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. 2011 Apr;7(4):e1002026.
doi: 10.1371/journal.ppat.1002026. Epub 2011 Apr 21.

A Yersinia effector with enhanced inhibitory activity on the NF-κB pathway activates the NLRP3/ASC/caspase-1 inflammasome in macrophages

Ying Zheng  1 , Sarit Lilo, Igor E Brodsky, Yue Zhang, Ruslan Medzhitov, Kenneth B Marcu, James B Bliska
Affiliations

A Yersinia effector with enhanced inhibitory activity on the NF-κB pathway activates the NLRP3/ASC/caspase-1 inflammasome in macrophages

Ying Zheng et al. PLoS Pathog. 2011 Apr.

Abstract

A type III secretion system (T3SS) in pathogenic Yersinia species functions to translocate Yop effectors, which modulate cytokine production and regulate cell death in macrophages. Distinct pathways of T3SS-dependent cell death and caspase-1 activation occur in Yersinia-infected macrophages. One pathway of cell death and caspase-1 activation in macrophages requires the effector YopJ. YopJ is an acetyltransferase that inactivates MAPK kinases and IKKβ to cause TLR4-dependent apoptosis in naïve macrophages. A YopJ isoform in Y. pestis KIM (YopJ(KIM)) has two amino acid substitutions, F177L and K206E, not present in YopJ proteins of Y. pseudotuberculosis and Y. pestis CO92. As compared to other YopJ isoforms, YopJ(KIM) causes increased apoptosis, caspase-1 activation, and secretion of IL-1β in Yersinia-infected macrophages. The molecular basis for increased apoptosis and activation of caspase-1 by YopJ(KIM) in Yersinia-infected macrophages was studied. Site directed mutagenesis showed that the F177L and K206E substitutions in YopJ(KIM) were important for enhanced apoptosis, caspase-1 activation, and IL-1β secretion. As compared to YopJ(CO92), YopJ(KIM) displayed an enhanced capacity to inhibit phosphorylation of IκB-α in macrophages and to bind IKKβ in vitro. YopJ(KIM) also showed a moderately increased ability to inhibit phosphorylation of MAPKs. Increased caspase-1 cleavage and IL-1β secretion occurred in IKKβ-deficient macrophages infected with Y. pestis expressing YopJ(CO92), confirming that the NF-κB pathway can negatively regulate inflammasome activation. K+ efflux, NLRP3 and ASC were important for secretion of IL-1β in response to Y. pestis KIM infection as shown using macrophages lacking inflammasome components or by the addition of exogenous KCl. These data show that caspase-1 is activated in naïve macrophages in response to infection with a pathogen that inhibits IKKβ and MAPK kinases and induces TLR4-dependent apoptosis. This pro-inflammatory form of apoptosis may represent an early innate immune response to highly virulent pathogens such as Y. pestis KIM that have evolved an enhanced ability to inhibit host signaling pathways.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Translocation of different YopJ isoforms and caspase-1 activation in macrophages infected with Y. pseudotuberculosis.
Y. pseudotuberculosis IP26 (IP2666ΔyopJ) carrying no pBAD plasmid as a control (CTL; lane 5) or pBAD vectors encoding the indicated YopJ-GSK isoforms (lanes 1–4), were grown under T3SS-inducing conditions in the presence of 0.2% of arabinose. Bacteria were added to BMDMs at an MOI of 20 and were allowed to infect for 2 hr. Arabinose (0.2%) was maintained in cell culture medium. Detergent lysates of infected macrophages were separated by SDS-PAGE and immunoblotting was performed with anti-phospho-GSK-3® antibody (A) and anti-caspase-1 antibody (B). Positions of molecular weight standards (kDa) are shown on the left and positions of YopJ- phospho-GSK and cleaved caspase-1 are shown on the right.
Figure 2
Figure 2. Cytokine secretion and cell death in macrophages infected with Y. pestis strains expressing different YopJ isoforms.
BMDMs were left uninfected (U), or infected with the indicated Yp-YopJ strains at an MOI of 10. Supernatants collected after 24 hr of infection were used to measure cell death by LDH release (A) and secretion of IL-1β (B), IL-18 (C) and TNF-α (D) by ELISA. Results shown are the average of three independent experiments. Error bars represent standard deviation. Bracketing indicates P values (ANOVA) between different conditions.
Figure 3
Figure 3. Measurement of IKKβ binding to different YopJ isoforms and phospho-IκBα levels in macrophages infected with different Y. pestis strains.
