Autoinducer-2-mediated communication network within human gut microbiota

Abstract

Quorum sensing (QS) is a chemical communication process that connects microbial members in various microbial systems. Bacterial communication networks mediated by QS play important roles in the regulation of intestinal microecological balance as well as nutrition and metabolism of the host. However, how human gut microbes utilize QS signals to communicate with one another remains largely unknown. In this study, we first examined the prevalence and abundance of genes encoding QS signal synthases in 3329 species representatives clustered from 289232 prokaryotic genomes in the Unified Human Gastrointestinal Genome collection. Our results show autoinducer-2 (AI-2) is the most prevalent QS signal within the human gut microbiota, with the synthase gene luxS being found in 2039 species mainly distributed within Firmicutes, Actinobacteriota, Bacteroidota, and Proteobacteria. Furthermore, 299 species carry genes encoding one or more types of AI-2 receptors (LuxP-, LsrB-, dCache_1-, and GAPES1-type). The dCache_1- and GAPES1-type receptors can function as methyl-accepting chemotaxis proteins, histidine kinases, c-di-GMP synthases and/or c-di-GMP-specific phosphodiesterases, serine phosphatases, and serine/threonine kinases, suggesting the diversity of AI-2-mediated interspecies communication modes among human gut microbiota. Metatranscriptomic analysis showed that a number of AI-2 synthase- and receptor-encoding genes can be expressed in the human gut in healthy and/or unhealthy states. The communication network analysis suggests that AI-2-mediated interactions widely occur among members of Firmicutes, Proteobacteria, Actinobacteriota, Campylobacterota, and Spirochaetota. Overall, this study deepens understanding of QS-mediated communication network among human gut microbiota, and provides guidance for engineering gut microbiota and constructing new synthetic microbial consortia based on complex microbial interactions.

Keywords: AI-2 receptors; Quorum sensing; autoinducer-2; communication network; human gut microbiota.

Figure 1

Hierarchical clustering of seven QS languages found in 543 human gut prokaryotic genera. 2353 prokaryotic species carrying genes encoding QS signal synthases are distributed in 10 bacterial phyla and two archaeal phyla and one representative species from each genus was selected for phylogenetic analysis. Bars in the outer seven layers represent the distribution of the QS languages AI-2, AIPs, AHLs, indole, HAQs, CAI-1, and DPO in the corresponding genomes, with their existence depicted in blue and absence in gray. This phylogenetic tree was constructed with the PanX software based on the genomes of 543 representative species. The scale bar represents 1.0 amino acid substitutions per site.

Figure 2

Phylogenetic analysis of LsrB-type AI-2 receptors in the human gut microbiota. This phylogenetic tree was constructed based on the amino acid sequences of 101 predicted LsrB proteins using FastTree software (v2.1.11). The scale bar represents 0.5 amino acid substitutions per site.

Figure 3

Widespread occurrence of the dCache_1-type AI-2 receptors in human gut microbiota. (A) Multiple sequence alignment and protein sequence logo of the 5919 dCache_1 domains within 5906 transmembrane proteins in 3329 human gut microbes. The five red arrows above the WebLogo denote five conserved sites corresponding to R126, W128, Y144, D146, and D173 of PctA. The sequence logos were created by WebLogo 3. (B) 12 dCache_1 domains are capable of retaining AI-2. Bioluminescence in V. harveyi MM32 was induced by addition of ligands released from purified proteins expressed in E. Coli strains with (white bars) or without the luxS gene (purple bars). PctA-LBD was used as a positive control. AI-2 activity is shown as fold induction relative to a buffer control. (C–I) AI-2 binds to seven dCache_1 domains with high affinity. The data are one representative of three independent experiments with similar results, with K_d and complex stoichiometry (n) presented as mean ± SD. (J) AI-2 induces the activity of MGYG000003144_03535 in c-di-GMP synthesis. MGYG000003144_03535 were incubated with GTP in the absence and presence of DPD/AI-2 (0, 100, and 200 μM) at 30°C before HPLC analysis. (K) AI-2 enhances the activity of MGYG000000018_00505 in c-di-GMP degradation. MGYG000000018_00505 were incubated with c-di-GMP in the absence and presence of DPD/AI-2 (0, 100, and 200 μM) at 30°C before HPLC analysis. (L) AI-2 inhibits the autophosphorylation activity of MGYG000002432_00530. MGYG000002432_00530 was incubated with ATP-γ-S in the absence and presence of DPD AI-2 (0, 2, and 10 μM) before autophosphorylation detection with anti-thiophosphate ester antibody (top). The amount of the protein was also determined by western blot with the anti-his antibody (bottom). The blots shown are one representative of three independent experiments with similar results. (B, J, K) Data are presented as mean ± SD of three independent experiments. (J, K) Statistical significance was evaluated using the Student's t-test. ^**P < 0.01; ^***P < 0.001.

