A Host-Produced Autoinducer-2 Mimic Activates Bacterial Quorum Sensing

Abstract

Host-microbial symbioses are vital to health; nonetheless, little is known about the role crosskingdom signaling plays in these relationships. In a process called quorum sensing, bacteria communicate with one another using extracellular signal molecules called autoinducers. One autoinducer, AI-2, is proposed to promote interspecies bacterial communication, including in the mammalian gut. We show that mammalian epithelia produce an AI-2 mimic activity in response to bacteria or tight-junction disruption. This AI-2 mimic is detected by the bacterial AI-2 receptor, LuxP/LsrB, and can activate quorum-sensing-controlled gene expression, including in the enteric pathogen Salmonella typhimurium. AI-2 mimic activity is induced when epithelia are directly or indirectly exposed to bacteria, suggesting that a secreted bacterial component(s) stimulates its production. Mutagenesis revealed genes required for bacteria to both detect and stimulate production of the AI-2 mimic. These findings uncover a potential role for the mammalian AI-2 mimic in fostering crosskingdom signaling and host-bacterial symbioses.

Figure 1

Mammalian epithelial cells produce an autoinducer-2 mimic (AI-2 mimic). (A) Schematic of the co-culture assay. Co-cultures were performed with the following mammalian cell lines for 5 h at 37°C, 5% CO₂: Caco-2, A549, HeLa, J774A.1, Jurkat, or U937 cells. (B and C) Bioluminescence from the V. harveyi reporter strain (B) TL26 (ΔluxN, ΔluxS, ΔcqsS) or (C) TL25 (ΔluxM, ΔluxPQ, ΔcqsS) following co-culture with the indicated mammalian cell line. Saturating, 1 μM AI-2 or 100 nM AI-1 were used as positive controls for V. harveyi TL26 and TL25, respectively. We note that the V. harveyi TL26 AI-2 detector strain displays intrinsically higher background bioluminescence than does the V. harveyi TL25 AI-1 detector strain. Relative light units (RLU) are counts per minute per mL per OD₆₀₀. (D) Total surviving V. harveyi after co-culture (CFU/mL). In all panels, error bars represent standard deviations (SD) for three replicates. See also Fig. S1 and S2.

Figure 2

Two-way signaling between epithelial and bacterial cells occurs during co-culture. Bioluminescence responses of V. harveyi TL26 during (A) direct or indirect co-culture and (B) basal or apical incubation with Caco-2 cells. Culture conditions as in Fig. 1. 1 μM AI-2 was included as a positive control. In all panels, error bars represent SD for three replicates. See also Fig. S3 and S4.

Figure 3

Caco-2 cells produce the AI-2 mimic when subjected to PBS-treatment. (A) Caco-2 cells were cultured for 48 h at 37°C, 5% CO₂ in the specified media. AI-2 mimic activity was measured using the V. harveyi TL26 bioluminescence assay. FBS is fetal bovine serum, Glc is glucose, Gln is glucosamine. (B) Caco-2 cells were cultured in DMEM, PBS, and water. AI-2 mimic activity was analyzed as in (A). (C) Caco-2 survival was assessed through Trypan blue staining. In panels (A) and (B), 1 μM AI-2 was included as the positive control. In all panels, error bars represent SD for three replicates. See also Fig. S4 and S5.

Figure 4

Detection of the AI-2 mimic requires the LuxP receptor in V. harveyi. (A) Preparations from PBS-cultured Caco-2 cells (denoted Mimic) were incubated with V. harveyi FED119 (ΔluxN, ΔluxPQ, ΔluxS) harboring wild-type LuxPQ (expressed from pFED368) or LuxP W167A and wild-type LuxQ (expressed from pFED408), and bioluminescence was measured. (B) Assessment of AI-2 mimic bound by recombinant LuxP. AI-2 mimic activity was assayed with V. harveyi TL26 as in Fig. 3. In both panels, additions to the protein (BSA or LuxP) are as follows: PBS, black; 1 μM AI-2, white; 10% v/v preparations from PBS-cultured Caco-2 cells (Mimic), gray. In all panels, error bars represent SD for three replicates. See also Fig. S6.

Figure 5

V. harveyi mutants defective in stimulation or detection of the AI-2 mimic. (A) Bioluminescence from V. harveyi TL26 Tn5 insertions mutants during co-culture with Caco-2 cells. (B) Bioluminescence of the same strains in response to 100 nM AI-2 was used to verify that mutants could quantitatively detect AI-2 at non-saturating levels. In all panels, error bars represent SD for three replicates. See also Fig. S7

Figure 6

Bacterial genes required for stimulation and detection of the mammalian AI-2 mimic. (A) AI-2 mimic activity in conditioned medium following co-culture of mutant V. harveyi strains with Caco-2 cells. (B) Bioluminescence from mutant V. harveyi strains in response to preparations from PBS-treated Caco-2 cells. (C) Cell-free culture fluids from LB-grown ΔluxS E. coli harboring the cloned apt gene or the empty vector were incubated with Caco-2 cells. We note that LB medium causes high endogenous background bioluminescence. In panels A, B, and C, AI-2 mimic activity was assessed using the V. harveyi TL26 bioluminescence assay, as in Fig. 3. (D and E) Bioluminescence of the specified V. harveyi strains following co-culture with Caco-2 cells: (D) V. harveyi TL26 tkt::Tn5, +/− ptkt and (E) V. harveyi TL26 hldE::Tn5 +/− phldE. We were unable to complement the V. harveyi apt and VIBHAR_02470 mutants because introduction of apt or VIBHAR_02470 on plasmids caused severe growth defects. In panels (B) and (C), 1 μM AI-2 was included as a positive control. In all panels, error bars represent SD of three replicates. See also Fig. S7.

Figure 7

Caco-2 tight junction disruption promotes production of AI-2 mimic. (A) AI-2 mimic activity in conditioned medium following incubation in DMEM, PBS, and 2.5% DSS. 1 μM AI-2 was included as the positive control. (B) Lucifer Yellow transfer and (C) Caco-2 survival was assessed as in Fig. 3. (D) Lactate dehydrogenase (LDH) release from Caco-2 cells cultured as in (A). In all panels, error bars represent SD for three replicates.