S. gordonii DL1 was grown in Brain Heart Infusion (BHI) broth (Difco, USA). F. nucleatum subsp. polymorphum (ATCC10953) was grown in BHI broth supplemented with 0.5% yeast extract (Difco, USA), 1 μg/ml vitamin K (Sigma-Aldrich, USA), and 5 μg/ml hemin (Sigma-Aldrich, USA) (Ding and Tan, 2016). Both bacterial strains were grown under anaerobic conditions (85% N₂, 10% H₂, 5% CO₂) at 37°C.

Intergeneric coaggregation was performed in coaggregation buffer (CAB) (150 mM NaCl, 1 mM Tris pH 8.0, 0.1 mM CaCl₂, 0.1 mM MgCl₂) as previously described (Cisar et al., 1979). F. nucleatum subsp. polymorphum and S. gordonii DL1 were grown until they reached late exponential growth phase. The optical density (OD_600nm) of F. nucleatum subsp. polymorphum reached 0.80 (~10⁹ CFU), and the OD_600nm of S. gordonii DL1 was assessed to be 0.65 (~10⁹ CFU). Bacterial cells of each species were harvested, washed twice, and resuspended in CAB to a final concentration of ~2×10⁹ cells/ml. In the coaggregation group, equal numbers of cells from each species were pooled together in a new sterile reaction tube. The reaction tube was vortexed for 20 seconds to maximize bacterial physical contact and incubated anaerobically at 37°C for 10 to 30 min for coaggregation. The cells were then pelleted by centrifugation at 100×g for 1 min. The supernatant containing non-aggregated bacterial cells was carefully collected and measured for OD_600nm (OD_{600nm( Sg – Fnp )}). In the autoaggregation groups, F. nucleatum subsp. polymorphum and S. gordonii cells were incubated separately in CAB to evaluate each species’ level of autoaggregation. The levels of interspecies binding at different time points were calculated as follows (Lima et al., 2017; Lima et al., 2019):

OD_{600nm( Sg )} and OD_{600nm( Fnp )} represents the optical density of each individual species at 600nm, and OD_{600nm( Sg-Fnp )} represents the optical density of the recovered supernatant after coaggregation. Pellets were frozen at -80°C and used for RNA extraction within 3 days. Three replicates were performed for this assay.

Total RNA of the coaggregated pellets and the monocultures of F. nucleatum subsp. polymorphum and S. gordonii DL1 was extracted using the hot phenol method and RNAprep Pure Tissue Kit according to the manufacturer’s instructions (Tiangen Biotech, Beijing, China). The extracted RNA was dissolved in 30-100 μl RNase-free water. RNA was quantified using NanoDrop-2000 (NanoDrop Technologies, DE, USA), and the integrity of RNA was determined using 2100 Bioanalyser (Agilent, CA, USA).

Library preparation and Illumina Hiseq sequencing were performed at Lianchuan Bio (Hangzhou, China). Five μg of total RNA from each sample was used. RNA-seq strand-specific libraries were prepared using the TruSeq™ RNA sample preparation kit (Illumina, CA, USA) according to the manufacturer’s instructions. Briefly, rRNA was removed using RiboZero rRNA removal kit (Illumina, CA, USA), and the remaining mRNA was fragmented using fragmentation buffer. After rRNA depletion, the first-strand cDNA was synthesized using random hexamer primers. Subsequently, the second-strand cDNA synthesis was performed. According to Illumina’s protocol, the fragments were then end repaired, added with poly(A) tails, and ligated to the Illumina-indexed adaptors. The second-strand cDNA was degraded using Uracil-DNA glycosylase (UNG). After purification, the product underwent PCR amplification using Phusion DNA polymerase (NEB) for 15 PCR cycles. The RNA-seq strand-specific library was then size-selected for cDNA target fragments of 200–300 bp on 2% low range ultra agarose. After quantified by TBS-380 Fluorometer (Turner BioSystems, CA, USA), the paired-end library was sequenced using Illumina NovaSeq 6000 platform (150bp×2).

