Figures and data in Enteropathogenic Escherichia coli-mediated fast and coordinated Ca²+ responses regulate NF-κB activation

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10 figures, 3 tables and 1 additional file

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Figure 1 with 1 supplement

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Enteropathogenic *Escherichia coli* (EPEC) induces isolated Ca²⁺ responses of limited amplitude in epithelial cells.

HeLa cells were loaded with the fluorescent indicator Cal-520, challenged with the indicated bacteria, and subjected to live-cell Ca²⁺ imaging at a frequency of one acquisition every 10 s (**A–C, F**) or fixed and processed for fluorescence microscopy analysis (**D–E**) (Materials and methods). (A) Representative traces of Ca²⁺ variations in single cells. The black arrowheads indicate the time of bacterial challenge. The blue arrowheads indicate stimulation with 3 µM histamine. (B) Response amplitude expressed as a percent of the maximal histamine response amplitude (N=3, n>63). (C) Percent of cells exhibiting Ca²⁺ responses (N=3, cells >66). (**D, E**) Cells challenged with red fluorescent protein (RFP)-expressing bacteria for 1 hr. (D) Representative confocal micrographs. Staining with DAPI (blue), phalloidin-Alexa 488 (green). The lower panels show a higher magnification of the insets in the top panels. Scale bar = 10 µm. (E) Percentage of bacteria-associated actin-rich pedestals (N=3, cells >273). (F) Average number of responses per cell during the first 30 min (0–30) and last 30 min (30-60) of bacterial challenge. Low MOI: 10 bacteria/cell. High multiplicity of infection (MOI): 50 bacteria/cell. Bar: mean. (N=3, cells >63). Mann–Whitney test. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

Figure 1—source data 1 All the source data for the graphs (B,C,E,F) in Figure 1.: https://cdn.elifesciences.org/articles/108953/elife-108953-fig1-data1-v1.xlsx
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Figure 1—figure supplement 1

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Enteropathogenic *Escherichia coli* (EPEC) induces Ca²⁺ responses with a frequency increasing during the time of infection.

Ca²⁺ imaging was performed on HeLa cells loaded with the Ca²⁺ fluorescent indicator Cal-520 and challenged with EPEC at a multiplicity of infection (MOI) of 100. (A) Traces of Ca²⁺ variations in single cells. (B) Trace corresponding to the average of traces shown in B (N=2, cells = 43). Note that cells do not show an increase in basal Ca²⁺ levels that could be mistakenly interpreted from the averaging of Ca²⁺ responses over the cell population.

Figure 2 with 2 supplements

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Enteropathogenic *Escherichia coli* (EPEC)-induced Ca²⁺ responses are elicited by ATP released by the type III secretion system (T3SS) translocon.

HeLa cells were loaded with the fluorescent indicator Cal-520 or with 200 μM suramin for 30 min, challenged with the indicated bacteria, and subjected to live-cell Ca²⁺ imaging for a 60 min-duration at a frequency of one acquisition every 10 s. (A) Representative traces of Ca²⁺ variations in single cells. The black arrowheads indicate the time of bacterial challenge. The blue arrowheads indicate stimulation with 3 µM histamine. (B) Percent of cells exhibiting Ca²⁺ responses (N=3, cells >70). (C) Average number of responses per cell. Low multiplicity of infection (MOI): 10 bacteria/cell. High MOI: 50 bacteria/cell. Bar: mean. (N=3, cells >30). Mann–Whitney test. **p<0.01; ***p<0.001; ****p<0.0001.

Figure 2—source data 1 All the source data for the graphs (B & C) in Figure 2.: https://cdn.elifesciences.org/articles/108953/elife-108953-fig2-data1-v1.xlsx
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Figure 2—figure supplement 1

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Enteropathogenic *Escherichia coli* (EPEC)-induced Ca²⁺ responses do not depend on Ca²⁺ influx but on Ca²⁺ release.

