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.

RNA-Sequencing Analyses of Small Bacterial RNAs and their Emergence as Virulence Factors in Host-Pathogen Interactions

“…Similar findings were obtained in a yersiniosis mouse model infected with Y. pseudotuberculosis and in which multiple sRNAs were mutated [85]. More than 180 sRNAs, including 37 new ones, were discovered upon deep RNA-seq of the Y. pestis transcriptome and sequence analysis of sRNA candidates [84]. A more prominent role for sRNAs in virulence is to be expected.…”

“…In bacterial species, such as Yersinia pestis (the infectious agent of bubonic plague), the T3SS is a significant virulence factor that is required to deliver mostly bacterial protein effectors to the host [83]. Functional studies on YsR40 sRNA (362 nt in length) have shown that its knock-down reduces infection efficiency in host cell culture and bacterial cell growth in response to stress [84]. Similar findings were obtained in a yersiniosis mouse model infected with Y. pseudotuberculosis and in which multiple sRNAs were mutated [85].…”

Expression of small RNAs of Bordetella pertussis colonizing murine tracheas

“…Most sRNAs post‐transcriptionally upregulate or downregulate downstream gene expression by affecting the stability and translational efficiency of target messenger RNAs (mRNAs) through base pairing with them 12,13 . A wide variety of physiological processes, including metabolism, stress responses, and the expression of virulence genes, are regulated by sRNAs 14–18 . In B. pertussis , many types of sRNAs have been identified or predicted by an in silico analysis and RNA‐seq on the bacteria grown in vitro 9,11,14 .…”

Discovering RNA-Based Regulatory Systems for Yersinia Virulence

“…Recent work has also identified multiple sRNAs of Y. pestis , which are upregulated within macrophages. Among them is Ysr170, which was shown to be important for intracellular replication (Li et al, 2016 ). Much more is known about conserved sRNAs and their impact on pathogenicity of Yersinia .…”