The raw paired-end reads from Illumina sequencing were saved in the form of FASTQ files. Adapter sequences and low-quality reads (quality score < 20) were removed by Trimmomatic with parameters (SLIDINGWINDOW:4:15 MINLEN:75) (version 0.36 http://www.usadellab.org/cms/uploads/supplementary/Trimmomatic). Then clean reads were separately aligned to the reference genome with orientation mode using Rockhopper (http://cs.wellesley.edu/~btjaden/Rockhopper/) software. Gene expression levels were also analyzed by Rockhopper with default parameters. Due to the incomplete database annotations in the reference genome of Fusobacterium nucleatum subsp. polymorphum ATCC 10953, reads from Fusobacterium nucleatum subsp. polymorphum monoculture were mapped to Fusobacterium nucleatum subsp. polymorphum NCTC10562, the closest NCBI reference genome for better mapping and further analysis, and the results were denoted as “Fnp.” Reads from S. gordonii DL1 monoculture were mapped to Streptococcus gordonii str. Challis substr. CH1 reference genome (NCBI), and the results were denoted as “Sg.” Samples from the dual-species coaggregation group were mapped to S. gordonii str. Challis substr. CH1 reference genome (NCBI) in the first mapping round, and the results were designated as “CAB_Sg.” In the second round of mapping, samples from the dual-species coaggregating group were mapped to Fusobacterium nucleatum subsp. polymorphum NCTC10562 reference genome (NCBI), and the results were designated as “CAB_Fnp”.

To identify differentially expressed genes (DEGs) induced by dual-species coaggregation, the expression level for each transcript was quantified using the Transcripts Per Million mapped reads method. Comparisons between Group CAB_Sg and Group Sg were performed to explore coaggregation-induced DEGs of S. gordonii DL1. Group CAB_Fnp and Group Fnp were compared as well to investigate coaggregation-induced DEGs of F. nucleatum subsp. polymorphum. edgeR (https://bioconductor.org/packages/release/bioc/html/edgeR.html) was used for differential expression analysis. The DEGs were selected using the following criteria: the |log2(fold change)| was greater than 1, and the false discovery rate (FDR) should be less than 0.05.

To explore the functions of the DEGs, Gene Ontology (GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were carried out by Goatools (https://github.com/tanghaibao/Goatools) and KEGG Orthology-Based Annotation System (http://kobas.cbi.pku.edu.cn/home.do), respectively. DEGs were considered significantly enriched in GO terms and KEGG pathways if their Benjaminiand Hochberg-corrected p values were less than 0.05. The STRING version 11.0 database (https://string-db.org/) was used to identify functional interactions among DEGs.

RT-qPCR was used to validate the dual RNA-seq data. Total RNA of the coaggregated pellets and the monocultures was extracted and examined as previously described. RNA reverse transcription process was performed according to the manufacturer’s instructions (Hifair^® II 1st Strand cDNA Synthesis Kit, Yeasen Biotech, Shanghai, China). RT-qPCR was performed using Hieff^® qPCR SYBR Green Master Mix (Low Rox Plus) (Yeasen Biotech, Shanghai, China) following the manufacturer’s protocol. Relative expression was calculated by normalizing to the corresponding 16S rRNA gene transcripts. Three biological replicates were performed for all RT-qPCR reactions. PCR primers used in the present study are listed in Supplementary Materials ( Table S1 ). Gene expression correlations between RT-qPCR results and RNA-seq results were evaluated by Pearson’s correlation coefficients using GraphPad Prism 8 (https://www.graphpad.com/scientific-software/prism/).

