Bacterial quorum sensing orchestrates longitudinal interactions to shape microbiota assembly
Microbiome volume 11, Article number: 241 (2023)
The mechanism of microbiota assembly is one of the main problems in microbiome research, which is also the primary theoretical basis for precise manipulation of microbial communities. Bacterial quorum sensing (QS), as the most common means for bacteria to exchange information and interactions, is characterized by universality, specificity, and regulatory power, which therefore may influence the assembly processes of human microbiota. However, the regulating role of QS in microbiota assembly is rarely reported. In this study, we developed an optimized in vitro oral biofilm microbiota assembling (OBMA) model to simulate the time-series assembly of oral biofilm microbiota (OBM), by which to excavate the QS network and its regulating power in the process.
By using the optimized OBMA model, we were able to restore the assembly process of OBM and generate time-series OBM metagenomes of each day. We discovered a total of 2291 QS protein homologues related to 21 QS pathways. Most of these pathways were newly reported and sequentially enriched during OBM assembling. These QS pathways formed a comprehensive longitudinal QS network that included successively enriched QS hubs, such as Streptococcus, Veillonella-Megasphaera group, and Prevotella-Fusobacteria group, for information delivery. Bidirectional cross-talk among the QS hubs was found to play critical role in the directional turnover of microbiota structure, which in turn, influenced the assembly process. Subsequent QS-interfering experiments accurately predicted and experimentally verified the directional shaping power of the longitudinal QS network in the assembly process. As a result, the QS-interfered OBM exhibited delayed and fragile maturity with prolonged membership of Streptococcus and impeded membership of Prevotella and Fusobacterium.
Our results revealed an unprecedented longitudinal QS network during OBM assembly and experimentally verified its power in predicting and manipulating the assembling process. Our work provides a new perspective to uncover underlying mechanism in natural complex microbiota assembling and a theoretical basis for ultimately precisely manipulating human microbiota through intervention in the QS network.
Human microbiomes have been empirically revealed to undergo nonrandom and repeatable community assembly and succession, such as the trait-based community assembly revealed in infant gut [1, 2]; as well as the quick recoverable assembly of human oral biofilm microbiome (OBM) after clinical scaling . Mechanisms underlying microbiota assembly are one of the main problems in microbiome research, which is also the primary theoretical basis for precise manipulation of microbial communities [4, 5]. Until now, several theories, such as priority effects , metabolic cooperation [7,8,9], interspecies bacterial competition , and hydrodynamic disturbance , have been proposed to uncover rules underlying community assembly from the perspective of metabolic interaction. However, metabolism in bacterial flora is extremely complex and redundant, so it is a big challenge to map the metabolic network for precise manipulation on both community structure and function . In this case, excavating an applicable ecological network with more universality, specificity, and regulatory power is of great significance for understanding assembly rules and realizing precise manipulation of bacterial flora.
Bacterial quorum sensing (QS), as the most common means for bacteria to exchange information and interact, is characterized by universality, specificity, and regulatory power and potentially influences the assembly processes of human microbiota. QS has been widely found from human microbiota such as oral cavity [12, 13], skin , lung , and gut [16, 17]. Among all the above flora, the assembly of OBM is more likely to be regulated by QS due to its compact spatial structure [18, 19] and the hydrophobic glycoproteins coat [20, 21], which promote a higher transmission efficiency of QS signals within it . Combined with its fast and repeatable assembly characteristics , OBM is an ideal target to study the role of QS in controlling microbiome assembly.
Due to current cultivation limitations, only three kinds of QS pathways have been experimentally identified in OBM, namely autoinducer peptide (AIP)-based QS represented by Streptococcus [23, 24], autoinducer-2 (AI-2)-based QS found in Fusobacterium nucleatum [25, 26], and recently discovered (acylated homoserine lactone) AHL-based QS pathway [27, 28]. High-throughput sequencing of target microbiome is beneficial to uncover QS pathways in uncultured microbes from human microflora . However, most studies are based on cross-sectional analysis, ignoring the potential changes and evolution of QS regulation, which limits our understanding of the true role of QS in community assembly. This is a key reason why QS manipulation is studied in simple synthetic microbial communities but is difficult to be widely utilized in larger multispecies communities.
