Targeting Fusobacterium nucleatum through Chemical Modifications of Host-Derived Transfer RNA Fragments

Abstract

ABSTRACTHost mucosal barriers possess an arsenal of defense molecules to maintain mucosal health. In addition to well-established defense molecules such as antimicrobial peptides and immunoglobulins, a subset of extracellular host-derived small RNAs (sRNAs) also exhibits antimicrobial functions in a cross-kingdom fashion. We recently uncovered the sRNA-mediated crosstalk between human normal oral keratinocytes and Fusobacterium nucleatum (Fn), an opportunistic oral pathobiont with increasing implications in extra-oral diseases. Notably, when challenged with Fn, oral keratinocytes released Fn-targeting tRNA-derived sRNAs (tsRNAs), an emerging class of noncoding sRNAs with diverse functions in gene regulation. Additionally, synthetic mimics of the Fn-targeting tsRNAs exhibited highly selective antimicrobial activity against Fn. However, excess synthetic tsRNAs (in the micromolar range) were required to achieve growth inhibition, which limits their potential as antimicrobials. Here, we chemically modify nucleotides of the anti-Fn tsRNAs, termed MOD-tsRNAs, and demonstrate their species- and sequence-specific inhibition in the nanomolar range in various Fn type strains and clinical tumor isolates. In contrast, the same MOD-tsRNAs do not inhibit two representative oral bacteria, Porphoromonas gingivalis (Pg) and Streptococcus mitis (Sm). Additionally, MOD-tsRNAs are internalized by different Fn strains while exhibiting minimal uptake by Pg and Sm. Further RNA sequencing and affinity pull-down assays implicate MOD-tsRNAs as potential ribosome-targeting antimicrobials against Fn. Taken together, our work provides a framework to target opportunistic pathobionts through co-opting host-derived extracellular tsRNAs, whose potential applications may have been limited by their intrinsic instability as well as our limited understanding of the inhibition mechanism.