rabbit polyclonal anti- s . typhi lps antibody  (sifin diagnostics)

Journal: Cell host & microbe

Article Title: Decoding a Salmonella Typhi regulatory network that controls typhoid toxin expression within human cells

doi: 10.1016/j.chom.2017.12.001

Figure Lengend Snippet: (A) Schematic diagram of the FAST-INSeq method. A random library of INSeq-compatible transposon mutants in the pltB:gfp reporter strain was used to infect Henle-407 cells for 18 hours. Bacteria from infected cells were sorted by FACS and the high and low fluorescence pools were analyzed by INSeq. Schematic of hypothetical results in which the abundance of individual transposon within genes that allow (black arrows) or prevent (red arrows) typhoid toxin expression are depicted. (B) FACS analysis of pltB:gfp in S. Typhi constitutively expressing of mCherry isolated from infected Henle- 407 cells. A histogram is shown depicting the GFP fluorescence intensities of individual bacteria 18 hs post-infection with gates showing the fraction of the population exhibiting high and low levels of GFP. (C) Circular diagram showing the distribution of transposon mutants within the S. Typhi genome in the mutant library. The outer track (black) shows the full S. Typhi genome, the next two tracks (red and blue) show the distribution of genes by DNA strand and the inner track (green) shows the distribution of transposon insertions in the library (inoculum pool) used for the screen. (D) Plot showing the normalized numbers of sequencing reads of transposon insertions within each S. Typhi gene in the inoculum pool versus the post-infection pool. Green and red dots represent genes required for chemotaxis and the function of the SPI-1 T3SS, respectively. See also Figure S1 and Tables S1–S4.

Article Snippet: Cells were then incubated in a 1:10,000 dilution of rabbit polyclonal anti- S . Typhi LPS antibody (Sifin) and a 1/200 dilution of a mouse anti-GM130 antibody (BD Biosciences) in HBSS for 15 minutes at 37°C (5% CO 2 incubator).

Techniques: Bacteria, Infection, Fluorescence, Expressing, Isolation, Mutagenesis, Sequencing, Chemotaxis Assay

Journal: Cell host & microbe

Article Title: Decoding a Salmonella Typhi regulatory network that controls typhoid toxin expression within human cells

doi: 10.1016/j.chom.2017.12.001

Figure Lengend Snippet: (A) Plot showing the normalized numbers of sequencing reads of transposon insertions within each S. Typhi gene in the high fluorescence vs. the low fluorescence pools. Open and black circles represent genes required for biotin biosynthesis or that were over-represented in the low fluorescence pool, respectively. (B) Expression of the pltB::lacZ reporter in wild-type (WT) S. Typhi and the indicated isogenic mutant strains. The levels of β-galactosidase activity in the inoculum and in bacteria isolated from infected cells were normalized by the number of CFU. Values represent the mean +/− standard deviation for three independent determinations. Asterisks indicate statistically significant differences from the wild-type control (** p<0.001, * p<0.05, n.s.s. not statistically significant). (C) FACS analysis of typhoid toxin expression of the indicated S. Typhi strains 18 hs post-infection. Histograms show the GFP fluorescence intensities of individual bacteria with gates showing high and low fluorescence populations. Data for the wild type sample, also shown in Figure 1B, is presented again for clarity. See also Figures S2 and S6 and Table S5.

Article Snippet: Cells were then incubated in a 1:10,000 dilution of rabbit polyclonal anti- S . Typhi LPS antibody (Sifin) and a 1/200 dilution of a mouse anti-GM130 antibody (BD Biosciences) in HBSS for 15 minutes at 37°C (5% CO 2 incubator).

Techniques: Sequencing, Fluorescence, Expressing, Mutagenesis, Activity Assay, Bacteria, Isolation, Infection, Standard Deviation, Control

Journal: Cell host & microbe

Article Title: Decoding a Salmonella Typhi regulatory network that controls typhoid toxin expression within human cells

doi: 10.1016/j.chom.2017.12.001

Figure Lengend Snippet: (A) Expression of the pltB::lacZ reporter in the indicated S. Typhi strains carrying a low copy plasmid encoding phoPQ or the empty vector (EV). The levels of β-galactosidase activity in the inoculum and bacteria isolated from infected Henle-407 cells 20 hours post infection (hpi) were normalized by the number of CFU. (B and C) β-galactosidase activity (Miller units) of the pltB:lacZ and cdtB:lacZ reporters in the indicated S. Typhi ΔphoPQ strains carrying a low copy plasmid encoding phoPQ, phoPQE232K, or the empty vector (EV) (B) or grown in defined growth media under conditions in which PhoPQ activity is either repressed (pH 7.2, 1 mM Mg2+), or stimulated (10 μM Mg2+, 5 μg/ml C18G, pH 4.9) (C). (D) PhoP binding to the promoter regions of the indicated genes in S. Typhi grown under conditions that stimulate (10 μM Mg2+) or repress (2 mM Mg2+) PhoPQ activity. The amount of DNA recovered for the indicated promoters was normalized to the input sample. Fold enrichment values represent the ratio of this value to that of the rpoD control promoter. Asterisks denote promoters that were significantly enriched compared to the rpoD control under the indicated conditions. (E) Expression of the pltB:lacZ and cdtB:lacZ reporters in the indicated S. Typhi strains. The levels of β-galactosidase activity in the inoculum and bacteria isolated from infected cells were normalized by the number of CFU. (F) β-galactosidase activity (Miller units) of the pltB:lacZ and cdtB:lacZ reporters in the indicated S. Typhi strains carrying a plasmid encoding slyA (slyA) or the empty vector (EV). Values represent the mean +/− standard deviation for three independent determinations. Statistical differences between the indicated samples are denoted as follows: **** p<0.0001, *** p<0.001, **p<0.01, *p<0.05, n.s.s. not statistically significant. See also Figure S3.