To gain a better understanding of the role of bacterial QS in the assembly of microbiota, we developed an in vitro model that mimics the process of OBM assembly. We conducted a thorough screening of QS pathways in OBM and analyzed their patterns of succession during assembly. We also proposed a longitudinal QS communicating network that deciphers the flow of QS signals. Based on this network, we experimentally verified the predictability and feasibility of interfering the QS network to control OBM assembly. These findings reveal an unprecedented QS network that drives microbiota assembly and provide novel perspectives on the precise manipulation of natural, multispecies microbiota.
In vitro modelling of Oral Biofilm Microbiota Assembling (OBMA)
Saliva samples were collected from 12 dentally healthy subjects, avoiding from antibiotic intakes for at least 3 months. Subjects were required to refrain from food or drink 1 h before saliva donation. Saliva samples were pooled together and processed for subsequent two purposes: as inoculating seeds and media component, separately. For the use as inoculating seeds, around 10 ml pooled saliva was centrifuged at 2600g for 10 min at 4 ℃ to remove large debris and eukaryotic cells, and the remaining supernatant with oral microbiota was mixed with same volume of 50% glycerol and stored at − 80 ℃ before use. For the use as media component, the rest of pooled saliva was treated with dithiothreitol (2.5 mM final concentration)  for 10 min and centrifuged at 17,500g for 30 min at 4 ℃. The supernatant was collected, filter-sterilized, and stored at − 20 ℃ before use.
The device for oral biofilm microbiota (OBM) assembling consisted of a 12-well polystyrene cell culture plate, optimized growth medium (OGM), and hydroxyapatite (HA) disks as previously described  as biofilm growth substrata (Fig. 1). HA discs were pre-coated with sterile saliva 1 day before OBM assembling (day − 1). To initiate assembly of OMB, the model was inoculated with 1% seeds in an optimized growth medium consisting of 30% sterile saliva, 60% modified mFUM (Guggenheim B et al., 2001) (supplemented with 1 mM L-arginine, 10 mg/L N-acetylmuramic acid, 1 mg/L hemin, 0.2 mg/L vitamin K, 0.1% sucrose, 0.1% glucose), 10% FBS, and 50 mmol/L PIPIES at pH 7.0 (day 0). Cultures were incubated with 8 replicates at 37 °C for 11 days in an anaerobic chamber (90% N2, 5% CO2 and 5% H2; Don Whitley Scientific Limited, UK) to favor the assembly of OBM. The culture medium was refreshed with OGM daily. Disks without inoculating treatment were conducted as negative controls.
OBM samples developed on the HA discs were collected daily. Briefly, the collected samples were “dip-washed” three times in sterile phosphate-buffered saline to remove the culturing suspension and the intact biofilm-discs were frozen at 80°C for further DNA extraction with the DNeasy PowerBiofilm Kit (Qiagen).
Quantitative PCR (qPCR) was performed as previously described  to assess the abundance of bacteria in OBM samples. qPCR reactions were performed in triplicate in a 20-µl system including 10 µl of 2 × SYBR Premix ExTaqII (TakaraBioInc), 0.2–0.4 µM of primer sets Eub338F/Eub518R, and 2 µl of 1/10 diluted template DNA. qPCR was performed with the CFX96 Touch (Bio-Rad) using the standard reaction conditions according to the standard operating manual.
