RadD from Fusobacterium nucleatum engages NKp46 to promote antitumor cytotoxicity
- The Concern Foundation Laboratories at the Lautenberg Center for Immunology and Cancer Research, Institute for Medical Research Israel Canada (IMRIC), Hebrew University Hadassah Medical School, Israel
- Institute of Medical Microbiology and Hygiene, Medical Centre University of Freiburg, Germany
- The Institute of Dental Sciences, The Hebrew University-Hadassah School of Dental Medicine, Israel
- Section of Biosystems and Function, Division of Oral and Systemic Health Sciences, UCLA School of Dentistry, United States
Figures
Figure 1 with 1 supplement
NKp46 expression modifies the prognostic effect of F. nucleatum in a tumor-type-specific manner.
(A) Kaplan–Meier survival curves for head and neck squamous cell carcinoma (HNSC) patients stratified by F. nucleatum status and NKp46 (NCR1) expression. Patients with concurrent F. nucleatum positivity (n=87) and high NKp46 expression exhibited significantly improved overall survival compared to those who were F. nucleatum-negative (n=44) but NKp46-positive (log-rank p<0.05). (B) Kaplan–Meier survival curves for colorectal cancer (CRC) patients stratified by the same criteria showed no significant difference in survival between F. nucleatum-positive (n=44) and F. nucleatum-negative (n=31) groups. (C) Table summarizing hazard ratios (HR) for F. nucleatum-negative cases among NKp46+ patients. In HNSC, absence of F. nucleatum was associated with a significantly poorer prognosis (HR = 2.08, 95% CI: 1.20–3.61), whereas in CRC, F. nucleatum absence showed no significant association with patient prognosis (HR = 0.71, 95% CI: 0.26–1.95). (D) Comparison of NKp46 expression across HNSC and CRC tumors. Log₂ expression levels of NKp46 mRNA were compared across HNSC and CRC cohorts, stratified by F. nucleatum positive and negative. Results were analyzed by one-way ANOVA with Bonferroni post hoc correction. ****p≤0.0001.
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Figure 1—source data 1
- https://cdn.elifesciences.org/articles/108439/elife-108439-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
SIGLEC7 and CEACAM1 expression and the prognostic effect of F. nucleatum in a tumor-type-specific manner.
Comparison of SIGLEC7 (A) and CEACAM1 (B) expression across HNSC and CRC tumors. Log2 expression levels of NKp46 mRNA were compared across HNSC and CRC cohorts, stratified by F. nucleatum positive and negative. Results were analyzed by one-way ANOVA with Bonferroni post hoc correction. *p<0.05, **p≤0.01, ***p≤0.001, and ****p≤0.0001.
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Figure 1—figure supplement 1—source data 1
The raw data for the SIGLEC7 and CEACAM1 mRNA expression and the prognostic effect of F. nucleatum in a tumor-type-specific manner and statistical tests used and significance.
- https://cdn.elifesciences.org/articles/108439/elife-108439-fig1-figsupp1-data1-v1.xlsx
Figure 2 with 1 supplement
Binding of Fusobacterium nucleatum to NKp46 and its D1 domain.
(A) The figure shows histograms of FITC-labeled F. nucleatum subsp. nucleatum ATCC 23726 (upper histograms) and ATCC 10953 (lower histograms) incubated with 2 μg of NKp46 Ig, D1 domain of NKp46 (D1 Ig), Ncr-1 Ig, and CD16 Ig fusion proteins. Representative staining from one of two independent experiments is shown. (B) An immunoprecipitation assay was performed using an NKp46–Ig fusion protein with F. nucleatum subsp. nucleatum ATCC 23726. Molecular weight markers are shown on the left. Lane 1 (Input) contains total lysates from the bacterial pellet (membrane protein fraction), showing a band corresponding to RadD (~350 kDa)-arrow. Lane 2 (RBD–Ig control) shows immunoprecipitation with 2.5 μg of control RBD–Ig, with no detectable band at ~350 kDa. Lane 3 (NKp46–Ig) shows immunoprecipitation with 2.5 μg of NKp46–Ig, revealing a band at ~350 kDa-arrow. Lanes 4–6 correspond to the supernatant fraction of the bacterial lysate. No bands are observed in lanes 5 and 6, indicating a lack of interaction in this fraction. Lanes 7 and 8 contain 2.5 μg of purified RBD–Ig and NKp46–Ig proteins, respectively.
