Fig 1.
Identification of NleA and NleE as inhibitors of host IL-1β secretion.
Differentiated THP-1 cells were infected for 1 hr followed by addition of gentamicin to terminate extracellular bacteria. Cells were further incubated for 6 hrs, and culture supernatant was collected for ELISA analysis. (A) IL-1β secretion from EPEC-infected cells. Cells were uninfected or infected with WT, ΔescF (T3SS-deficient strain) and TOE-A6 (non-LEE effector-deleted strain), and the amount of IL-1β in the culture supernatant was measured. (B) TOE-A4 and TOE-A5 isogenic mutants lost their inhibitory property on IL-1β secretion. Cells were infected with WT and TOE-A strains (A1-A6), followed by measurement of the amount of IL-1β secreted in culture supernatants. (C and D) Complementation test for TOE-A4 or TOE-A5 identified NleE and NleA as inhibitors. TOE-A4 possessing nleE, or nleB, or espL were used for infection of THP-1 cells along with the parental strain (C). Alternatively, TOE-A5 possessing nleA, or nleF, or nleH2 were used for infection of THP-1 cells along with the parental strain (D). * p < 0.05 by Student’s t-test. Experiments were done in triplicate and repeated independently three times for (A) and (B); two times for (C) and (D); and one of the representative experiments with similar results is shown.
Fig 2.
NleA suppresses caspase-1 activation.
(A) NleA does not interrupt IκB degradation during bacterial infection. dTHP-1 were uninfected or infected with indicated strains as described in the section of Method with modifications. Cells were centrifuged for 10 min and infected for 1.5 hrs. Then cells were washed once with PBS and directly lysed in 1x SDS sampler buffer. Proteins were separated by SDS-PAGE and probed with anti-IκB and anti-α-tubulin. (B and C) NleA does not inhibit NF-κB nuclear translocation. Differentiated THP-1 cells were uninfected (UI) or infected with wild type (WT), TOE-A5, and TOE-A5/nleA strains. After 1 hr of infection and 30 min of gentamicin-treatment, cells were washed, fixed and immunostained with anti-RelA antibody (green) and DAPI (blue). Images from nine random fields were captured using confocal microscopy. Cells with intense nuclear staining of RelA were counted. The percentage of such cells was calculated by dividing the total number of cells in each given field of view. Student’s t-test * p < 0.05 (v.s WT). (D and E) NleA inhibits secretion of IL-1β but not TNF-α. Differentiated THP-1 cells were uninfected (UI) or were infected by WT, TOE-A5, and TOE-A5/nleA for 1 hr, and the culture supernatants at 6 hr post-infection were analyzed by ELISA. Student’s t-test * p < 0.05 (D: vs WT). (F and G) NleA inhibits caspase-1 activation. After 1 hr of infection followed by (F) 6 hrs or (G) 3 hrs of incubation, culture supernatant from cells uninfected (UI) or infected with indicated strains were centrifuged and the proteins in the supernatant were TCA-precipitated. Immunoblottings were performed to assess the amount of active caspase-1 (caspase-1 p20) by anti-caspase-1 antibody. SN, supernatant; CL, cell lysate.
Fig 3.
NleA suppresses formation of the inflammasome.
Differentiated THP-1 cells were uninfected (UI) or infected with TOE-A5 and TOE-A5/nleA for 1 hr and further incubated for 3 and 6 hrs. Cells at the indicated times were immunostained, followed by visualization using a confocal microscope. The foci are indicated by arrowheads. (A) Foci formed with active caspase-1 in infected cells. The cells were stained with FAM-YVAD-FMK FLICA for active caspase-1 (Green) and DAPI (Blue). (B) Foci formed with total caspase-1 in infected cells. The cells were stained with anti-caspase-1 (Red) and DAPI (Blue). (C) Foci formed with ASC in infected cells. The cells were immunostained with anti-ASC (Red) and DAPI (Blue). (D, E and F) Quantification of respective foci formation. From nine random fields, the percentages of active caspase-1-, total caspase-1-, or ASC- foci were calculated as (number of identified speck-like structures)/(number of cells in a given field of view)x100%. Student’s t-test, * p < 0.05. n.d, not determined. (G) NleA reduces ASC oligomer formation. After 1 hr of infection and 3 hrs of further incubation, the cell lysates from uninfected (UI) or cells infected with TOE-A5 or TOE-A5/nleA were enriched for the inflammasome-containing fraction. The fraction was further treated with a DSS protein-protein cross-linker for 30 min at room temperature, and the reaction was terminated by the direct addition of 2x SDS sample buffer. The samples were separated by SDS-PAGE, and an anti-ASC antibody was used to detect ASC species. CL, cytosolic lysate.
Fig 4.
NleA reduces formation of mature NLRP3 inflammasomes.