Shotgun sequencing and bioinformatics
The DNA extracted from the OBM samples and control samples was quantified using a Qubit 4.0 Fluorometer (Life Technologies, Grand Island, NY). High-quality DNA samples from OBM were used for subsequent metagenomic sequencing. Sequencing of paired-end libraries was generated using the Next®Ultra™ DNA Library Prep Kit for Illumina ® (New England Biolabs, MA, USA) following the manufacturer’s recommendations. The resulting libraries were then sequenced on one 2 × 150 bp lane of an Illumina NovaSeq 6000 platform.
The raw data was processed using Trimmomatic (v.0.36) to acquire the clean data for subsequent analysis. Clean reads were assembled into contigs using MEGAHIT (v.1.2.9) with default parameters. Open reading frames (ORFs) were predicted based on assembled contigs using metaProdigal (v.2.6.3) with a minimum length of 100 nt. A non-redundant gene catalog was constructed using CD-HIT (v.4.8.1) with thresholds of 95% identity and 90% coverage.
The taxonomic assignment of each unigene was performed based on the result of gene annotation using MEGAN (v.6.21.7) with LCA algorithm. To calculate the relative abundance of each gene, the number of reads assigned to a specific gene was divided by the length of the gene and subsequently compared to the sum of divided read number of all genes using the BWA-MEM program .
Workflow for the retrieval of QS proteins from OBM metagenomes
Construction of a reference database of QS synthases and receptors
According to the Sigmol  and the Quorum Peps  databases, proteins participating in synthesizing or sensing 26 different types of QS signals have been experimentally characterized and summarized into a list of QS systems in a previous study . Protein sequences of the listed QS proteins were obtained from NCBI (https://www.ncbi.nlm.nih.gov/) and UniProt (https://www.uniprot.org/) and were used as training sequences to create a QS reference database.
Putative homologues of QS proteins in OBM metagenomes were retrieved using the BLASTP command implemented in DIAMOND against the QS reference database with the following thresholds: sequence identity ≥ 30%; alignment coverage ≥ 50%; e-value ≤ 1e−5. All retrieved homologues were further submitted on the Conserved Domain Database (CDD)  and the non-reductant protein database (NR) at NCBI. Those sequences without conserved domains (CDs) and annotations similar to reference QS proteins were discarded.
Relative abundance of each QS homologues from OBM metagenomes was calculated by summing up the abundances of all sequences affiliated to the specific QS homologue, which was subsequently divided by the abundance of a single-copy housekeeping gene recA to get normalized abundance of each QS homologues [38, 39]. The normalized abundance represented the averaging copies of each homologue in an individual cell. The complete workflow for retrieval of QS proteins from the OBM metagenomes is exhibited in Fig. S1.
To assess the power of QS in driving the assembly of OBM, another verification experiment with a control group (3 replicates) and an AI-2 signal interfering group (3 replicates) was conducted using the OBMA model (Fig. S2). All cultures were incubated at 37 °C for 7 days to fulfill a complete assembly of OBM. Differently, the interfering group was additionally supplemented with D-ribose, a frequently used AI-2 inhibitor [40, 41], to interrupt the AI-2 based signaling from day 2 to day 7. The OBM disks were sampled to extract DNA and quantified as previously mentioned. Community structures of the collected OBM samples were analyzed by sequencing 16S rRNA gene amplicons in Magigene (Guangzhou, China). Briefly, the variable region four (V4) of bacteria 16S rRNA gene was amplified using primer set 515F/806R  and sequenced on one Illumina Hiseq2500 PE250 platform. All the obtained paired-end reads of 16S rRNA gene amplicons were analyzed using QIIME  to obtain their taxonomic assignments and calculate their relative abundances. All libraries were rarefied to an even depth based on the smallest sample.
All statistical analyses were performed in RStudio (v.1.4.1103) with R (v.4.2.1). Shannon index was calculated to characterize microbial diversity using the vegan package . Differences and taxonomy classification in microbial communities were calculated and visualized by a PCA ordination plot and a histogram plot respectively using the MicrobiotaProcess package . Nonparametric Spearman’s correlation test was used to test the associations between genus and annotated metabolic pathways of OBM. Time periods when key genera were differentially abundant between the AI-2 interfering and control group were evaluated using the “MetaDprof” R package with smoothing spline ANOVA (ssANOVA) . All the analyses were unpaired.
