Physiologically relevant coculture model for oral microbial-host interactions

Abstract

Understanding microbial-host interactions in the oral cavity is essential for elucidating oral disease pathogenesis and its systemic implications. In vitro bacteria-host cell coculture models have enabled fundamental studies to characterize bacterial infection and host responses in a reductionist yet reproducible manner. However, existing in vitro coculture models fail to establish conditions that are suitable for the growth of both mammalian cells and anaerobes, thereby hindering a comprehensive understanding of their interactions. Here, we present an asymmetric gas coculture system that simulates the oral microenvironment by maintaining distinct normoxic and anaerobic conditions for gingival epithelial cells and anaerobic bacteria, respectively. Using a key oral pathobiont, Fusobacterium nucleatum, as the primary test bed, we demonstrate that the system preserves bacterial viability and supports the integrity of telomerase-immortalized gingival keratinocytes. Compared to conventional models, this system enhanced bacterial invasion, elevated intracellular bacterial loads, and elicited more robust host pro-inflammatory responses, including increased secretion of CXCL10, IL-6, and IL-8. In addition, the model enabled precise evaluation of antibiotic efficacy against intracellular pathogens. Finally, we validate the ability of the asymmetric system to support the proliferation of a more oxygen-sensitive oral pathobiont, Porphyromonas gingivalis. These results underscore the utility of this coculture platform for studying oral microbial pathogenesis and screening therapeutics, offering a physiologically relevant approach to advance oral and systemic health research.

Keywords

Biological Sciences / Microbiology

References

[1] AttarN. Microbial ecology: FISHing in the oral microbiota. Nat. Rev. Microbiol., 2016, 14: 132-133.

[2] SignatB, RoquesC, PouletP, DuffautD. Role of Fusobacterium nucleatum in periodontal health and disease. Curr. Issues Mol. Biol., 2011, 13: 25-36

[3] MeiF, et al. . Porphyromonas gingivalis and its systemic impact: current status. Pathogens, 2020, 9: 1-23.

[4] GaoL, et al. . Oral microbiomes: more and more importance in oral cavity and whole body. Protein Cell, 2018, 9: 488-500.

[5] Le BarsP, et al. . The oral cavity microbiota: between health, oral disease, and cancers of the aerodigestive tract. Can. J. Microbiol., 2017, 63: 475-492.

[6] Fofanova, T. Y. et al. A novel system to culture human intestinal organoids under physiological oxygen content to study microbial-host interaction. PLoS One19, e0300666 (2024).

[7] ZhangJ, et al. . Primary human colonic mucosal barrier crosstalk with super oxygen-sensitive Faecalibacterium prausnitzii in continuous culture. Med, 2021, 2: 74-98.e9.

[8] Jalili-FiroozinezhadS, et al. . A complex human gut microbiome cultured in an anaerobic intestine-on-a-chip. Nat. Biomed. Eng., 2019, 3: 520-531.

[9] MountcastleSE, et al. . A review of co-culture models to study the oral microenvironment and disease. J. Oral. Microbiol., 2020, 12: 1773122.

[10] XuX, et al. . Oral cavity contains distinct niches with dynamic microbial communities. Environ. Microbiol., 2015, 17: 699-710.

[11] AlbenbergL, et al. . Correlation between intraluminal oxygen gradient and radial partitioning of intestinal microbiota. Gastroenterology, 2014, 147: 1055-1063.e8.

[12] MakkarH, et al. . Modeling crevicular fluid flow and host-oral microbiome interactions in a gingival crevice-on-chip. Adv. Healthc. Mater., 2023, 12: 1-13.

[13] AdelfioM, et al. . Three-dimensional humanized model of the periodontal gingival pocket to study oral microbiome. Adv. Sci., 2023, 10: 1-13.

[14] LaiY, MiJ, FengQ. Fusobacterium nucleatum and malignant tumors of the digestive tract: a mechanistic overview. Bioengineering, 2022, 9: 1-12.

[15] Stokowa-SołtysK, WojtkowiakK, JagiełłoK. Fusobacterium nucleatum—friend or foe?. J. Inorg. Biochem., 2021, 224: 111586.

[16] HowKY, SongKP, ChanKG. Porphyromonas gingivalis: an overview of periodontopathic pathogen below the gum line. Front. Microbiol., 2016, 7: 1-14.

[17] ZhangZ, et al. . The role of Porphyromonas gingivalis outer membrane vesicles in periodontal disease and related systemic diseases. Front. Cell. Infect. Microbiol., 2021, 10: 1-12.

