Journal: Nature Communications

Article Title: Identification of TFPI as a receptor reveals recombination-driven receptor switching in Clostridioides difficile toxin B variants

doi: 10.1038/s41467-022-33964-9

Figure Lengend Snippet: a Schematic diagram of the CRISPR-Cas9 screen process. b Genes identified by NGS were analyzed with the MAGeCK program and plotted based on the log 2 value of fold change of NGS reads and statistical significance (shown as log 10 value of RRA p -value and plotted as the y -axis). The genes involved in the GPI (glycosylphosphatidylinositol) biosynthetic pathway are colored red. c Schematic diagram of human TFPIβ (TFPI), a GPI-anchored protein with two BPTI/Kunitz protease inhibitor domains (K1 and K2). N, N-termini; C, C-termini. HeLa ( d ) or A549 ( e ) KO cells lacking TFPI, TFPI2 (a homolog of TFPI), PIGS, or PIGV were generated via the CRISPR-Cas9 approach. UGP2-KO cells were also analyzed as a control. Cells were exposed to recombinant TcdB4.2 for 24 h. Their CR 50 values are normalized to WT and plotted in a bar-chart ( f ). Error bars indicate mean ± s.d.; N = 3 (biologically independent experiments); *, p < 0.05; **, p < 0.01 (Student’s t -test, two-sided). g – i HeLa ( g ) or 5637 ( h ) cells overexpressing triple-HA-tagged TFPI, TFPI2, or mouse TFPI (mTFPI) via lentiviral transduction were exposed to TcdB4.2 for 24 h. The percentages of rounded cells were plotted over toxin concentrations. Their CR 50 values are normalized to WT and plotted in a bar-chart ( i ). Error bars indicate mean ± s.d.; N = 3 (biologically independent experiments); **, p < 0.01 (Student’s t -test, two-sided). j Binding of TcdB4.2 (500 nM) to Fc-tagged TFPI, TFPI2, and mTFPI (immobilized onto capture biosensors) was examined using biolayer interferometry (BLI) assays. Fc-tagged extracellular domains of FZD2 (CRD2), SEMA6A, and IgG were used as controls. Representative sensorgrams from one of three independent experiments are shown. k HeLa cells were exposed to either TcdB4.2 alone (4 pM) or TcdB4.2 pre-incubated with Fc-tagged TFPI, TFPI2, or mTFPI at the indicated molar ratios (1:250 ~ 1:20,000) on ice for 1 h. The percentage of cell-rounding at 6 h incubation was plotted. Error bars indicate mean ± s.d.; N = 3 (biologically independent experiments); *, p < 0.05; **, p < 0.01 (Student’s t -test, two-sided). l HeLa-WT, TFPI-KO, and TFPI2-KO cells were exposed to recombinant TcdB2.1, TcdB7.2, TcdB12.1, TcsL, or culture supernatants of C. difficile strains expressing TcdB10.1 or TcdB11.2 for 24 h. The percentages of rounded cells were plotted over toxin concentrations are shown in Supplementary Fig. . Their CR 50 values were normalized to WT and plotted here. Error bars indicate mean ± s.d.; N = 3 (biologically independent experiments). m – p HeLa-WT, two CSPG4 KO single clones (CSPG4#1 and CSPG4#8), and two CSPG4/TFPI double KO cells (CSPG4#1-TFPI-KO and CSPG4#8-TFPI-KO) were exposed to TcdB4.2 ( m ), TcdB2.1 ( n ), or TcdB2.2 ( o ) for 24 h. The percentages of rounded cells were plotted over toxin concentrations. Their relative CR 50 values are plotted in a bar-chart ( p ). Error bars indicate mean ± s.d.; N = 3 (biologically independent experiments); **, p < 0.01 (Student’s t -test, two-sided). q HeLa cells overexpressing HA-tagged TFPI, TFPI2, or mTFPI via lentiviral transduction were exposed to recombinant TcdB2.1, TcdB2.2, TcdB7.2, TcdB12.1, TcsL, or culture supernatants of C. difficile strains expressingTcdB10.1 or TcdB11.2 for 24 h. The percentages of rounded cells were plotted over toxin concentrations and are shown in Supplementary Fig. . Their CR 50 values are normalized to WT and plotted here. Error bars indicate mean ± s.d.; N = 3 (biologically independent experiments); **, p < 0.01 (Student’s t -test, two-sided). Source data are provided as a Source Data file.

Article Snippet: The cDNAs of TFPI were obtained from the indicated vendors: TFPI (Horizon Discovery, MHS6278-202756867), TFPI2 (Horizon Discovery, MHS6278-202839472), and mouse TFPI (Sino Bio, MG50131-M).