In vitro OBMA model is robust to simulate the assemble process of OBM
The in vitro OBMA model uses pooled saliva as the inoculum seed and a modified OGM, which can well simulate the naturally complex conditions for OBM assembly. During 11 consecutive days of culture, we observed the complete assembly process of OBM from attachment, expansion to maturation using the OBMA model (Fig. 2A). Based on the successional characteristics of the assembled OBM, we divided the whole process into three periods: the adapting phase (AP) from day 0 to day 2, the growing phase (GP) from day 2 to day 5, and the mature phase (MP) from day 5 to day 11.
During AP, oral bacteria started to colonize with the smallest biomass, which was hardly observed on the surface of the HA discs (Fig. 2A). We noticed that, even no significant difference of biomass in former 2 days, the Shannon index of the OBM on day 2 had a distinct increment compared to that in day 1 (Fig. 2B). This implies that a large and diverse group of bacteria were recruited before rapid development of OBM. In this case, we subdivided the AP phase into AP1 stage (day 0 ~ day 1) and AP2 stage (day 1 ~ day 2). During the period of AP (especially at AP1 stage), Streptococcus showed absolute abundance advantage in OBM (Fig. 2C and D; around 30 ~ 70%) and assumed more responsibilities for amino acid and nucleotide biosynthesis (Fig. 2E). After that, the assembly of OBM grew exponentially in GP (Fig. 2A), during which the richness was slightly increased (Fig. 2B). At this stage, the community structure was relatively stable (Fig. 2C) and was mainly characterized by the increment of Veillonella and Megasphaera (Fig. 2C and D). Once assembled into MP, Prevotella and Fusobacteria were further enriched (Fig. 2C and D) and the function of MP-OBM demonstrated more mature and invasive features such as peptidoglycan and secondary metabolite biosynthesis (Fig. 2E); we therefore concluded that the complete assembly of OBM was reproduced in vitro using the OBMA model.
The whole assembly process followed the trajectory of “adaptation-rapid proliferation-maturation,” during which the core genus succeed from Streptococcus, passing by Veillonella and Megasphaera, finally to Prevotella and Fusobacterium (Fig. 2C and D). The assembly process simulated by the OBMA model is consistent with the observed development of human oral plaque in previous studies , which reflects the reliability of the OBMA model in simulating the real assembly process and provides a reliable basis for studying the assembly rules and mechanisms of complex multispecies microbiota.
Diverse QS pathways were identified through mining OBM genomes
In this study, we created a QS reference database, containing 415 reference protein sequences linked to 26 types of QS pathways. This database was used as a subject data in a BLASTP search for homologues of QS proteins in OBM genomes. Using stringent criteria, 7531 putative protein sequences were identified from the non-redundant gene catalog of OBM genomes. After careful screening for correct functional CDs and annotations corresponding to the reference QS proteins, 2291 homologues were identified that were associated with 21 distinct QS signals. Among these, only 10 QS pathways had both signal-synthesizing and signal-sensing proteins (Table 1), while the remaining QS pathways were incomplete with only a signal-synthesizing or signal-sensing protein (Table S1). To ensure the necessity for signal delivery, only the 10 complete QS pathways were selected for further analysis. The roles of these pathways, summarized in Table 1, are mainly involved in regulating metabolisms such as bacteriocin production, biofilm formation, virulence, and more, through intraspecies, interspecies, and even interkingdom communications. Importantly, this study provides the first comprehensive picture of QS-based communicating in OBM.
