Single-Copy Green Fluorescent Protein Gene FusionsAllow Accurate Measurement of
            <i>Salmonella</i>
            Gene Expression InVitro and during Infection of MammalianCells

Hautefort, Isabelle; Proença, Maria José; Hinton, Jay C. D.

doi:10.1128/aem.69.12.7480-7491.2003

Cited by 226 publications

(249 citation statements)

References 66 publications

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“…Eight of nine single-copy constructs expressed GFP levels exceeding the detection threshold of Ϸ8,000 copies per Salmonella cell in infected mice ( Fig. 2; and Table 2, which is published as supporting information on the PNAS web site), thus confirming the high in vivo activity of the respective promoters (30).…”

Section: Identification Of Highly In Vivo Expressed Salmonella Antigensmentioning

confidence: 59%

“…Randomly sheared fragments of Salmonella genomic DNA were inserted upstream of a plasmidencoded promoterless gfp ova gene, and the resulting promotertrap librar y was transformed into S. enterica serovar Typhimurium. We retained the original episomal strategy (28) for promoter screening instead of inserting gfp at random positions in the chromosome to prevent inactivation of essential virulence genes (30) and loss of the corresponding promoter fusions during pool infections.…”

Section: Identification Of Highly In Vivo Expressed Salmonella Antigensmentioning

confidence: 99%

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Antigen selection based on expression levels during infection facilitates vaccine development for an intracellular pathogen

Rollenhagen

Sörensen

Rizos

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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Vaccines effective against intracellular pathogens could save the lives of millions of people every year, but vaccine development has been hampered by the slow largely empirical search for protective antigens. In vivo highly expressed antigens might represent a small attractive antigen subset that could be rapidly evaluated, but experimental evidence supporting this rationale, as well as practical strategies for its application, is largely lacking because of technical difficulties. Here, we used Salmonella strains expressing differential amounts of a fluorescent model antigen during infection to show that, in a mouse typhoid fever model, CD4 T cells preferentially recognize abundant Salmonella antigens. To identify a large number of natural Salmonella antigens with high expression levels during infection, we used a quantitative in vivo screening strategy. Immunization studies with five particularly attractive candidates revealed two highly protective antigens that might permit the development of an improved typhoid fever vaccine. In conclusion, we have established a rationale and an experimental strategy that will substantially facilitate vaccine development for Salmonella and possibly other intracellular pathogens.

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Section: Identification Of Highly In Vivo Expressed Salmonella Antigensmentioning

confidence: 59%

Section: Identification Of Highly In Vivo Expressed Salmonella Antigensmentioning

confidence: 99%

Antigen selection based on expression levels during infection facilitates vaccine development for an intracellular pathogen

Rollenhagen

Sörensen

Rizos

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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“…25 Control animals received empty bacteria or recombinant bacteria carrying the same plasmid but with reporter gene expression under control of the constitutive rpsM promoter. 21 In a first set of experiments, we aimed to evaluate the presence of reporter protein in different tissues. At 7 days following injection, tumor and normal tissues were excised and homogenates prepared to quantify colonization and expression of the GFP reporter.…”

Section: Resultsmentioning

confidence: 99%

“…Fragments for subcloning were isolated from low melting agarose using the QiaQuick gel extraction kit (Westburg, Hilden, Germany) and restriction sites incorporated into the primers were then used to subclone fragments into the pSP-Luc+NF expression vector (Promega Corp, Madison, WI) and pZEP-08. 21 Transformation into E. coli TG1 (supE hsd∆5 thi ∆ [lac-proAB]) was performed using chemocompetent cells (Gibco BRL) obtained with the RbCl method. Introduction of recombinant plasmids into attenuated Salmonella was done by electroporation [25 µF, 400 and 2.5 kV,] using 0.2 mm cuvettes.…”

Section: Methodsmentioning

confidence: 99%

Development of a flexible and potent hypoxia-inducible promoter for tumor-targeted gene expression in attenuated salmonella

