EMBO/soda-nel huggingface dataset:
A Brucella effector modulates the Arf6-Rab8a GTPase cascade to promote intravacuolar replication (2021)
Figure 2

Figure 2. BspF targets the tubular recycling endosome-to-TGN transport pathway.

(A) Quantification of ss-eGFP-FKBPF36M trafficking in HeLa(M)-C1 cells transfected for 24 h with pmCherry (mCherry) or pmCherry-BspF (mCherry-BspF). Rapamycin was added to initiate secretory traffic of ss-eGFP-FKBPF36M and its colocalization with calnexin (ER), ERGIC-53 (ERGIC), GM130 (Golgi), p230 (TGN), or secretory vesicles (SV) was scored over a 60 min time course. Data are means ± SD from n=3 independent experiments. Asterisks indicate statistically significant differences between mCherry- and mCherry-BspF-expressing cells as determined by a two-way ANOVA with Sidak's multiple comparisons test (P<0.05).

(B) Representative confocal fluorescence micrographs of HeLa cells co-transfected for 24 h to produce GFP-TGN38 and mCherry-BspF and stained for F-actin with AlexaFluor™647-phalloidin. Cells were left untreated or treated with Cytochalasin D (200 nM) for 30 min prior to fixation. Scale bars: 10 and 1 µm (insets).

(C) Quantification of colocalization between mCherry-BspF and GFP-TGN38 in untreated (-CytoD) and Cytochalasin D-treated (+CytoD) HeLa cells. Regions of Interests (ROI, representative shown as insets in panels B) were randomly selected and a Pearson's correlation coefficient was calculated using NIH Fiji image analysis software and Coloc_2 plug-in. Data are means ± SD from n=3 independent experiments in which 2 ROIs from 10 cells (n=20) were analyzed per experiment. The asterisk indicates a statistically significant difference between treatments as determined by a Mann-Whitney test (P<0.05).

(D) Representative Western blot analysis of HeLa cells transfected for 24 h to produce HA-BspF, separated into saponin-, Triton X-100- and SDS-soluble fractions and probed for HA-BspF, Hsp27 (cytosol), Calnexin (membranes) and Lamin A/C (nucleus).

(E) Schematic depicting key host proteins that control transport pathways associated with the TGN-RE-plasma membrane compartment. Protein colors depict their compartmentalized functions.

(F) Representative confocal fluorescence micrographs of HeLa cells co-transfected for 24 h to produce mCherry-BspF and either GFP-Rab6a, GFP-Rab8a or Arf6-GFP and treated with Cytochalasin D (200 nM) for 30 min prior to fixation. Scale bars: 10 and 2 µm (insets).

(G) Quantification of localization of GFP-Rab6a, GFP-Rab8a and Arf6-GFP on mCherry-BspF-labelled tubules in transfected HeLa cells. Data are means ± SD from n=3 independent experiments, in which at least 300 individual cells per experiment were analyzed.
A Brucella effector modulates the Arf6-Rab8a GTPase cascade to promote intravacuolar replication (2021)
Figure 4

Figure 4. BspF interacts with the Arf6 GTPase activating protein ACAP1.

(A) Yeast two-hybrid mating screen showing interaction of BspF with a fragment of ACAP1 (amino-acid residues 460-740; ACAP1Lib) or full-length ACAP1 (ACAP1FL), compared to positive (p53/T antigen) and negative (empty vectors) control matings plated on permissive double dropout (DDO) or selective quadruple dropout (QDO) media. ACAP1 schematic indicates the region of interaction initially identified (ACAP1Lib).

(B) Representative co-immunoprecipitations of HA-BspF and myc-ACAP1 in HeLa cells. HeLa cells were transfected to either co-produce or individually produce HA-BspF and myc-ACAP1 and either HA-BspF or myc-ACAP1 were immunoprecipitated using either anti-HA-conjugated (upper panel) or anti-myc-conjugated (lower panel) magnetic beads following cross-linking (+DSP) or not (-DSP) with dithiobis[succinimidylpropionate]. Input lysates (10% of the post-nuclear supernatant) and co-immunoprecipitates were separated by SDS-PAGE and probed for HA-BspF and myc-ACAP1 by Western blotting.

