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 seconds (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, n > 66). (D, E) Cells challenged with RFP-expressing bacteria for 1 hour. 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, n > 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 MOI: 50 bacteria / cell. Bar: mean. (N = 3, n > 63). Mann-Whitney test. *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001.

EPEC-induced Ca2+ responses are elicited by ATP released by the T3SS translocon

HeLa cells were loaded with the fluorescent indicator Cal-520 or with 200 μM suramin for 30 minutes, 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 seconds. 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, n > 66). C, Average number of responses per cell. Low MOI: 10 bacteria / cell. High MOI: 50 bacteria / cell. Bar: mean. (N = 3, n > 30). Mann-Whitney test. **: p < 0.01; ***: p < 0.001; ****: p < 0.0001.

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 seconds. A, Representative time-serie 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 image 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, n > 256). + 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, n > 20). 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 seconds following infection with the indicated bacterial strain as depicted the scheme. The average number of responses per cell is indicated (N > 3, n > 29).

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 2 s (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 serie of fluorescent micrographs pseudocolored using the “glow” Fiji lookup table, where the blue pixel 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 area inferred from the dampening of the response amplitude. The dashed red and green lines point at the aggregation of responses throughout the cell.

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 60s 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.

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-E, H, Densitometry analysis of the indicated antibody signal normalized to that of HSP90 (B, C, F) or total P65 (E). p-P65: anti-phospho P65 antibody. P-IκB: anti-phospho IκB antibody. ANCOVA test. *: p < 0.05; **: p < 0.01; ***: p < 0.001.

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 or 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.

EPEC induces Ca 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 MOI of 100. A, Traces of Ca2+ variations in single cells. B, Trace corresponding to the average of traces shown in B (n=32). 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.

EGTA does not inhibit EPEC-induced Ca2+ responses.

HeLa cells were challenged with a high MOI of EPEC wild-type (WT) or a low MOI of the ΔespC mutant. + EGTA: cells treated with 4 mM EGTA. A, Percent of cells showing Ca2+ responses. B, C, Frequency of Ca2+ responses per cell.

Suramin does not affect EPEC-Induced actin pedestals.

HeLa cells were challenged with the indicated RFP-expressing bacterial strains in the presence or absence of 200 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 MOI: 50 bacteria / cell. Low MOI ΔespC: 10 bacteria / cell. (N=3, n > 273). Mann Whitney test. ns: not significant.

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 2 subcellular regions of the same cell. No Ca2+ responses were observed for cells treated with BAPTA-AM (N = 3, > 65 cells). B, Percent of cells exhibiting Ca2+ responses (N > 3, n > 62). C, average number of responses per cell. (N > 3, n > 62). Mann-Whitney test. **: p < 0.01; ***: p < 0.001.

CCRICs induced by EPEC result from Ca2+ released by highly transient or mobile IP3R clusters.

Time series of HeLa cells loaded with Cal-520 and challenged with wild type EPEC at a MOI of 50 bacteria / cell. Time is indicated in ms. Traces: variations of Ca2+ in ROI depicted at T = 0 in the corresponding color. The red arrow points at the response peak illustrated by the time series. Images framed in purple are taken at 57 ms interval. The marks inside the frame aim at better highlighting cluster / channel mobility. Cal-520 intensity in pseudocolor. A, Globally Coordinated responses associated with small and highly mobile clusters. Note the bigger and less mobile cluster (purple arrow). B, Puff-like response.

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.