Journal: The EMBO Journal

Article Title: A bipartite bacterial virulence factor targets the complement system and neutrophil activation

doi: 10.1038/s44318-024-00342-8

Figure Lengend Snippet: ( A , B ) co-IP assays. For this experiment, dHL-60 cell lysates were incubated with or without His-tagged N0362 protein (10 µg) for 3 h at 4 °C. Then, 5 µl Ni-NTA resin slurry was added to the mixture and further incubated at 4 °C for overnight. Following the incubation, the mixture was washed five times with PBS containing 0.05% Tween 20. The final pellets were suspended in Laemmli sample buffer, boiled for 5 min, and then briefly centrifuged to collect the supernatants, which were then subjected to immunoblotting analysis probed against different antibodies, including mAb-N7, a monoclonal antibody that recognizes N0362, anti-FPR1 (αFPR1), anti-CXCR1 (αCXCR1), anti-CXCR2 (αCXCR2), anti-CD11b (αCD11b), and anti-C5αR (αC5αR, Fig. ). ( C , D ) Immunofluorescence analysis of dHL-60 cells. The cells were incubated with Alexa Fluor 488-labeled N0362 proteins for 2 h, stained with αFPR1 or αCXCR1, followed by staining with Alexa Fluor 594. The micrographs were captured under fluorescence microscopy using FITC, TRITC, and DAPI emission filters, and the images were merged. The scale bars represent 20 µm. ( E , F ) Downregulation of FPR1 or CXCR1 expression using siRNA impairs the binding of N0362 to dHL-60 cells. For this study, dHL-60 cells were treated using siRNA specific to either ( E ) FPR1 or ( F ) CXCR1 for 96 h, followed by immunofluorescence analysis as above described. The scale bars represent 20 µm. .

Article Snippet: dHL-60 cells (1 × 10 6 ) were seeded into 24-well cell culture plates and transfected with 0.5 μg of either CXCR1 siRNA (sc-40026), FPR1 siRNA (sc-40121) or scramble siRNA (sc-37007, Santa Cruz Biotechnology) using Lipofectamine LTX with PLUS reagent (Invitrogen) for 96 h at 37 °C and 5% CO 2 .

Techniques: Co-Immunoprecipitation Assay, Incubation, Western Blot, Immunofluorescence, Labeling, Staining, Fluorescence, Microscopy, Expressing, Binding Assay

Journal: The EMBO Journal

Article Title: A bipartite bacterial virulence factor targets the complement system and neutrophil activation

doi: 10.1038/s44318-024-00342-8

Figure Lengend Snippet: ( A – C ) For this experiment, DMSO-differentiated HL-60 cell (dHL-60) lysates were incubated with or without His-tagged N0362 protein (10 µg) for 3 h at 4 °C, followed by precipitation using Ni-NTA resin. The resulted co-IP samples were subjected to immunoblotting analysis probed against mAb-N7, a monoclonal antibody that recognizes N0362. ( A ) anti-CXCR2 (αCXCR2), ( B ) anti-CD11b (αCD11b), and ( C ) anti-C5aR (αC5aR). ( D ) siRNA knockdown of FPR1 or CXCR1 reduces the binding of N0362 to HL-60 cells. For this experiment, activated HL-60 cells were transfected with scramble siRNA (control), FPR1 siRNA or CXCR1 siRNA for 3 days and then the cells were co-incubated with His-tagged N0362 for 2 h. Cells were then fixed and stained anti-His antibody. Fluorescent cells were counted and recorded as percentage of positive cells in relative to total cells. * P value < 0.05. ( E , F ) Detection of FPR1 and CXCR1 by immunoblotting analysis. Lane 1: scramble siRNA; Lane siRNA-FPR1 or CXCR1 72 h after the knockdown; and Lane 3: siRNA-FPR1 or CXCR1 96 h. β-actin was used as a loading control.

Article Snippet: dHL-60 cells (1 × 10 6 ) were seeded into 24-well cell culture plates and transfected with 0.5 μg of either CXCR1 siRNA (sc-40026), FPR1 siRNA (sc-40121) or scramble siRNA (sc-37007, Santa Cruz Biotechnology) using Lipofectamine LTX with PLUS reagent (Invitrogen) for 96 h at 37 °C and 5% CO 2 .

