scholarly journals Genome-Wide Screen of Salmonella Genes Expressed during Infection in Pigs, Using In Vivo Expression Technology

2007 ◽  
Vol 73 (23) ◽  
pp. 7522-7530 ◽  
Author(s):  
Yanyan Huang ◽  
Christopher L. Leming ◽  
Mitsu Suyemoto ◽  
Craig Altier
Keyword(s):  
Genomic Library ◽  
Clinical Signs ◽  
Great Majority ◽  
Dna Fragments ◽  
Environmental Signals ◽  
Genome Wide ◽  
Serovar Typhimurium ◽  
Genes Encoding ◽  
Elevated Expression

ABSTRACT Pigs are a food-producing species that readily carry Salmonella but, in the great majority of cases, do not show clinical signs of disease. Little is known about the functions required by Salmonella to be maintained in pigs. We have devised a recombinase-based promoter-trapping strategy to identify genes with elevated expression during pig infection with Salmonella enterica serovar Typhimurium. A total of 55 clones with in vivo-induced promoters were selected from a genomic library of ∼10,000 random Salmonella DNA fragments fused to the recombinase cre, and the cloned DNA fragments were analyzed by sequencing. Thirty-one genes encoding proteins involved in bacterial adhesion and colonization (including bcfA, hscA, rffG, and yciR), virulence (metL), heat shock (hscA), and a sensor of a two-component regulator (hydH) were identified. Among the 55 clones, 19 were isolated from both the tonsils and the intestine, while 23 were identified only in the intestine and 13 only in tonsils. High temperature and increased osmolarity were identified as environmental signals that induced in vivo-expressed genes, suggesting possible signals for expression.

scholarly journals CRISPR-Cas9 Screen Reveals PSMB3 Contributes to Gliomagenesis Through Proteasome-Dependent and Independent Mechanisms

2021 ◽  
Author(s):  
Shivani Baisiwala ◽  
Shreya Budhiraja ◽  
Andrew Zolp ◽  
Khizar Nandoliya ◽  
Li Chen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Brain Tumor ◽  
Significant Positive Correlation ◽  
Patient Survival ◽  
Genome Wide ◽  
A Genome ◽  
Elevated Expression ◽  
A Subunit ◽  
Cancer Phenotype

Glioblastoma (GBM) is the most common adult malignant brain tumor, with a median survival of 21 months and a 100% recurrence rate. Even though many of the critical oncogenic drivers for GBM have been identified, the basis of gliomagenesis is still under investigation. To identify novel genes that contribute to GBM progression, we performed a genome-wide CRISPR-Cas9 knockout screen. We identified four previously unstudied genes – PSMB3, CHCHD4, SPDYE5, HSPA1 – which had elevated expression in cancer and demonstrated a significant positive correlation with respect to GBM growth and patient survival in vivo and patient datasets. Furthermore, overexpression of PSMB3 and HSPA5 in neural stem cells resulted in transformation to a cancer phenotype. Further investigation of PSMB3, a subunit of the proteasome, allowed us to identify both ubiquitin-mediated and non-ubiquitin-mediated mechanisms of oncogenesis. Ultimately, the data from our CRISPR screens suggests that these genes drive tumor progression, making them promising therapeutic targets for GBM.

scholarly journals Genetic determinants inSalmonella entericaserotype Typhimurium required for overcoming stressors in the host environment

2019 ◽  
Author(s):  
Rabindra K. Mandal ◽  
Tieshan Jiang ◽  
Young Min Kwon
Keyword(s):  
Drug Target ◽  
Defense Mechanisms ◽  
Disease Burden ◽  
Vaccine Development ◽  
Genetic Determinants ◽  
Innate Defense ◽  
Serovar Typhimurium ◽  
Genes Encoding

AbstractSalmonella entericaserovar Typhimurium (S. Typhimurium), a non-typhoidalSalmonella(NTS), result in a range of diseases, including self-limiting gastroenteritis, bacteremia, enteric fever, and focal infections representing a major disease burden worldwide. There is still a significant portion ofSalmonellagenes whose functional basis to overcome host innate defense mechanisms, consequently causing disease in host, largely remains unknown. Here, we have applied a high-throughput transposon sequencing (Tn-seq) method to unveil the genetic factors required for the growth or survival of S. Typhimurium under various host stressors simulatedin vitro. A highly saturating Tn5 library ofS. Typhimurium 14028s was subjected to selection during growth in the presence of short chain fatty acid (100 mM propionate), osmotic stress (3% NaCl) or oxidative stress (1 mM H2O2) or survival in extreme acidic pH (30 min in pH3) or starvation (12 days in 1X PBS). We have identified an overlapping set of 339 conditionally essential genes (CEGs) required byS. Typhimurium to overcome these host insults. Interestingly, entire eight genes encoding F0F1-ATP synthase subunit proteins were required for fitness in all five stresses. Intriguingly, total 88 genes inSalmonellapathogenicity island (SPI), including SPI-1, SPI-2, SPI-3, SPI-5, SPI-6 and SPI-11 are also required for fitness under thein vitroconditions evaluated in this study. Additionally, by comparative analysis of the genes identified in this study and the genes previously shown to be required forin vivofitness, we identified novel genes (marBCT,envF,barA,hscA,rfaQ,rfbIand putative proteins STM14_1138, STM14_3334, STM14_4825, and STM_5184) that has compelling potential to be exploited as vaccine development and/or drug target to curb theSalmonellainfection.

