s typhi antibodies  (Aviva Systems)

Journal: eLife

Article Title: Typhoid toxin sorting and exocytic transport from Salmonella Typhi-infected cells

doi: 10.7554/eLife.78561

Figure Lengend Snippet: ( a and b ) Purified FLAG-tagged wild-type typhoid toxin or its PltB S35A mutant unable to bind glycosylated receptor proteins ( a ) were used in affinity purification experiments (outlined in a) to identify typhoid toxin-interacting proteins, which led to the identification of CI-M6PR (see ). ( b ) The interaction between typhoid toxin and CI-M6PR was verified in Salmonella Typhi-infected cells. Henle-407 cells were infected with S . Typhi expressing FLAG-tagged CdtB for 24 hr and the interaction between typhoid toxin and endogenous CI-M6PR was probed by affinity purification with a FLAG antibody (directed to the CdtB subunit of typhoid toxin) and western blot (with antibodies to both FLAG and anti-CI-M6PR). ( c and d ) Co-localization of the S. Typhi-containing vacuole and CI-M6PR. Henle-407 cells were infected with S . Typhi for the indicated times and examined by immunofluorescence with differentially labeled antibodies to S. Typhi and CI-M6PR. The quantification of the co-localization is shown in ( d ). Values (Mander’s overlap coefficient) represent the degree of co-localization between CI-M6PR and S . Typhi and are the mean ± SEM. ****: p<0.0001. Scale bar = 5 µm. SCV: Salmonella -containing vacuole; CI-M6PR: cation-independent mannose-6-phosphate receptor. WCL: whole-cell lysates; IP: immunoprecipitation. Figure 1—source data 1. Unprocessed coomassie stain of the protein gel. Figure 1—source data 2. Interacting proteins of cation-independent mannose-6-phosphate receptor (CI-M6PR) identified by immunoprecipitation-mass spectrometry (IP-MS). Figure 1—source data 3. Unprocessed CdtB and cation-independent mannose-6-phosphate receptor (CI-M6PR) western blots. Figure 1—source data 4. Raw data of .

Article Snippet: Antibody , Anti- S. Typhi (rabbit polyclonal) , Sifin , TS1605 , IF (1:10,000).

Techniques: Purification, Mutagenesis, Affinity Purification, Infection, Expressing, Western Blot, Immunofluorescence, Labeling, Immunoprecipitation, Staining, Mass Spectrometry, Protein-Protein interactions

Journal: eLife

Article Title: Typhoid toxin sorting and exocytic transport from Salmonella Typhi-infected cells

doi: 10.7554/eLife.78561

Figure Lengend Snippet: ( a ) Immunoblot analyses of CI-M6PR expression in parental HEK293T and CI-M6PR-deficient cells generated by CRISPR/Cas9 genome editing. ( b ) Intracellular survival of Salmonella Typhi in parental HEK293T and CI-M6PR-deficient cells. Cells were infected with S . Typhi with a multiplicity of infection of 30, and CFUs were enumerated at 4, 24, and 48 hr after infection. Values are the mean ± SD of three independent experiments. CFU = colony-forming units; SD = standard deviation. ( c ) Toxicity of typhoid toxin in parental HEK293T and CI-M6PR-deficient cells. Cells were treated with a serial dilution of purified typhoid toxin and the typhoid toxin intoxication was evaluated by examining the proportion of cells in G2/M as a consequence of typhoid toxin-mediated DNA damage. The data shown are the mean ± SD of three independent experiments. ( d and e ) Typhoid toxin transport carrier formation in parental HEK293T and CI-M6PR-deficient cells. Cells were infected with a S . Typhi strain expressing 3xFLAG epitope-tagged CdtB and stained with antibodies against the FLAG epitope (green) and S . Typhi LPS (red) ( d ). Scale bar, 5 μm. The quantification of typhoid toxin-associated fluorescent puncta, a measure of typhoid toxin carrier intermediates in infected cells, is shown in ( e ). Values represent relative fluorescence intensity and are the mean ± SEM of one of three independent experiments. ****: p<0.0001, unpaired two-sided t test. The results of two additional experiments are shown in . ( f–h ) Quantification of typhoid toxin export into the infection medium. Infection media obtained from S . Typhi-infected HEK293T parental ( f ) or CI-M6PR-deficient cells ( g ) were serially diluted as indicated and applied to uninfected HEK293T cells. The cell cycle profile of treated cells was analyzed by flow cytometry, and the percentage of cells at the G2/M phase, a measure of typhoid toxin toxicity, was determined. Values are the mean ± SD of three independent experiments. The relative toxicity of the different samples, shown in ( h ), was measured by determining the percentage of cells in the G2/M phase from the results of the dilution of infection media experiments (shown in f and g) fitted by nonlinear regression. Values were normalized relative to those of the parental cells, which was considered to be 100 and are the mean ± SD of three independent experiments. ****: p<0.0001, unpaired two-sided t test. CI-M6PR: cation independent mannose-6-phosphate receptor; TT: typhoid toxin. Figure 2—source data 1. Unprocessed CdtB western blot. Figure 2—source data 2. Raw data of .

