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Search Results: 1 - 10 of 2041 matches for " Steffen Porwollik "
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Coordinated Regulation of Virulence during Systemic Infection of Salmonella enterica Serovar Typhimurium
Hyunjin Yoon,Jason E. McDermott,Steffen Porwollik,Michael McClelland,Fred Heffron
PLOS Pathogens , 2009, DOI: 10.1371/journal.ppat.1000306
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.
Virulence of 32 Salmonella Strains in Mice
Matthew C. Swearingen, Steffen Porwollik, Prerak T. Desai, Michael McClelland, Brian M. M. Ahmer
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0036043
Abstract: Virulence and persistence in the BALB/c mouse gut was tested for 32 strains of Salmonella enterica for which genome sequencing is complete or underway, including 17 serovars within subspecies I (enterica), and two representatives of each of the other five subspecies. Only serovar Paratyphi C strain BAA1715 and serovar Typhimurium strain 14028 were fully virulent in mice. Three divergent atypical Enteritidis strains were not virulent in BALB/c, but two efficiently persisted. Most of the other strains in all six subspecies persisted in the mouse intestinal tract for several weeks in multiple repeat experiments although the frequency and level of persistence varied considerably. Strains with heavily degraded genomes persisted very poorly, if at all. None of the strains tested provided immunity to Typhimurium infection. These data greatly expand on the known significant strain-to-strain variation in mouse virulence and highlight the need for comparative genomic and phenotypic studies.
The Fur regulon in anaerobically grown Salmonella enterica sv. Typhimurium: identification of new Fur targets
Bryan Troxell, Ryan C Fink, Steffen Porwollik, Michael McClelland, Hosni M Hassan
BMC Microbiology , 2011, DOI: 10.1186/1471-2180-11-236
Abstract: Microarray analysis of anaerobically grown Δfur S. Typhimurium identified 298 differentially expressed genes. Expression of several genes controlled by Fnr and NsrR appeared to be also dependent on Fur. Furthermore, Fur was required for the activity of the cytoplasmic superoxide disumutases (MnSOD and FeSOD). The regulation of FeSOD gene, sodB, occurred via small RNAs (i.e., the ryhB homologs, rfrA and rfrB) with the aid of the RNA chaperone Hfq. The transcription of sodA was increased in Δfur; however, the enzyme was inactive due to the incorporation of iron instead of manganese in SodA. Additionally, in Δfur, the expression of the gene coding for the ferritin-like protein (ftnB) was down-regulated, while the transcription of the gene coding for the nitric oxide (NO·) detoxifying flavohemoglobin (hmpA) was up-regulated. The promoters of ftnB and hmpA do not contain recognized Fur binding motifs, which indicated their probable indirect regulation by Fur. However, Fur activation of ftnB was independent of Fnr. In addition, the expression of the gene coding for the histone-like protein, H-NS (hns) was increased in Δfur. This may explain the observed down-regulation of the tdc operon, responsible for the anaerobic degradation of threonine, and ftnB in Δfur.This study determined that Fur is a positive factor in ftnB regulation, while serving to repress the expression of hmpA. Furthermore, Fur is required for the proper expression and activation of the antioxidant enzymes, FeSOD and MnSOD. Finally, this work identified twenty-six new targets of Fur regulation, and demonstrates that H-NS repressed genes are down-regulated in Δfur.The Ferric uptake regulator (Fur) is a metal-dependent regulator of transcription and post-transcription in bacteria, which senses metal concentration and/or the redox state of the cells (reviewed in [1]). The classical model of the regulatory role of Fur depicts transcriptional repression through ferrous iron that results in Fur-Fe2+ binding to
Nitric Oxide Antagonizes the Acid Tolerance Response that Protects Salmonella against Innate Gastric Defenses
Travis J. Bourret, Steffen Porwollik, Michael McClelland, Rui Zhao, Todd Greco, Harry Ischiropoulos, Andrés Vázquez-Torres
PLOS ONE , 2008, DOI: 10.1371/journal.pone.0001833