(C) Representative confocal micrographs of HeLa cells transfected to produce mCherry-BspF and GFP-ACAP1 and treated with Cytochalasin D (200 nM) for 30 min and quantification of colocalization between mCherry-BspF and GFP-ACAP1. Arrows indicate areas of BspF and ACAP1 colocalization. Scale bars: 10 µm and 2 µm (insets). Data are means ± SD from n=3 independent experiments in which 10 cells were analyzed per experiment. Pearson's correlation coefficients were calculated from whole cells using NIH Fiji image analysis software and Coloc_2 plug-in.
A Brucella effector modulates the Arf6-Rab8a GTPase cascade to promote intravacuolar replication (2021)
Figure 5

Figure 5. BspF interferes with ACAP1 to modulate Arf6 activity.

(A) Representative confocal micrograph of HeLa cells transfected to produce either mCherry (red), GFP-ACAP1 (green) and Arf6-HA (blue; left hand panels) or mCherry-BspF (red), GFP-ACAP1 (green) and HA-Arf6 (blue; right hand panels) and treated with Cytochalasin D (200 nM) for 30 min prior to fixation. Scale bars: 10 µm and 2 µm (insets).

(B) Representative Western blot analysis of co-immunoprecipitations of myc-ACAP1 and Arf6-HA in the presence or absence of HA-BspF. HeLa cells were transfected to produce Arf6-HA and combinations of myc-ACAP1 and HA-BspF, or not, and myc-ACAP1 was immunoprecipitated using anti-myc-conjugated magnetic beads. Input lysates (6% of post-nuclear supernatants) and co-immunoprecipitates were separated by SDS-PAGE and probed for Arf6-HA, HA-BspF and myc-ACAP1 by Western blotting. Quantification of the Arf6/ACAP1 ratio was performed by densitometric analysis. Data are means ± SD of 3 independent experiments. The asterisk indicates a statistically significant difference (P = 0.0017, unpaired Student's t test) between BspF-producing and control conditions.

(C) Quantification of Arf6 activity (GTP-Arf6) in HeLa cells transfected to produce either mCherry and Arf6-HA or mCherry-BspF and Arf6-HA by G-LISA. Data are means ± SD of n=3 independent experiments, normalized to mCherry-producing controls. The asterisk indicates a statistically significant difference (P = 0.0026, unpaired Student's t test) between BspF-producing and control conditions.

(D) Bacterial replication in BMMs transduced to either produce GFP, Arf6Q67L-GFP or Arf6T27N-GFP and infected with either wild type (2308), ΔbspF, or complemented ∆bspF (ΔbspF::bspF) bacteria for 24 h. Data are means ± SD of n=4 independent experiments, in which at least 100 cells were analyzed per experiment. Grey dots represent individual cells analyzed (n>300); black dots indicate means of individual experiments. Asterisks indicate statistically significant differences (P<0.05, two-way ANOVA followed by Dunnett's multiple comparisons test) between test and control conditions.

(E) Bacterial replication in BMMs transduced to either produce GFP, GFP-ACAP1 or GFP-ACAP1R448Q and infected with either wild type (2308), ΔbspF, or complemented ∆bspF (ΔbspF::bspF) bacteria for 24 h. Data are means ± SD of n=3 independent experiments, in which at least 100 cells were analyzed per experiment. Grey dots represent individual cells analyzed (n>300); black dots indicate means of individual experiments. Asterisks indicate statistically significant differences (P<0.05, two-way ANOVA followed by Dunnett's multiple comparisons test) between test and control conditions.
SARS-CoV-2 Evades Immune Detection in Alveolar Macrophages (2020)
Figure 1

Figure 1: Alveolar macrophages are highly responsive to IFN-λ and good producers of IFNs. Alveolar macrophages from different donors were purified from BAL fluid by allowing them to attach to the culture dish for 2 hours. Subsequently, they were pre-treated overnight with either type I or type III IFNs (1 ng/mL IFN-λ1 and 10 U/mL IFN-α2, respectively) before infection with Sendai virus (SeV) and influenza A virus (IAV, strain PR8) for 6 hours as indicated. The column labelled c shows control samples collected directly after purification. (A) Total RNA was harvested and subjected to qPCR for quantification of the IFN response measured by IFNA2, IFNB1, and IFNL1. (B) ISG response measured by ISG15 and RSAD2. (C) Quantification of the presence of SeV or IAV. (A-C) Each colour corresponds to a donor; 9 different donors were analysed. The dashed line represents the detection limit. An ordinary one-way-ANOVA test was used for the statistical analysis: ns, not significant; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.
SARS-CoV-2 Evades Immune Detection in Alveolar Macrophages (2020)
Figure 2

