A type III effector protease NleC from enteropathogenic Escherichia coli targets NF-κB for degradation

Abstract

Many bacterial pathogens utilize a type III secretion system (T3SS) to inject virulence effector proteins into host cells during infection. Previously, we found that enteropathogenic Escherichia coli (EPEC) uses the type III effector, NleE, to block the inflammatory response by inhibiting IκB degradation and nuclear translocation of the p65 subunit of NF-κB. Here we screened further effectors with unknown function for their capacity to prevent p65 nuclear translocation. We observed that ectopic expression of GFP-NleC in HeLa cells led to the degradation of p65. Delivery of NleC by the T3SS of EPEC also induced degradation of p65 in infected cells as well as other NF-κB components, c-Rel and p50. Recombinant His(6) -NleC induced p65 and p50 cleavage in HeLa cell lysates and mutation of a consensus zinc metalloprotease motif, HEIIH, abrogated NleC proteolytic activity. NleC inhibited IL-8 production during prolonged EPEC infection of HeLa cells in a protease activity-dependent manner. A double nleE/nleC mutant was further impaired for its ability to inhibit IL-8 secretion than either a single nleE or a single nleC mutant. We conclude that NleC is a type III effector protease that degrades NF-κB thereby contributing the arsenal of bacterial effectors that inhibit innate immune activation.

Fig. 1

Effect of ectopically expressed NleC on p65 immunostaining. Representative immunofluorescence fields of p65 degradation using anti-p65 (red) in HeLa cells transfected with pEGFP–C2 (GFP), pGFP–NleC (GFP–NleC), pGFP–NleE, pGFP–NleD, pGFP–NleF and pGFP–NleG (green) and left unstimulated or stimulated with TNF for 30 min where indicated. Cell nuclei were stained with DAPI (blue). Transfections and staining were performed independently at least three times per GFP–effector fusion. Arrows indicate transfected cells with poor p65 staining.

Fig. 2

Effect of NleC on p65 during EPEC infection. Representative immunofluorescence fields showing p65 staining (green) in FAS-positive HeLa cells (red) uninfected or infected for 4 h with wild-type EPEC E2348/69, a ΔPP4/IE6 double island mutant, ΔPP4/IE6 carrying the overexpression vector pNleC or EPEC E2348/69 carrying the overexpression vector pNleC as indicated, and stained for nucleic acid with DAPI (blue). Infections and staining were performed independently at least three times per EPEC E2348/69 derivative. Arrows indicate FAS-positive lesions.

Fig. 3

Effect of NleC on inflammatory signalling mediators.A. Alignment of the regions of NleC and NleD containing the putative zinc metalloprotease motif (underlined).B and C. Representative immunoblots showing degradation of inflammatory signalling mediators in HeLa cells infected with derivatives of EPEC E2348/69 for 4 h. Cells were harvested for immunoblotting and host proteins were detected with antibodies to p65, p50, c-Rel, STAT1 and Erk. Infections and immunoblotting were performed independently at least three times per EPEC E2348/69 derivative. Antibodies to actin were used as loading control. UI, uninfected.

Fig. 4

Effect of recombinant NleC on the degradation of NF-κB. Representative immunoblot showing degradation of Rel proteins in HeLa cells incubated with increasing concentrations of recombinant His⁶NleC and His⁶NleC_AEIIA (0.01, 0.1, 0.5 and 1 µg) for 4 h. Incubations were performed independently at least three times per His⁶NleC derivative. Cells were harvested for immunoblotting and Rel proteins were detected with antibodies to p65 and p50. Antibodies to calnexin were used as loading control for HeLa lysates. His⁶-tagged proteins were detected with anti-His antibodies.

Fig. 5

Effect of EPEC infection on p65 degradation.A. Immunoblot showing degradation of p65 in HeLa cells infected with derivatives of EPEC E2348/69 for 2, 4 and 6 h. Infections and immunoblotting were performed independently at least three times per EPEC E2348/69 derivative and per time point. Cells were harvested for immunoblotting and detected with antibodies to p65. Antibodies to actin were used as loading control. UI, uninfected.B. Mean band intensity of immunoblots performed on HeLa cells infected with derivatives of EPEC E2348/69 and detected with antibodies to p65 and actin as indicated. UI, uninfected. Results are expressed as the mean ± SEM of three independent experiments. *P < 0.05 compared with uninfected, unstimulated cells at each time point, one-way anova.

Fig. 6

Effect of prolonged EPEC infection on p65 degradation.A. Immunoblot showing degradation of p65 in HeLa cells infected with derivatives of EPEC E2348/69 for 6 h. Infections and immunoblotting were performed independently at least three times per EPEC E2348/69 derivative. Cells were harvested for immunoblotting and detected with antibodies to p65. Antibodies to actin were used as loading control. UI, uninfected.B. Mean band intensity of immunoblots performed on HeLa cells infected with derivatives of EPEC E2348/69 and detected with antibodies to p65 and actin as indicated. Results are expressed as the mean ± SEM of three independent experiments. *P < 0.05 compared with uninfected, unstimulated cells at each time point, one-way anova.

Fig. 7

Effect of nleE and nleC mutations on IL-8 secretion from infected HeLa cells.A. HeLa cells were infected with derivatives of EPEC E2348/69 for 4 h and left unstimulated (light grey bars) or stimulated with TNF for 24 h (dark grey bars). Results are the mean ± SEM of at least three independent experiments carried out in duplicate. *P < 0.0001 compared with uninfected, unstimulated cells, one-way anova.B. HeLa cells were infected with derivatives of EPEC E2348/69 for 6 h and left unstimulated (light grey bars). Results are the mean ± SEM of at least three independent experiments carried out in duplicate. *P < 0.0001 compared with uninfected, unstimulated cells, one-way anova. #ΔPP4/IE6 significantly greater than nleE/C double mutant P < 0.0001, unpaired two-tailed t-test.