NOD-like receptor activation by outer membrane vesicles from Vibrio cholerae non-O1 non-O139 strains is modulated by the quorum-sensing regulator HapR

Abstract

Vibrio cholerae is an inhabitant of aquatic systems and one of the causative agents of severe dehydrating diarrhea in humans. It has also emerged as an important cause of different kinds of inflammatory responses, and in particular, V. cholerae strains of the non-O1 non-O139 serogroups (NOVC) have been associated with such infections in human. We analyzed the potential of outer membrane vesicles (OMVs) derived from the NOVC strain V:5/04 to induce inflammatory responses in human host cells. V:5/04 OMVs were taken up by human epithelial cells and induced inflammatory responses. Small interfering RNA (siRNA)-mediated gene knockdown revealed that the inflammatory potential of NOVC OMVs was partially mediated by the nucleotide-binding domain-, leucine-rich repeat-containing family member NOD1. Physiochemical analysis of the content of these OMVs, in conjunction with NOD1 and NOD2 reporter assays in HEK293T cells, confirmed the presence of both NOD1 and NOD2 active peptidoglycan in the OMVs. Furthermore, we show that deletion of the quorum-sensing regulator HapR, which mimics an infective life style, specifically reduced the inflammatory potential of the V:5/04 OMVs and their ability to activate NOD1 and NOD2. In conclusion, our study shows that NOVC OMVs elicit immune responses mediated by NOD1 and NOD2 in mammalian host cells. Moreover, we provide evidence that the quorum-sensing machinery plays an important regulatory role in this process by attenuating the inflammatory potential of OMVs under infective conditions. This work thus identifies a new facet of how Vibrio affects host immune responses and defines a role for the quorum-sensing machinery in this process.

FIG. 1.

Structure and composition of OMVs from the NOVOC strains used. (A) EM pictures of preparations of OMVs derived from NOVC V:5/04 and V:5/04 ΔhapR bacteria. Bars, 200 nm. (B) Coomassie blue-stained polyacrylamide gel showing the protein contents of V:5/04 and V:5/04 ΔhapR OMVs. Detection of OmpU by immunoblot analysis is presented in the lower panel; 1.5 μg total protein was loaded for each sample. (C) Silver-stained SDS-polyacrylamide gel showing the LPS contents of V:5/04 and V:5/04 ΔhapR OMVs.

FIG. 2.

NOVC-derived OMVs are taken up by epithelial cells. HeLa cells were inoculated with V:5/04 OMVs or medium as a control. At 8 h after inoculation, cells were fixed and subjected to indirect immunofluorescence analysis. OMVs were stained using an OmpU-specific antibody (red). A merge image with actin stained in green and DNA in blue is shown (upper panel), as well as the OmpU signal alone in gray scale (lower panel). Bar = 10 μm.

FIG. 3.

OMVs activate inflammatory responses in different human host cells. (A) HeLa cells were treated with vesicles derived from NOVC V:5/04 or V:5/04 ΔhapR (1 μg/ml). Stimulation with 0.1 μg/ml LPS served as control for reactivity of the cells. At 8 h after inoculation, the IL-8 release was measured in the supernatant by ELISA. Means and SDs (n = 3) are shown. (B) THP1 cells were treated for 16 h with NOVC V:5/04 or V:5/04 ΔhapR OMVs (0.05 μg/ml) or with MDP (1 μM) or LPS (0.1 μg/ml) as controls. IL-8 release was measured in the supernatant by ELISA. Means and SDs (n = 3) are shown. (C) THP1-Blue NF-κB reporter cells were treated for 16 h with NOVC V:5/04 or V:5/04 ΔhapR OMVs (0.5 μg/ml) or with MDP (10 μM) or LPS (0.1 μg/ml) as controls. NF-κB activation was determined by measuring SEAP activity of the supernatant. Mean fold activation relative to the untreated control (set to 1) and SDs (n = 3) are shown. n.s., not stimulated. (D) Assay as for panel C, showing MDP, TriDAP, and LPS as controls. NOVC V:5/04 OMVs (0.5 μg/ml) were applied in the absence or presence of polymyxin B (25 μg/ml).

FIG. 4.

OMVs trigger NOD1- and NOD2-dependent NF-κB activation. HEK293T cells were transiently transfected with plasmids encoding NOD1 (left panel) or NOD2 (right panel) together with a NF-κB (A and B) or IL-8 (C and D) luciferase reporter system. Cells were stimulated with the indicated OMVs (0.5 μg/ml) or MDP (50 nM) and TriDAP (500 nM) as controls. RPS, NF-κB activity of OMVs (0.5 μg/ml) in cells transfected only with the reporter system. NF-κB activity was determined by measuring luciferase activity of the cell lysates after 16 h of incubation. Means and SDs (n = 3) are shown. n.s., not stimulated.

FIG. 5.

OMVs contain NOD1/2 active peptidoglycan. (A) The NOD1 or NOD2 gene was silenced in THP1-Blue cells by transfecting specific siRNA duplexes for 72 h. Cells were subsequently stimulated with NOVC V:5/04 OMVs (1 μg/ml) or with TriDAP (10 μg/ml) or MDP (10 μM) as controls. NF-κB activity was determined by measuring SEAP activity in the supernatant after 16 h and is given as mean fold over background from two independent experiments. Error bars indicate standard errors of the means (n = 4). n.s., not stimulated. (B) Quantitative PCR of NOD1 and NOD2 mRNAs prepared from samples used for panel A. Mean expression relative to GAPDH is shown normalized to NOD1/2 expression in cells treated with nontargeting siRNA (siCTRL) (set to 1). Error bars indicate SDs (n = 3). (C) The chemical composition of vesicle samples was determined using multivariate analysis of X-ray photoelectron spectroscopy (XPS) as described in Materials and Methods. The near-surface (<10 nm) composition of polysaccharide, lipid, and protein/peptidoglycan for each sample is shown (means and SDs; n = 3).

FIG. 6.

Differential NOD1/2 response to bacterial cell lysate and OMVs. (A) Cell viability assay (XTT) of HeLa cells exposed to the indicated culture supernatants. The mean percentage of viable cells compared to the control (set to 100%) and SD (n = 3) are shown. (B) HeLa cells were treated with bacterial lysate or LPS (0.2 μg/ml) as a control. IL-8 release was measured in the supernatant by ELISA at 8 h postexposure. (C) Western blot of equal amounts of bacterial lysates probed with an RpoS-specific antibody. (D) THP1-Blue cells were treated for 16 h with the indicated bacterial lysate (0.05 μg/ml) or with MDP (10 μM), LPS (0.1 μg/ml), or a combination of both as controls. NF-κB activity was determined by measuring SEAP activity in the supernatant. Activity is given as mean fold over background. Error bars indicate SDs (n = 3). (E) HEK293T cells were transiently transfected with a plasmid encoding NOD2 together with an NF-κB luciferase reporter system. Cells were stimulated with bacterial lysate (0.5 μg/ml) or with MDP (50 nM) as a control. NF-κB activity was determined by measuring luciferase activity in the cell lysates after 16 h of incubation. Means and SDs (n = 3) are shown. n.s., not stimulated.