Techniques: CRISPR, Protease Inhibitor, Generated, Recombinant, Transduction, Binding Assay, Incubation, Expressing, Clone Assay

Journal: Nature Communications

Article Title: Identification of TFPI as a receptor reveals recombination-driven receptor switching in Clostridioides difficile toxin B variants

doi: 10.1038/s41467-022-33964-9

Figure Lengend Snippet: a Schematic diagrams of TcdB4.2, TcdB4.2 1286–1805 , TcdB4.2 1835–2367 , TcdB1.1-FBD. TcdB4.1(B1.1) and TcdB1.1(B4.2) represent two mutant fragments exchanging 29 residues in the region 1432– 1600 that differ between TcdB4.1 and TcdB1.1. The numbers indicate the position of amino acid residues. GTD, glucosyltransferase domain; CPD, cysteine protease domain; DRBD, delivery/receptor-binding domain; CROPs, combined repetitive oligopeptides. Binding of 500 nM TcdB4.2 1286– 1805 ( b ) or TcdB4.2 1835– 2367 ( c ) to Fc-tagged TFPI and mTFPI was examined using BLI assays. Fc-tagged TFPI2, CRD2, SEMA6A, and IgG were used as controls. Representative sensorgrams from one of three independent experiments are shown. d HeLa cells transiently transfected with TFPI, TFPI2, mTFPI, SEMA6A, or FZD2 were exposed to FLAG-tagged TcdB4.2 1286– 1805 (5 µg/mL) on ice for 60 min, washed, fixed, permeabilized, and subjected to immunostaining analysis. Expression of exogenous proteins was confirmed by detecting fused HA or 1D4 tag. Nuclei were labeled with DAPI (blue). Scale bar, 5 µm. Representative images were from one of three independent experiments. Binding of 500 nM TcdB1.1-FBD, TcdB4.2 1286– 1805 , TcdB4.2(B1.1), and TcdB1.1(B4.2) to Fc-tagged CRD2 ( e ) and TFPI ( f ) was examined using BLI assays. Representative sensorgrams from one of three independent experiments are shown. g Schematic diagram of TFPIβ, TFPI-K1, and TFPI-K2 fragments. Sig, signal peptide; N, N-terminal domain; K1, BPTI/Kunitz inhibitor domain 1; L1, loop 1; K2, BPTI/Kunitz inhibitor domain 2; L2, loop 2; β, GPI anchor sequence for TFPIβ. h Binding of 500 nM TcdB4.2 1286-1805 to Fc-tagged TFPI, TFPI-K1, and TFPI-K2 was examined using BLI assays. Representative sensorgrams from one of three independent experiments are shown. i , j TcdB4.2 binding to TFPI-K2 prevents TFPI-K2 binding to its natural ligand coagulation factor Xa (FXa). FXa (0.5 ng/mL) cleaves its fluorescently labeled substrate and generates increasing fluorescent signal (measured as relative light unit, RLU, y -axis) over time ( x -axis). FXa’s enzymatic activity was inhibited by TFPI-K2 ( i , 7.5 ng/mL). The inhibitory effect of TFPI was blocked by adding TcdB4.2 1286– 1805 in a dose-dependent manner (1:1, 1:3, or 1:10 molar ratio). Representative curve from one of three independent experiments is shown. FXa activity was quantified by measuring the slope of RLU curves (1–10 min) and plotted as a bar-chart ( j ). The same experiments were also carried out for TFPI-Fc and mTFPI-Fc, with their curves shown in Supplementary Fig. and quantification in ( j ). Error bars indicate mean ± s.d.; N = 3; *, p < 0.05; **, p < 0.01 (Student’s t -test, two-sided). Source data are provided as a Source Data file.

Article Snippet: The cDNAs of TFPI were obtained from the indicated vendors: TFPI (Horizon Discovery, MHS6278-202756867), TFPI2 (Horizon Discovery, MHS6278-202839472), and mouse TFPI (Sino Bio, MG50131-M).