Transcriptional regulation of sdiA by cAMP-receptor protein, LeuO, and environmental signals in Salmonella enterica serovar Typhimurium

2012 ◽  
Vol 58 (1) ◽  
pp. 10-22 ◽  
Author(s):  
Amy L. Turnbull ◽  
Wook Kim ◽  
Michael G. Surette
Keyword(s):  
Salmonella Enterica ◽  
Salmonella Enterica Serovar Typhimurium ◽  
Receptor Protein ◽  
Camp Receptor Protein ◽  
Late Stationary Phase ◽  
Environmental Signals ◽  
Serovar Typhimurium ◽  
Elevated Expression ◽  
Transcriptional Changes ◽  
Camp Receptor

The sdiA gene encodes for a LuxR-type transcription factor, which is active when bound to N-acyl homoserine lactones (AHLs). Because Salmonella enterica serovar Typhimurium does not produce AHLs, SdiA senses signals produced by other organisms. SdiA is not expressed constitutively, and response is limited to conditions in which elevated expression occurs, but little is known about the regulation of sdiA expression. Here we map the sdiA promoter and define several regulators that directly or indirectly act on the promoter. The major activator of sdiA expression is cAMP-receptor protein (CRP), and we define the CRP operator in the sdiA promoter using promoter and crp mutants. LeuO activates sdiA expression to a lesser extent than does CRP. We demonstrate that LeuO directly binds the sdiA promoter and the Rcs phosphorelay represses sdiA expression. In this study, NhaR, IlvY, and Fur affected sdiA expression indirectly and weakly. Expression in late-stationary phase depended on RpoS. AHL-dependent expression of the SdiA-regulated gene rck correlated to the observed sdiA transcriptional changes in regulator mutants. The data demonstrate that regulation of sdiA involves integration of multiple environmental and metabolic signals.

scholarly journals Transcription of IVIAT and Virulence Genes in Photobacterium damselae subsp. piscicida Infecting Solea senegalensis

2018 ◽  
Vol 6 (3) ◽  
pp. 67 ◽  
Author(s):  
José Núñez-Díaz ◽  
Milena Fumanal ◽  
Ana do Vale ◽  
Catalina Fernández-Díaz ◽  
Miguel Moriñigo ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Iron Acquisition ◽  
Disease Outbreaks ◽  
Head Kidney ◽  
Solea Senegalensis ◽  
Marine Aquaculture ◽  
Environmental Signals ◽  
Genes Encoding ◽  
Photobacterium Damselae

Photobacterium damselae subsp. piscicida (Phdp) is responsible for disease outbreaks in marine aquaculture worldwide. Solea senegalensis, a valuable fish species for aquaculture in the south of Europe, is frequently affected by this pathogen. It is well established that bacteria respond to environmental signals and, in the case of pathogens, this ability may determine the outcome of their interaction with the host. Determination of gene expression under in vivo conditions constitutes a valuable tool in the assessment of microbial pathogenesis. Considering that different hosts may represent different environments for the pathogen, expression of Phdp virulence and in vivo induced antigen (IVIAT) genes during S. senegalensis infection has been determined in the present work. Increased transcription of genes encoding proteins involved in iron acquisition (Irp1, Irp2, HutB and HutD), oxidative stress defence (AhpC and Sod), adhesion (PDP_0080), toxins (AIP56) and metabolism (Impdh, Shmt and AlaRS) were detected in Phdp infecting S. senegalensis head kidney or liver. The highest increases corresponded to genes involved in survival under iron limiting conditions and oxidative stress, indicating their essential role during infection of sole. Results obtained give insight into Phdp virulence strategies and contribute to the identification of promising targets for the control of photobacteriosis.

scholarly journals TheIn VitroRedundant Enzymes PurN and PurT Are Both Essential for Systemic Infection of Mice in Salmonella enterica Serovar Typhimurium