HeLa cells were loaded with the fluorescent indicator Cal-520, challenged with a high multiplicity of infection (MOI) of EPEC wild-type (WT) or a low MOI of the *ΔespC* mutant and subjected to live-cell Ca²⁺ imaging at a frequency of one acquisition every 10 s (**A-C**). (A) Representative traces of Ca²⁺ variations in single cells. The black arrowheads indicate the time of bacterial challenge. The blue arrowheads indicate stimulation with 3 µM histamine. (B) Percent of cells exhibiting Ca²⁺ responses. Cells challenged with: *ΔespC* (N=4, cells >400); +U73122: *ΔespC* in the presence of 10 μM U73122 (N=4, cells >160);+U73343: *ΔespC* in the presence of 10 μM U73343 (N=2, cells = 230); +PPADS: *ΔespC* in the presence of 20 μM PPADS (N=4, cells >400). (C) Cells challenged with wild-type EPEC in the absence or presence of +hexokinase (200 units/ml) and 5 mM glucose (+HexoK, N=3, cells >120). (**D–F**) +EGTA: cells treated with 4 mM EGTA. (N=3, cells >30). (D) Percent of cells showing Ca²⁺ responses. (E, F) Frequency of Ca²⁺ responses per cell. Mann–Whitney test. ns: not significant. **p<0.01; ****p<0.0001.

Figure 2—figure supplement 1—source data 1 All the source data for the graphs (B,C,D,E,F) in Figure 2—figure supplement 1.: https://cdn.elifesciences.org/articles/108953/elife-108953-fig2-figsupp1-data1-v1.xlsx
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Figure 2—figure supplement 2

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Suramin does not affect enteropathogenic *Escherichia coli* (EPEC)-induced actin pedestals.

HeLa cells were challenged with the indicated red fluorescent protein (RFP)-expressing bacterial strains in the presence or absence of 100 μM suramin. Samples were fixed and processed for fluorescence staining. (A) Representative micrographs. Blue: DAPI; green: Phalloidin-Alexa488; red: bacteria. Scale bar = 10 µm. (B) Percent of cells exhibiting actin-rich pedestals. High multiplicity of infection (MOI): 50 bacteria/cell. Low MOI *ΔespC*: 10 bacteria/cell. (N=3, n>150). Mann–Whitney test. ns: not significant.

Figure 2—figure supplement 2—source data 1 Source data for the graph B in Figure 2—figure supplement 2.: https://cdn.elifesciences.org/articles/108953/elife-108953-fig2-figsupp2-data1-v1.xlsx
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Figure 3

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Enteropathogenic *Escherichia coli* (EPEC) induces rapid and coordinated elementary Ca²⁺ responses.

HeLa cells were loaded with the fluorescent indicator Cal-520, challenged with the indicated bacteria, and subjected to high-speed Ca²⁺ imaging at a frequency of one acquisition every 57 ms for a duration of 110 s. (A) Representative time-series of pseudocolored fluorescent micrographs of cells challenged with wild-type EPEC. The numbers indicate the elapsed time in ms from an arbitrarily determined origin. Scale bar = 10 µm. (**B, D**) Traces of Ca²⁺ variations in 2 subcellular regions of the same cell. (B) WT: Traces corresponding to the regions depicted in the Figure 0 of panel A. The arrowheads point to the Ca²⁺ responses shown in Panel A with the corresponding color. (C) Percent of cells exhibiting Ca²⁺ responses (N>3, cells >134). +Suramin: treatment with 200 μM Suramin. +U73122: treatment with 10 μM U73122. (E) Response amplitude expressed as a percent of the maximal response amplitude induced by treatment with 3 μM histamine (N=3, cells >166). (F) Average number of responses per cell. (**C, E**) Bar: mean. Mann–Whitney test. **p<0.01; ****p<0.0001. (F) High-speed Ca²⁺ imaging was performed every 5 min for 110 s following infection with the indicated bacterial strain as depicted the scheme. The average number of responses per cell is indicated (N>3, cells >29).

Figure 3—source data 1 All the source data for the graphs (C, E) in Figure 3.: https://cdn.elifesciences.org/articles/108953/elife-108953-fig3-data1-v1.xlsx
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Figure 4 with 4 supplements

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Enteropathogenic *Escherichia coli* (EPEC)-induced coordinated elementary Ca²⁺ responses are reproduced by low ATP levels.