Article Snippet: Antibody , Anti- S. Typhi (rabbit polyclonal) , Sifin , TS1605 , IF (1:10,000).

Techniques: Western Blot, Expressing, Generated, CRISPR, Infection, Standard Deviation, Serial Dilution, Purification, Staining, FLAG-tag, Fluorescence, Flow Cytometry

Journal: eLife

Article Title: Typhoid toxin sorting and exocytic transport from Salmonella Typhi-infected cells

doi: 10.7554/eLife.78561

Figure Lengend Snippet: ( a ) Quantification of the intensity of typhoid toxin-associated fluorescent puncta associated with typhoid toxin carrier intermediates in parental HEK293T and CI-M6PR-deficient-cell clones (c29 and c33) infected with Salmonella Typhi. Values represent relative fluorescence intensity and are the mean ± SEM of two independent experiments. ( b and c ) Quantification of typhoid toxin export into the infection medium of parental HEK293T and CI-M6PR-deficient cell clones (c29 and c33) infected with S . Typhi. ( b ) Infection media were serially diluted as indicated and applied to uninfected HEK293T cells. The cell cycle profile of treated cells was analyzed by flow cytometry, and the percentage of cells at the G2/M phase, a measure of typhoid toxin toxicity, was determined. Values are the mean ± SD of three independent experiments. ( c ) The relative toxicity of the different samples was measured by determining the percentage of cells in the G2/M phase from the results of the dilution of infection media experiments (shown in b) fitted by nonlinear regression. Values were normalized relative to those of the parental cells, which was considered to be 100 and are the mean ± SD of three independent experiments. ****: p<0.0001, unpaired two-sided t test. CI-M6PR: cation independent mannose-6-phosphate receptor; TT: typhoid toxin.

Article Snippet: Antibody , Anti- S. Typhi (rabbit polyclonal) , Sifin , TS1605 , IF (1:10,000).

Techniques: Clone Assay, Infection, Fluorescence, Flow Cytometry

Journal: eLife

Article Title: Typhoid toxin sorting and exocytic transport from Salmonella Typhi-infected cells

doi: 10.7554/eLife.78561

Figure Lengend Snippet: ( a ) Quantification of the intensity of fluorescent puncta associated with typhoid toxin carrier intermediates in parental HEK293T and CLTC-deficient-cell clones (c8 and c31) infected with Salmonella Typhi. Values represent relative fluorescence intensity and are the mean ± SEM. ( b and c ) Quantification of typhoid toxin export into the infection medium of parental HEK293T and CLTC (clone c8)- or CLTB (clone c2)-deficient-cells infected with S . Typhi. ( b ) Infection media were serially diluted as indicated and applied to uninfected HEK293T cells. The cell cycle profile of treated cells was analyzed by flow cytometry, and the percentage of cells at the G2/M phase, a measure of typhoid toxin toxicity, was determined. ( c ) Relative toxin export was determined by measuring the relative toxicity of the different samples, which was determined by determining the percentage of cells in the G2/M phase from the results of the dilution of infection media experiments (shown in b) fitted by nonlinear regression. Values were normalized relative to those of the parental cells, which was considered to be 100.

Article Snippet: Antibody , Anti- S. Typhi (rabbit polyclonal) , Sifin , TS1605 , IF (1:10,000).

Techniques: Clone Assay, Infection, Fluorescence, Flow Cytometry

Journal: eLife

Article Title: Typhoid toxin sorting and exocytic transport from Salmonella Typhi-infected cells

doi: 10.7554/eLife.78561

Figure Lengend Snippet: ( a ) Typhoid toxin export into the infection medium. The quantification of the levels of typhoid toxin in the infection media was carried out by serial dilutions as indicated in the legend for . Infection media obtained from Salmonella Typhi-infected HEK293T parental and the indicated deficient cells were serially diluted and applied to uninfected HEK293T cells. The cell cycle profile of treated cells was analyzed by flow cytometry, and the percentage of cells in the G2/M phase, a measure of typhoid toxin toxicity, was determined. The relative toxicity was determined by the percentage of cells at the G2/M phase after treatment with the different of the infection media fitted by nonlinear regression. Values were normalized relative to wild-type cells, which was considered to be 100 and are the mean ± SD of three independent experiments. **: p<0.01, unpaired two-sided t test. n.sd: differences not statistically significant. ( b ) Western blot analysis of the expression of typhoid toxin in parental HEK293T and Rab11B-deficient cells. Cells were infected with S . Typhi expressing FLAG-tagged CdtB, lysed 24 hr after infection and analyzed by western blot with antibodies directed to the FLAG epitope and the Salmonella protein RecA. ( c and f ) Quantification of the intensity of fluorescent puncta associated with typhoid toxin carrier intermediates in parental HEK293T and Rab11B- ( c ) or Rip11-deficient ( f ) cells. Values represent relative fluorescence intensity and are the mean ± SEM. n.s: differences not statistically significant. The results of an additional independent experiment are shown in . ( e ) Western blot analysis of the expression of typhoid toxin in parental HEK293T and Rip11-deficient cells carried out as indicated in ( b ). ( d ) Relative typhoid toxin export in Rip11-deficient cells. Relative toxin export was determined as indicated in ( a ). Values were normalized relative to parental cells, which was considered to be 100, and are the mean ± SEM. ****: p<0.0001, unpaired two-sided t test. TT: typhoid toxin. Figure 5—source data 1. Raw data of . Figure 5—source data 2. Unprocessed CdtB and RecA western blots. Figure 5—source data 3. Unprocessed CdtB and RecA western blots.