2016 ◽  
Vol 84 (7) ◽  
pp. 2076-2085 ◽  
Author(s):  
Lotte Jelsbak ◽  
Mie I. B. Mortensen ◽  
Mogens Kilstrup ◽  
John E. Olsen
Keyword(s):  
Salmonella Enterica ◽  
Single Gene ◽  
Systemic Infection ◽  
Gene Deletions ◽  
Content Type ◽  
Serovar Typhimurium ◽  
Genes Encoding ◽  
Glycine Cleavage

Metabolic enzymes show a high degree of redundancy, and for that reason they are generally ignored in searches for novel targets for anti-infective substances. The enzymes PurN and PurT are redundantin vitroinSalmonella entericaserovar Typhimurium, in which they perform the third step of purine synthesis. Surprisingly, the results of the current study demonstrated that single-gene deletions of each of the genes encoding these enzymes caused attenuation (competitive infection indexes [CI] of <0.03) in mouse infections. While the ΔpurTmutant multiplied as fast as the wild-type strain in cultured J774A.1 macrophages, net multiplication of the ΔpurNmutant was reduced approximately 50% in 20 h. The attenuation of the ΔpurTmutant was abolished by simultaneous removal of the enzyme PurU, responsible for the formation of formate, indicating that the attenuation was related to formate accumulation or wasteful consumption of formyl tetrahydrofolate by PurU. In the process of further characterization, we disclosed that the glycine cleavage system (GCV) was the most important for formation of C1unitsin vivo(CI = 0.03 ± 0.03). In contrast, GlyA was the only important enzyme for the formation of C1unitsin vitro. The results with the ΔgcvTmutant further revealed that formation of serine by SerA and further conversion of serine into C1units and glycine by GlyA were not sufficient to ensure C1formation inS. Typhimuriumin vivo. The results of the present study call for reinvestigations of the concept of metabolic redundancy inS. Typhimuriumin vivo.

scholarly journals Genome-Wide Expression and Location Analyses of the Candida albicans Tac1p Regulon

2007 ◽  
Vol 6 (11) ◽  
pp. 2122-2138 ◽  
Author(s):  
Teresa T. Liu ◽  
Sadri Znaidi ◽  
Katherine S. Barker ◽  
Lijing Xu ◽  
Ramin Homayouni ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Clinical Isolates ◽  
Azole Resistance ◽  
Dependent Manner ◽  
Major Mechanism ◽  
Zinc Cluster ◽  
New Genes ◽  
Genome Wide ◽  
Genes Encoding

ABSTRACT A major mechanism of azole resistance in Candida albicans is overexpression of the genes encoding the ATP binding cassette transporters Cdr1p and Cdr2p due to gain-of-function mutations in Tac1p, a transcription factor of the zinc cluster family. To identify the Tac1p regulon, we analyzed four matched sets of clinical isolates representing the development of CDR1- and CDR2-mediated azole resistance by using gene expression profiling. We identified 31 genes that were consistently up-regulated with CDR1 and CDR2, including TAC1 itself, and 12 consistently down-regulated genes. When a resistant strain deleted for TAC1 was examined similarly, expression of almost all of these genes returned to levels similar to those in the matched azole-susceptible isolate. Using genome-wide location (ChIP-chip) analysis (a procedure combining chromatin immunoprecipitation with hybridization to DNA intergenic microarrays), we found 37 genes whose promoters were bound by Tac1p in vivo, including CDR1 and CDR2. Sequence analysis identified nine new genes whose promoters contain the previously reported Tac1p drug-responsive element (CGGN4CGG), including TAC1. In total, there were eight genes whose expression was modulated in the four azole-resistant clinical isolates in a TAC1-dependent manner and whose promoters were bound by Tac1p, qualifying them as direct Tac1p targets: CDR1, CDR2, GPX1 (putative glutathione peroxidase), LCB4 (putative sphingosine kinase), RTA3 (putative phospholipid flippase), and orf19.1887 (putative lipase), as well as IFU5 and orf19.4898 of unknown function. Our results show that Tac1p binds under nonactivating conditions to the promoters of its targets, including to its own promoter. They also suggest roles for Tac1p in regulating lipid metabolism (mobilization and trafficking) and oxidative stress response in C. albicans.

scholarly journals Novel Phage Lysin Capable of Killing the Multidrug-Resistant Gram-Negative Bacterium Acinetobacter baumannii in a Mouse Bacteremia Model

2015 ◽  
Vol 59 (4) ◽  
pp. 1983-1991 ◽  
Author(s):  
Rolf Lood ◽  
Benjamin Y. Winer ◽  
Adam J. Pelzek ◽  
Roberto Diez-Martinez ◽  
Mya Thandar ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Genomic Library ◽  
Multidrug Resistant ◽  
Clinical Isolates ◽  
Gram Negative ◽  
Content Type ◽  
Highly Active ◽  
Genes Encoding