(**A–C**) HeLa cells were loaded with the fluorescent indicator Cal-520, challenged with 150 nM ATP and subjected to Ca²⁺ imaging. Image acquisition every 52 ms (A) or 22 ms (**B, C**). (A) Traces of Ca²⁺ variations corresponding to a single cell (red trace), or subcellular regions within the same cell (inset). (A) arrowhead: challenge with 2 μM ATP. (B) Time series of fluorescent micrographs pseudocolored using the ‘glow’ Fiji lookup table, where the blue pixels correspond to an arbitrarily set threshold value. The numbers indicate the elapsed time in seconds. Blue: high-intensity pixels showing the large top cell area with CCRICs and the local lower puff area. Scale bar = 10 µm. (C) Traces corresponding to Ca²⁺ variations in the subcellular regions depicted in Panel B. The responses are labelled 1–4, with the response 1 corresponding to the puff (Panel B, red ROI) impulsing the response 3 in the same region. Responses 2 and 4 correspond to CCRICs in Panel B, blue and green ROIs. Note the diffusion of the responses from the initial release area in other areas inferred from the dampening of the response amplitude.

Figure 4—figure supplement 1

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Low concentrations of histamine also induce small and fast coordinated Ca²⁺ responses.

HeLa cells were loaded with the fluorescent indicator Cal-520, challenged with 100 nM histamine and subjected to Ca²⁺ imaging, with image acquisition performed every 57 ms. Left: fluorescence micrograph of the single cell with black and red ROIs corresponding to the traces of Ca²⁺ variations in matching color. The box corresponds to a higher magnification of the inset in traces shown at the bottom. Arrowhead: challenge with 10 μM histamine. Traces are representative of 340 cells analyzed from two independent experiments, with a frequency of 4.5±0.4 peaks·min⁻¹ (mean ± SEM) and peak duration of 4.45±0.19 s (mean ± SEM).

Figure 4—figure supplement 2

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Enteropathogenic *Escherichia coli* (EPEC) induces CRICCs in polarized intestinal cells via eATP release.

Polarized TC7 cells were loaded with the fluorescent indicator Cal-520 were challenged with the EPEC *ΔespC* strain expressing the red fluorescent protein (RFP) (**A-E**) or with ATP (**F, G**). (A) Representative confocal micrographs. Samples were fixed and processed for fluorescence microscopy analysis. Green: ZO-1 staining. Red: RFP fluorescence. Scale bar = 10 µm. (**B-G**) Samples were subjected to live-cell Ca²⁺ imaging at a frequency of one acquisition every 5 s. (**B, D**) Representative traces of Ca²⁺ variations in single cells challenged with the indicated strain and inhibitor. The black arrowheads indicate the time of bacterial challenge. The blue arrowhead indicates stimulation with 3 µM histamine. (**C, E**) Percent of cells exhibiting Ca²⁺ responses (C) (N=4, cells >241) or CCRICs (E) (N=3, cells >66) following challenge with the indicated bacterial strain and inhibitor. Each value corresponds to a replicate. Mann–Whitney test. ****p<0.0001. (F) Representative traces of Ca²⁺ variations following challenge with 150 nM ATP (TC7+ATP) or in buffer alone (TC7). The pink and green traces correspond to Ca²⁺ variations in ROIs within the same cell. (G) Percent of cells exhibiting Ca²⁺ responses following challenge with the indicated ATP concentration. Each value represents the mean ± SEM of at least 140 cells in three independent experiments.

Figure 4—figure supplement 2—source data 1 All the source data for the graphs (C,E,G) in Figure 4—figure supplement 2.: https://cdn.elifesciences.org/articles/108953/elife-108953-fig4-figsupp2-data1-v1.xlsx
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Figure 4—figure supplement 3

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CCRICs induced by enteropathogenic *Escherichia coli* (EPEC) result from Ca²⁺ released by highly transient or mobile IP₃R clusters.