Article Snippet: Cells were then incubated in a 1:10,000 dilution of rabbit polyclonal anti- S . Typhi LPS antibody (Sifin) and a 1/200 dilution of a mouse anti-GM130 antibody (BD Biosciences) in HBSS for 15 minutes at 37°C (5% CO 2 incubator).

Techniques: Expressing, Plasmid Preparation, Activity Assay, Bacteria, Isolation, Infection, Binding Assay, Control, Standard Deviation

Journal: Cell host & microbe

Article Title: Decoding a Salmonella Typhi regulatory network that controls typhoid toxin expression within human cells

doi: 10.1016/j.chom.2017.12.001

Figure Lengend Snippet: (A) H-NS binding to the promoter regions of the indicated S. Typhi genes after growth in a medium not permissive for typhoid toxin expression (pH 7.2, 2 mM Mg2+). Included in the analysis are the PhoP-activated genes pagC and mgtA as positive and negative controls, respectively. The amount of DNA recovered was normalized to the amount in the input sample. Fold enrichment values represent the ratio of this value to that of the rpoD control promoter. Asterisks denote promoters that were significantly enriched compared to the rpoD control. (B and C) β-galactosidase activity (Miller units) of the pltB:lacZ reporter in the indicated S. Typhi reporter strains grown under non-inducing conditions. Expression of the reporter in five independently-generated Δhns/ΔphoPQ mutant strains is shown (B). Expression of the reporter in the Δhns/ΔphoPQ strain carrying a plasmid encoding hns or the empty vector (EV) (C). Asterisks denote statistical significance when compared to the wild-type sample. (D) Relative mRNA levels of the indicated genes in wild-type or Δhns/ΔphoPQ S. Typhi mutant strains grown under non-inducing conditions for toxin expression. PhoP-activated genes that are silenced by H-NS (pagC) or independent of H-NS (mgtA, slyB) were included as controls. Values shown represent the mean +/− standard deviation for three independent determinations. **** p<0.0001, *** p<0.001, **p<0.01, *p<0.05, n.s.s. not statistically significant.

Article Snippet: Cells were then incubated in a 1:10,000 dilution of rabbit polyclonal anti- S . Typhi LPS antibody (Sifin) and a 1/200 dilution of a mouse anti-GM130 antibody (BD Biosciences) in HBSS for 15 minutes at 37°C (5% CO 2 incubator).

Techniques: Binding Assay, Expressing, Control, Activity Assay, Generated, Mutagenesis, Plasmid Preparation, Standard Deviation

Journal: Cell host & microbe

Article Title: Decoding a Salmonella Typhi regulatory network that controls typhoid toxin expression within human cells

doi: 10.1016/j.chom.2017.12.001

Figure Lengend Snippet: (A and B) Henle-407 cells were infected with pltB:gfp S. Typhi constitutively expressing mCherry. At 18 hours post infection (hpi) the samples were fixed and S. Typhi were visualized using the mCherry (red) signal, while typhoid toxin expression was visualized using the GFP signal (green). A representative field in which all S. Typhi exhibit high levels of typhoid toxin expression is shown (A). A rare infected cell that contains large numbers of S. Typhi, distributed throughout much of the cell, that do not express the pltB:gfp typhoid toxin reporter is also shown (B). (C) HeLa cells were infected with a S. Typhi strain expressing pltB:gfp. At 12 hpi the samples were fixed and the cells were selectively permeabilized using digitonin. The nuclei of HeLa cells were visualized using DAPI staining (blue), which also faintly stained the S. Typhi chromosomal DNA. S. Typhi localized within the cell cytosol was visualized using an antibody against S. Typhi LPS (red) and typhoid toxin expression was visualized using the GFP signal (green). The image shown is a representative example of a rare infected cell that contains large numbers of cytoplasmic S. Typhi that exhibit little or no typhoid toxin expression. Scale bars: 10 μm. For all panels, brightness and contrast were optimized for each of the individual color channels to maximize visual clarity. See also Figures S4 and S5.

Article Snippet: Cells were then incubated in a 1:10,000 dilution of rabbit polyclonal anti- S . Typhi LPS antibody (Sifin) and a 1/200 dilution of a mouse anti-GM130 antibody (BD Biosciences) in HBSS for 15 minutes at 37°C (5% CO 2 incubator).

Techniques: Infection, Expressing, Staining