Innate immune detection of the type III secretion apparatus through the NLRC4 inflammasome

Abstract

The mammalian innate immune system uses Toll-like receptors (TLRs) and Nod-LRRs (NLRs) to detect microbial components during infection. Often these molecules work in concert; for example, the TLRs can stimulate the production of the proforms of the cytokines IL-1beta and IL-18, whereas certain NLRs trigger their subsequent proteolytic processing via caspase 1. Gram-negative bacteria use type III secretion systems (T3SS) to deliver virulence factors to the cytosol of host cells, where they modulate cell physiology to favor the pathogen. We show here that NLRC4/Ipaf detects the basal body rod component of the T3SS apparatus (rod protein) from S. typhimurium (PrgJ), Burkholderia pseudomallei (BsaK), Escherichia coli (EprJ and EscI), Shigella flexneri (MxiI), and Pseudomonas aeruginosa (PscI). These rod proteins share a sequence motif that is essential for detection by NLRC4; a similar motif is found in flagellin that is also detected by NLRC4. S. typhimurium has two T3SS: Salmonella pathogenicity island-1 (SPI1), which encodes the rod protein PrgJ, and SPI2, which encodes the rod protein SsaI. Although PrgJ is detected by NLRC4, SsaI is not, and this evasion is required for virulence in mice. The detection of a conserved component of the T3SS apparatus enables innate immune responses to virulent bacteria through a single pathway, a strategy that is divergent from that used by plants in which multiple NB-LRR proteins are used to detect T3SS effectors or their effects on cells. Furthermore, the specific detection of the virulence machinery permits the discrimination between pathogenic and nonpathogenic bacteria.

Fig. 1.

PrgJ activates NLRC4. (A) WT or NLRC4^−/− BMM were primed with LPS for 4 h, then infected with flagellin mutant (fliC fljB) or SPI1 mutant (prgJ) S. typhimurium strain under SPI1-inducing conditions at the indicated multiplicity of infection (MOI) for 2 h and IL-1β secretion determined by ELISA. (B) Cross-sectional schematic of structure of flagellar and SPI1 T3SS, with location of flagellar filament (FliC, blue), needle (PrgI, green), and internal rod (PrgJ, red) indicated. (C) Published structures of FliC (1UCU) and PrgI (2JOW) are presented in comparison with our predicted structure of PrgJ. The N-terminal and C-terminal regions of PrgI are not well resolved; the top three models from the published data are shown superimposed. The N-terminal region of PrgJ is not well resolved and is indicated only for reference. (D) Quantities of 8, 16, or 31 ng/well purified FliC, PrgI, or PrgJ or no protein control (Ø) were delivered to the cytosol of LPS primed BMM using a protein transfection reagent (Profect P1). IL-1β secretion was determined by ELISA 1 h later. Standard deviations are indicated. (E) BMM from WT or NLRC4 null mice were transfected with purified FliC, PrgJ, or PrgI protein for 1 h, and caspase 1 processing was determined by Western blot. (F) To perform a retroviral lethality screen, BMM were infected with transgenic retroviruses expressing GFP alone, or prgJ followed by an IRES-GFP element. GFP positive cells were identified by flow cytometry 2 days after infection. (G) Quantitation of F.

Fig. 2.

PrgJ and FliC share sequence identity within an NLRC4 activating motif. (A) Alignment of FliC and PrgJ starting at the indicated amino acid through the carboxy terminal residue. Identity (black) and similarity (gray) are indicated. (B) 6xHis-tagged PrgJ or protein carrying the indicated mutations were purified and delivered to the cytosol of LPS primed BMM. IL-1β secretion was determined 1 h later. (C) CHO cells expressing TLR5 were stimulated with the indicated protein and expression of firefly luciferase under the control of NF-κB was measured.

Fig 3.

PrgJ homologs activate NLRC4. (A) Alignment of the carboxy terminal regions of PrgJ (S. typhimurium SPI1), BsaK (B. pseudomallei), MxiI (S. flexneri), EprJ (EHEC), PscI (P. aeruginosa), EscI (EHEC and EPEC), and SsaI (S. typhimurium SPI2) from the indicated amino acid residue through the carboxy terminal residue. Residues identical in four of the seven sequences (black) or similar residues (gray) are indicated. (B and C) Retrovirus lethality screen as described in Fig. 1; percentage of GFP-positive cells was determined by flow cytometry. (D and E) WT or NLRC4 null BMM were infected with WT, flagellin (fliC), or T3SS (escN) mutant strains of EPEC at the indicated MOI for 1 h. Gentamicin was added to the media, and the infection was continued for 3 h. IL-1β secretion was determined by ELISA. Standard deviations are shown.

Fig. 4.

Lack of detection of SPI2 T3SS by NLRC4 is beneficial for S. typhimurium. (A) BMM were infected with S. typhimurium grown under SPI1 or SPI2 inducing conditions for the indicated time and MOI before IL-1β secretion was determined. (B) A 63-ng quantity of the indicated purified proteins was delivered to LPS primed BMM by protein transfection, and IL-1β secretion was determined. (C) Retroviral lethality screen was performed as in Fig. 3 for prgJ, ssaI, ssaI-prgJ, and ssaI-escI hybrid proteins. (D) BMM were infected with S. typhimurium grown under SPI2-inducing conditions for 8 h (1 h infection followed by 7 h treatement with gentamicin) at MOI 50, and IL-1β secretion was determined by ELISA. (E) Mice were infected with equal numbers of wild-type S. typhimurium carrying empty vector (pWSK129, kanamycin resistant) or a plasmid-expressing SPI2 controlled prgI or prgJ (ampicillin resistant). Competitive index was determined 48 h after i.p.injection of wild-type (BL6) or NLRC4 null mice by determining colony forming units in the spleen. The ratio of ampicillin/kanamycin-resistant colonies is shown with standard deviation from three mice (ratio of 0.01 represents a 100-fold reduction in recovered ampicillin-resistant bacteria compared with wild-type).