HeLa cells were loaded with Cal-520 and challenged with wild-type EPEC at a multiplicity of infection (MOI) of 50 bacteria/cell. (**A, B**) Globally coordinated responses associated with small and highly mobile clusters. **Time series micrographs**, Cal-520 intensity depicted in pseudocolor. Numbers: time in ms. Lower panels are magnification of the purple box in the upper panel, with a time interval of 57 ms. Scale bar = 5 μm. Note the high mobility of small clusters/channels. The black arrows point at larger and less mobile clusters. **Traces**, variations of Ca²⁺ in ROI depicted at T=0 in the micrographs in the corresponding color. The black arrow points at the response peak illustrated by the time series. (**C, D**) micrographs, Cal-520 fluorescence depicted in the Fiji red lute. Images were taken every 22 ms. Solid circles: ROIs where the variations of Ca2+ are shown in traces in the corresponding panel on the right. The blue ROI corresponds to the Ca2+ release source based on the higher response amplitude, and the green ROI to a distal region. The number indicates the distance between the ROIs shown by the dashed line. (C) Arrows point at the peak of the response in the ROI with the corresponding color. Note the delay in response elicitation between the source- (blue) and distal (green) ROI. (D) Puff-like responses (black arrowhead). Note the absence of Ca²⁺ variations in the distal region (Top), and the simultaneous elicitation of a puff and CCRICs in different ROIs (Bottom).

Figure 4—figure supplement 4

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Intracellular Ca²⁺ chelation inhibits CCRICs.

HeLa cells were loaded with the fluorescent indicator Cal-520 in the presence or absence of 20 μM EGTA-AM or BAPTA-AM. Samples were stimulated with 150 nM ATP and subjected to high-speed Ca²⁺ imaging at a frequency of one acquisition every 22 ms. (A) Representative traces of Ca²⁺ variations in two subcellular regions of the same cell in samples treated in the presence or absence of the indicated inhibitor. No Ca²⁺ responses were observed for cells treated with BAPTA-AM (N=3, >65 cells). EGTA-AM treatment led to an inhibition of Ca²⁺ responses, associated with small variations in the Ca²⁺ baseline that were arbitrarily scored as flattened Ca²⁺ pseudo-responses (ATP + EGTA AM, red arrows). (B) Percent of cells exhibiting Ca²⁺ responses (N>3, cells >62). (C) Average number of responses per cell. (N>3, n>62). Mann–Whitney test. **p<0.01; ***p<0.001.

Figure 4—figure supplement 4—source data 1 All the source data for the graphs (B,C) in Figure 4—figure supplement 4.: https://cdn.elifesciences.org/articles/108953/elife-108953-fig4-figsupp4-data1-v1.xlsx
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Figure 5

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Modeling of coordinated elementary Ca²⁺ responses.

**Top**, Ca²⁺ variations in subcellular area within a single cell are represented in pseudocolor. Shown are the maximum values of Δ[Ca²⁺]/[Ca²⁺]_b reached in each compartment during a 60 s simulation. Empty white squares: IP₃R clusters. **Graphs**: traces correspond to Ca²⁺ variations in the region with the matching color. Colored arrows: Ca²⁺ response due to the activation of an IP₃ cluster in the region with the matching color. Colored arrowhead and dashed red and blue lanes: Ca²⁺ variations due to the diffusion of a Ca²⁺ response from or nearby to the region with the matching color. Black arrows: Ca²⁺ response due Ca²⁺-activated Ca²⁺ release. (A) Low density of IP₃R clusters with local responses detected. (**B, C**) Empty black box: area with a high density IP₃R clusters. (C) similar to B, but following IP₃R cluster sensitization due to increased Ca²⁺ responses.

Figure 6 with 1 supplement

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Low ATP levels dampen NF-kappaB activation.