[18] YangM, et al. . Targeting Fusobacterium nucleatum through chemical modifications of host-derived transfer RNA fragments. ISME J., 2023, 17: 880-890.

[19] KenneyEB, AshMM. Oxidation reduction potential of developing plaque, periodontal pockets and gingival sulci. J. Periodontol., 1969, 40: 630-633

[20] MettrauxGR, GusbertiFA, GrafH. Oxygen tension (pO2) in untreated human periodontal pockets. J. Periodontol., 1984, 55: 516-521.

[21] BaileyJ, OliveriA, LevinE. Establishment and characterization of a telomerase immortalized human gingival epithelial cell line Catherine. Bone, 2013, 23: 1-7

[22] Koskinen HolmC, QuC. Engineering a 3D in vitro model of human gingival tissue equivalent with genipin/cytochalasin D. Int. J. Mol. Sci., 2022, 23: 7401.

[23] LeonardoTR, et al. . Differential expression and function of bicellular tight junctions in skin and oral wound healing. Int. J. Mol. Sci., 2020, 21: 1-17.

[24] KasperSH, et al. . Colorectal cancer-associated anaerobic bacteria proliferate in tumor spheroids and alter the microenvironment. Sci. Rep., 2020, 10: 1-13.

[25] CasasantaMA, et al. . Fusobacterium nucleatum host-cell binding and invasion induces IL-8 and CXCL1 secretion that drives colorectal cancer cell migration. Sci. Signal., 2020, 13: 1-12.

[26] UdayasuryanB, et al. . Fusobacterium nucleatum induces proliferation and migration in pancreatic cancer cells through host autocrine and paracrine signaling. Sci. Signal., 2022, 15: eabn4948.

[27] LiY, et al. . Intracellular Fusobacterium nucleatum infection attenuates antitumor immunity in esophageal squamous cell carcinoma. Nat. Commun., 2023, 14: 1-16

[28] KoidoS, et al. . The stimulatory effect of Fusobacteria on dendritic cells under aerobic or anaerobic conditions. Sci. Rep., 2022, 12: 1-13.

[29] HanYW, et al. . Interactions between periodontal bacteria and human oral epithelial cells: Fusobacterium nucleatum adheres to and invades epithelial cells. Infect. Immun., 2000, 68: 3140-3146.

[30] HuangR, LiM, GregoryRL. Bacterial interactions in dental biofilm. Virulence, 2011, 2: 435-444.

[31] LiuH, LiuY, FanW, FanB. Fusobacterium nucleatum triggers proinflammatory cell death via Z-DNA binding protein 1 in apical periodontitis. Cell Commun. Signal., 2022, 20: 1-15.

[32] UdayasuryanB, et al. . Fusobacterium nucleatum infection modulates the transcriptome and epigenome of HCT116 colorectal cancer cells in an oxygen-dependent manner. Commun. Biol., 2024, 7: 551.

[33] Robinson, A., Wilde, J. & Allen-Vercoe, E. In Colorectal Neoplasia and the Colorectal Microbiome, Vol 1 (eds Floch, H. M.) Ch. 6 (Elsevier, 2020).

[34] MeyerDH, MintzKP, Fives-TaylorPM. Models of invasion of enteric and periodontal pathogens into epithelial cells: A comparative analysis. Crit. Rev. Oral. Biol. Med., 1997, 8: 389-409.

[35] FinlayBB, FalkowS. Common themes in microbial pathogenicity. Microbiol. Rev., 1989, 53: 210-230.

[36] FinlayBB, CossartP. Exploitation of mammalian host cell functions by bacterial pathogens. Science, 1997, 276: 718-725.

[37] GursoyUK, PöllänenM, KönönenE, UittoV. Biofilm formation enhances the oxygen tolerance and invasiveness of Fusobacterium nucleatum in an oral mucosa culture model. J. Periodontol., 2010, 81: 1084-1091.

[38] MuchovaM, et al. . Fusobacterium nucleatum subspecies differ in biofilm forming ability in vitro. Front. Oral. Heal., 2022, 3: 1-12

[39] GroegerS, ZhouY, RufS, MeyleJ. Pathogenic mechanisms of Fusobacterium nucleatum on oral epithelial cells. Front. Oral. Heal., 2022, 3: 1-11

[40] Dabija-WolterG, et al. . Fusobacterium nucleatum enters normal human oral fibroblasts in vitro. J. Periodontol., 2009, 80: 1174-1183.