Techniques: Mutagenesis, Binding Assay, Transfection, Immunostaining, Expressing, Labeling, Sequencing, Coagulation, Activity Assay

Journal: Nature Communications

Article Title: Identification of TFPI as a receptor reveals recombination-driven receptor switching in Clostridioides difficile toxin B variants

doi: 10.1038/s41467-022-33964-9

Figure Lengend Snippet: a Cultured undifferentiated human enteroids (in growth medium), differentiated human rectoids (in differentiation medium), and mouse intestinal organoids were exposed to either TcdB4.2 alone (10 pM) or TcdB4.2 pre-incubated with Fc-tagged TFPI, TFPI2, or mTFPI (100 nM) for 8 h. PBS was used as control (Ctrl). Stars indicate the dissociated organoids with released luminal contents; arrows indicate shrunken organoids; scale bar, 50 µm. Representative images are from one of three independent experiments. b Experiments were carried out as described in panel ( a ). After exposure to toxin for 3 days, cell viability was measured using the MTT assay and plotted as a bar-chart. Error bars indicate ± s.d.; N = 3 (biologically independent experiments); *, p < 0.05; **, p < 0.01 (Student’s t -test, two-sided). c , d Accumulation of fluid in the thoracic cavity occurred within 15 h after intraperitoneal injection of TcdB4.2 into mice (50 ng per 25 g bodyweight). Injection of TcdB4.2 pre-incubated with Fc-tagged TFPI or mTFPI at 1:2000 molar ratios showed less fluid accumulation. Co-injection of TcdB4.2 with Fc-tagged TFPI2 at 1:2000 molar ratio did not affect fluid accumulation. Injection of saline was included as a control. The range of boxes indicates ± s.e.m.; whiskers indicate ± s.d.; percentiles indicate median; **, p < 0.01 (Student’s t -test, two-sided). e Experiments were carried out as described in panel ( c ) and the edema in lung tissues was evaluated by calculating dry-to-wet weight ratios. TcdB4.2 reduced dry-to-wet weight ratio of lung tissue more than in the saline group. Co-injection of TcdB4.2 with TFPI-Fc or mTFPI-Fc prevented this reduction, whereas co-injection with TFPI2-Fc showed no protection from TcdB4.2. The range of boxes indicates ± s.e.m.; whiskers indicate ± s.d.; percentiles indicate median; *, p < 0.05; **, p < 0.01 (Student’s t -test, two-sided). f , g The sensitivity of HUVECs transfected with siRNAs targeting TFPI to TcdB1.1 or TcdB4.2 was analyzed using the 24 h cell-rounding assay. HUVECs transfected with non-targeting scrambled siRNAs served as a control. Dose-response curves are plotted in ( f ), and their relative CR 50 are plotted in a bar chart ( g ). Error bars indicate ± s.d.; N = 3 (biologically independent experiments); **, p < 0.01; NS not significant (Student’s t -test, two-sided). Source data are provided as a Source Data file.

Article Snippet: The cDNAs of TFPI were obtained from the indicated vendors: TFPI (Horizon Discovery, MHS6278-202756867), TFPI2 (Horizon Discovery, MHS6278-202839472), and mouse TFPI (Sino Bio, MG50131-M).

Techniques: Cell Culture, Incubation, MTT Assay, Injection, Transfection

Journal: Nature Communications

Article Title: Identification of TFPI as a receptor reveals recombination-driven receptor switching in Clostridioides difficile toxin B variants

doi: 10.1038/s41467-022-33964-9

Figure Lengend Snippet: a Amino acids across all 206 known TcdB sequences and 6 TcsL sequences were aligned and visualized using a haplotype coloring algorithm we recently developed , showing variation patterns across TcdB members. The first sequence (TcdB1.1) is assigned black color, and all other sequences colored black if they share the same residues. Unique residues in the second sequence (TcdB2.1) are colored green, followed by unique residues in the third sequence (TcdB4.2) colored red. The region 1460 to 1626 is enlarged for TcdB2/4/7 members. A unique B4/B7-haplotype can be visualized with residues in red color, with their position marked. Residue A1518 is unique in TcdB7 members and is colored gray. b The sequences of TcdB2.11, TcdB2.22, TcdB7.2, and TcdB7.5 were analyzed using a sliding window comparison with TcdB1.1, 2.1, 4.2, and 7.1, revealing their recombination patterns. Below each sliding window plot is a graphical summary depicting the recombination pattern. The location surrounding the TFPI/FZD-binding site (specificity-determining region) is marked. HeLa-WT, TFPI-KO, and TFPI2-KO cells were exposed to recombinant TcdB7.1 ( c ) or the culture supernatant of C. difficile strain expressing TcdB7.9 ( d ) for 24 h. The percentages of round-shaped cells were plotted over toxin or supernatant dilutions. Their CR values are normalized to WT and plotted in a bar-chart ( e ). Error bars indicate mean ± s.d.; N = 3 (biologically independent experiments); **, p < 0.01 (Student’s t -test, two-sided). HeLa cells overexpressing HA-tagged TFPI, TFPI2, or mTFPI via lentiviral transduction were exposed to recombinant TcdB7.1 ( f ) or the culture supernatant of C. difficile strain expressing TcdB7.9 ( g ) for 24 h. The percentages of round-shaped cells were plotted over toxin or supernatant dilutions. Their CR values were normalized to WT and plotted in a bar-chart ( h ). Error bars indicate mean ± s.d.; N = 3 (biologically independent experiments); **, p < 0.01 (Student’s t -test, two-sided). Source data are provided as a Source Data file.