HeLa cells were stimulated with 10 ng/ml TNF-α alone or in the presence of 150 nM ATP or 20 μM BAPTA-AM (**F–H**). At the indicated time points, cell lysates were analyzed by Western blot using the indicated antibodies. (**A, D, F, G**) Representative blots. (**B, C, E, H**) Densitometry analysis of the indicated antibody signal normalized to that of HSP90 (**B, C**) or total P65 (**E, H**), expressed as fold-increase to basal levels of p-IκB (B), IκB (C), or p-p65 (**E, H**) at time = 0. Values correspond to the mean ± SEM of three or four independent experiments. p-p65: anti-phospho P65 antibody. p-IκB: anti-phospho IκB antibody. ANCOVA test. *p<0.05; **p<0.01; ***p<0.001.

Figure 6—source data 1 PDF file containing original Western blots for Figure 6A, D, F and G, indicating the relevant bands and treatments.: https://cdn.elifesciences.org/articles/108953/elife-108953-fig6-data1-v1.zip
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Figure 6—source data 2 Original files for western blot analysis displayed in Figure 6A, D, F and G.: https://cdn.elifesciences.org/articles/108953/elife-108953-fig6-data2-v1.zip
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Figure 6—source data 3 All the source data for the graphs (B,C,E,H) in Figure 6.: https://cdn.elifesciences.org/articles/108953/elife-108953-fig6-data3-v1.xlsx
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Figure 6—figure supplement 1

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Effects of ATP on TNF-α-induced profiles of O-GlcNacylation in HeLa cells.

HeLa cells were stimulated with 10 ng/ml TNF-α alone or co-stimulated with 10 ng/ml TNF-α and 150 nM ATP. Representative blots of cell lysates analyzed by Western blot at the time points indicated in minutes using the indicated antibodies.

Figure 6—figure supplement 1—source data 1 PDF file containing original Western blots for Figure 6—figure supplement 1 indicating the relevant bands and treatments.: https://cdn.elifesciences.org/articles/108953/elife-108953-fig6-figsupp1-data1-v1.zip
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Figure 6—figure supplement 1—source data 2 Original files for Western blot analysis displayed in Figure 6—figure supplement 1.: https://cdn.elifesciences.org/articles/108953/elife-108953-fig6-figsupp1-data2-v1.zip
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Figure 7

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Low ATP levels down-regulate NF-kB O-GlcNAcylation in a Ca²⁺-dependent manner.

HeLa cells were stimulated with 10 ng/ml TNF-α alone or in the presence of 150 nM ATP with or without 20 μM BAPTA-AM for 12 min. Cell lysates were subjected to P65 immunoprecipitation. (A) Representative blots with the indicated antibodies. IP: immunoprecipitates; L: total cell lysates. (B) Densitometry analysis of the O-GlucNAc signal in P65 immunoprecipitates normalized to that of TNF-α alone. Mann–Whitney test. N=4. *p<0.05. ns: not significant.

Figure 7—source data 1 PDF file containing original Western blots for Figure 7 (IP and Input) indicating the relevant bands and treatments.: https://cdn.elifesciences.org/articles/108953/elife-108953-fig7-data1-v1.zip
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Figure 7—source data 2 Original files for Western blot analysis displayed in Figure 7 (IP and Input).: https://cdn.elifesciences.org/articles/108953/elife-108953-fig7-data2-v1.zip
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Appendix 1—figure 1

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Model describing the dynamics of a cluster of IP₃Rs.

From Ornelas-Guevara et al., 2023. Ca²⁺ diffusion is modeled as a stochastic jump between adjacent compartments (Kraus et al., 1996). The propensity of a Ca²⁺ ion to move depends on the deterministic diffusion coefficient (100  µm²/s) and concentration gradients, assuming isotropic and homogeneous diffusion. All reaction and diffusion events are simulated using the Gillespie algorithm. At each step, an event is selected probabilistically based on its propensity (See propensity functions in Appendix 1—table 1).

Appendix 2—figure 1

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Dependence of the effective Ca2+ diffusion coefficient on [Ca2+] in thepresence of cytosolic buffers and impact on [Ca2+] profiles.