The dominant bacteria play a key role as carriers of QS signaling within OBM
The QS pathways identified in this study were found to be sequentially enriched and converted during the AP-, GP-, and MP-assembling periods of OBM (Fig. S3). Specific QS pathways, such as those based on AIP_Agr-Fsr_Like, Lantibiotics, and Bacteriocin-II, were found to be AP-specific, with enrichment during AP but a sharp decrease once assembling into GP (Fig. 3A and B). The genus Streptococcus was the main participant involved in the AP-specific QS communications, utilizing these pathways to regulate adhesion , competence , biofilm formation , toxin production , and other related metabolisms involved in interference competition, preventing the invasion of other species, and establishing itself in a new community. The AP-QS communications enhanced the dominant colonization by Streptococcus to initiate the assembly of OBM. Similarly, MP-specific QS pathways, such as those based on AHK, AHL, and ethanolamine (Fig. 3A), were enriched during the MP period and mediated signal transmission among MP bacteria, especially Prevotella and Fusobacterium, and Bacteroides (Fig. 3B). MP-QS pathways regulate biofilm formation [72, 73], antibiotic resistance , virulence expression , and interspecies and even interkingdom cross-signaling [71, 73, 74], and stimulate the immune system of the host [76, 77], playing a crucial role in the formation of mature OBM.
The other four QS pathways based on AI-2, AIP_RRNPP, DSF, and HAQ were classified as GP-specific and had relatively stable abundances throughout the assembling process (Fig. 3B). GP-QS pathways have been reported to mediate interspecies cross-talk [60, 61, 66,67,68,69], facilitating collaborative multispecies biofilm formation (Table 1). More importantly, the GP-QS pathways undertook longitudinal transmission of QS signals from initial colonizers to late colonizers, thereby promoting steady assembly of OBM from AP to MP period.
The longitudinal QS network shaped the direction of OBM assembly
By doing BLASTP, we identified key QS signal synthesis and signal reception proteins involved in QS pathways and annotated them to respective species (Fig. 3, Figure S5 and Table 1). Species that possess QS signal synthase have the ability to produce and release QS signals, which are then sensed by other species that possess receptors. We refer to this type of interaction as “cross-talk”. The sequentially dominant Streptococcus, Megasphaera and Veillonella, Prevotella, and Fusobacterium were found to be responsible for multiple QS signals synthesizing and sensing during the assembly of OBM by possessing the related signal synthases and receptors. That means the interspecies cross-talk will be intensive among these core QS generalists. Two types of cross-talk were identified among the QS hubs: forward and reverse. The forward cross-talk delivered QS signal from the OBM of the previous period to the later period, such as the transmission of AI-2 from Streptococcus to Megasphaera and Veillonella. Conversely, the reverse type conveyed QS signal from the OBM of the later period to the former period, such as the transmission of AI-2 from Fusobacterium to previously colonized Streptococcus (Fig. 4B). Both types of cross-talk were expected to notify responders in time to cooperate with their corresponding signal synthesizers. However, the difference between them is that the forward cross-talk was found to be conducive to the colonization and reproduction of later arrivals, while the reverse type inhibited the further proliferation of former colonists (Fig. 4A). Overall, bidirectional cross-talk among QS hubs was identified as a key factor in shaping the directional transformation of bacterial structure during OBM assembly.
AI-2 interfering experiment verified that the longitudinal QS network plays a crucial role in shaping OBM assembly
According to the longitudinal QS network, AI-2-based QS is the only one mediating both forward and reverse cross-talk across the whole assembling process (Fig. 4B). Therefore, AI-2 based subnetwork is more potentially involved in promoting OBM assembly than the others. If the delivery of AI-2 in OBM was blocked, according to our assumption, the proliferation of Megasphaera and Veillonella would be directly delayed, thereby delaying the conversion of downstream OBM; meanwhile, the reverse AI-2-based cross-talk would be deactivated, that is, AI-2 synthesized from Prevotella, Fusobacterium can no longer act backwards on initially colonized Streptococcus, resulting in a longer proliferation time and higher abundance of Streptococcus than those without AI-2 interference. This will also inhibit community conversion and normal assembly of OBM (Fig. S5).