Article Snippet: Antibody , Anti- S. Typhi (rabbit polyclonal) , Sifin , TS1605 , IF (1:10,000).

Techniques: Infection, Flow Cytometry, Western Blot, Expressing, FLAG-tag, Fluorescence

Journal: eLife

Article Title: Typhoid toxin sorting and exocytic transport from Salmonella Typhi-infected cells

doi: 10.7554/eLife.78561

Figure Lengend Snippet: ( a ) Quantification of typhoid toxin export into the infection medium. Infection media obtained from Salmonella Typhi-infected HEK293T parental and the indicated deficient cells were serially diluted and applied to uninfected HEK293T cells. The cell cycle profile of treated cells was analyzed by flow cytometry, and the percentage of cells at the G2/M phase, a measure of typhoid toxin toxicity, was determined. The relative toxicity was determined by the percentage of cells at the G2/M phase from the dilution of infection media fitted by nonlinear regression. Values were normalized relative to parental cells, which was considered to be 100 and are the mean ± SD. ***: p<0.001; **: p<0.01, unpaired two-sided t test. ( b ) Western blot analysis of the expression of typhoid toxin in parental HEK293T and the SNAP-23-, VAMP7-, and STX4-deficient cells. Cells were infected with S . Typhi expressing FLAG-tagged CdtB, lysed after 24 hr of infection, and analyzed by western blot with antibodies directed to FLAG and the Salmonella protein RecA. ( c ) Quantification of the intensity of fluorescent puncta associated with typhoid toxin carrier intermediates in parental HEK293T and the indicated deficient cells. Cells were infected with a S . Typhi strain expressing FLAG-tagged CdtB and the levels of fluorescence associated with typhoid toxin carriers were determined 24 hr after infection. Values represent relative fluorescence intensity and are the mean ± SEM. n.s: differences not statistically significant. The results of an additional independent experiment are shown in . TT: typhoid toxin. Figure 6—source data 1. Raw data of . Figure 6—source data 2. Unprocessed CdtB and RecA western blots.

Article Snippet: Antibody , Anti- S. Typhi (rabbit polyclonal) , Sifin , TS1605 , IF (1:10,000).

Techniques: Infection, Flow Cytometry, Western Blot, Expressing, Fluorescence

Journal: eLife

Article Title: Typhoid toxin sorting and exocytic transport from Salmonella Typhi-infected cells

doi: 10.7554/eLife.78561

Figure Lengend Snippet: Quantification of the intensity of fluorescent puncta associated with typhoid toxin carrier intermediates in parental HEK293T and the indicated deficient cells. Cells were infected with a Salmonella Typhi strain expressing FLAG-tagged CdtB (to visualize typhoid toxin) and the levels of fluorescence associated with typhoid toxin carriers were determined 24 hr after infection. Values represent relative fluorescence intensity and are the mean ± SEM. *: p<0.05, unpaired two-sided t test. The results shown are from replicates of the experiments shown in .

Article Snippet: Antibody , Anti- S. Typhi (rabbit polyclonal) , Sifin , TS1605 , IF (1:10,000).

Techniques: Infection, Expressing, Fluorescence

Journal: eLife

Article Title: Typhoid toxin sorting and exocytic transport from Salmonella Typhi-infected cells

doi: 10.7554/eLife.78561

Figure Lengend Snippet: Mediated by the action of effector proteins of its two type III secretion systems, Salmonella Typhi gains access to host cells and builds an intracellular niche that allows its replication and survival. Within this intracellular niche, S . Typhi expresses typhoid toxin, which is secreted to the lumen of the Salmonella -containing vacuole by a type X secretion system. Though interaction with its glycan-receptor-binding B subunit, typhoid toxin engages cation-independent mannose-6-phosphate receptor (CI-M6PR), which acts as its sorting receptor and mediates its packaging into vesicle transport carriers aided by COPII complex and Rab11b. The vesicle carriers are transported to the cell periphery by motor proteins engaging the Rab11b-interacting protein Rip11. On the cell periphery, the vesicle carriers undergo exocytosis by fusing to the plasma membrane, a process mediated by SNARE proteins VAMP7 on the vesicle carriers and SNAP23 and STX4 on the plasma membrane.

Article Snippet: Antibody , Anti- S. Typhi (rabbit polyclonal) , Sifin , TS1605 , IF (1:10,000).

Techniques: Glycoproteomics, Binding Assay, Clinical Proteomics, Membrane

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