(A) Effective diffusion coefficient of Ca²⁺ (D_eff) as a function of free cytosolic [Ca²⁺] (c) for a mixture of three buffers. Buffer 1 is immobile and low-affinity (K_d = 10 µM, total concentration 15 µM, diffusion coefficient 0 µm²/s). Buffer 2 is mobile and has moderate affinity (K_d = 10 µM, total concentration 85 µM, diffusion coefficient 15 µm²/s). Buffer 3 is highly mobile and has a high affinity (K_d = 0.1 µM, total concentration 15 µM, diffusion coefficient 120 µm²/s). (**B, C**) Spatial profiles of free [Ca²⁺] 10 ms after a constant point-source release in the centre of a 10×10 µm² domain representing a cell, obtained from a two-dimensional reaction–diffusion simulation with explicit Ca²⁺–buffer binding using the same buffer parameters as in (A). Only Ca²⁺ release and binding to buffers were included in the simulation. The rate of Ca²⁺ release is 10 times larger in C than in B. [Ca²⁺] peaks at 0.25 µM (B) and 2.5 µM (C).

Appendix 3—figure 1

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Global Ca²⁺ response at higher [IP₃].

(A) Time course of the averaged cytosolic [Ca²⁺] obtained from the stochastic IP₃R cluster model for a uniform [IP₃]=0.1 µM and an effective diffusion coefficient $D_{eff} = 40 µ m^{2} / s$ . All other parameters are identical to those used in the simulations shown in Figure 5. Under these conditions, as opposed to CCRICs, the model produces a ‘classical’ global Ca²⁺ response with larger amplitude and long duration. (B) 2D cell geometry (10×10 µm²) used in the simulations. Squares indicate the random positions of IP₃R clusters.

Tables

Key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Strain, strain background (Wild-type Enteropathogenic Escherichia coli)	EPEC WT	Levine et al., 1985, DOI: https://doi.org.10.1093/infdis/152.3.550	Strains WT E2348/69
Strain, strain background (Enteropathogenic Escherichia coli escN mutant)	ΔescN	Garmendia et al., 2004, DOI: https://doi.org/10.1111/j.1462-5822.2004.00459.x		escN isogenic mutant of WT E2348/69
Strain, strain background (Enteropathogenic Escherichia coli espC mutant)	ΔespC	Guignot et al., 2015, DOI: https://doi.org.10.1371/journal.ppat.1005013		espC isogenic mutant of WT E2348/69
Cell line (Homo sapiens)	HeLa	ATCC	ATCC CCL2	RRID:CVCL_0030
Cell line (Homo sapiens)	Caco2/TC7	Chantret et al., 1994, DOI: https://doi.org/10.1242/jcs.107.1.213
Antibody	anti-Z0-1 polyclonal antibody	Thermo Fisher Scientific	Cat# 40–2200 RRID:AB_2533456	IF (1:50)
Antibody	NF-κB p65 Rabbit monoclonal antibody	Cell Signaling Technology	Cat# 8242 RRID:AB_10859369	WB (1:1000)
Antibody	Phospho-NF-κB p65 Rabbit monoclonal antibody	Cell Signaling Technology	Cat# 5733 RRID:AB_10706937	WB (1:1000)
Antibody	Phospho-IκB-α Mousse monoclonal antibody	Cell Signaling Technology	Cat# 4088 RRID:AB_1904009	WB (1:1000)
Antibody	IκB-alpha Rabbit polyclonal antibody	Cell Signaling Technology	Cat# 9242 RRID:AB_331623	WB (1:1000)
Antibody	anti-rabbit IgG-Alexa 488	Thermo Fisher Scientific	Cat# A-11034 RRID:AB_2576217	IF (1:200)
Antibody	HSP90 Mousse monoclonal antibody	Santa Cruz Biotechnologies	#sc-13119 RRID:AB_675659	WB (1:1000)
Antibody	NF-kB p65 Rabbit polyclonal antibody	Abcam	ab16502 RRID:AB_443394	WB (1:1000)
Antibody	O-GlcNAc Mousse monoclonal antibody	Abcam	ab2739 RRID:AB_303264
Antibody	Actin Rabbit polyclonal antibody	Cell Signaling Technology	Cat# 4967 RRID:AB_330288	(1:200)
Other	pRFP	Addgene	# 26924 RRID:Addgene_26924	Plasmid
Chemical compound, drug	Alexa Fluor 488 Phalloidin	Cell Signaling	#8878	Fluorescent indicator dye (1:400)
Chemical compound, drug	Cal-520	AAT Bioquest	AAT #21130	Fluorescent indicator dye
Software, algorithm	Prism7	Graphpad	RRID:SCR_002798
Other	DAPI stain	Merck	#102362276001	(1 µg/ml)