Supernatant samples of four different culture groups (monoculture, co-culture, and coaggregation) were collected. In the monoculture groups, bacterial cells of each species were harvested, washed twice, resuspended in CAB, and cultured anaerobically for 30 min. Bacterial cells were then pelleted by centrifugation at 12,000×g for 5 min. The supernatant was carefully discarded, and fresh growth media (BHI broth supplemented with 0.5% yeast extract, 1 μg/ml vitamin K, and 5 μg/ml hemin) was added to a final concentration of ~1×10⁹ cells/ml. In the co-culture group, equal numbers of both species were harvested, mixed together, washed twice, resuspended in PBS, and cultured anaerobically for 30 min. Bacterial cells were then pelleted and cultured in fresh growth media (~1×10⁹ cells/ml). In the coaggregation group, bacterial cells of each species were harvested and coaggregated as previously described. After 30-min coaggregation, pellets were collected by centrifugation at 100×g for 1 min, and the supernatant was carefully removed. Fresh growth media was added to a final concentration of ~1×10⁹ cells/ml. All groups were cultured anaerobically at 37°C for another 3 h. Supernatant samples were then collected by centrifugation at 12,000×g for 5 min and stored at -80°C and sent to APPLIED PROTEIN TECHNOLOGY (Shanghai, China) for GC-MS analysis which measured the concentrations of various types of SCFAs, including acetic acid, propanoic acid, isobutyric acid, butyric acid, hexanoic acid, and valeric acid.

The human monocyte cell line THP-1 was kindly provided by Stem Cell Bank, Chinese Academy of Sciences (Shanghai, China). THP-1 monocytes were grown in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) (Australian origin, Thermo Fisher Scientific, United States) in a CO₂ incubator (5% CO₂ humidified atmosphere at 37°C). Cells were treated with 50 ng/ml phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich, United States) for 48 h for macrophage differentiation. The THP-1 derived macrophages (dTHP-1 cells) were then grown in fresh RPMI 1640 supplemented with 10% FBS for 24 h in a CO₂ incubator.

Bacterial invasion assay and intercellular bacterial survival assay were performed as described previously (Croft et al., 2018; Xue et al., 2018). dTHP-1 cells were seeded in sterile Corning™ Costar™ flat bottom 6-well cell culture plates (1.0×10⁶ cell/well) and incubated for 12 h before use. dTHP-1 cells were infected with S. gordonii DL1 alone, F. nucleatum subsp. polymorphum alone, co-culture of the two species, and coaggregates of the two species, respectively (multiplicity of infection [MOI] was 10:1). We conducted a preliminary test to determine the CFUs of the coaggregates when the same volume of bacterial cells was used across all groups. In detail, the supernatant of the coaggregation group containing non-aggregating cells was removed. The pellets (coaggregates) were resuspended in PBS, and the tube was vigorously vortexed until no pellet was visible. A 100-μl aliquot of the solution was taken, subjected to serial dilutions in PBS (1:10), and seeded onto sheep blood agar plates as well as BHI agar plates. After the preliminary test, the volumes of bacterial cells used in the coaggregation group were adjusted to ensure that the final aggregates had similar numbers of bacterial cells with the mono-culture groups and met the MOI (10:1) requirement. After initial centrifugation (5 min at 250×g) to increase interactions between dTHP-1 cells and bacterial cells, the culture plates were incubated for 30 min in a CO₂ incubator at 37°C. Cells were further divided into two groups: one is for bacterial invasion assay, and the other is for intercellular bacterial survival assay.

For bacterial invasion assay, dTHP-1 cells were washed five times extensively with sterile PBS. The supernatants were collected, spread onto 5% sheep blood agar plates, and incubated anaerobically for 3 days to validate that extracellular bacteria were wiped out. Cells were immediately lysed using 0.1% Triton X-100 (Beyotime, Shanghai, China). The sample of each well was serially diluted (1:10 dilution), spread onto 5% sheep blood agar plates and BHI agar plates, and incubated for 16-48 h to quantify the colony-forming units (CFUs) of “invading bacterial cells”.