(A) Mature NLRP3 foci in infected THP-1 cells. Differentiated THP-1 cells were uninfected (UI) or infected with TOE-A5 or TOE-A5/nleA. After 1 hr of infection and 3 hrs of further incubation, the cells were processed for immunostaining with an anti-NLRP3 antibody (Green), total caspase-1 (Red) and DAPI (Blue). A mature NLRP3 focus was identified as strong co-localization of the signals between NLRP3 and total caspase-1 (arrowheads). (B) Quantification of mature NLRP3 foci. Using a confocal microscope, nine random fields were investigated, and the number of mature NLRP3 foci was counted. The percentage of foci was expressed as (number of identified speck-like structures)/(number of cells in a given field of view) x100%. Student’s t-test, * p < 0.05. (C) NleA interferes with the de-ubiquitin modification of NLRP3. Differentiated THP-1 cells were primed with LPS (1 μg/ml) for 2 hrs prior to infection. Cells were then uninfected (UI) or infected with TOE-A5 or TOE-A5/nleA for 1 hr and then incubated for 3 hrs. The cell lysates were subjected to immunoprecipitation by an anti-NLRP3 antibody, and the precipitated products were analyzed by immunoblotting with an anti-ubiquitin antibody. NLPR3 in cell lysate (CL) used for immunoprecipitation was also detected.
Fig 5.
NLRP3 directly interacts with NleA.
(A) NleA associates with endogenous NLRP3. Columns packed with resins of bacterial expressed MBP (8 μg) or MBP-NleA (8 μg) were used to pulldown endogenous proteins of the THP-1 lysates prepared from approximately 1x108 cells. The bound proteins were eluted with a high salt elution buffer and analyzed with the indicated antibodies. (B) NleA interacts with NLRP3 in cells. HeLa cells were seeded in 6-well plates and transfected with plasmids expressing the indicated fusion proteins for 24 hrs. The cell lysates were immunoprecipitated (IP) with anti-GFP-coupled magnetic beads. The pulldown products were analyzed by immunoblotting (IB) using anti-NLRP3 and anti-GFP antibodies. (C) NleA directly binds NLRP3. Purified GST or GST-NLRP3-bound resins (2.5 μg each) were packed in columns to pull down the purified MBP-NleA (250 μl of 5 μg/ml). The bound proteins were eluted with a high salt elution buffer and analyzed by IB using an anti-MBP antibody. (D) Domains of NLRP3. The amino acid sequences containing one of three domains were fused with GST for purification. (E) NleA binds to the PYD and LRR domains of NLRP3. GST or GST-PYD, GST-NACHT, and GST-LRR fusion proteins were purified and immobilized on resins at approximately 2.5 μg each. An equal amount of purified MBP-NleA (500 μl of 5 μg/ml) was applied for direct binding. The columns were extensively washed with column buffer. The eluted products were probed with an anti-MBP antibody.
Fig 6.
NleA binds to ubiquitinated NLRP3 and represses de-ubiquitination.
(A) NleA binds to ubiquitinated NLRP3. Lysates of HeLa cells expressing combinations of proteins as indicated were subjected to immunoprecipitation by an anti-GFP antibody, and the NLRP3 fusion proteins and GFP fusion proteins were detected by immunoblotting using anti-KGC and anti-GFP antibodies. (B) In vitro de-ubiquitination assay of NLRP3. The total KGC-NLRP3 (including HA-ubiquitin modified KGC-NLRP3) was purified and immobilized on magnetic beads using an anti-KGC antibody. Non-treated or MBP- or MBP-NleA-treated immobilized KGC-NLRP3 was resuspended in the assay buffer and LPS-primed dTHP-1 lysate was added at 1/100 of reaction volume. The reaction was performed at 0°C or 37°C for 1 hr and was terminated by first washing the beads with the assay buffer, followed by the addition of 1x SDS sampler buffer. The amount of HA-ubiquitinated NLRP3 was analyzed by immunoblot using an anti-HA antibody. Please refer to the Methods section for more detailed descriptions.
Fig 7.
Proposed function of NleA as an inhibitor of NLRP3.
Stimuli including PAMPs and DAMPs trigger the onset of various upstream signaling pathways, and these pathways converge to activate the NLRP3 inflammasome (left panel). The upstream positive signals promote the de-ubiquitination of NLRP3 and allow NLRP3 to begin recruitment of ASC and caspase-1. The formed mature NLRP3 inflammasome then becomes competent for enzymatically processing pro-IL-1β into IL-1β by active caspase-1. EPEC and other A/E pathogens use NleA to subvert the normal pathway leading to the assembly of the mature NLRP3 inflammasome. This blockade of the pathway occurs at stage of NLRP3 activation where NleA directly associated ubuiqtinated and non-ubiqutiinated NLRP3, which would prohibit the subsequent inflammasome assembly, resulting in dampening of the processing of pro-IL-1β for secretion.