Appendix 1—table 1

Propensity functions and action of each process.

$N_{c a, i}$ and $N_{c a, j}$ represent the number of ions in the current box (i) and in an adjacent box (j) selected randomly.

Process	Propensity function	Action
1. Ca²⁺ diffusion	$4 \frac{D_{C}}{Δ x^{2}} N_{c a, i}$	$N_{c a, i} - 1$ $N_{c a, j} + 1$
2. SERCA pumps	$Ω \frac{v_{p} N_{c a, i} (N_{c a, i} - 1)}{{(K_{p} Ω)}^{2} + N_{c a, i} (N_{c a, i} - 1)}$	$N_{c a, i} - 1$
3. Leak from the ER	$Ω \frac{v_{p} {[{C a}^{2 +}]}_{b}^{2}}{K_{p}^{2} + {[{C a}^{2 +}]}_{b}^{2}}$	$N_{c a, i} + 1$
4. $C \to O$	$k_{c o} \frac{N_{c a, i}}{Ω} {(\frac{[I P_{3}]}{[I P_{3}] + K_{D}})}^{4} (N_{V c} - N_{o, i} - N_{i 1, i} - N_{i 2, i})$	$N_{o, i} = 1$ $N_{c, i} = 0$
5. $O \to i_{1}$	$k_{o i 1} N_{o, i} \frac{N_{c a, i} (N_{c a, i} - 1) (N_{c a, i} - 2)}{Ω^{3}}$	$N_{o, i} = 0$ $N_{i 1, i} = 1$
6. $O \to i_{2}$	$k_{o i 1} N_{o, i}$	$N_{o, i} = 0$ $N_{i 2, i} = 1$
7. $i_{1} \to C$	$k_{i 1 c} N_{i 1, i}$	$N_{i 1, i} = 0$ $N_{c, i} = 1$
8. $i_{2} \to C$	$k_{i 2 c} N_{i 2, i}$	$N_{i 2, i} = 0$ $N_{c, i} = 1$
9. Ca²⁺ release	$Ω Σ \frac{N_{o, i}}{N_{V c}}$	$N_{c a, i} + 1$

Appendix 1—table 2

Parameter values used for the simulations shown in Figure 5.

$k_{c o}$	$12.5 {μ M}^{- 1} s^{- 1}$	$C \to O$
$k_{o i 1}$	$0.0125 {μ M}^{- 3} s^{- 1}$	$O \to i_{1}$
$k_{o i 2}$	$10 s^{- 1}$	$O \to i_{2}$
$k_{i 1 c}$	$0.00125 s^{- 1}$	$i_{1} \to C$
$k_{i 2 c}$	$0.5 s^{- 1}$	$i_{2} \to C$
$v_{p}$	$0.225 μ M s^{- 1}$	Maximal rate of SERCA
$K_{p}$	$0.1 μ M$	SERCA binding constant
$K_{D}$	$0.1 μ M$	Affinity of IP₃Rs for IP₃
[Ca²⁺]_b	$0.04 μ M$	Basal cytosolic [Ca²⁺]
$D_{C}$	$100 {μ m}^{2} s^{- 1}$	Ca²⁺ diffusion coefficient
$Δ x$	$0.5 μ m$	Length of a compartment
$V_{c}$	$10^{- 16} L$	Volume of a compartment

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Fangrui Guo
Roberto Ornelas Guevara
Linda Oussaedine
Geneviève Dupont
Laurent Combettes
Guy Tran Van Nhieu

(2026)

Enteropathogenic Escherichia coli-mediated fast and coordinated Ca^²+ responses regulate NF-κB activation

eLife 14:RP108953.

https://doi.org/10.7554/eLife.108953.3

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Cite this article

Enteropathogenic Escherichia coli (EPEC) induces isolated Ca2+ responses of limited amplitude in epithelial cells.