Article Snippet: The cDNAs of TFPI were obtained from the indicated vendors: TFPI (Horizon Discovery, MHS6278-202756867), TFPI2 (Horizon Discovery, MHS6278-202839472), and mouse TFPI (Sino Bio, MG50131-M).

Techniques: Sequencing, Binding Assay, Recombinant, Expressing, Transduction

Journal: Nature Communications

Article Title: Identification of TFPI as a receptor reveals recombination-driven receptor switching in Clostridioides difficile toxin B variants

doi: 10.1038/s41467-022-33964-9

Figure Lengend Snippet: Binding of 500 nM TcdB1.1-FBD, TcdB2.1 1285– 1804 , TcdB2.11 1286– 1805 , TcdB4.2 1286– 1805 , TcdB7.2 1286– 1805 , TcdB7.5 1286– 1805 , TcdB10.1 1285– 1804 , TcdB11.2 1285– 1804 , TcdB12.1 1285– 1804 , and TcsL 1285– 1804 to Fc-tagged TFPI ( a ), mTFPI ( b ), or CRD2 ( c ) was examined using BLI assays. Representative sensorgrams from one of three independent experiments are shown. d , e HeLa-WT, FZD1/2/7-KO, CSPG4-KO, and UGP2-KO cells were exposed to the culture supernatant of a C. difficile strain expressing TcdB2.22 for 24 h. The percentages of round-shaped cells were plotted over supernatant dilution ( d ). The relative CR 50 values in different cell lines were normalized to the WT and plotted as a bar-chart ( e ). Error bars indicate mean ± s.d.; N = 3 (biologically independent experiments); *, p < 0.05; **, p < 0.01 (Student’s t -test, two-sided). f , g HeLa-WT, TFPI-KO, and TFPI2-KO cells were exposed to the culture supernatant of a C. difficile strain expressing TcdB2.22 for 24 h. The percentages of round-shaped cells were plotted over supernatant dilutions ( f ). The relative CR50 values in different cell lines were normalized to the WT and plotted as bar-chart ( g ). Error bars indicate mean ± s.d.; N = 3 (biologically independent experiments). h There are six residues that are different between TFPI-binding TcdB4.2, 2.11, 7.9 versus TcdB2.1 and 7.2 (1495, 1505, 1509, 1547, 1596, and 1599, marked as MT site 1–6). Mutagenesis studies were performed to replace the indicated residues on TcdB2.1 1285-1804 with the corresponding residues found in TcdB4.2. The binding of the indicated mutant proteins (500 nM) to immobilized TFPI-Fc was analyzed using BLI assays. TcdB2.11 1286– 1805 and TcdB2.1 1285– 1804 were analyzed as controls. Representative sensorgrams from one of three independent experiments are shown. i – k A TcdB1.1-FBD-5M mutant proteins were generated by replacing five residues in TcdB1.1 with the corresponding residues found in TcdB4.2 (positions 1495, 1505, 1547, 1596, and 1599). The binding of this mutant protein to immobilized TFPI-Fc ( i ), mTFPI-Fc ( j ), and CRD2 ( k ) was analyzed using BLI assays. TcdB1.1-FBD and TcdB4.2 1285– 1804 were analyzed as controls. Source data are provided as a Source Data file.

Article Snippet: The cDNAs of TFPI were obtained from the indicated vendors: TFPI (Horizon Discovery, MHS6278-202756867), TFPI2 (Horizon Discovery, MHS6278-202839472), and mouse TFPI (Sino Bio, MG50131-M).

Techniques: Binding Assay, Expressing, Mutagenesis, Generated

Journal:

Article Title: Local gene transfer of tissue factor pathway inhibitor regulates intimal hyperplasia in atherosclerotic arteries

doi: 10.1073/pnas.061004098

Figure Lengend Snippet: TFPI secretion in canine VSMC. Human TFPI was measured by the ELISA in the conditioned medium of dog VSMC. (a) Time course of TFPI (in ng/106 cells/24 h) secreted by VSMC infected for 6 h with Ad-TFPI or Ad-RR (identical construct minus the foreign gene) at moi 500 (see Methods). (b) Viral dose response of TFPI secretion in VSMC (ng/106 cells/24 h) 4 days after a 6-h infection with Ad-TFPI. No TFPI was detected in VSMC infected with Ad-RR. (c) Anti-TF/factor VIIa activity of human TFPI secreted by dog VSMC. TFPI activity in the conditioned medium is expressed in TFPI units (where 1 unit corresponds to about 55 ng TFPI in human plasma; see Methods).