Figure 4

Phylogenetic analysis of 300 dCache_1 domains and the functional types of the 300 dCache_1-containing AI-2 receptors in 221 human gut bacterial species. This phylogenetic tree was constructed based on the amino acid sequences of 300 dCache_1 domains with the five conserved residues using FastTree software (v2.1.11). The outermost circle represents the functional type corresponding to each dCache_1-type AI-2 receptor. Among them, red indicates MCPs, black indicates CSPs, blue indicates HKs, green indicates STKs, purple indicates SPs, and brown indicates uncharacterized function. The scale bar represents 1.0 amino acid substitutions per site.

Figure 5

The analysis and validation of the GAPES1-type AI-2 receptors in human gut microbiota. (A) Amino acid sequence alignment of 31 GAPES1 domains and that of the YeaJ protein from S. Typhimurium. The conserved residues in the two positions corresponding to Y210 and D239 of YeaJ are highlighted in yellow and nonconserved residues are highlighted in purple. (B) Two GAPES1 domains are capable of retaining AI-2. Ligands released from the purified GAPES1 domains, which were expressed in E. Coli BL21 (DE3) with or without luxS, were used to induce bioluminescence in V. harveyi MM32. PctA-LBD served as a positive control. AI-2 activity is reported as fold induction relative to light production induced by a buffer control, with values presented as mean ± SD of three independent experiments. (C) Two GAPES1 domains interact with AI-2 with high affinity. The data shown are one representative of three independent experiments with similar results. (D) AI-2 enhances the activities of MGYG000002534_01118 and MGYG000002506_01176 in c-di-GMP synthesis. The two GAPES1-containing proteins were incubated with GTP in the absence and presence of DPD/AI-2 (0, 100, and 200 μM) at 30°C before HPLC analysis. The data shown are mean ± SD of three independent experiments and the Student's t-test was used for statistical analyses. ^**P < 0.01; ^***P < 0.001. (E) Phylogenetic analysis of 30 GAPES1-type AI-2 receptors in 29 human gut bacterial species. The phylogenetic tree was constructed based on the amino acid sequences of the 30 predicted GAPES1-type AI-2 receptors using FastTree software (v2.1.11). The scale bar represents 1.0 amino acid substitutions per site.

Figure 6

The distribution and abundance of AI-2 synthase and receptors within the 2105 human gut prokaryotic species. Blocks in the inner cycle indicate microbial taxa (kingdom, phylum, class, order, family, and genus). The red bar closest to the inner cycle indicate the distribution and number of LuxS in the corresponding prokaryotic species (2067 in 2039 species). The blue, green, orange, and purple bars in the outer layers represent the distribution and number of the LsrB-type (101 in 101 species), LuxP-type (six in six species), GAPES1-type (30 in 29 species), and dCache_1-type (300 in 221 species) AI-2 receptors, respectively, in the corresponding species. This sunburst diagram was created by ape R and visualized by iTOL.

Figure 7

AI-2 mediates widespread communication in the human gut microbiota. (A) AI-2-mediated interspecies communications network for 2105 species carrying the AI-2 synthase and/or receptors within human gut microbiota. The nodes in this network represent the 2104 bacterial species and one archaeal species. The color of the node represents the presence of LuxS and AI-2 receptors within this species. The edges symbolize the potential communication processes (the synthesis and sensing of the AI-2 signal). (B) AI-2-mediated communication network among 233 bacterial species with both LuxS and AI-2 receptors. In this network, nodes represent species with their different colors indicating the phylum to which the species belongs, whereas the size of the nodes reflects the number of LuxS or AI-2 receptors of each functional type in the species. Green edges symbolize the synthesis of AI-2 by species, and the orange, blue, pink, and gray-purple edges indicate the perception of AI-2 by the four types of receptors to activate the functional modules (LuxP, LsrB, MCPs, CSPs, HKs, and SPs).