flagellin (monomers comprising the flagella filaments), particularly the FliC subunit, which is highly expressed on the bacterial surface, and lipopolysaccharide (LPS).Oxygen availability is limited in the inflamed gut whereas other compounds, such as hydrogen sulfide (H2S) and H2 are abundantly produced by the colonic bacteria. In addition, nutrients are also limited inside the SCV, while reactive oxygen as wellas nitrogen species may be found . In anaerobic conditions, Salmonella is able to survive by using alternative energy sources, such as nitrate or fumarate, through the use of specific enzymes: nitrate and nitrite reductases, fumarate reductase , DMSO reductase and respiratory hydrogenases.In a previous study, we compared transcriptomes of a clinical isolate of S. Typhimurium (strain 50-wt) and its derivative-multidrug-resistant mutant (strain 50-64) using microarrays . In addition to showing antimicrobial resistance, strain was also less invasive. In the present study, we focused on the genes of unknown function that showed impaired expression. We further selected those genes with a putative role in the acquired resistance phenotype or in the observed repressed virulence observed. Here, 4 putative membrane proteins and the yedF of unknown function were investigated to evaluate their involvement inthese two phenotypes.

Project description:Background: Biofilm formation by Salmonella species enhances the capacity of these pathogenic bacteria to survive stresses that are commonly encountered within food processing and during host infection. The persistence of Salmonella within the food chain has become a major health concern, as biofilms can serve as a reservoir to contaminate food products. While the molecular mechanisms required for the survival of bacteria on surfaces are not fully understood, transcriptional studies of other bacteria have demonstrated that biofilm growth triggers the expression of specific sets of genes, compared with planktonic cells. Until now, most transcriptomic studies of Salmonella have focused on the effect of infection-relevant stressors on virulence and on comparing mutant and wild-type (WT). However little is known about the physiological responses taking place inside a Salmonella biofilm. Results: We have determined the transcriptomic and proteomic profiles of biofilms of Salmonella enterica serovar Typhimurium. We discovered that 124 of detectable proteins were differentially expressed in the biofilm compared with planktonic cells, and that 10% (433 genes) of S. Typhimurium genes showed a biofilm-specific pattern of transcription (i.e. a 2-fold or more change in the biofilm compared with planktonic cells). The genes that were significantly up-regulated implicated specific cellular processes in biofilm development including amino acid metabolism, cell motility, global regulation and tolerance to stress. We found that the most highly down-regulated genes in the biofilm were located on Salmonella Pathogenicity Island 2 (SPI2), but that a functional SPI2 secretion system regulator (ssrA) was required for WT biofilm formation. We identified STM0341 as a gene of unknown function that was needed for WT biofilm growth. Genes involved in tryptophan (trp) biosynthesis and transport were up-regulated in the biofilm. Deletion of trpE led to a decrease in bacterial attachment and we show that this biofilm defect is restored by exogenous tryptophan or indole. Conclusion: Biofilm growth of S. Typhimurium causes distinct changes in gene and protein expression. Our results show that aromatic amino acids make an important contribution to biofilm formation, and reveal that SPI2 genes, required for virulence in host cells, show opposing patterns of expression to biofilm-specific genes in S. Typhimurium. Overnight cultures of SL1344 grown in CFA broth were standardized to achieve an initial concentration of 10^6 CFU ml-1 and injected into a stirring influent flask containing 5 L of pre-warmed (25C) sterile CFA medium. The inoculated influent was then pumped through silicon tubing (5 mm internal diameter, Samco Silicon Products Ltd.) at 60 ml h-1 using a peristaltic pump (Minipulse 3, Gilson). The bacteria were allowed to flow through the closed system and either attach to a vertical piece of silicon tubing (1 meter length, 16 mm internal diameter, Samco Silicon Products Ltd.) or flow out into a waste reservoir. The tubing was positioned vertically to collect biofilm cells adherent to the tubing and minimise collecting bacteria that had simply sedimented. Possible backflow of media or bacteria into the influent from the silicon tubing was eliminated using media breaks. Samples taken to determine the pH of the medium or bacterial cell counts were removed via a media port located near the effluent and influent vessel, respectively. Planktonic cells were removed after 72 h of growth from the influent vessel to avoid any contamination with biofilm cells. The entire system was kept at a constant temperature of 25C and biofilm cells were isolated after 72 h of growth. All planktonic and biofilm samples were isolated from the same experimental system and used for both RNA and protein isolation. For this study, four biological replicates were performed at four different dates (Jun25, Jul5, Jul16, Oct11; as indicated in the sample title). For the dual-colour microarray hybridisations, we used Salmonella genomic DNA as the comparator which also acted as the control for spot quality. Individual labelled cDNA samples (RNA) were hybridised up to 4 times (technical replicates).

Dataset Information

Transcriptional profiling of 14 regulators required for Salmonella virulene

Publications

Coordinated regulation of virulence during systemic infection of Salmonella enterica serovar Typhimurium.

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