Article Snippet: TFPI activity is expressed in TFPI units, whereby 1 TFPI unit corresponds to 55 ng of TFPI, the mean concentration of TFPI found in 1 ml of plasma in 300 human volunteers (American Diagnostica; data available on request).

Techniques: Enzyme-linked Immunosorbent Assay, Infection, Construct, Activity Assay

Journal: Blood

Article Title: Activated protein C cofactor function of protein S: a critical role for Asp95 in the EGF1-like domain

doi: 10.1182/blood-2009-11-256610

Figure Lengend Snippet: Effect of APC and protein S on thrombin generation. Thrombin generation was performed in protein S–deficient plasma with 100nM inhibitory antibodies against TFPI. Up to 10nM APC had no effect on thrombin generation in the absence of protein S. All concentrations generate lines that are superimposable (A). After addition of 120nM protein S (at 0-10nM APC), an APC dose-dependent effect was observed (B). The top single line represents 0 to 10nM APC in the absence of protein S. Protein S in the presence of no or 2.5nM APC generated lines that were superimposable. Conditions used are noted adjacent to the peaks to which they refer. The anticoagulant effect of 10nM APC and 120nM protein S was inhibited by polyclonal antibodies against protein S (C) or against protein C (D). PS indicates protein S; PC, protein C. Representative experiments are shown (n = 3).

Article Snippet: A polyclonal antibody against TFPI (Haematologic Technologies Inc), 100nM, was used to inhibit any protein S cofactor activity toward TFPI.

Techniques: Clinical Proteomics, Generated

Journal: Blood

Article Title: Activated protein C cofactor function of protein S: a critical role for Asp95 in the EGF1-like domain

doi: 10.1182/blood-2009-11-256610

Figure Lengend Snippet: Effect of WT protein S, D95A, D95N, D78A, and Q79A variants on thrombin generation. Thrombin generation was measured in protein S–deficient plasma supplemented with 9nM APC, 100nM antibodies against TFPI, and 0 to 120nM WT protein S (A), protein S D95A (B), protein S D95N (C), or 90nM purified WT (dashed line) or purified protein S D95A (dotted line; D). Protein S concentrations are positioned adjacent to the peaks to which they refer. The cofactor activity of 60nM WT protein S and protein S variants D95A, D78A, and Q79A was compared at 9nM APC (E). Typical experiments are shown (n = 3). Whereas the cofactor activity of WT protein S is highly dependent on the APC concentration used (Figure 1B), that of protein S D95A is not, explaining the difference in fold activity between WT protein S and protein S D95A in Figures 2 and ​and3.3. Dose-response data from titrations with WT protein S, protein S D95A, and protein S D95N in the presence of 9nM APC are shown in panel F (data are expressed as mean ± SD of 2 independent experiments performed in duplicate). Inset in panel B shows recognition of WT protein S and protein S D95A in media by polyclonal antibodies and a monoclonal antibody recognizing only γ-carboxylated Gla domains. Inset in panel D shows the SeeBlue-prestained marker, plasma-purified protein S from Enzyme Research Laboratories Ltd (lane 1), purified recombinant WT protein S (lane 2), and purified protein S D95A (lane 3) visualized with silver staining.

Article Snippet: A polyclonal antibody against TFPI (Haematologic Technologies Inc), 100nM, was used to inhibit any protein S cofactor activity toward TFPI.

Techniques: Clinical Proteomics, Purification, Activity Assay, Concentration Assay, Marker, Recombinant, Silver Staining

Journal: Blood

Article Title: Activated protein C cofactor function of protein S: a critical role for Asp95 in the EGF1-like domain

doi: 10.1182/blood-2009-11-256610

Figure Lengend Snippet: Binding of protein S to phospholipid surfaces. Protein S (0-120nM) was incubated in a plate coated with 25 μg/mL phospholipids. Bound protein S was detected with an HRP-conjugated polyclonal antibody against protein S. A representative experiment is shown. The apparent Kd values, 5.69 ± 1.24 and 9.54 ± 2.26nM for WT protein S and protein S D95A, respectively, were obtained by calculating the mean ± SD of 3 independent experiments performed in duplicate. PL indicates phospholipids.

Article Snippet: A polyclonal antibody against TFPI (Haematologic Technologies Inc), 100nM, was used to inhibit any protein S cofactor activity toward TFPI.

Techniques: Binding Assay, Incubation