To verify our hypothesis, D-ribose, a frequently used substrate competition inhibitor of AI-2, was supplied to deactivate AI-2 singling during OBM assembling (Fig. S2). We then analyzed changes in OBM biomass, growth rate of key QS hubs to evaluate the power of AI-2 subnetwork in shaping OBM assembly. The results turned out that OBM developed in the AI-2 interference group was more fragile compared to the control (Fig. 5A). Specifically, time needed for conversion of OBM from GP to MP was 1 day delayed when AI-2 signal was interfered (Fig. 5A), which was coincident with delayed proliferation of Veillonella (Fig. 5B). Moreover, Streptococcus proliferation was enhanced in AI-2 interference group (Fig. 5B). Its maximum biomass doubled, and its proliferation was prolonged compared with the control group (Fig. 5B, Fig. S6). In the meantime, growth of downstream QS hubs Prevotella and Fusobacterium were suppressed with significant lower biomass than the control group (Fig. 5B, Fig. S6). It can be seen that AI-2-based QS controlled the turnover of OBM community structure via timely regulation of the growth rate of key QS hubs, thereby shaping OBM assembly.
In this study, we have successfully developed an optimized in vitro oral biofilm model (OBMA) by incorporating human saliva and an optimized growth medium. This model not only demonstrates greater diversity but also provides growth conditions that closely resemble a real oral biofilm, thus yielding more realistic and reliable outcomes compared to previous in vitro models. By mixing saliva from multiple individuals, the aim is to create an environment conducive to studying the common characteristics of oral biofilm microbiota within hosts, while still accounting for individual specificity in the oral microbiome. Additionally, our findings from cohort studies provide supporting evidence for the reliability of reconstructing human oral biofilm using the OBMA model. Specifically, both the oral biofilms developed in the human oral cavity and in our OBMA model exhibited similar proliferation patterns, showing a rapid proliferation phase followed by reaching a stable state . Secondly, a consistent pattern was observed during the transition of microbial community structures. Specifically, the initial population of human oral microbiome is facultative anaerobic Streptococcus and was ultimately replaced by gram-negative anaerobic Fusobacteria, Prevotella, and Porphyromonas along with the formation of oral biofilm [3, 78]. Additionally, the core genus Veillonella observed in the growing period in this study has been confirmed as a bridging species critical in guiding the development of multispecies biofilms in the human oral microenvironment [79, 80]. Above all, our constructed OBMA model is reliable to restore the maturation trajectory of human oral biofilm. The OBMA model developed in this study not only holds potential for exploring the regulatory mechanisms of oral biofilm assembly but also can be utilized in early-stage clinical research for screening and evaluating the effectiveness of drugs in controlling oral biofilms.
Our study explores the regulatory role of bacterial QS in the assembly of OBM regardless of host factors. Using the OBMA model, we can observe the entire assembly process while consuming fewer resources and manpower compared to cohort studies.
In contrast to previous strategies based on metabolic collaboration and competition, our study suggests that QS-based interactions regulate microbiota assembly ahead of the onset of other forms of metabolic interactions. QS signals are released and accumulated in the logarithmic growth phase of bacteria to mediate interspecific interactions before most enzymes and metabolites synthesized in stationary growth phase. These signals recruit bacteria clustering with kinship to facilitate microbiota assembly . QS is also responsible for biofilm matrix formation, affecting the microbiota’s structure and function. QS is the fundamental underlying mechanism regulating microbiota assembly.