Figure 1—source data 1

Enteropathogenic Escherichia coli (EPEC) induces Ca2+ responses with a frequency increasing during the time of infection.

Enteropathogenic Escherichia coli (EPEC)-induced Ca2+ responses are elicited by ATP released by the type III secretion system (T3SS) translocon.

Figure 2—source data 1

Enteropathogenic Escherichia coli (EPEC)-induced Ca2+ responses do not depend on Ca2+ influx but on Ca2+ release.

Figure 2—figure supplement 1—source data 1

Suramin does not affect enteropathogenic Escherichia coli (EPEC)-induced actin pedestals.

Figure 2—figure supplement 2—source data 1

Enteropathogenic Escherichia coli (EPEC) induces rapid and coordinated elementary Ca2+ responses.

Figure 3—source data 1

Enteropathogenic Escherichia coli (EPEC)-induced coordinated elementary Ca2+ responses are reproduced by low ATP levels.

Low concentrations of histamine also induce small and fast coordinated Ca2+ responses.

Enteropathogenic Escherichia coli (EPEC) induces CRICCs in polarized intestinal cells via eATP release.

Figure 4—figure supplement 2—source data 1

CCRICs induced by enteropathogenic Escherichia coli (EPEC) result from Ca2+ released by highly transient or mobile IP3R clusters.

Intracellular Ca2+ chelation inhibits CCRICs.

Figure 4—figure supplement 4—source data 1

Modeling of coordinated elementary Ca2+ responses.

Low ATP levels dampen NF-kappaB activation.

Figure 6—source data 1

Figure 6—source data 2

Figure 6—source data 3

Effects of ATP on TNF-α-induced profiles of O-GlcNacylation in HeLa cells.

Figure 6—figure supplement 1—source data 1

Figure 6—figure supplement 1—source data 2

Low ATP levels down-regulate NF-kB O-GlcNAcylation in a Ca2+-dependent manner.

Figure 7—source data 1

Figure 7—source data 2

Model describing the dynamics of a cluster of IP3Rs.

Dependence of the effective Ca2+ diffusion coefficient on [Ca2+] in thepresence of cytosolic buffers and impact on [Ca2+] profiles.

Global Ca²+ response at higher [IP3].

Propensity functions and action of each process.

Parameter values used for the simulations shown in Figure 5.

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Enteropathogenic Escherichia coli (EPEC) induces isolated Ca²⁺ responses of limited amplitude in epithelial cells.

Enteropathogenic Escherichia coli (EPEC) induces Ca²⁺ responses with a frequency increasing during the time of infection.

Enteropathogenic Escherichia coli (EPEC)-induced Ca²⁺ responses are elicited by ATP released by the type III secretion system (T3SS) translocon.

Enteropathogenic Escherichia coli (EPEC)-induced Ca²⁺ responses do not depend on Ca²⁺ influx but on Ca²⁺ release.

Enteropathogenic Escherichia coli (EPEC) induces rapid and coordinated elementary Ca²⁺ responses.

Enteropathogenic Escherichia coli (EPEC)-induced coordinated elementary Ca²⁺ responses are reproduced by low ATP levels.

Low concentrations of histamine also induce small and fast coordinated Ca²⁺ responses.

CCRICs induced by enteropathogenic Escherichia coli (EPEC) result from Ca²⁺ released by highly transient or mobile IP₃R clusters.

Intracellular Ca²⁺ chelation inhibits CCRICs.

Modeling of coordinated elementary Ca²⁺ responses.

Low ATP levels down-regulate NF-kB O-GlcNAcylation in a Ca²⁺-dependent manner.

Model describing the dynamics of a cluster of IP₃Rs.

Global Ca²⁺ response at higher [IP₃].