Abstract: Background Reactive nitrogen species (RNS) derived from dietary and salivary inorganic nitrogen oxides foment innate host defenses associated with the acidity of the stomach. The mechanisms by which these reactive species exert antimicrobial activity in the gastric lumen are, however, poorly understood. Methodology/Principal Findings The genetically tractable acid tolerance response (ATR) that enables enteropathogens to survive harsh acidity was screened for signaling pathways responsive to RNS. The nitric oxide (NO) donor spermine NONOate derepressed the Fur regulon that controls secondary lines of resistance against organic acids. Despite inducing a Fur-mediated adaptive response, acidified RNS largely repressed oral virulence as demonstrated by the fact that Salmonella bacteria exposed to NO donors during mildly acidic conditions were shed in low amounts in feces and exhibited ameliorated oral virulence. NO prevented Salmonella from mounting a de novo ATR, but was unable to suppress an already functional protective response, suggesting that RNS target regulatory cascades but not their effectors. Transcriptional and translational analyses revealed that the PhoPQ signaling cascade is a critical ATR target of NO in rapidly growing Salmonella. Inhibition of PhoPQ signaling appears to contribute to most of the NO-mediated abrogation of the ATR in log phase bacteria, because the augmented acid sensitivity of phoQ-deficient Salmonella was not further enhanced after RNS treatment. Conclusions/Significance Since PhoPQ-regulated acid resistance is widespread in enteric pathogens, the RNS-mediated inhibition of the Salmonella ATR described herein may represent a common component of innate host defenses.
The Accuracy of Survival Time Prediction for Patients with Glioma Is Improved by Measuring Mitotic Spindle Checkpoint Gene Expression
Li Bie, Gang Zhao, Pui Cheng, Gaelle Rondeau, Steffen Porwollik, Yan Ju, Xiao-Qin Xia, Michael McClelland
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0025631
Abstract: Identification of gene expression changes that improve prediction of survival time across all glioma grades would be clinically useful. Four Affymetrix GeneChip datasets from the literature, containing data from 771 glioma samples representing all WHO grades and eight normal brain samples, were used in an ANOVA model to screen for transcript changes that correlated with grade. Observations were confirmed and extended using qPCR assays on RNA derived from 38 additional glioma samples and eight normal samples for which survival data were available. RNA levels of eight major mitotic spindle assembly checkpoint (SAC) genes (BUB1, BUB1B, BUB3, CENPE, MAD1L1, MAD2L1, CDC20, TTK) significantly correlated with glioma grade and six also significantly correlated with survival time. In particular, the level of BUB1B expression was highly correlated with survival time (p<0.0001), and significantly outperformed all other measured parameters, including two standards; WHO grade and MIB-1 (Ki-67) labeling index. Measurement of the expression levels of a small set of SAC genes may complement histological grade and other clinical parameters for predicting survival time.
Gene set analyses for interpreting microarray experiments on prokaryotic organisms
Nathan L Tintle, Aaron A Best, Matthew DeJongh, Dirk Van Bruggen, Fred Heffron, Steffen Porwollik, Ronald C Taylor
BMC Bioinformatics , 2008, DOI: 10.1186/1471-2105-9-469
Abstract: We extend five methods of gene set analysis from use on experiments with multiple replicates, for use on experiments with few replicates. We then use simulated and real data to compare these methods with each other and with the Fisher's exact test (FET) method. As a result of the simulation we find that a method named MAXMEAN-NR, maintains the nominal rate of false positive findings (type I error rate) while offering good statistical power and robustness to a variety of gene set distributions for set sizes of at least 10. Other methods (ABSSUM-NR or SUM-NR) are shown to be powerful for set sizes less than 10. Analysis of three sets of experimental data shows similar results. Furthermore, the MAXMEAN-NR method is shown to be able to detect biologically relevant sets as significant, when other methods (including FET) cannot. We also find that the popular GSEA-NR method performs poorly when compared to MAXMEAN-NR.MAXMEAN-NR is a method of gene set analysis for experiments with few replicates, as is common for prokaryotes. Results of simulation and real data analysis suggest that the MAXMEAN-NR method offers increased robustness and biological relevance of findings as compared to FET and other methods, while maintaining the nominal type I error rate.DNA microarrays measuring gene expression continue to grow in popularity, furthering our understanding of the genetic operation of organisms spanning humans to prokaryotes. Questions remain, however, about how best to interpret the wealth of gene-by-gene transcriptional levels measured in microarrays. Over the past few years, many statistical methods of analyzing gene expression data in the context of gene sets have been proposed to simplify and increase the impartiality of gene expression data analysis. Gene set methods are designed to aid the investigator in making biological sense of gene expression data by viewing genes under study in the context of a priori identified, biologically relevant, gene sets. Gene sets are gro