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Figure 2—source data 1
NKp46 immunoprecipitation with Fusobacterium nucleatum ATCC 23726 lysates.
- https://cdn.elifesciences.org/articles/108439/elife-108439-fig2-data1-v1.zip
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Figure 2—source data 2
An original Western blot gel as well as the labeled and uncropped one, which was used in validating the interaction between Fusobacterium nucleatum and NKp46.
- https://cdn.elifesciences.org/articles/108439/elife-108439-fig2-data2-v1.zip
Figure 2—figure supplement 1
Ccm-1 Ig binding to Fusobacterium ATCC 23726 and ATCC 10953.
Histograms show binding of mCcm1-Ig, NKp46, and Ncr-1 fusion proteins to the two Fusobacterium strains. Gray represents secondary antibody controls.
Figure 3
RadD is the bacterial ligand for NKp46.
(A, B) Density plot of FITC labeled ATCC 10953 (A) and its ∆fadI mutant derivative ATCC 10953 ∆Fad-I (B) stained with the various fusion proteins (listed in the X axis). (C) Schematic representation of ATCC 10953 wild type (WT) strain and RadD surface expression (left) compared to ATCC 10953 ∆Fad-I (right). (D) Density plot of the FITC-labeled ∆RadD mutant strain of ATCC 10953 stained with various fusion proteins (listed in the X axis). The figure shows data from one representative experiment out of three to five independent experiments. (E) Fold change quantification of FITC-labeled bacteria binding to the fusion proteins Ccm1-Ig, NKp46 Ig, and Ncr-1 Ig in ATCC 10953 (left) and ATCC 23726 (right). Summary of three to five independent experiments. The mean value ± SD of the experiments is presented. *p<0.05, **p≤0.01, ***p≤0.001, and ****p≤0.0001.
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Figure 3—source data 1
Raw data for median fluorescent intensity (MFI) measurements corresponding to Figure 3E, including quantification for Fusobacterium nucleatum strains ATCC 10953 and ATCC 23726, as described in the Figure 3E legend.
- https://cdn.elifesciences.org/articles/108439/elife-108439-fig3-data1-v1.xlsx
Figure 4 with 2 supplements
NKp46-02 antibody and arginine block ATCC 10953 binding to NKp46.
(A) Quantification of median fluorescent intensity (MFI) of FITC-labeled ATCC 10953 binding to Ccm1-Ig, NKp46-Ig, and Ncr-1 Ig, without or with 5 and 10 mM of L-Arginine. Data combined from three to four independent experiments are presented. (B) NKp46-Ig (2 µg) was pre-incubated with 1 µg of a control anti-PVR antibody and NKp46 monoclonal antibodies (9E2, 461-G1, and 02) to evaluate the blocking of ATCC 10953 interaction with the NKp46 receptor. (C) Shows the quantification results of histograms depicted in (B). The mean value ± SD of the experiments (n=8) is presented. *p<0.05, **p≤0.01, ***p≤0.001, and ****p≤0.0001.
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Figure 4—source data 1
Median fluorescence intensity (MFI) quantification of fusion protein binding to Fusobacterium nucleatum ATCC 10953 corresponding to Figure 4A.
The source data also include raw MFI values for the quantification shown in Figure 4C.
- https://cdn.elifesciences.org/articles/108439/elife-108439-fig4-data1-v1.xlsx
Figure 4—figure supplement 1
Fusobacterium binding inhibition by L-Arginine.
The figure shows the binding of Ccm-1 Ig (left panel), NKp46-Ig (middle panel), and Ncr1-Ig (right panel) in the presence of 0 mM (black), 5 mM (red), and 10 mM (blue) L-arginine.
Figure 4—figure supplement 2
Arginine inhibition of NKp46-Ig and Ncr1-Ig binding in F. nucleatum ΔFadI.
