Genome-Wide Analysis of Small RNAs Expressed by Yersinia pestis Identifies a Regulator of the Yop-Ysc Type III Secretion System

Abstract

bSmall noncoding RNA (sRNA) molecules are integral components of the regulatory machinery for many bacterial species and are known to posttranscriptionally regulate metabolic and stress-response pathways, quorum sensing, virulence factors, and more. The Yop-Ysc type III secretion system (T3SS) is a critical virulence component for the pathogenic Yersinia species, and the regulation of this system is tightly controlled at each step from transcription to translocation of effectors into host cells. The contribution of sRNAs to the regulation of the T3SS in Yersinia has been largely unstudied, however. Previously, our lab identified a role for the sRNA chaperone protein Hfq in the regulation of components of the T3SS in the gastrointestinal pathogen Yersinia pseudotuberculosis. Here we present data demonstrating a similar requirement for Hfq in the closely related species Yersinia pestis. Through deep sequencing analysis of the Y. pestis sRNA-ome, we found 63 previously unidentified putative sRNAs in this species. We identified a Yersinia-specific sRNA, Ysr141, carried by the T3SS plasmid pCD1 that is required for the production of multiple T3SS proteins. In addition, we show that Ysr141 targets an untranslated region upstream of yopJ to posttranscriptionally activate the synthesis of the YopJ protein. Furthermore, Ysr141 may be an unstable and/or processed sRNA, which could contribute to its function in the regulation of the T3SS. The discovery of an sRNA that influences the synthesis of the T3SS adds an additional layer of regulation to this tightly controlled virulence determinant of Y. pestis.

Functional and Structural Analysis of a Highly-Expressed Yersinia pestis Small RNA following Infection of Cultured Macrophages

“…Overall, 68% of the reads from the extracellular and 11% from the intracellular pathogen fractions mapped to the reference genome. We compared the 421 sRNA predictions in RefSeq, Rfam and three previous RNA sequencing studies[10, 13, 14] using coverage plots (S1 Fig). We merged 96 duplicate predictions like sR007 and ysr151 that match the highly expressed RNaseP (Table 1).…”

“…We also discovered another 37 novel sRNAs on the chromosome, bringing the total of sRNAs analyzed in our study to 180 (143 previously-identified and 37 novel sRNAs, S2 Table). Following the ysr (for Yersinia small RNA) numbering system in previous studies[13, 23], we designated these novel sRNAs as Ysr252 to Ysr288.…”

“…Differential sRNA expression in Y . pestis has been observed in response to temperature shift from 26°C to 37°C, which simulates host infection[10, 13], and in the lungs of infected mice. [14] The deletion of one sRNA candidate, ysr35, resulted in the attenuation of Y .…”

“…We retrieved 421 sRNA predictions from RefSeq, the Rfam database version 12.0[12] and three previous RNA sequencing studies. [10, 13, 14] For the two studies that reported sRNA coordinates for different Y . pestis strains, KIM6[10] and 201[14], we used megablast to find matching positions on the CO92 RefSeq genome.…”

RNA-based mechanisms of virulence control in Enterobacteriaceae

“…5,117 Expression of Ysr141 stimulates the production of multiple T3SS/Yop proteins (e.g. YopE, YscF, YopK) as well as their main activator LcrF, and seems to control yopJ mRNA translation through basepairing with its 5 0 -UTR.…”