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Mycobacterium tuberculosis infection drives mitochondria-biased dysregulation of host tRNA-derived fragments

Monika M. Looney1#,2, Yin Lu2, Grant Butschek3, Anne Hamacher-Brady,3 Marc K. Halushka2*, Petros C. Karakousis1*

*Co-senior authors

#Corresponding author: Monika M. Looney, mlooney2@jhmi.edu

1 Department of Medicine, Division of Infectious Disease, Johns Hopkins University School of Medicine, 

Baltimore, MD 21287 USA

2 Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD 21205 USA

3 Department of Molecular Microbiology and Immunology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD 21205 USA

The ability of Mycobacterium tuberculosis (Mtb) to suppress and evade host immune responses is directly related to its success as a human pathogen. Many critical host defense pathways targeted by Mtb are controlled by small non-coding regulatory RNAs, such as microRNAs (miRNAs). Emerging evidence suggests that a novel class of small non-coding RNAs, tRNA-derived fragments (tRFs), may regulate such pathways similarly to miRNAs. tRFs have been shown to be dysregulated in various disease conditions, but have not yet been assessed in the context of bacterial infections. Here, we develop and apply a novel tool for small RNA sequencing analysis, tRFcluster, and a combination of original and publicly available RNA sequencing datasets, to analyze dysregulation of tRFs in host cells infected with Mtb and other intracellular bacterial pathogens. We find that tRFs are dysregulated in a pathogen-specific manner. Specifically, tRFs show broad dysregulation, and primarily upregulation, in infection with Mtb and Listeria monocytogenes, but not in infection with BCG, Salmonella typhimurium, or Yersinia pseudotuberculosis. Additionally, Mtb uniquely overproduces tRFs from tRNAs derived from the host mitochondrial genome (mtRFs). We also find that in Mtb infection, the RNase responsible for cleaving intact tRNAs into tRFs, Angiogenin, is robustly recruited to the mitochondria in infected host macrophages. Finally, we observe a similar pattern on mitochondrial recruitment of an apoptosis suppressor, XIAP, in Mtb-infected cells. These data reveal a novel phenomenon which may suggest tRF involvement in the regulation of host mitochondrial responses to infection with Mtb.