Histograms show binding to F. nucleatum ATCC 10953 ΔFadI (A) and to F. nucleatum ATCC 23726 ΔFadI (B) following exposure to 5 mM and 10 mM L-Arginine. Panels display the mean fluorescence intensity (MFI) quantification. *p<0.05, **p≤0.01, ***p≤0.001, and ****p≤0.0001.
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Figure 4—figure supplement 2—source data 1
Raw data showing the mean fluorescence intensity (MFI) for ATCC 10953 ∆FadI (A) and ATCC 23726 ∆FadI (B), including the corresponding statistical analyses and significance levels.
- https://cdn.elifesciences.org/articles/108439/elife-108439-fig4-figsupp2-data1-v1.xlsx
Figure 5
Cytotoxicity and tumor growth is RadD and Ncr1-dependent.
(A) Schematic diagram showing the design of the NK cells cytotoxicity assay against breast cancer cell lines T47D and MCF7. 1. Tumor cells were stained with Calcein-AM dye and then incubated either with tumor cells (T47D or MCF7) only, tumor + NK, tumor +bacteria (ATCC 10953 WT and ATCC 10953 ∆RadD)+NK with/without preincubation with 02 antibody. 2. Killing assays were performed in a 37°C incubator for 4 hours. 3. The fluorescence intensity of Calcein was measured to determine cell viability using a spectrophotometer (Tecan Spark). Summary of NK cytotoxicity against T47D (B) and MCF7 (C) breast cancer cell lines. Combined results from five independent experiments. (D) C57BL/6 or NCR1-KO mice were shaved and AT3 cells (1 × 106 cells in 100 μl PBS) were injected 1 day later into the mammary fat pad. When tumors reached a size of about 500 mm3, mice were inoculated intravenously with 5 × 107 ATCC 10953 WT and 5 × 107 ATCC 10953 ∆RadD bacteria. Eight days later, mice were sacrificed and tumor weight was determined. (E) The tumor weight of C57BL/6 or NCR1-KO (F) mice. The figure shows the combination of 4–5 experiments performed. The mean value ± SD of the experiments is presented. NK + cells + ATCC10953 RadD is the ATCC10953 deleted for RadD. *p<0.05, **p≤0.01, ***p≤0.001, and ****p≤0.0001.
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Figure 5—source data 1
Raw data for cytotoxicity quantification shown in Figure 5B and C.
The source data also include tumor weight measurements from C57BL/6 mice and NCR1-KO mice corresponding to Figure 5E and F.
- https://cdn.elifesciences.org/articles/108439/elife-108439-fig5-data1-v1.xlsx
Figure 6
Postulated model for the RadD-NKp46 interaction impact on NK cytotoxicity and tumor growth.
NKp46 interaction with RadD expressed by Fusobacterium nucleatum triggers NK cell cytotoxicity. This activation enhances tumor cell killing in vitro and in vivo. Conversely, the absence of RadD or the blocking of NKp46 impairs NK cell activity, leading to tumor. This figure was created using BioRender.com.
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Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | APC α-human NKp46 ‘mouse monoclonal’ | Biolegend | Cat#331917; RRID:AB_2561649 | 0.2 ug |
| Antibody | Purified anti-human CD335 (NKp46) Antibody (Clone 9E2) ‘mouse monoclonal‘ | Biolegend | Cat#331902; RRID:AB_1027637 | 0.2 ug |
| Antibody | α-human NKp46- 461-G1 ‘mouse monoclonal‘ | In-house Berhani et al., 2019 | In-house | 2 ug for blocking |
| Antibody | α-human NKp46- 02mAb ‘mouse monoclonal‘ | In-house Berhani et al., 2019 | In-house | 2 ug for blocking |
| Antibody | Human CD155/PVR Antibody ‘mouse monoclonal’ | R&D Systems | Catalog #: MAB25301 RRID:AB_2174021 | 2 ug for blocking |
