Use of Genetically Encoded Fluorescent Aptamers for Visualization of Mycobacterium tuberculosis Small RNA MTS1338 in Inf
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HEMISTRY, BIOPHYSICS, AND MOLECULAR BIOLOGY
Use of Genetically Encoded Fluorescent Aptamers for Visualization of Mycobacterium tuberculosis Small RNA MTS1338 in Infected Macrophages O. S. Bychenkoa,*, Yu. V. Skvortsovaa, A. S. Grigorova, and T. L. Azhikinaa Presented by Academician S. A. Luk’yanov Received March 19, 2020; revised April 16, 2020; accepted April 16, 2020
Abstract—The possibility to visualize small bacterial RNAs inside macrophages infected with Mycobacterium tuberculosis was demonstrated for the first time. A macrophage cell line was infected with the M. tuberculosis strain expressing small noncoding mycobacterial RNA MTS1338 fused with an RNA aptamer, which could bind a fluorophore and trigger its fluorescence. As a result, treatment of the infected macrophages with the DFHBI-1T fluorophore allowed fluorescence-based detection of the aptamer-labeled MTS1338 both in mycobacteria and in the host cell cytoplasm. This system can significantly aid in revealing the role of small M. tuberculosis RNAs in the pathogenesis of tuberculosis through identification of their secretion routes and eukaryotic targets and elucidation of the associated molecular pathways. Keywords: Mycobacterium tuberculosis, small noncoding RNA, MTS1338, Broccoli aptamer, infection DOI: 10.1134/S1607672920040055
Small noncoding bacterial RNAs modulate a wide range of physiological responses and regulate the key life stages in a number of pathogens [1, 2]. Currently, the idea that small RNAs of intracellular pathogenic bacteria can not only adapt their own transcriptome to changing conditions but also interact with the transcriptome of the infected organism, thereby interfering with antibacterial defense processes, has been discussed. The identification and study of the molecular chains of adaptation of pathogenic bacteria to the host’s immune defense during persistence in macrophages is an important scientific problem. In this work, we studied the small noncoding RNA MTS1338 of Mycobacterium tuberculosis, which is specific only for the Mycobacterium tuberculosis complex. Its in vivo expression level is extremely high and is approximately 10–1 relative to the 16S rRNA expression level [3]. It was previously shown that MTS1338 makes a significant contribution to the interaction of M. tuberculosis with macrophages, adapting the bacterial transcriptom to an aggressive intramacrophagous environment. The changes in the bacterial metabolism caused by overexpression of this small RNA lead to a modulation of the immune response [4]. a Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia *e-mail: [email protected]
There are examples of how noncoding bacterial RNAs of intracellular pathogens can be secreted into the cytoplasm of infected cells and modulate the immune response during infection [5, 6]. It is believed that small RNA MTS1338 of M. tuberculosis can also be secreted and affect the protective cells of the immune system. To study this process, a system for visualizing small bacte
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