Mengesha

Dubois²,

Lambin³

et al. 2006

Cancer Biology & Therapy

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“…The backbone of pLS203 was used to construct plasmids for expressing heterologous genes in L. salivarius and other Lactobacillus species. The luxABCDE loci (50) and the gfp ϩ gene from Aequoria victoria (23) were chosen for expression in Lactobacillus. A native promoter (cysKp) of L. salivarius UCC118 was chosen as a constitutive promoter because it was ranked, by global transcriptional analysis, among the top 3% of highly expressed genes during exponential and early stationary growth phase (M. W. Mangan and P. W. O'Toole, unpublished data).…”

Section: Methodsmentioning

confidence: 99%

Characterization of Endogenous Plasmids from Lactobacillus salivarius UCC118

Fang

Flynn²,

et al. 2008

Appl Environ Microbiol

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The genome of Lactobacillus salivarius UCC118 comprises a 1.83-Mb chromosome, a 242-kb megaplasmid (pMP118), and two smaller plasmids of 20 kb (pSF118-20) and 44 kb (pSF118-44). Annotation and bioinformatic analyses suggest that both of the smaller plasmids replicate by a theta replication mechanism. Furthermore, it appears that they are transmissible, although neither possesses a complete set of conjugation genes. Plasmid pSF118-20 encodes a toxin-antitoxin system composed of pemI and pemK homologs, and this plasmid could be cured when PemI was produced in trans. The minimal replicon of pSF118-20 was determined by deletion analysis. Shuttle vector derivatives of pSF118-20 were generated that included the replication region (pLS203) and the replication region plus mobilization genes (pLS208). The plasmid pLS203 was stably maintained without selection in Lactobacillus plantarum, Lactobacillus fermentum, and the pSF118-20-cured derivative strain of L. salivarius UCC118 (strain LS201). Cloning in pLS203 of genes encoding luciferase and green fluorescent protein, and expression from a constitutive L. salivarius promoter, demonstrated the utility of this vector for the expression of heterologous genes in Lactobacillus. This study thus expands the knowledge base and vector repertoire of probiotic lactobacilli.Lactic acid bacteria and in particular members of the genus Lactobacillus are the most common microbes used as probiotics. They may beneficially affect the host upon ingestion by a variety of potential or proven mechanisms (13,41). Similarly to bifidobacteria, lactobacilli are normal inhabitants of human and animal intestines. Among the more than 100 species of the genus Lactobacillus that have been identified, those that are used as . rhamnosus, and L. salivarius (32). Genome sequence availability considerably facilitates the identification of probiotic characteristics of these bacteria and the prediction of their behaviors in the human gastrointestinal (GI) tract. To date, 11 Lactobacillus genome sequences have been published, and at least 11 additional sequencing projects are in progress (6). This information has dramatically improved our understanding of the metabolic processes and genetics of these microorganisms, as well as their potential roles in health promotion of their hosts. However, for the targeted analysis of genes that contribute to probiotic characteristics, the development of molecular tools for these lactobacilli is of paramount importance.Plasmids, autonomously replicating extrachromosomal genetic elements, are widely present in the genus Lactobacillus. About 38% of the species in this genus contain plasmids (60). Endogenous plasmids from Lactobacillus are of interest because of the traits they confer upon the host. For example, these plasmids may harbor genes encoding resistance to antibiotics (39, 54) and metal ions (55), genes encoding bacteriocins (30, 44), gene clusters for conjugation (55), genes involved in adherence and biotin metabolism (10), and genes encoding toxin-antitoxin (TA) pr...

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Single-Copy Green Fluorescent Protein Gene FusionsAllow Accurate Measurement of Salmonella Gene Expression InVitro and during Infection of MammalianCells

Cited by 226 publications

References 66 publications

Antigen selection based on expression levels during infection facilitates vaccine development for an intracellular pathogen

Antigen selection based on expression levels during infection facilitates vaccine development for an intracellular pathogen

Development of a flexible and potent hypoxia-inducible promoter for tumor-targeted gene expression in attenuated salmonella

Characterization of Endogenous Plasmids from Lactobacillus salivarius UCC118

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