Figure 2: Alveolar macrophages do not produce an IFN response upon SARS-CoV-2 challenge. Alveolar macrophages from different donors were purified from BAL fluid and pre-treated with either Poly(I:C) or IFN-λ1 (+; 1 ng/mL, or ++; 10 ng/mL) before infection with SARS-CoV-2. (A) Total RNA was harvested and subjected to qPCR for quantification of the IFN response measured by IFNA2, IFNB1, and IFNL1 (Donors marked in orange and blue were not quantifiable in all test conditions). (B) ISG response measured by ISG15, RSAD2, and IFIT1. (C) Quantification of the presence of SARS-CoV-2. (D) An overview of the timeline of the experiment. (A-D) Each colour corresponds to a donor; 6 different donors were analysed. The dashed line represents the detection limit. An ordinary one-way-ANOVA test was used for statistical analysis: ns, not significant; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.
SARS-CoV-2 Evades Immune Detection in Alveolar Macrophages (2020)
Figure 3

Figure 3: SARS-CoV-2 challenge does not affect the IFN response in alveolar macrophages. Alveolar macrophages from different donors were purified from BAL fluid and infected with SARS-CoV-2 before treatment with either Poly(I:C) or IFN-λ1 (1 ng/mL). (A) Timeline of the experiment. (B) Total RNA was harvested and subjected to qPCR for quantification of the IFN response measured by IFNA2, IFNB1, and IFNL1. (C) ISG response measured by ISG15, RSAD2, and IFIT1 (Donors marked in green and red were not quantifiable in all test conditions). (D) Quantification of the presence of SARS-CoV-2. (E) Western blots were performed using samples from two donors. (A-D) Each colour corresponds to a donor; 5 different donors were analysed. The dashed line represents the detection limit. An ordinary one-way-ANOVA test was used for statistical analysis: ns, not significant; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.
SARS-CoV-2 Evades Immune Detection in Alveolar Macrophages (2020)
Figure 4

Figure 4: SARS-CoV-2 nucleocapsid phosphoprotein decreases over time. Alveolar macrophages from different donors were purified from BAL and treated with SARS-CoV-2 for either 2 or 24 hours. (A) The amount of SARS-CoV-2 nucleocapsid phosphoprotein was quantified using LC-MS/MS. (B) After subtraction of the background (unchallenged AMs), the ratio between the amount of SARS-CoV-2 nucleocapsid phosphoprotein at 24 and 2 hours was calculated. Each colour corresponds to a donor; 3 different donors were analyzed. An ordinary one-way-ANOVA test was used for statistical analysis: ns, not significant; *, P ≤ 0.05.
SARS-CoV-2 Evades Immune Detection in Alveolar Macrophages (2020)
Figure 5

Figure 5: Determination of viral infection effectivity. (A) Total RNA was harvested from AMs and primary human airway epithelial cells (HAE) and subjected to qPCR for quantification of the ACE2 receptor. Each colour corresponds to a donor. The dashed line represents the detection limit. (B) The presence of nucleoprotein mRNA of SARS-CoV-2 in AMs and primary HAE was also quantified after SARS-CoV-2 treatment. Each colour corresponds to a donor. An unpaired t-test test was used for statistical analysis: ****, P≤0.0001. (C) Primer efficiency was tested using serial dilutions of positive samples using qPCR. The Ct values were plotted on a logarithmic scale along with the corresponding dilutions before performing a linear regression in order to calculate the slope of the trend line through the data points. This analysis revealed an amplification factor of 1.92, 1.91, and 1.85 for the SARS-CoV-2, IAV, and SeV primer set, respectively. The different colours correspond to different primer sets.
Immunomodulatory activity of extracellular Hsp70 mediated via paired receptors Siglec‐5 and Siglec‐14 (2015)
Figure 1

