Manipulation of small Rho GTPases is a pathogen-induced process detected by NOD1

Abstract

Our innate immune system distinguishes microbes from self by detecting conserved pathogen-associated molecular patterns. However, these are produced by all microbes, regardless of their pathogenic potential. To distinguish virulent microbes from those with lower disease-causing potential the innate immune system detects conserved pathogen-induced processes, such as the presence of microbial products in the host cytosol, by mechanisms that are not fully resolved. Here we show that NOD1 senses cytosolic microbial products by monitoring the activation state of small Rho GTPases. Activation of RAC1 and CDC42 by bacterial delivery or ectopic expression of SopE, a virulence factor of the enteric pathogen Salmonella, triggered the NOD1 signalling pathway, with consequent RIP2 (also known as RIPK2)-mediated induction of NF-κB-dependent inflammatory responses. Similarly, activation of the NOD1 signalling pathway by peptidoglycan required RAC1 activity. Furthermore, constitutively active forms of RAC1, CDC42 and RHOA activated the NOD1 signalling pathway. Our data identify the activation of small Rho GTPases as a pathogen-induced process sensed through the NOD1 signalling pathway.

Figure 1. SopE-induced NF-κB activation requires Rip2 and Nod1

(A–D) HEK293 cells transfected with a NF-κB-luciferase reporter were treated as indicated. (A and C) Cells were infected with S. Typhimurium (S. Tm) wild type (WT) or mutants. (A–D) Mean luciferase activity ± s.e.m., N = 3. (E–F) Streptomycin-pretreated Nod-1-deficient mice and wild-type littermates (for N see Figure S5B) were infected and the cecum collected after 24 hours. (E) Average histopathology score. Whisker plots represent the second and third quartiles (boxes) and the first and fourth quartiles (lines). (F) Average number of neutrophils per microscopic field. (A–F) NS, not significantly different.

Figure 2. NOD1 senses the activation state of small Rho GTPases

HEK293 cells were transfected as indicated, together with a NF-κB luciferase reporter. (A–E) Luciferase activity was measured 48 hours after transfection. (F–G) Cells were stimulated with C12-iE-DAP or FliC as indicated and luciferase activity was measured 5 hours thereafter. (A–E) Data are presented as mean ± s.e.m. from at least three independent experiments. Brackets indicate the significance of differences. NS, not significantly different.

Figure 3. SopE forms a multi-protein complex with Rac1, Cdc42, Nod1 and Hsp90

(A–C and F) Ectopically expressed SopE-HA (A, C and F), SopE_G168A-HA (B) and endogenous Nod1 (A–B), Rac1 (C), and Hsp90 (F) were detected by confocal microscopy. (D and E) Whole cell lysates and anti-Flag immunoprecipitates were separated by SDS-PAGE and subjected to immunoblotting. (G) HEK293 cells transfected with a NF-κB luciferase reporter were pretreated with the Hsp90 inhibitor geldanamycin (100 nm). Data are presented as mean ± s.e.m. of at least three independent experiments. Brackets indicate the significance of differences. NS, not significantly different.