Identification of a Fusobacterial RNA-binding protein involved in host small RNA-mediated growth inhibition

Abstract

Host-derived small RNAs are emerging as critical regulators in the dynamic interactions between host tissues and the microbiome, with implications for microbial pathogenesis and host defense. Among these, transfer RNA-derived small RNAs (tsRNAs) have garnered attention for their roles in modulating microbial behavior. However, the bacterial factors mediating tsRNA interaction and functionality remain poorly understood. In this study, using RNA affinity pull-down assay in combination with mass spectrometry, we identified a putative membrane-bound protein, annotated as P-type ATPase transporter (PtaT) in Fusobacterium nucleatum (Fn), which binds Fn-targeting tsRNAs in a sequence-specific manner. Through targeted mutagenesis and phenotypic characterization, we showed that in both the Fn type strain and a clinical tumor isolate, deletion of ptaT led to reduced tsRNA intake and enhanced resistance to tsRNA-induced growth inhibition. Global RNA sequencing and label-free Raman spectroscopy revealed the phenotypic differences between Fn wild type and PtaT-deficient mutant, highlighting the functional significance of PtaT in purine and pyrimidine metabolism. Furthermore, AlphaFold 3 prediction provides evidence supporting the specific binding between PtaT and Fn-targeting tsRNA. By uncovering the first RNA-binding protein in Fn implicated in growth modulation through interactions with host-derived small RNAs (sRNAs), our study offers new insights into sRNA-mediated host-pathogen interplay within the context of microbiome-host interactions.

References

[1] ChaplinDD. Overview of the immune response. J. Allergy Clin. Immunol., 2010, 125: S3-S23

[2] ZhengD, LiwinskiT, ElinavE. Interaction between microbiota and immunity in health and disease. Cell Res., 2020, 30: 492-506

[3] BelkaidY, HandTW. Role of the microbiota in immunity and inflammation. Cell, 2014, 157: 121-141

[4] MalardF, DoreJ, GauglerB, MohtyM. Introduction to host microbiome symbiosis in health and disease. Mucosal Immunol., 2021, 14: 547-554

[5] LiM, et al.. Symbiotic gut microbes modulate human metabolic phenotypes. Proc. Natl Acad. Sci. USA, 2008, 105: 2117-2122

[6] Katiyar-AgarwalS, JinH. Role of small RNAs in host-microbe interactions. Annu. Rev. Phytopathol., 2010, 48: 225-246

[7] ChandanK, GuptaM, SarwatM. Role of host and pathogen-derived microRNAs in immune regulation during infectious and inflammatory diseases. Front. Immunol., 2020, 103081

[8] HuangC-Y, WangH, HuP, HambyR, JinH. Small RNAs – Big players in plant-microbe interactions. Cell Host Microbe, 2019, 26: 173-182

[9] Ahmadi BadiS, et al.. Small RNAs in outer membrane vesicles and their function in host-microbe interactions. Front. Microbiol., 2020, 111209

[10] KoeppenK, et al.. A novel mechanism of host-pathogen interaction through sRNA in bacterial outer membrane vesicles. PLOS Pathog., 2016, 12e1005672

[11] ChoiJ-W, KimS-C, HongS-H, LeeH-J. Secretable small RNAs via outer membrane vesicles in periodontal pathogens. J. Dent. Res., 2017, 96: 458-466

[12] ZhangL, LiuJ, HouY. Classification, function, and advances in tsRNA in non-neoplastic diseases. Cell Death Dis., 2023, 14748

[13] KimHK, YeomJ-H, KayMA. Transfer RNA-derived small RNAs: Another layer of gene regulation and novel targets for disease therapeutics. Mol. Ther., 2020, 28: 2340-2357

[14] LiuB, et al.. Deciphering the tRNA-derived small RNAs: origin, development, and future. Cell Death Dis., 2021, 1324

[15] KrishnaS, et al.. Dynamic expression of tRNA-derived small RNAs define cellular states. EMBO Rep., 2019, 20e47789

[16] HeX, et al.. Human tRNA-derived small RNAs modulate host–oral microbial interactions. J. Dent. Res., 2018, 97: 1236-1243

[17] PandeyKK, et al.. Regulatory roles of tRNA-derived RNA fragments in human pathophysiology. Mol. Ther. Nucleic Acids, 2021, 26: 161-173

[18] HanYW. Fusobacterium nucleatum: a commensal-turned pathogen. Curr. Opin. Microbiol., 2015, 23: 141-147

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

[20] WangN, FangJ-Y. Fusobacterium nucleatum, a key pathogenic factor and microbial biomarker for colorectal cancer. Trends Microbiol., 2023, 31: 159-172

[21] HongM, et al.. Fusobacterium nucleatum aggravates rheumatoid arthritis through FadA-containing outer membrane vesicles. Cell Host Microbe, 2023, 31: 798-810.e797

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

[23] LiuP, et al.. Detection of Fusobacterium Nucleatum and fadA adhesin gene in patients with orthodontic gingivitis and non-orthodontic periodontal inflammation. PLoS ONE, 2014, 9e85280

[24] BullmanS, et al.. Analysis of Fusobacterium persistence and antibiotic response in colorectal cancer. Science, 2017, 358: 1443-1448

[25] KosticAD, et al.. Fusobacterium nucleatum potentiates intestinal tumorigenesis and modulates the tumor-immune microenvironment. Cell Host Microbe, 2013, 14: 207-215

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

[27] JazurekM, CiesiolkaA, Starega-RoslanJ, BilinskaK, KrzyzosiakWJ. Identifying proteins that bind to specific RNAs - focus on simple repeat expansion diseases. Nucleic Acids Res., 2016, 44: 9050-9070