Identification of sialic acid‐independent Siglec‐5 ligands

Human tissue sections were probed with Siglec‐5 Fc and Siglec‐5 R119A Fc to determine the presence of potential sialic acid‐independent ligands. Positive signals (in blue) for such ligands were identified in the lumen of the lung bronchioles and prostate ducts, as well as in pancreatic tissues.
Pancreatic tissue lysate or lung BAL were incubated with Siglec‐5 R119A Fc (Sig5‐RA Fc) or Siglec‐7 Fc (Sig7Fc) immobilized onto Protein G beads. The proteins captured by these complexes were identified by mass spectrometric analysis. Hsp70 was the only protein that was captured by the Sig5‐RA Fc bead complex from pancreas lysate and bronchoalveolar lavage, but not captured by Sig7Fc bead complex.
Immunomodulatory activity of extracellular Hsp70 mediated via paired receptors Siglec‐5 and Siglec‐14 (2015)
Figure 2

Co‐localization of Hsp70 with Siglec‐5 and Siglec‐14 ligands in lung bronchioles

Immunofluorescence of human lung and prostate sections probed with Siglec‐5 R119A Fc or Siglec‐14 R119A Fc (red) with an anti‐Hsp70 antibody (green). The co‐localized signals for the Siglec‐Fc probes with an anti‐Hsp70 antibody suggest that Hsp70 is one of the possible sialic acid‐independent ligands of Siglec‐5 and Siglec‐14 in the lung bronchiole. White dashes mark the lung bronchiole epithelium. All images are representative of at least 2–3 replicates.
Immunomodulatory activity of extracellular Hsp70 mediated via paired receptors Siglec‐5 and Siglec‐14 (2015)
Figure 3

Direct interaction between Hsp70 and Siglec‐5 and Siglec‐14

Immunoblot probed with Siglec‐5 Fc and Siglec‐14 Fc to detect direct binding to Hsp70. B6N domain of GBS β‐protein was used as a positive control, and clusterin was used as the negative control.
ELISA demonstrates direct binding of Siglec‐5 Fc and Siglec‐14 Fc, but not Siglec‐7 Fc, to Hsp70. Bovine serum albumin (BSA) was used as the negative control.
Siglec‐5 or Siglec‐14 Fc immobilized on magnetic beads was incubated with heat‐shocked HEK293A cell lysates. A Western blot of the captured proteins using an anti‐Hsp70 antibody demonstrates Hsp70 endogenously produced by HEK293A cells interacts with Siglec‐5 and Siglec‐14.
HEK293A cells were transfected with expression plasmids for Siglec‐7, Siglec‐5 or Siglec‐14, and then subsequently incubated with (red) or without (gray) recombinant Hsp70 (bottom panels). Flow cytometry analysis reveals Hsp70 binding to cell surfaces of HEK293A cells expressing Siglec‐5 and Siglec‐14, but not Siglec‐7. Histogram of Siglec expression after transfection is shown as blue lines (top panels).
Using flow cytometry, undifferentiated or PMA‐differentiated THP1 cells with empty vector (EV), Siglec‐5 (Sig5), or Siglec‐14 (Sig14) overexpression were assessed for the presence of endogenously secreted Hsp70 bound on the cell surface. The mean fluorescence intensity (MFI) of signals from an anti‐Hsp70 antibody on unpermeabilized THP1 cells is shown.

Data information: Data are representative of at least 2–3 replicates. Graphs display mean ± SD. *P 0.05, as determined by unpaired t‐test.
Immunomodulatory activity of extracellular Hsp70 mediated via paired receptors Siglec‐5 and Siglec‐14 (2015)
Figure 4

Hsp70 interacts with V‐set domain of Siglec‐5 and Siglec‐14

A comparison of the relative binding affinities between Siglec‐5 WT versus R119A Fc, and Siglec‐14 WT versus R119A Fc to Hsp70 by ELISA. The arginine‐mutated Siglecs display lower affinity for Hsp70 in comparison with their wild‐type sequence.
A lysine‐to‐alanine mutation in residue 134 in Siglec‐14 Fc (K134A) also abrogates its affinity for Hsp70, as determined by ELISA. Lysine 134 was identified as a possible residue that changes binding affinity by aligning the sequences of Siglec‐5 and Siglec‐14 with Siglec‐7. The Siglec‐14 R119A and K134A double mutant (DM) Fc reduces the interaction with Hsp70 to background levels.
Antibodies against Siglec‐5 and Siglec‐14 neutralized the interaction between Siglec‐14 Fc to Hsp70, as determined by ELISA.
Co‐incubation of B6N peptide with Siglec‐5 and Siglec‐14 Fc decreased binding of each Siglec to Hsp70 in an ELISA.

