Human tRNA-Derived Small RNAs Modulate Host-Oral Microbial Interactions

Abstract

Coevolution of the human host and its associated microbiota has led to sophisticated interactions to maintain a delicate homeostasis. Emerging evidence suggests that in addition to small molecules, peptides, and proteins, small regulatory noncoding RNAs (sRNAs) might play an important role in cross-domain interactions. In this study, we revealed the presence of diverse host transfer RNA-derived small RNAs (tsRNAs) among human salivary sRNAs. We selected 2 tsRNAs (tsRNA-000794 and tsRNA-020498) for further study based on their high sequence similarity to specific tRNAs from a group of Gram-negative oral bacteria, including Fusobacterium nucleatum, a key oral commensal and opportunistic pathogen. We showed that the presence of F. nucleatum triggers exosome-mediated release of tsRNA-000794 and tsRNA-020498 by human normal oral keratinocyte cells. Furthermore, both tsRNA candidates exerted a growth inhibition effect on F. nucleatum, likely through interference with bacterial protein biosynthesis, but did not affect the growth of Streptococcus mitis, a health-associated oral Gram-positive bacterium whose genome does not carry sequences bearing high similarity to either tsRNA. Our data provide the first line of evidence for the modulatory role of host-derived tsRNAs in the microbial-host interaction.

Keywords: antimicrobials; cross-domain interactions; microbial-host interaction; oral microbiome; sRNAs; tsRNAs.

Figure 1.

Salivary host-derived tsRNAs. (A) Relative abundance of host-derived tsRNAs in total sRNA reads among 4 subjects (SK3S_I1, MY2S_I2, DA1S_I1, and CD4S_I1). (B) A subset of host-derived tsRNAs have substantial matches to bacteria, particularly Gram-negative oral bacterial tRNA sequences. (C) Alignment of tsRNA-000794 and tsRNA-020498 with partial sequences of specific tRNAs from oral Gram-negative bacterial species. Letters in red indicate the nucleotides conserved between or among tsRNA and partial tRNA sequences from selected oral bacterial species. sRNA, small regulatory noncoding RNA; tRNA, transfer RNA; tsRNA, tRNA-derived small sRNA.

Figure 2.

tsRNA-000794 and tsRNA-020498 induced growth inhibition in Fusobacterium nucleatum. (A) Different concentrations of tsRNA-000794, tsRNA-020498, tsRNA-016926, tsRNA-018570, piRNA-016792, and piRNA-006465 were added to F. nucleatum cultures. Cultures were incubated overnight at 37 ºC under anaerobic condition before OD₆₀₀ was measured. The growth inhibition is expressed as percentage of absorbance to that of the negative control (no addition of tRF). (B) tsRNA-000794 and tsRNA-020498 were pretreated with RNase A before being added to the F. nucleatum culture, and impact on bacterial growth was similarly monitored. tsRNA-000794 (100 µM), tsRNA-020498 (100 µM), piRNA-016792 (100 μM), or scrambled control RNA (100 μM) was added to F. nucleatum (C) and Streptococcus mitis (D), and the growth curves were measured. Assays were performed in triplicates, and mean ± SEM values are shown. ns, P > 0.05; *P < 0.05; **P < 0.01; ****P < 0.0001. All the columns without “ns” or stars are nonsignificant according to statistical tests. piRNA, PIWI-interacting RNA; tsRNA, transfer RNA–derived small sRNA; tRF, tRNA-derived fragment.

Figure 3.

tsRNA-000794 and tsRNA-020498 affect protein synthesis in Fusobacterium nucleatum. The effect of the addition of tsRNA-000794 and tsRNA-020498 on protein synthesis in F. nucleatum and Streptococcus mitis (A and B) and the effect of kanamycin on protein synthesis in F. nucleatum (C). The left panels show representative fluorescence images of differentially treated cultures. The right panel shows the quantification of fluorescence intensity per area unit covered by bacterial cells. au, arbitrary units. Data are expressed as mean ± SEM. *P < 0.05; **P < 0.01; ****P < 0.0001. All the columns with no “ns” or stars are nonsignificant according to statistical tests. tsRNA, transfer RNA–derived small sRNA.

Figure 4.

Fusobacterium nucleatum specifically induces the secretion of tsRNA-000794 and tsRNA-020498 from human oral keratinocytes. Human oral keratinocyte NOKSI cells were cocultured with F. nucleatum or Streptococcus mitis at a multiplicity of infection of 100. (A) The level of exosome-associated sRNAs in NOKSI medium when cells were challenged with F. nucleatum was measured by ddPCR. The fold changes were calculated per the comparison with nonchallenged keratinocytes. (B) The levels of exosome-associated sRNAs in NOKSI medium when cells were challenged with S. mitis were measured by ddPCR. The fold changes were calculated per the comparison with nonchallenged keratinocytes. Assays were performed in triplicates, and mean ± SEM values are shown. *P < 0.05. All the columns with no “ns” or stars are nonsignificant according to statistical tests. ddPCR, droplet digital polymerase chain reaction; NOKSI, normal oral keratinocyte; sRNA, small regulatory noncoding RNA; tsRNA, transfer RNA–derived small sRNA.