For intercellular bacterial survival assay, dTHP-1 cells with invading bacteria were incubated for another 2 h before cell lysis. Briefly, dTHP-1 cells were washed twice with sterile PBS and cultured in fresh RPMI 1640 containing 10% FBS, gentamicin (300 μg/ml), and metronidazole (200 μg/ml) (Solarbio, Beijing, China) for 2 h to exterminate extracellular bacteria. dTHP-1 cells were then washed three times with sterile PBS. The supernatant of each group was collected and spread onto 5% sheep blood agar plates, and the plates were incubated anaerobically for 3 days to validate the killing of extracellular bacteria. dTHP-1 cells were lysed using 0.1% Triton X-100 (Beyotime, Shanghai, China). The sample of each well was vortexed for 10 seconds, serially diluted (1:10 dilution), spread onto 5% sheep blood agar plates and BHI agar plates, and incubated for 16-48 h to quantify the CFUs of “surviving bacterial cells”. All experiments were repeated at least three times.

dTHP-1 cells were seeded in 96-well plates (1×10⁴ cells/well) and infected with 0.5 μl/ml lipopolysaccharide (LPS) (Solarbio, Beijing, China), PBS (negative control), the monoculture, the co-culture, and the coaggregates of S. gordonii and F. nucleatum subsp. polymorphum, respectively. After 2 h or 4 h, the plates were washed with PBS and cultured in fresh RPMI 1640 containing 10% FBS, gentamicin (300 μg/ml), and metronidazole (200 μg/ml) (Solarbio, Beijing, China) to kill extracellular bacteria. After 2 h, the plates were cultured in fresh RPMI 1640 containing 10% FBS, gentamicin (60 μg/ml), and metronidazole (40 μg/ml). Cell viability assay was performed using Cell Counting Kit-8 (CCK-8, Dojindo Laboratories, Kumamoto, Japan), and 10 μl CCK-8 was added into each well following the manufacturer’s instructions. The absorbance of the supernatant was recorded at 450nm using a microplate reader (Tecan, Reading, UK). dTHP-1 cells exposed to PBS served as negative control, and fresh RPMI 1640 containing 10% FBS, gentamicin (60 μg/ml), and metronidazole (40 μg/ml) without cells or bacteria served as blank controls. All groups were assessed in quintuplicate.

dTHP-1 cells were seeded in 6-well plates (1×10⁶ cells/well) and were infected with 0.5 μl/ml LPS (Solarbio, Beijing, China), PBS, the monoculture, the co-culture, and the coaggregates of S. gordonii and F. nucleatum subsp. polymorphum, respectively. After 2 h or 4 h, the plates were washed with PBS and cultured in fresh RPMI 1640 containing 10% FBS, gentamicin (300 μg/ml), and metronidazole (200 μg/ml) (Solarbio, Beijing, China) to kill extracellular bacteria. After 2 h, the plates were cultured in fresh RPMI 1640 containing 10% FBS, gentamicin (60 μg/ml), and metronidazole (40 μg/ml). Supernatants of samples collected at different time points were collected by centrifugation at 3,000 rpm (20 min, 4°C). Pro-inflammatory cytokines IL-1β, IL-6, and TNF-α were analyzed using ELISA kits (Neobioscience, Shenzhen, China) according to the manufacturer’s instructions.

Regarding results other than the transcriptional analysis, statistically significant differences (p < 0.05) were determined by One-Way analysis of variance with post-hoc Dunnett’s test.

Coaggregation of F. nucleatum with other bacterial species is a highly specific process, and its subspecies exhibited varied coaggregation abilities (Hojo et al., 2009; Guo et al., 2017). The level of physical adherence between F. nucleatum subsp. polymorphum and S. gordonii was assessed in the study. Results of the coaggregation assay showed that the interspecies binding between F. nucleatum subsp. polymorphum and S. gordonii cells in CAB occurred quickly within 10 min and remained stable for the next 20 min ( Figure 1 ). In addition, the level of dual-species coaggregation was high (~91%), while the level of autoaggregation of each species was markedly low (~10%). The present results indicated that F. nucleatum subsp. polymorphum and S. gordonii cells could extensively adhere to each other.