(A) Binding of IKKβ to different YopJ isoforms as determined using a GST pull down procedure and lysates of transfected HEK293T cells. Purified proteins corresponding to GST (lane 2) or the indicated GST-YopJ fusion proteins (lanes 3–5) were immobilized on beads and incubated in cell lysates containing overexpressed IKKβ. After washing, proteins bound to the beads were detected and the signals quantified by immunoblotting using antibodies to IKKβ or GST and an Odyssey imaging system. Lane 1 contains a sample of the input transfected cell lysate (Input). Positions of molecular weight standards (kDa) are shown on the left and positions of IKKβ, GST-YopJ, and GST proteins are shown on the right. (B) Ratios of the signals for IKKβ and GST obtained by immunoblotting are presented in bar graph format, with values representing averages of two independent experiments. (C) BMDMs were left uninfected (U) or infected with Yp-YopJKIM, Yp-YopJC172A or Yp-YopJCO92 at an MOI of 50. At 1 hr post infection lysates of the infected macrophages were prepared and subjected to ELISA to determine levels of phospho (p)-IκBα. Results show p-IκBα values normalized to arbitrary units by setting uninfected to 1. Results were averaged from six (uninfected and Yp-YopJKIM) or three (Yp-YopJC172A and Yp-YopJCO92) independent experiments and error bars represent standard deviations. P value<0.05 (t test) as compared to Yp-YopJKIM condition is indicated by (*).
Figure 4
Figure 4. IL-1β and TNF-α secretion in IkkβF/F or IkkβΔ macrophages infected with Y. pestis strains expressing different YopJ isoforms.
IkkβF/F or IkkβΔ macrophages were left uninfected (U) or infected with the indicated Yp-YopJ strains at an MOI of 10. Twenty-four hr post infection, cell supernatants were collected. Secreted TNF-α (A) and IL-1β (B) were measured by ELISA. Results were averaged from three independent experiments, and error bars represent standard deviation. P values (t test) are indicated by bracketing (P<0.05 (*), P<0.01 (**), P<0.001(***).
Figure 5
Figure 5. Caspase-1 activation in IkkβF/F or IkkβΔ macrophages infected with Y. pestis strains expressing different YopJ isoforms.
IkkβF/F or IkkβΔ BMDMs were left uninfected (U) or infected with the indicated Yp-YopJ strains at an MOI of 20 (A) or 10 (B and C), or treated with LPS for 3 hr and then exposed to ATP (LPS/ATP). (A) Caspase-1 cleavage was determined at 1 hr post ATP treatment or 2 hr post infection. In (A) samples of detergent lysates were separated by SDS-PAGE and immunoblotted with anti-caspase-1 antibody (upper panel) or anti-actin antibody antibody (lower panel). Positions of molecular weight standards (kDa) are shown on the left and positions of cleaved caspase-1 and actin are shown on the right. In (B) uninfected or infected macrophages on coverslips were incubated with FLICA reagent (FAM-YVAD-FMK) at 9 hr post infection to stain for active caspase-1 (green fluorescence). The samples were fixed, mounted on slides, and light microscopy was used to detect phase (a–d, i–l) or fluorescence (e-h, m-p) signals. Representative images of uninfected or infected cells were captured by digital photomicroscopy. White arrows point to FLICA positive cells. In (C), average percentages (error bars show standard deviation) of FLICA positive cells counted from three random fields per coverslip in three independent experiments is shown. P values comparing results of infection in IkkβΔ to IkkβF/F BMDMs was determined (P<0.01, **; P<0.001, ***).
Figure 6
Figure 6. Measurement of phospho-MAPK levels in macrophages infected with Y. pestis strains expressing different YopJ isoforms.
BMDMs were left uninfected (U), or infected with the indicated Yp-YopJ strains at an MOI of 20. At 30 or 60 min post infection lysates of the infected macrophages were prepared and subjected to ELISA to determine levels of phospho-ERK (A), -p38 (B) or –SAPK/JNK (C). Results show OD450 values averaged from three (uninfected, Yp-YopJKIM and Yp-YopJCO92) or two (Yp-YopJC172A) independent experiments and error bars represent standard deviations. P value (*, <0.05) (t test) is indicated by bracket.
Figure 7
Figure 7. Determination of the importance of inflammasome components for cytokine secretion and cell death in infected macrophages.
Wild-type BMDMs, or BMDMs deficient for NLRC4 (Ipaf), ASC (ASC) or NLRP3 (Nalp3) were left uninfected (U) or infected with Yp-YopJKIM or Yp-YopJC172A at an MOI of 10. Supernatants were collected at 24 hr post-infection and analyzed by ELISA to quantify amounts of secreted IL-1β (A, B) or TNF-α (C, D). Cell death was measured by LDH release (E, F). Results shown are the average of three independent experiments. Error bars represent standard deviation. Statistical significance compared to YP-YopJKIM-infected wild-type BMDMs was determined (ANOVA; P<0.05, *; P<0.01, **).
Figure 8
Figure 8. Determination of the effect of exogenous KCl on cytokine expression and secretion in infected macrophages.
BMDMs were left uninfected (U) or infected at an MOI of 10 with Yp-YopJKIM or Yp-YopJC172A and treated with 30 mM KCl (K+), 30 mM NaCl (Na+) or left untreated (Media) as indicated. Supernatants were collected at 8 hr (A, B) or 24 hr (C, D) post-infection, and secreted IL-1β (A, C) and TNF-α (B, D) were measured by ELISA. (E) Pro-IL-1β in cell lysates prepared at 8 hr post-infection was detected by immunoblotting with anti-IL-1β antibody. Actin in the same samples was detected by immunoblotting as a loading control. Results shown in A–D are the average of three independent experiments. Error bars represent standard deviation. Statistical significance compared to YP-YopJKIM-infected BMDMs in media alone was determined (ANOVA; P<0.01, **; P<0.001, ***).
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