Broad detection of bacterial type III secretion system and flagellin proteins by the human NAIP/NLRC4 inflammasome

Abstract

Inflammasomes are cytosolic multiprotein complexes that initiate host defense against bacterial pathogens by activating caspase-1-dependent cytokine secretion and cell death. In mice, specific nucleotide-binding domain, leucine-rich repeat-containing family, apoptosis inhibitory proteins (NAIPs) activate the nucleotide-binding domain, leucine-rich repeat-containing family, CARD domain-containing protein 4 (NLRC4) inflammasome upon sensing components of the type III secretion system (T3SS) and flagellar apparatus. NAIP1 recognizes the T3SS needle protein, NAIP2 recognizes the T3SS inner rod protein, and NAIP5 and NAIP6 recognize flagellin. In contrast, humans encode a single functional NAIP, raising the question of whether human NAIP senses one or multiple bacterial ligands. Previous studies found that human NAIP detects both flagellin and the T3SS needle protein and suggested that the ability to detect both ligands was achieved by multiple isoforms encoded by the single human NAIP gene. Here, we show that human NAIP also senses the Salmonella Typhimurium T3SS inner rod protein PrgJ and that T3SS inner rod proteins from multiple bacterial species are also detected. Furthermore, we show that a single human NAIP isoform is capable of sensing the T3SS inner rod, needle, and flagellin. Our findings indicate that, in contrast to murine NAIPs, promiscuous recognition of multiple bacterial ligands is conferred by a single human NAIP.

Keywords: NAIP; NLRC4; flagellin; inflammasome; type III secretion system.

Fig. 1.

Salmonella Typhimurium induces T3SS-dependent, flagellin-independent inflammasome responses in primary human macrophages. hMDMs were primed with LPS for 3 h and treated with PBS (mock), WT Salmonella (WT ST), ΔsipB ST, or ΔfliCfljB ST at a multiplicity of infection of 20 for 4 h. (A) Cell death (percentage cytotoxicity) was measured by lactate dehydrogenase release assay and normalized to mock infected cells. (B and C) IL-1α and IL-1β supernatant levels were measured by ELISA. (D) Immunoblot analysis was performed on supernatants (sup) for mature IL-1β and on lysates for pro–IL-1β and β-actin as a loading control (representative of two donors). Each data point represents the mean of triplicate infected wells for each of five different human donors. Shaded bars represent the overall mean of the donors. NS, not significant. *P < 0.05 by paired t test; **P < 0.01 by paired t test.

Fig. 2.

L. monocytogenes-mediated delivery of the T3SS inner rod protein PrgJ activates the inflammasome in primary human macrophages. hMDMs were primed with Pam3CSK4 for 3 h and infected with PBS (mock), WT Listeria (Lm), or Lm strains expressing PrgJ and PrgI at a multiplicity of infection of five for 16 h. (A) Cell death (percentage cytotoxicity) was measured by lactate dehydrogenase release assay and normalized to mock infected cells. (B and C) IL-1α and IL-1β supernatant levels were measured by ELISA. (D) Immunoblot analysis of supernatants (sup) for mature IL-1β and lysates for pro—IL-1β and β-actin as a loading control (representative of two donors). Each data point represents the mean of triplicate infected wells for each of seven different human donors. Shaded bars represent the overall mean of the donors. *P < 0.05 by paired Wilcoxon signed rank test.

Fig. 3.

Anthrax toxin-mediated delivery of the T3SS inner rod protein PrgJ induces robust inflammasome activation in primary human macrophages. hMDMs were primed with Pam3CSK4 for 4 h and treated with PA alone, LFn-FlaA^310–475 alone, LFn-PrgJ alone, PA+LFn-FlaA^310–475 (FlaTox), or PA+LFn-PrgJ (PrgJTox) for 16 h. (A) Cell death (percentage cytotoxicity) was measured by lactate dehydrogenase release assay and normalized to mock infected cells. (B and C) IL-1α and IL-1β supernatant level were measured by ELISA. (D) Immunoblot analysis of supernatants (sup) for mature IL-1β and lysates for pro–IL-1β and β-actin as a loading control (representative of two donors). Each data point represents the mean of triplicate infected wells for each of four different human donors. Shaded bars represent the overall mean of the donors. **P < 0.01 by paired t test.

Fig. 4.

Human NAIP is required for maximal inflammasome responses to flagellin and the T3SS inner rod protein PrgJ. Primary hMDMs were transfected with control siRNA or siRNA against NAIP for 48 h and primed with Pam3CSK4 for 4 h. (A and B) Cells were treated with PA+LFn-PrgJ (PrgJTox) or PA+LFn-FlaA^310–475 (FlaTox) for 5 h. (C and D) Cells were treated with LPS+Nigericin for 5 h. IL-1α and IL-1β supernatant levels were measured by ELISA. Each data point represents the mean of triplicate infected wells for six different human donors. Shaded bars represent the overall mean of the donors. NS, not significant. *P < 0.05 by paired t test; **P < 0.01 by paired t test.

Fig. 5.

T3SS inner rod proteins from multiple bacterial species activate the human inflammasome, whereas the Salmonella Typhimurium SPI-2 T3SS inner rod protein, SsaI, evades immune recognition. hMDMs were primed with Pam3CSK4 for 3 h and infected with WT Listeria (Lm) or strains ectopically expressing PrgJ, PrgI, BsaK MxiI, CprJ, or SsaI at a multiplicity of infection of five for 16 h. Cells were treated with PBS for the mock control. (A and C) IL-1β supernatant levels were measured by ELISA. (B and D) Immunoblot analysis of supernatants (sup) for mature IL-1β and lysates for pro–IL-1β and β-actin as a loading control. Each data point represents the mean of triplicate infected wells for each of 8–10 different human donors. Shaded bars represent the overall mean of the donors. NS, not significant. **P < 0.01 by paired Wilcoxon signed rank test.

Fig. 6.

A single NAIP isoform is sufficient for inflammasome responses to flagellin, the T3SS inner rod protein, and the T3SS needle protein. (A and B) THP-1 cells were primed with Pam3CSK4 and treated with PA alone, LFn-FlaA^310–475 alone, LFn-PrgJ alone, PA+LFn-FlaA^310–475 (FlaTox), or PA+LFn-PrgJ (PrgJTox) for 5 h. IL-1α and IL-1β supernatant levels were measured by ELISA. Bar graphs display the mean ± SD of triplicate wells. Representative of three independent experiments. ***P < 0.001 by unpaired t test; ****P < 0.0001 by unpaired t test. (C) HEK293 cells were transfected with expression vectors encoding NLRC4, caspase-1, and IL-1β. Where indicated, cells were also transfected with vectors encoding NAIP* (+) or empty vector control (−). After 18 h, cells were treated with PA+LFn-PrgJ, PA+LFn-FlaA^310–475, PA+LFn-YscF, or PA alone for 9 h. Immunoblot analysis was performed on cell lysates for mature and pro–IL-1β, NAIP*, NLRC4, caspase-1, and β-actin as a loading control. Representative of three independent experiments.