Hypochlorous acid and hydrogen peroxide-induced negative regulation of Salmonella enterica serovar Typhimurium ompW by the response regulator ArcA
Eduardo H Morales, Iván L Calderón, Bernardo Collao, Fernando Gil, Steffen Porwollik, Michael McClelland, Claudia P Saavedra
BMC Microbiology , 2012, DOI: 10.1186/1471-2180-12-63
Abstract: H2O2 and HOCl influx was determined both in vitro and in vivo. A S. Typhimurium ompW mutant strain (?ompW) exposed to sub-lethal levels of H2O2 and HOCl showed a decreased influx of both compounds as compared to a wild type strain. Further evidence of H2O2 and HOCl diffusion through OmpW was obtained by using reconstituted proteoliposomes. We hypothesized that ompW expression should be negatively regulated upon exposure to H2O2 and HOCl to better exclude these compounds from the cell. As expected, qRT-PCR showed a negative regulation in a wild type strain treated with sub-lethal concentrations of these compounds. A bioinformatic analysis in search for potential negative regulators predicted the presence of three ArcA binding sites at the ompW promoter region. By electrophoretic mobility shift assay (EMSA) and using transcriptional fusions we demonstrated an interaction between ArcA and one site at the ompW promoter region. Moreover, qRT-PCR showed that the negative regulation observed in the wild type strain was lost in an arcA and in arcB mutant strains.OmpW allows the influx of H2O2 and HOCl and is negatively regulated by ArcA by direct interaction with the ompW promoter region upon exposure to both toxic compounds.Hydrogen peroxide (H2O2) and hypochlorous acid (HOCl) are reactive oxygen species that are part of the oxidative burst encountered by S. Typhimurium upon internalization by phagocytic cells. Under acidic conditions, such as those found inside the phagosome, H2O2 is generated spontaneously by the reaction of two superoxide anion (O2?) molecules [1]. Moreover, S. Typhimurium encodes both periplasmic and cytoplasmic superoxide dismutases that catalyze O2? dismutation to generate H2O2 and molecular oxygen [2-4]. HOCl is produced by the action of myeloperoxidase (MPO) in a reaction that depends on H2O2, Cl?and acidic conditions [5,6]. Taken together, H2O2 and HOCl react with thiol and heme groups, copper and iron salts generating the reactive hydroxyl radica
Genome-wide analysis of the PreA/PreB (QseB/QseC) regulon of Salmonella enterica serovar Typhimurium
Massimo Merighi, Alecia N Septer, Amanda Carroll-Portillo, Aditi Bhatiya, Steffen Porwollik, Michael McClelland, John S Gunn
BMC Microbiology , 2009, DOI: 10.1186/1471-2180-9-42
Abstract: To determine the PreA/PreB regulon in S. Typhimurium, we performed DNA microarrays comparing the wild type strain and various preA and/or preB mutants in the presence of ectopically expressed preA (qseB). These data confirmed our previous findings of the negative effect of PreB on PreA gene regulation and identified candidate PreA-regulated genes. A proportion of the activated loci were previously identified as PmrA-activated genes (yibD, pmrAB, cptA, etc.) or were genes located in the local region around preA, including the preAB operon. The transcriptional units were defined in this local region by RT-PCR, suggesting three PreA activated operons composed of preA-preB, mdaB-ygiN, and ygiW-STM3175. Several putative virulence-related phenotypes were examined for preAB mutants, resulting in the observation of a host cell invasion and slight virulence defect of a preAB mutant. Contrary to previous reports on this TCS, we were unable to show a PreA/PreB-dependent effect of the quorum-sensing signal AI-2 or of epinephrine on S. Typhimurium with regard to bacterial motility.This work further characterizes this unorthadox OmpR/EnvZ class TCS and provides novel candidate regulated genes for further study. This first in-depth study of the PreA/PreB regulatory system phenotypes and regulation suggests significant comparative differences to the reported function of the orthologous QseB/QseC in E. coli.Salmonella spp. have a broad host range and antibiotic resistant isolates are on the rise [1]. Salmonellae infections of humans result in two primary clinical manifestations: enteric (typhoid) fever and gastroenteritis. The latter is characterized by a local infection primarily of the small intestine and involves massive neutrophil transmigration into the intestinal lumen. Typhoid fever is a systemic infection in which the bacterium is carried from the intestinal submucosa to distal organs primarily within host cells such as macrophages.Two-component signal transduction is critic