Data information: Data are representative of at least 2–3 replicates. Graphs display mean ± SD.
Immunomodulatory activity of extracellular Hsp70 mediated via paired receptors Siglec‐5 and Siglec‐14 (2015)
Figure 5

Hsp70 inhibits inflammation through Siglec‐5 in THP1 cells

Secretion of pro‐inflammatory cytokine TNFα by THP1 cells transfected with an empty vector (EV) or Siglec‐5 (Sig5) expression plasmid was evaluated by ELISA after concurrent exposure with LPS and 10 μg/ml Hsp70 or LPS alone. Secretion of TNFα was reduced only in THP1 Sig5 cells exposed to both LPS and Hsp70 but not LPS alone.
Similarly, IL‐8 secretion from THP1 EV and Sig5 cells was evaluated by ELISA after concurrent exposure with LPS and 10 μg/ml Hsp70. Secretion of IL‐8 was also reduced in only THP1 Sig5 cells exposed to both LPS and Hsp70 but not LPS alone.
THP1 Sig5 cells were incubated with Hsp70, and the levels of SHP‐1 recruitment to Siglec‐5 were evaluated by immunoprecipitation of Siglec‐5 and Western blot for SHP‐1. Higher levels of SHP‐1 were co‐immunoprecipitated with Siglec‐5 when the cells were incubated with Hsp70.
Western blots demonstrate phosphorylated p38 (p‐p38) was reduced in THP1 Siglec‐5 cells when exposed to both LPS and Hsp70 in comparison with LPS alone. This reduction was also greater in THP1 Siglec‐5 cells in comparison with the control THP1 cells. Numbers below immunoblots indicate densitometric analysis of each band normalized to the respective cell line's unstimulated control group, divided by the respective loading control (total p38).
Secretion of pro‐inflammatory cytokine IL‐8 by PMA‐differentiated THP1 EV or THP Sig5 cells was evaluated by ELISA after concurrent exposure with 10 ng/ml LPS with an anti‐Hsp70 antibody or isotype‐matched antibody. Secretion of IL‐8 was increased in only THP1 Sig5 cells exposed to LPS and an anti‐Hsp70 antibody in comparison with the isotype control antibody, but this increase was not replicated in THP1 EV cells.

Data information: Data are representative of at least 2–3 replicates. Graphs display mean ± SD. *P 0.05, as determined by unpaired t‐test.
Immunomodulatory activity of extracellular Hsp70 mediated via paired receptors Siglec‐5 and Siglec‐14 (2015)
Figure 6

Hsp70 augments inflammation through Siglec‐14 in THP1 cells

Secretion of TNFα by THP1 cells transfected with a Siglec‐5 or Siglec‐14 expression vector was evaluated by ELISA after exposure to Hsp70 at the various concentrations in the presence of endotoxin‐neutralizing agent polymyxin B. Only THP1 Siglec‐14 cells exhibited significant secretion of TNFα in an Hsp70 dose‐dependent manner.
IL‐8 secretion was also evaluated after THP1 Siglec‐5 and Siglec‐14 cells were exposed to Hsp70. Similarly, only THP1 Siglec‐14 cells exhibited significant secretion of IL‐8 in an Hsp70 dose‐dependent manner.
Western blot analysis demonstrates a greater increase in phosphorylated p38 (p‐p38) from THP1 Siglec‐14 cells after exposure to Hsp70 in comparison with control THP1 EV cells in a time‐dependent manner. Graph below immunoblots indicates densitometric analysis of each band normalized to the respective cell line's unstimulated control group, divided by the respective loading control.
Exposure to Hsp70 also yielded greater degradation of the NF‐κB inhibitor, IκBα, in THP1 Siglec‐14 cells in comparison with control THP1 cells, augmentation of the pro‐inflammatory response. Graph below immunoblots indicates densitometric analysis of each band normalized to the respective cell line's unstimulated control group, divided by the respective loading control.
Secretion of pro‐inflammatory cytokine IL‐8 by PMA‐differentiated THP1 EV or THP Sig 14 cells was evaluated by ELISA after concurrent exposure with an anti‐Hsp70 antibody or isotype‐matched antibody. Secretion of IL‐8 was decreased in only THP1 Sig 14 cells exposed to LPS and an anti‐Hsp70 antibody in comparison with the isotype control antibody, but this change was not observed in THP1 EV cells.