| Antibody | Alexa Fluor 647 AffiniPure F(ab') Fragment Donkey Anti-Human IgG | Jackson ImmunoResearch | Cat#709-606-098; RRID:AB_2340580 | (1:200) |
| Antibody | Alexa Fluor 647 AffiniPure F(ab') Fragment Goat Anti-Mouse IgG (H+L) | Jackson ImmunoResearch | Cat#115-606-146; RRID:AB_2338930 | (1:200) |
| Antibody | PE Mouse IgG1, κ Isotype Ctrl Antibody (MOPC-21) ‘mouse monoclonal’ | Biolegend | Cat#400112; RRID:AB_2847829 | 0.2 ug |
| Antibody | APC Mouse IgG1, κ Isotype Ctrl Antibody (MOPC-21) ‘mouse monoclonal’ | Biolegend | Cat#400120; RRID:AB_2888687 | 0.2 ug” |
| Other | F. nucleatum ATCC23726 | ATCC | N/A | F. nucleatum strain maintained in Ofer Mandelboim’s lab |
| Other | F. polymorphum ATCC 10953 | ATCC | N/A | F. nucleatum strain maintained in Ofer Mandelboim’s lab |
| Other | F. nucleatum ATCC 23726 ΔRadD | Kaplan et al., 2009, Mol Microbiol | N/A | F. nucleatum strain maintained in Ofer Mandelboim’s lab |
| Other | F. polymorphum ATCC 10953 ΔRadD | Guo et al., 2024 Mol Oral Microbiol | N/A | F. nucleatum strain maintained in Ofer Mandelboim’s lab |
| Other | F. nucleatum ATCC 23726 ΔfadI | Shokeen et al., 2020 Microorganisms | N/A | F. nucleatum strain maintained in Ofer Mandelboim’s lab |
| Other | F. polymorphum ATCC 10953 ΔfadI | Bhattacharyya et al., 2016 | N/A | F. nucleatum strain maintained in Ofer Mandelboim’s lab |
| Peptide, recombinant protein | NKp46 fusion protein- (NKp46 Ig) (human) | In-house | N/A | |
| Peptide, recombinant protein | Ncr-1 fusion protein (Ncr-1 Ig) (mouse) | In-house | N/A | |
| Peptide, recombinant protein | D1 domain of NKp46 fusion protein- (D1 Ig) | In-house | N/A | |
| Peptide, recombinant protein | CD16 fusion protein- CD16 Ig | In-house | N/A | |
| Peptide, recombinant protein | Ccm-1 fusion protein- Ccm-1 Ig | In-house | N/A | |
| Cell line (Homo-sapiens) | HEK293T | In-house | ATCC: CRL-3216 | Human embryonic kidney cell line Cell line maintained in Ofer Mandelboim’s lab |
| Cell line (Homo sapiens) | MCF7 | In-house | ATCC: HTB-22 | Breast cancer cell line Cell line maintained in Ofer Mandelboim’s lab |
| Cell line (Homo sapiens) | T47D | In-house | ATCC: CRL-2865 | Breast cancer cell line Cell line maintained in Ofer Mandelboim’s lab |
| Commercial assay or kit | Fluorescein-Isothiocyanate Isomer I (FITC) | Sigma | Cat#7250 | |
| Commercial assay or kit | Arginine | Sigma | Cat#A8094 | |
| Commercial assay or kit | Calcein AM | Thermo Fisher | Cat#C1413 | |
| Commercial assay or kit | Protein A/G-Sepharose affinity Chromatography | (Sigma) | GE17-0405-01 | |
| Commercial assay or kit | EasySep Human NK Cell Isolation Kit | (STEMCELL Technologies) | Cat#17955 | |
| Software, algorithm | Prism 8 | GraphPad | RRID:SCR_002798 | |
| Software, algorithm | FCS Express | De Novo Software | RRID:SCR_016431 | |
| Software, algorithm | BioRender | RRID:SCR_018361 | ||
| Other (female mice) | C57BL/6 | Envigo | RRID:MGI:2159769 | C57BL/6 inbred mice (C57BL/6JOlaHsd) https://www.inotiv.com/research-model/c57bl-6jolahsd |
| Other (female mice) | Ncr-1 | In-house Gazit et al., 2006 Nature Immunology | RRID:MGI:5699740 | See the results section in Gazit et al., 2006 |
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RadD from Fusobacterium nucleatum engages NKp46 to promote antitumor cytotoxicity
eLife 14:RP108439.
https://doi.org/10.7554/eLife.108439.2