Global Systems-Level Analysis of Hfq and SmpB Deletion Mutants in Salmonella: Implications for Virulence and Global Protein Translation
Charles Ansong, Hyunjin Yoon, Steffen Porwollik, Heather Mottaz-Brewer, Brianne O. Petritis, Navdeep Jaitly, Joshua N. Adkins, Michael McClelland, Fred Heffron, Richard D. Smith
PLOS ONE , 2009, DOI: 10.1371/journal.pone.0004809
Abstract: Using sample-matched transcriptomics and proteomics measurements it is now possible to begin to understand the impact of post-transcriptional regulatory programs in Enterobacteria. In bacteria post-transcriptional regulation is mediated by relatively few identified RNA-binding protein factors including CsrA, Hfq and SmpB. A mutation in any one of these three genes, csrA, hfq, and smpB, in Salmonella is attenuated for mouse virulence and unable to survive in macrophages. CsrA has a clearly defined specificity based on binding to a specific mRNA sequence to inhibit translation. However, the proteins regulated by Hfq and SmpB are not as clearly defined. Previous work identified proteins regulated by hfq using purification of the RNA-protein complex with direct sequencing of the bound RNAs and found binding to a surprisingly large number of transcripts. In this report we have used global proteomics to directly identify proteins regulated by Hfq or SmpB by comparing protein abundance in the parent and isogenic hfq or smpB mutant. From these same samples we also prepared RNA for microarray analysis to determine if alteration of protein expression was mediated post-transcriptionally. Samples were analyzed from bacteria grown under four different conditions; two laboratory conditions and two that are thought to mimic the intracellular environment. We show that mutants of hfq and smpB directly or indirectly modulate at least 20% and 4% of all possible Salmonella proteins, respectively, with limited correlation between transcription and protein expression. These proteins represent a broad spectrum of Salmonella proteins required for many biological processes including host cell invasion, motility, central metabolism, LPS biosynthesis, two-component regulatory systems, and fatty acid metabolism. Our results represent one of the first global analyses of post-transcriptional regulons in any organism and suggest that regulation at the translational level is widespread and plays an important role in virulence regulation and environmental adaptation for Salmonella.
Analysis of Pools of Targeted Salmonella Deletion Mutants Identifies Novel Genes Affecting Fitness during Competitive Infection in Mice
Carlos A. Santiviago equal contributor,M. Megan Reynolds equal contributor,Steffen Porwollik,Sang-Ho Choi,Fred Long,Helene L. Andrews-Polymenis ,Michael McClelland
PLOS Pathogens , 2009, DOI: 10.1371/journal.ppat.1000477
Abstract: Pools of mutants of minimal complexity but maximal coverage of genes of interest facilitate screening for genes under selection in a particular environment. We constructed individual deletion mutants in 1,023 Salmonella enterica serovar Typhimurium genes, including almost all genes found in Salmonella but not in related genera. All mutations were confirmed simultaneously using a novel amplification strategy to produce labeled RNA from a T7 RNA polymerase promoter, introduced during the construction of each mutant, followed by hybridization of this labeled RNA to a Typhimurium genome tiling array. To demonstrate the ability to identify fitness phenotypes using our pool of mutants, the pool was subjected to selection by intraperitoneal injection into BALB/c mice and subsequent recovery from spleens. Changes in the representation of each mutant were monitored using T7 transcripts hybridized to a novel inexpensive minimal microarray. Among the top 120 statistically significant spleen colonization phenotypes, more than 40 were mutations in genes with no previously known role in this model. Fifteen phenotypes were tested using individual mutants in competitive assays of intraperitoneal infection in mice and eleven were confirmed, including the first two examples of attenuation for sRNA mutants in Salmonella. We refer to the method as Array-based analysis of cistrons under selection (ABACUS).
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