Data information: All figures are representative of at least 2–3 replicates. Graphs display mean ± SD. *P 0.05, as determined by unpaired t‐test.
Immunomodulatory activity of extracellular Hsp70 mediated via paired receptors Siglec‐5 and Siglec‐14 (2015)
Figure 7

Hsp70 modulation of human monocytes is dependent on the SIGLEC14 genotype

TNFα secretion from SIGLEC14+/+ monocytes was measured after exposure to LPS or Hsp70 in the presence or absence of endotoxin‐neutralizing compound polymyxin B. Hsp70 was able to induce secretion of TNFα even in the presence of polymyxin B, suggesting a mechanism independent of endotoxin contaminants. DnaK, the E. coli homolog of human Hsp70, did not induce significant secretion of TNFα above background levels.
TNFα secretion from SIGLEC14+/+ monocytes by Hsp70 was reduced when the cells were exposed to an anti‐Siglec‐5/Siglec‐14 blocking antibody.
Human monocytes isolated from SIGLEC14+/+, SIGLEC14+/−, and SIGLEC14−/− individuals were exposed to varying concentrations of Hsp70, and TNFα secretion was evaluated. The amount of TNFα secreted was normalized to the respective cells’ response to 1 ng/ml LPS to minimize the impact of other possible polymorphisms and factors that contribute to variation between individuals.
TNFα secretion from SIGLEC14−/− monocytes stimulated with 15 ng/ml LPS and various concentrations of Hsp70. TNFα secretion was reduced with increasing concentrations of Hsp70.

Data information: Data are representative of at least 2–3 replicates. Graphs display mean ± SD. *P 0.01, as determined by unpaired t‐test.
Regulators of complement activity mediate inhibitory mechanisms through a common C3b-binding mode (2016)
Figure 1

Figure 1 - Structural overview of C3b-regulator complexes. A. From left to right, Surface representation of the three-dimensional structures of C3b-MCP (CCP1-4) (blue), C3b-SPICE (CCP1-4) (green), C3b-FH (CCP1-4) (orange), C3b-CR1 (CCP15-17) (dark red), C3b-DAF (CCP2-4) (purple). B. Cartoon representation of the five regulators. The width of the cartoon is proportional to the B-factors observed in the structural data. The B-factor coloring (from blue to red) is in relative scale according to the resolution of the crystal structures. C. Cartoon representation of the five C3b molecules in the C3b-regulator complexes. Coloring and style as in B. D. Highlight of electron density maps (2Fo-Fc, blue, contour level 1.2σ; Fo-Fc, green and red, contour level 3σ and -3σ, resp.) for the regions of the C3b-regulator complexes showing flexibility. From left to right: the putative site for MCP CCP2, SPICE CCP4, CR1 CCP15, and DAF CCP2.
Regulators of complement activity mediate inhibitory mechanisms through a common C3b-binding mode (2016)
Figure 2

Figure 2 - Domain organization in the C3b-regulator complexes and molecular interactions. A. "Open-book" surface representation of C3b and regulator molecules, highlighting their domain organizations using different colors as shown in the neighboring schemes. Domains in complement regulators are labeled using domain numbering from CCPi to CCPiv based on their contact sites on C3b. The corresponding CCP domains in each regulator described in this work are shown in the bottom-left scheme. Domains CCP1-2 of MCP are shown in shaded grey because they are not involved in C3b binding. The table reports the extent of the regulator contact surface on C3b and the associated buried surface area. B. Opened view of the molecular footprint of the regulator-C3b interaction (as shown in Fig. 2A), highlighting the C3b domains on the regulator surface (top) and the regulator domains on C3b (bottom).