Techniques: Incubation, Co-Immunoprecipitation Assay, Western Blot, Knockdown, Binding Assay, Transfection, Control, Staining

Journal: The EMBO Journal

Article Title: A bipartite bacterial virulence factor targets the complement system and neutrophil activation

doi: 10.1038/s44318-024-00342-8

Figure Lengend Snippet: ( A , B ) Determination of affinity constants between N0362 and CXCR1 using surface plasmon resonance (SPR). For this study, recombinant GST-CXCR1 proteins were coupled to CM5 sensors which were used for the binding experiment using six different concentrations of N0362 proteins (ranging from 0.05 µM to 3 µM) diluted in the running buffer. ( A ) Representative sensorgram of CXCR1-N0362 binding plots with kinetic fit (1:2 binding model). ( B ) Dose-dependent saturation binding of N0362 to CXCR1. The equilibrium disassociation constant K D ( k d / k a ) was calculated. ( C ) IL-8 reporter assays using ready-to-assay CXCR1 chemokine receptor cells (Chem-1 cells). For this experiment, Chem-1 cells were first treated with different recombinant N0362 proteins (5 μg) as labeled for 30 min and then stimulated with IL-8 (10 nM). The fluorescence intensity was monitored at Ex/Em = 340/510 nm and Ex/Em = 380/510 nm. The data are presented as the mean of relative fold change based on the fluorescence ratio of 340/380 nm ± standard error of the mean (SEM). The sample with only IL-8 is set as the baseline (onefold). N 0 , N0362 (23–204 aa); N 1 , N0362 (23–114 aa); and N 2 , N0362 (115–204 aa). Five biological samples ( n = 5) were included. Statistical analysis was determined using unpaired t test. .

Article Snippet: dHL-60 cells (1 × 10 6 ) were seeded into 24-well cell culture plates and transfected with 0.5 μg of either CXCR1 siRNA (sc-40026), FPR1 siRNA (sc-40121) or scramble siRNA (sc-37007, Santa Cruz Biotechnology) using Lipofectamine LTX with PLUS reagent (Invitrogen) for 96 h at 37 °C and 5% CO 2 .

Techniques: SPR Assay, Recombinant, Binding Assay, Labeling, Fluorescence

Journal: The EMBO Journal

Article Title: A bipartite bacterial virulence factor targets the complement system and neutrophil activation

doi: 10.1038/s44318-024-00342-8

Figure Lengend Snippet: TDE0362 is expressed, secreted presumably through either bacterial β-barrel assembly machinery (BAM) or tripartite ABC-type toxin complex system (TCS), and then cleaved by PrtP, generating at least two functional units: N0362 with two bacterial Ig-like domains (Big_1 and Big_2) and C0362 with a Mac-1 domain. N0362 binds to FPR1 and CXCR1/2 receptors and blocks neutrophil chemotaxis and activation. C0362 is secreted into extracellular milieu and functions as a cysteine protease that disarms the complement system via degrading key complement factors such as C3 and C4. Collectively, this dual domain protein annihilates both the complement system and neutrophils and renders bacteria protection from these two innate defense mechanisms.

Article Snippet: dHL-60 cells (1 × 10 6 ) were seeded into 24-well cell culture plates and transfected with 0.5 μg of either CXCR1 siRNA (sc-40026), FPR1 siRNA (sc-40121) or scramble siRNA (sc-37007, Santa Cruz Biotechnology) using Lipofectamine LTX with PLUS reagent (Invitrogen) for 96 h at 37 °C and 5% CO 2 .

Techniques: Functional Assay, Chemotaxis Assay, Activation Assay, Bacteria

Journal: The EMBO Journal

Article Title: A bipartite bacterial virulence factor targets the complement system and neutrophil activation

doi: 10.1038/s44318-024-00342-8

Figure Lengend Snippet: Reagents and tools table

Article Snippet: dHL-60 cells (1 × 10 6 ) were seeded into 24-well cell culture plates and transfected with 0.5 μg of either CXCR1 siRNA (sc-40026), FPR1 siRNA (sc-40121) or scramble siRNA (sc-37007, Santa Cruz Biotechnology) using Lipofectamine LTX with PLUS reagent (Invitrogen) for 96 h at 37 °C and 5% CO 2 .

Techniques: Isolation, Recombinant, Plasmid Preparation, Sequencing, Labeling, Staining, Transfection, Software, Binding Assay, Microscopy, Electron Microscopy