In total, 57.35 million raw reads and 38.75 million clean reads were produced by the three coaggregated F. nucleatum subsp. polymorphum and S. gordonii biological replicates. Among these coaggregated biological replicates, an average of 32.54% clean reads were mapped to the F. nucleatum subsp. polymorphum reference genome, and 58.47% clean reads were mapped to the S. gordonii reference genome. In monoculture samples, three biological replicates of F. nucleatum subsp. polymorphum produced 47.26 million raw reads and 31.69 million clean reads, and an average of 88.12% clean reads were mapped to reference genomes of F. nucleatum subsp. polymorphum. Three biological replicates of S. gordonii yielded 45.0 million raw reads and 32.18 million clean reads, and an average of 92.76% clean reads were mapped to reference genomes of S. gordonii.

Analysis of differential gene expression revealed that a total of 267 S. gordonii genes and 211 F. nucleatum subsp. polymorphum genes were differentially expressed (p < 0.05). Volcano plots of DEGs in the two different comparisons illustrated distinct transcriptional profiles ( Figure 2 ).

A group of DEGs of each species was examined by RT-qPCR ( Figure 3 ). Gene expression levels were normalized to 16S rRNA expression of the corresponding species. The patterns of gene expression by dual RNA-seq and RT-qPCR were similar. There was a strong correlation between the two data sets in each species (Pearson’s correlation coefficients were 0.9993 for S. gordonii DEGs and 0.9952 for F. nucleatum subsp. polymorphum DEGs, p<0.02).

S. gordonii and F. nucleatum subsp. polymorphum do not automatically coaggregate in the co-culture group which serves as control. We previously treated S. gordonii and F. nucleatum subsp. polymorphum with CAB buffer lacking calcium, and these two strains still coaggregated extensively. The coaggregation levels were similar to those occurring in the original CAB buffer. Coaggregation between S. gordonii and F. nucleatum is likely mediated via multiple pairs of macromolecules, including adhesins and protein-receptors (Kaplan et al., 2009; Lima et al., 2017). Calcium might play a less important role in the coaggregation between these two species. PBS is a buffer solution commonly used in biological research, and S. gordonii and F. nucleatum subsp. polymorphum did not automatically coaggregate in PBS. Therefore, PBS, instead of CAB without calcium, was used as a buffer in the control group. Results showed that, compared with the mono-species groups and dual-species co-culture group, the dual-species coaggregation group exhibited significantly decreased levels of propanoic acid and butyric acid ( Figure 6 ). Results indicated that dual-species coaggregation greatly curtailed the overall production of propanoic acid and butyric acid.

To investigate the effect of dual-species coaggregation on both species’ invading and intracellular survival ability, we treated dTHP-1 cells with live S. gordonii cells, live F. nucleatum subsp. polymorphum cells, co-culture of the two species, and coaggregates of the two species, respectively. The bacterial cells that invaded and survived within dTHP-1 cells were counted, respectively. F. nucleatum subsp. polymorphum formed pinpoint circular colonies on blood agar plates, while S. gordonii formed grayish-white, larger, and smooth colonies on blood agar plates. In addition, we reconfirmed the CFUs of S. gordonii by counting the number of bacterial colonies on BHI agar plates because only S. gordonii could grow on the surfaces of BHI agar plates. Results revealed that significantly more F. nucleatum subsp. polymorphum cells and S. gordonii cells invaded macrophages in the coaggregation group compared with the co-culture group and the mono-species groups ( Figure 7A ). In addition, the survival rates of both species were significantly higher in the coaggregation group compared with other groups ( Figure 7B ).

Different time points were chosen according to relevant published studies (Bor et al., 2016; Xu et al., 2018; Periselneris et al., 2019). After 2 h or 4 h incubation time, extracellular bacteria were wiped out, and macrophages were incubated for another 6 h or 24 h. 6 h represents the early stage of cytokine production, and 24 h refers to the late stage of cytokine production. The time points chosen in the host cell viability assay was matched with those used in the cytokine production assay. Results of CCK-8 assay revealed that dTHP-1 cells infected with the coaggregates of F. nucleatum subsp. polymorphum and S. gordonii had similar cell viabilities with those infected with monocultures or the co-culture of both species at both 6 h and 24 h post infection ( Figure 8 ). ELISA results demonstrated that dTHP-1 cells infected with the coaggregates of F. nucleatum subsp. polymorphum and S. gordonii secreted significantly lower levels of IL-1β and IL-6 at 24 h post infection compared with those infected with the co-culture of both species. The levels of TNF-α were similar between the co-culture group and the co-aggregation group at 24 h post infection ( Figure 9 ).

