Host tRNA-derived small RNAs (tsRNAs) mediate interactions between host and oral microbes

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Host mucosal surfaces are highly specialized and possess a complex array of innate and adaptive immunity. They provide the first line of protection against infectious agents by initiating protective responses to potential pathogens. Furthermore, the symbiotic relationship of the hundreds of microbial species with the host requires a fine-tuned response at the mucosal surface that prevents overgrowth of opportunistic pathogens, but sparing beneficial microbes. While small RNAs (sRNAs) have been regarded as a class of intracellular regulatory elements, emerging studies in plant-pathogen and host-gut microbiome interactions uncover that hosts can exploit sRNAs, such as transfer RNA (tRNA)-derived sRNAs (termed “tsRNAs”), via encapsulation in exosomes, as a new mode of bacterial gene modulation or defense mechanism. Yet, it remains to be elucidated whether such sRNA-mediated inter-kingdom gene modulation or defense mechanism exist in the context of host-oral microbiome interaction. To address this knowledge gap, the present R01 application will focus on host-derived, exosome-borne, salivary tsRNAs, of which the biological functions have remained largely elusive to date. Speficically, we recently demonstrated that human Normal Oral Keratinocyte- Spontaneously Immortalized (NOKSI) cells released two exosome-borne tsRNAs, tsRNA-000794 and tsRNA- 020498, when challenged with Fusobacterium nucleatum (Fn), a key oral commensal and opportunistic periodontal pathogen. Importantly, these two tsRNAs can be readily detected from saliva in healthy human subjects. Intriguingly, both tsRNAs exhibit highly selective, Fn-targeting antimicrobial activity—directly adding synthetic mimics of these two tsRNAs, but not scramble RNA, to bacterial culture inhibits the growth of Fn, but not that of Porphyromonas ginigivalis (Pg), a gram-negative periodontal pathogen or Streptococcus mitis (Sm), a health-associated oral bacterium. In preliminary work, we further took a multi-facet approach to identify a putative RNA-specific membrane transporter as well as candidate intracellular bacterial protein targets of Fn- targeting tsRNAs. Building on our comprehensive preliminary data, the goal of this application is two-fold: (Part I) achieve mechanistic understanding of the cross-kingdom trafficking of host-derived Fn-targeting tsRNAs and their modulating effect on Fn growth during NOKSI-Fn interaction, through exosome tracking and in-depth dissection of the tsRNAs transporter and intracellular targets in Fn; (Part II) expand our work to profile and compare salivary tsRNAs between healthy and periodontitis subjects, with a focus on demonstrating the broad implication of host-generated tsRNAs as a conserved mechanism to achieve host-microbial homeostasis. The realization of this application will not only address a fundamental question by defining tsRNAs as a new class of host defense molecules in maintaining host-microbiome homeostasis via targeted microbial modulation, but also will pave the way for a new therapeutic strategy against oral diseases, considering the already successful trajectory of nucleic acid therapeutics in recent years.