[28] PelusoEA, ScheibleM, Ton-ThatH, WuC. Genetic manipulation and virulence assessment of Fusobacterium nucleatum. Curr. Protoc. Microbiol., 2020, 57e104

[29] 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, 13eaba9157

[30] AbranchesJ, ChenY-YM, BurneRA. Galactose metabolism by Streptococcus mutans. Appl. Environ. Microbiol., 2004, 70: 6047-6052

[31] TianJ, et al.. Acquisition of the arginine deiminase system benefits epiparasitic Saccharibacteria and their host bacteria in a mammalian niche environment. Proc. Natl Acad. Sci. USA, 2022, 119e2114909119

[32] JaishankarJ, SrivastavaP. Molecular basis of stationary phase survival and applications. Front. Microbiol., 2017, 82000

[33] VeselovskyVA, et al.. The gene expression profile differs in growth phases of the Bifidobacterium longum culture. Microorganisms, 2022, 101683

[34] BathkeJ, KonzerA, RemesB, McIntoshM, KlugG. Comparative analyses of the variation of the transcriptome and proteome of Rhodobacter sphaeroides throughout growth. BMC Genomics, 2019, 20358

[35] ObaY, et al.. Identifying the wide diversity of extraterrestrial purine and pyrimidine nucleobases in carbonaceous meteorites. Nat. Commun., 2022, 132008

[36] EgliM, ManoharanM. Chemistry, structure and function of approved oligonucleotide therapeutics. Nucleic Acids Res., 2023, 51: 2529-2573

[37] ChengJ-X, XieXS. Vibrational spectroscopic imaging of living systems: an emerging platform for biology and medicine. Science, 2015, 350aaa8870

[38] ButlerHJ, et al.. Using Raman spectroscopy to characterize biological materials. Nat. Protoc., 2016, 11: 664-687

[39] DongP-T, et al.. Polarization-sensitive stimulated Raman scattering imaging resolves amphotericin B orientation in Candida membrane. Sci. Adv., 2021, 7eabd5230

[40] Gardner-LubbeS. Linear discriminant analysis for multiple functional data analysis. J. Appl. Stat., 2021, 48: 1917-1933

[41] Xanthopoulos, P., Pardalos, P. M. & Trafalis, T. B. In: Robust Data Mining 27–33 (Springer New York, 2013).

[42] XuJ, et al.. Artificial intelligence-aided rapid and accurate identification of clinical fungal infections by single-cell Raman spectroscopy. Front. Microbiol., 2023, 141125676

[43] EmberKJI, et al.. Raman spectroscopy and regenerative medicine: a review. npj Regener. Med., 2017, 212

[44] RubinsteinMR, et al.. Fusobacterium nucleatum promotes colorectal cancer by inducing Wnt/catenin modulator Annexin A1. EMBO Rep., 2019, 20e47638

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

[46] Zhou, P., Bibek, G. C. & Wu, C. Development of a conditional plasmid for gene deletion in non-model Fusobacterium nucleatum strains. bioRxivhttps://doi.org/10.1101/2024.09.09.612158 (2024).

[47] AbramsonJ, et al.. Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature, 2024, 30: 493-500

[48] KühlbrandtW. Biology, structure and mechanism of P-type ATPases. Nat. Rev. Mol. Cell Biol., 2004, 5: 282-295

[49] SalustrosN, et al.. Structural basis of ion uptake in copper-transporting P_1B-type ATPases. Nat. Commun., 2022, 135121

[50] TsudaT, ToyoshimaC. Nucleotide recognition by CopA, a Cu⁺-transporting P-type ATPase. EMBO J., 2009, 28: 1782-1791

[51] Dar, D. & Sorek, R. Bacterial noncoding RNAs excised from within protein-coding transcripts. mBio 9: https://doi.org/10.1128/mbio.01730-18 (2018).

[52] LambertM, BenmoussaA, ProvostP. Small non-coding RNAs derived from eukaryotic ribosomal RNA. Non-Coding RNA, 2019, 516

[53] YamamuraS, Imai-SumidaM, TanakaY, DahiyaR. Interaction and cross-talk between non-coding RNAs. Cell. Mol. Life Sci., 2018, 75: 467-484

[54] LiuS, et al.. The host shapes the gut microbiota via fecal microRNA. Cell Host Microbe, 2016, 19: 32-43

[55] LiM, ChenW-D, WangY-D. The roles of the gut microbiota–miRNA interaction in the host pathophysiology. Mol. Med., 2020, 26101

[56] FeinbergEH, HunterCP. Transport of dsRNA into cells by the transmembrane protein SID-1. Science, 2003, 301: 1545-1547

[57] ChenQ, et al.. SIDT1-dependent absorption in the stomach mediates host uptake of dietary and orally administered microRNAs. Cell Res., 2021, 31: 247-258

[58] NguyenTA, et al.. SIDT2 transports extracellular dsRNA into the cytoplasm for innate immune recognition. Immunity, 2017, 47: 498-509.e496

[59] ChanH, et al.. The P-Type ATPase superfamily. J. Mol. Microbiol. Biotechnol., 2010, 19: 5-104

[60] PalmgrenM. P-type ATPases: many more enigmas left to solve. J. Biol. Chem., 2023, 299105352

[61] WangP, et al.. ANT2 functions as a translocon for mitochondrial cross-membrane translocation of RNAs. Cell Res., 2024, 34: 504-521

[62] GCB, ZhouP, WuC. HicA toxin-based counterselection marker for allelic exchange mutations in Fusobacterium nucleatum. Appl. Environ. Microbiol., 2023, 89: e00091-00023

Funding

U.S. Department of Health & Human Services | NIH | National Institute of Dental and Craniofacial Research (NIDCR)(DE030943)

RIGHTS & PERMISSIONS

The Author(s)