The lack of efficient genetic and molecular systems has hindered the further study of F. nucleatum at the molecular level. Although shuttle plasmid pHS17 was constructed to study properties of F. nucleatum subsp. polymorphum (ATCC 10953), transformation efficiency of pHS17 was low (Haake et al., 2000; Kinder Haake et al., 2006). Only a limited number of studies constructed trait-specific isogenic mutants to validate F. nucleatum gene functions; hence there was insufficient research to aid the further transcriptome analysis of many F. nucleatum subsp. polymorphum DEGs.

Genes that encode ABC transporters were differentially regulated following coaggregation with S. gordonii. Five DEGs that encode different types of ABC transporters were identified ( Table S5 ). Among them, genes AT688_RS00240 and AT688_RS00245 were significantly up-regulated, and they might have multiple functions such as participating in cell division, hemin transport, and acetoin utilization (Yoshida et al., 2000; Bouige et al., 2002; Friedman et al., 2006). The other three DEGs were significantly down-regulated with fold changes ranged from -2.14 to -4.20. One of them encodes LPS export system permeases (AT688_RS05820, LptF/LptG family permease), facilitating LPS assembly (Ruiz et al., 2008), and the downregulation of the gene might hinder the process of bacterial LPS assembly in F. nucleatum subsp. polymorphum.

The innate immune system is actively against microbial pathogens. Macrophages are common innate immune cells that respond to microbial infection, recognize and engulf microbial pathogens (phagocytosis), and create an internal compartment where pathogens may be killed (phagosomes) (Gordon, 2016). Past research showed that bacterial species could use multiple strategies to survive within macrophages. For instance, pathogens could reduce immunogenicity to hide from macrophage defense, bond with inhibitory receptors to mitigate host immune responses, or subvert phagosome maturation (Sarantis and Grinstein, 2012; Bor et al., 2016; Mitchell et al., 2016; Thakur et al., 2019). Bor et al. discovered that the interspecies coaggregation between Candida albicans (C. albicans) and F. nucleatum attenuated both species’ virulence, contributing to a long-term commensal lifestyle and prolonged survival of both species within macrophages (Bor et al., 2016). To survive and even replicate within macrophages, Candida glabrata could mask immunostimulatory cell-wall components and induce poor host cell activation, causing low levels of pro-inflammatory cytokines upon infection (Kasper et al., 2015). Mycobacterium tuberculosis has learned to counter-balance the macrophage killing by reducing inflammasome activation and facilitating immune evasion of the bacterium (Weiss and Schaible, 2015).

The present study revealed the transcriptional profiling of S. gordonii and F. nucleatum subsp. polymorphum in response to the short-term dual-species coaggregation. Further studies are encouraged to investigate transcriptional changes of coaggregated species over a longer period to observe the trends of gene regulation in partner species. Nevertheless, in this study, 30 minutes of coaggregation between S. gordonii and F. nucleatum subsp. polymorphum has induced significant transcriptional changes in multiple DEGs related to the biosynthesis and export of peptidoglycan and LPS as well as carbohydrate and pyruvate metabolism. In addition, GC-MS analysis revealed that the coaggregates of S. gordonii and F. nucleatum subsp. polymorphum produced significantly reduced levels of propanoic acid and butyric acid compared with the co-culture of both species. Furthermore, the dual-species coaggregation group exhibited significantly better intracellular survival within macrophages and induced markedly lower production of IL-6 and IL-1β compared with the dual-species co-culture group. Transcriptome results together with GC-MS analysis indicated that both species following coaggregation are likely to resist macrophage killing via mitigation of the immune responses. Intergeneric coaggregation between F. nucleatum subsp. polymorphum and S. gordonii prepared both opportunistic pathogens for better resilience against macrophage killing.