While it has been suggested QS can promote or inhibit biofilm formation of monocultures, it is challenging to predict how QS plays a role in the assembly of a complex microbial community with hundreds of species based solely on monoculture test results. Our study uncovers the successions of community structure and QS pathways during OBM assembly. We constructed a longitudinal QS network based on this and find it possessing more diverse and complex QS pathways than ever reported. These pathways sequentially enrich and play roles in initial colonizing, exponential proliferation, and maturation, respectively. QS signals in OBM transmit bidirectionally, allowing key QS hubs to regulate transformation of community structure to drive OBM assembly. The construction and regulatory effects of the QS network in the assembly of complex microbiota are unprecedented. Our study provides a new perspective for exploring the underlying mechanism of flora assembly.
The longitudinal QS network proposed in our study provides new possibilities for the application of QS regulation mechanisms in the manipulation of complex microbiota. Based on this network, we accurately predicted and verified the reverse effects of the forward and reverse AI-2 signaling and their respective target QS hubs. Our research results suggest that the exploration of QS regulation can be extended to other human microbiota research, such as the evolution and assembly of colorectal cancer microbiota. By targeting the core QS signals and QS hubs, we can regulate the proliferation of microbiota members, reshape the microbiota in a targeted manner, and ultimately develop new strategies to prevent or treat microbiota-related diseases.
Our results establish a longitudinal QS network scaffolded by dominant bacteria in shaping OBM assembly. This network demonstrates the predictability and controllability of QS interference in regulating the microbiota, thereby expanding our understanding of the mechanism governing microflora assembly, and boosting confidence in applying QS networks to modulate assembly and development of human microbiota.
Availability of data and materials
The sequencing data of metagenome used in this study have been deposited in the GenBank Short Read Archive with accession number PRJNA983519. The accession number can be found below: http://www.ncbi.nlm.nih.gov/sra. The scripts for the retrieval of QS proteins from OBM metagenomes are available in the following Git repository: https://github.com/TaoDing/QS_01.
Oral biofilm microbiota
Oral biofilms microbiota assembling
Optimized growth medium
Conserved domain database
Diffusible signal factors
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We thank Cancan Fan of Hospital of Stomatology, Sun Yat-Sen University, for the assistance with sample collection. We thank the volunteers who participated in this study for providing samples.
This project was supported by the Guangdong Basic and Applied Basic Research Foundation (Grant No. 2022A1515011912 and 2021A1515110810 to Y.S.), the China Postdoctoral Science Foundation (Grant No. 2021M703758 to Y.S.), the National Natural Science Foundation of China (Grant number 81971906 to T.D.), and the Guangdong Key Area Research and Development Program (Grant number 2019B020228001 to T.D.).
Ethics approval and consent to participate
The study was approved by the ethical committee of Zhongshan School of Medicine, Sun Yat-Sen University (ZSSOM-SYSU IRB protocol 2021–028). Informed written consent was obtained from all participants.
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Workflow for retrieval of QS proteins from OBM metagen. Fig. S2. The study design of QS-interfering experiment. Fig. S3. Profiles of QS pathways of OBM shown in robust principal component analysis (PCA) biplot, whose transition correlated with the turn over of the assembly phase of OBM. Fig. S4. The dominant genera that participate in QS signal receptions during AP, GP, and MP stages of OBM assembly. Fig. S5. Diagram of dynamical responses of key QS hubs in the AI-2 interfering experiment. A) Expected proliferation trends of the five QS hubs during OBM assembly when AI-2 signaling gets interfered. B) Diagram of changes in the bidirectional cross talk among the QS hubs when AI-2 signaling gets interfered. Fig. S6. Validation of driving role by AI-2 based QS subnetwork in the assembly of OBM. Longitudinal differential analysis using MetaLonDA revealed temporal changes in absolute abundances of core QS hubs between control and AI-2 interfering groups, with the gray shaded area indicating the significant time interval during which differences were observed.
Proposed roles for the homologs of reference QS proteins found within the oral biofilm microbiota.
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Su, Y., Xu, My., Cui, Y. et al. Bacterial quorum sensing orchestrates longitudinal interactions to shape microbiota assembly. Microbiome 11, 241 (2023). https://doi.org/10.1186/s40168-023-01699-4