title: Functional and Structural Analysis of a Highly-Expressed Yersinia pestis Small RNA following Infection of Cultured Macrophages.
abstract: Non-coding small RNAs (sRNAs) are found in practically all bacterial genomes and play important roles in regulating gene expression to impact bacterial metabolism, growth, and virulence. We performed transcriptomics analysis to identify sRNAs that are differentially expressed in Yersinia pestis that invaded the human macrophage cell line THP-1, compared to pathogens that remained extracellular in the presence of host. Using ultra high-throughput sequencing, we identified 37 novel and 143 previously known sRNAs in Y. pestis. In particular, the sRNA Ysr170 was highly expressed in intracellular Yersinia and exhibited a log2 fold change ~3.6 higher levels compared to extracellular bacteria. We found that knock-down of Ysr170 expression attenuated infection efficiency in cell culture and growth rate in response to different stressors. In addition, we applied selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) analysis to determine the secondary structure of Ysr170 and observed structural changes resulting from interactions with the aminoglycoside antibiotic gentamycin and the RNA chaperone Hfq. Interestingly, gentamicin stabilized helix 4 of Ysr170, which structurally resembles the native gentamicin 16S ribosomal binding site. Finally, we modeled the tertiary structure of Ysr170 binding to gentamycin using RNA motif modeling. Integration of these experimental and structural methods can provide further insight into the design of small molecules that can inhibit function of sRNAs required for pathogen virulence.
authors.affiliation: Bioscience Division, Los Alamos National Laboratory, Los Alamos, New Mexico, United States of America.
authors.forename: Nan
authors.initials: N
authors.lastname: Li
authors.affiliation: Theoretical Division, Los Alamos National Laboratory, Los Alamos, New Mexico, United States of America.
authors.forename: Scott P
authors.initials: SP
authors.lastname: Hennelly
authors.affiliation: Bioscience Division, Los Alamos National Laboratory, Los Alamos, New Mexico, United States of America.
authors.forename: Chris J
authors.initials: CJ
authors.lastname: Stubben
authors.affiliation: Bioscience Division, Los Alamos National Laboratory, Los Alamos, New Mexico, United States of America.
authors.forename: Sofiya
authors.initials: S
authors.lastname: Micheva-Viteva
authors.affiliation: Bioscience Division, Los Alamos National Laboratory, Los Alamos, New Mexico, United States of America.
authors.forename: Bin
authors.initials: B
authors.lastname: Hu
authors.affiliation: Bioscience Division, Los Alamos National Laboratory, Los Alamos, New Mexico, United States of America.
authors.forename: Yulin
authors.initials: Y
authors.lastname: Shou
authors.affiliation: Bioscience Division, Los Alamos National Laboratory, Los Alamos, New Mexico, United States of America.
authors.forename: Momchilo
authors.initials: M
authors.lastname: Vuyisich
authors.affiliation: Theoretical Division, Los Alamos National Laboratory, Los Alamos, New Mexico, United States of America.
authors.forename: Chang-Shung
authors.initials: CS
authors.lastname: Tung
authors.affiliation: Bioscience Division, Los Alamos National Laboratory, Los Alamos, New Mexico, United States of America.
authors.forename: Patrick S
authors.initials: PS
authors.lastname: Chain
authors.affiliation: Theoretical Division, Los Alamos National Laboratory, Los Alamos, New Mexico, United States of America.
authors.forename: Karissa Y
authors.initials: KY
authors.lastname: Sanbonmatsu
authors.affiliation: Bioscience Division, Los Alamos National Laboratory, Los Alamos, New Mexico, United States of America.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Hong-Geller
mesh_terms: D001483:Base Sequence; D020869:Gene Expression Profiling; D015964:Gene Expression Regulation, Bacterial; D016680:Genome, Bacterial; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008264:Macrophages / Q000378:metabolism* / Q000382:microbiology / Q000473:pathology; D009690:Nucleic Acid Conformation; D010930:Plague / Q000235:genetics / Q000382:microbiology*; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics; D058727:RNA, Small Untranslated / Q000737:chemistry* / Q000235:genetics*; D014774:Virulence / Q000235:genetics*; D015010:Yersinia pestis / Q000235:genetics* / Q000302:isolation & purification / Q000472:pathogenicity
chemical_list: D012329:RNA, Bacterial; D058727:RNA, Small Untranslated