Identification of P-type ATPase as a bacterial transporter for host-derived small RNA

Abstract

ABSTRACTHost-derived small RNAs represent a finely tuned host response to maintain the host-microbial homeostasis. Among these, an emerging class known as transfer RNA-derived small RNA (tsRNA) has been implicated in modulating microbial-host interaction. Our previous study showed that when challenged with an oral pathobiont,Fusobacterium nucleatum (Fn), an immortalized human oral keratinocyte cell line releases certainFn-targeting tsRNAs that selectively inhibit the growth ofFnvia their ribosome-targeting function. We also revealed the sequence- and energy-dependent uptake of tsRNAs byFn. However, the mechanism underlying the tsRNA uptake at the molecular level remains elusive. In this study, using RNA affinity pull-down assay in combination with Mass Spectrometry, we identified a putative P-type ATPase transporter (PtaT) inFn, which bindsFn-targeting tsRNAs in a sequence-specific manner. AlphaFold 3 simulation provides further evidence supporting the specific binding between PtaT and tsRNA compared to the scrambled control and the DNA counterpart. Through targeted mutagenesis and phenotypic characterization, we demonstrated the important role of PtaT in the uptake and antimicrobial capacity of tsRNAs againstFnin both ATCC 23726 type strain and a clinical tumor isolate (FnCTI). Furthermore, global RNA sequencing and label-free Raman spectroscopy revealed the phenotypic differences betweenFnwild type and PtaT-deficient mutant, highlighting the functional significance of PtaT in purine and pyrimidine metabolism. Collectively, our work identifies a bacterial transporter for tsRNAs and provides critical information for a fundamental understanding of how the host-generated tsRNAs specifically interact with its targeted bacteria at the molecular level.