ABSTRACTAcinetobacter baumannii, a Gram-negative multidrug-resistant (MDR) bacterium, is now recognized as one of the more common nosocomial pathogens. Because most clinical isolates are found to be multidrug resistant, alternative therapies need to be developed to control this pathogen. We constructed a bacteriophage genomic library based on prophages induced from 13A. baumanniistrains and screened it for genes encoding bacteriolytic activity. Using this approach, we identified 21 distinct lysins with different activities and sequence diversity that were capable of killingA. baumannii. The lysin (PlyF307) displaying the greatest activity was further characterized and was shown to efficiently kill (>5-log-unit decrease) all testedA. baumanniiclinical isolates. Treatment with PlyF307 was able to significantly reduce planktonic and biofilmA. baumanniibothin vitroandin vivo. Finally, PlyF307 rescued mice from lethalA. baumanniibacteremia and as such represents the first highly active therapeutic lysin specific for Gram-negative organisms in an array of native lysins found inAcinetobacterphage.

scholarly journals Delineation of the Salmonella enterica Serovar Typhimurium HilA Regulon through Genome-Wide Location and Transcript Analysis

2007 ◽  
Vol 189 (13) ◽  
pp. 4587-4596 ◽  
Author(s):  
Inge M. V. Thijs ◽  
Sigrid C. J. De Keersmaecker ◽  
Abeer Fadda ◽  
Kristof Engelen ◽  
Hui Zhao ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Target Genes ◽  
Salmonella Enterica Serovar Typhimurium ◽  
Pathogenicity Island ◽  
Secretion System ◽  
Effector Proteins ◽  
Transcript Analysis ◽  
Genome Wide ◽  
Serovar Typhimurium ◽  
Genes Encoding

ABSTRACT The Salmonella enterica serovar Typhimurium HilA protein is the key regulator for the invasion of epithelial cells. By a combination of genome-wide location and transcript analysis, the HilA-dependent regulon has been delineated. Under invasion-inducing conditions, HilA binds to most of the known target genes and a number of new target genes. The sopB, sopE, and sopA genes, encoding effector proteins secreted by the type III secretion system on Salmonella pathogenicity island 1 (SPI-1), were identified as being both bound by HilA and differentially regulated in an HilA mutant. This suggests a cooperative role for HilA and InvF in the regulation of SPI-1-secreted effectors. Also, siiA, the first gene of SPI-4, is both bound by HilA and differentially regulated in an HilA mutant, thus linking this pathogenicity island to the invasion key regulator. Finally, the interactions of HilA with the SPI-2 secretion system gene ssaH and the flagellar gene flhD imply a repressor function for HilA under invasion-inducing conditions.

scholarly journals Hha Is a Negative Modulator of Transcription ofhilA, the Salmonella enterica Serovar Typhimurium Invasion Gene Transcriptional Activator

2001 ◽  
Vol 183 (22) ◽  
pp. 6620-6629 ◽  
Author(s):  
Thomas F. Fahlen ◽  
Rebecca L. Wilson ◽  
Jennifer D. Boddicker ◽  
Bradley D. Jones
Keyword(s):  
Salmonella Enterica ◽  
Transcriptional Activator ◽  
Negative Influence ◽  
Chromosomal Dna ◽  
Invasive Phenotype ◽  
Environmental Signals ◽  
Serovar Typhimurium ◽  
Suboptimal Condition

ABSTRACT An early step in the establishment of Salmonella enterica serovar Typhimurium murine infection is the penetration of the intestinal mucosa of the small intestine. The majority of the genes responsible for the Salmonellainvasive phenotype are encoded on Salmonellapathogenicity island 1, and their transcription is controlled by thehilA transcriptional activator. The expression ofhilA is regulated by environmental signals including oxygen, osmolarity, pH, and growth phase such that the presence of any one suboptimal condition results in repression of hilAexpression and the invasive phenotype. We have conducted a search for negative regulators of hilA by introduction of aSalmonella enterica serovar Typhimurium chromosomal DNA gene bank into a Salmonella enterica serovar TyphimuriumhilA::Tn5lacZY reporter strain. This screen has identified the hha gene as a regulator that exerts a negative influence on hilA expression. Plasmid-encoded hha significantly reduceshilA::Tn5lacZY chromosomal expression, as well as expression of the invasion genesinvF, prgH, and sipC. Anhha null mutation results in substantial derepression of both chromosomally encoded and plasmid-encodedhilA::Tn5lacZY expression. Introduction of plasmid-encoded hha into strain SL1344 results in attenuation of invasion using in vitro and in vivo assays. Importantly, purified Hha protein was found to bind to ahilA DNA promoter fragment, suggesting that the regulatory activity of the Hha protein occurs at thehilA promoter. These data add detail to the developing model of the regulation of Salmonella invasion genes.

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