[41] NeurathN, KestingM. Cytokines in gingivitis and periodontitis: from pathogenesis to therapeutic targets. Front. Immunol., 2024, 15: 1435054.

[42] WrightHJ, ChappleILC, MatthewsJB, CooperPR. Fusobacterium nucleatum regulation of neutrophil transcription. J. Periodontal Res., 2011, 46: 1-12.

[43] OkadaH, MurakamiS. Cytokine expression in periodontal health and disease. Crit. Rev. Oral. Biol. Med., 1998, 9: 248-266.

[44] KagnoffMF, EckmannL. Epithelial cells as sensors for microbial infection. J. Clin. Invest., 1997, 100: 6-10.

[45] JangJY, et al. . Immunologic characteristics of human gingival fibroblasts in response to oral bacteria. J. Periodontal Res., 2017, 52: 447-457.

[46] SheikhiM, GustafssonA, JarstrandC. Cytokine, elastase and oxygen radical release by Fusobacterium nucleatum-activated leukocytes: a possible pathogenic factor in periodontitis. J. Clin. Periodontol., 2000, 27: 758-762.

[47] BickelM. The role of interleukin-8 in inflammation and mechanisms of regulation. J. Periodontol., 1993, 64: 456-460

[48] BrennanCA, GarrettWS. Fusobacterium nucleatum—symbiont, opportunist and oncobacterium. Nat. Rev. Microbiol., 2019, 17: 156-166.

[49] AnyiamIV, OkelueF. Effects of metronidazole and amoxicillin on selected anaerobes from oral infections. Afr. J. Biol. Med. Res., 2024, 7: 29-46.

[50] Dabija-WolterG, et al. . The effect of metronidazole plus amoxicillin or metronidazole plus penicillin V on periodontal pathogens in an in vitro biofilm model. Clin. Exp. Dent. Res., 2018, 4: 6-12.

[51] FeresM, et al. . Microbiome changes in young periodontitis patients treated with adjunctive metronidazole and amoxicillin. J. Periodontol., 2021, 92: 467-478.

[52] BradshawDJ, MarshPD, WatsonGK, AllisonC. Role of Fusobacterium nucleatum and coaggregation in anaerobe survival in planktonic and biofilm oral microbial communities during aeration. Infect. Immun., 1998, 66: 4729-4732.

[53] DiazPI, ZilmPS, RogersAH. Fusobacterium nucleatum supports the growth of Porphyromonas gingivalis in oxygenated and carbon-dioxide-depleted environments. Microbiology, 2002, 148: 467-472.

[54] XuW, ZhouW, WangH, LiangS. Roles of Porphyromonas gingivalis and its virulence factors in periodontitis. Adv. Protein Chem. Struct. Biol., 2020, 120: 45-84.

[55] BuguenoIM, et al. . Porphyromonas gingivalis bypasses epithelial barrier and modulates fibroblastic inflammatory response in an in vitro 3D spheroid model. Sci. Rep., 2018, 8: 1-13.

[56] LamontRJ, et al. . Porphyromonas gingivalis invasion of gingival epithelial cells. Infect. Immun., 1995, 63: 3878-3885.

[57] JangJY, BaekKJ, ChoiY, JiS. Relatively low invasive capacity of Porphyromonas gingivalis strains into human gingival fibroblasts in vitro. Arch. Oral. Biol., 2017, 83: 265-271.

[58] XuM, et al. . FadA from Fusobacterium nucleatum utilizes both secreted and nonsecreted forms for functional oligomerization for attachment and invasion of host cells. J. Biol. Chem., 2007, 282: 25000-25009.

[59] AbedJ, et al. . Fap2 mediates Fusobacterium nucleatum colorectal adenocarcinoma enrichment by binding to tumor-expressed Gal-GalNAc. Cell Host Microbe, 2016, 20: 215-225.

[60] WelchJLM, et al. . Biogeography of a human oral microbiome at the micron scale. Proc. Natl Acad. Sci. USA, 2016, 113: E791-E800

[61] RobinsonW, et al. . Identification of intracellular bacteria from multiple single-cell RNA-seq platforms using CSI-Microbes. Sci. Adv., 2024, 10: 1-18.

[62] HashimotoK, et al. . The effects of coating culture dishes with collagen on fibroblast cell shape and swirling pattern formation. J. Biol. Phys., 2020, 46: 351-369.

Funding

U.S. Department of Health & Human Services | NIH | National Institute of Dental and Craniofacial Research (NIDCR)(R03DE031329)

National Science Foundation (NSF)(2238972)

RIGHTS & PERMISSIONS

The Author(s)