Pivotal Roles for Ribonucleases inStreptococcus pneumoniaePathogenesis

Abstract

RNases perform indispensable functions in regulating gene expression in many bacterial pathogens by processing and/or degrading RNAs. Despite the pivotal role of RNases in regulating bacterial virulence factors, the functions of RNases have not yet been studied in the major human respiratory pathogen Streptococcus pneumoniae (pneumococcus). Here, we sought to determine the impact of two conserved RNases, the endoribonuclease RNase Y and exoribonuclease polynucleotide phosphorylase (PNPase), on the physiology and virulence of S. pneumoniae serotype 2 strain D39. We report that RNase Y and PNPase are essential for pneumococcal pathogenesis as both deletion mutants showed strong attenuation of virulence in murine models of invasive pneumonia. Genome-wide transcriptomic analysis revealed that nearly 200 mRNA transcripts were significantly up-regulated, whereas the abundance of several pneumococcal sRNAs, including the Ccn (CiaR Controlled Noncoding RNA) sRNAs, were altered in the Δrny mutant relative to the wild-type strain. Additionally, lack of RNase Y resulted in pleiotropic phenotypes that included defects in pneumococcal cell morphology and growth in vitro. In contrast, Δpnp mutants showed no growth defect in vitro, but differentially expressed a total of 40 transcripts including the tryptophan biosynthesis operon genes and numerous 5’-cis-acting regulatory RNAs, a majority of which were previously shown to impact pneumococcal disease progression in mice using the serotype 4 strain TIGR4. Altogether our data suggest that RNase Y exerts a global impact on pneumococcal physiology, while PNPase-mediates virulence phenotypes, likely through sRNA regulation.IMPORTANCEStreptococcus pneumoniae is a notorious human pathogen that adapts to conditions in distinct host tissues and responds to host cell interactions by adjusting gene expression. Ribonucleases (RNases) are key players that modulate gene expression by mediating the turnover of regulatory and protein-coding transcripts. Here, we characterized two highly conserved RNases, RNase Y and PNPase, and evaluated their impact on the S. pneumoniae transcriptome for the first time. We show that PNPase influences the levels of a narrow set of mRNAs, but a large number of regulatory RNAs primarily implicated in virulence control, whereas RNase Y has a more sweeping effect on gene expression, altering levels of transcripts involved in diverse cellular processes including cell division, metabolism, stress response, and virulence. This study further reveals that RNase Y regulates expression of genes governing competence by mediating the turnover of CiaR-controlled-noncoding (Ccn) sRNAs.

A Small Non-Coding RNA Modulates Expression of Pilus-1 Type in Streptococcus pneumoniae

“…Differential cleavage of the 5 -UTR of rrgA could be the result of a concert action between endoand exo-ribonucleases, thus generating either a functional rrgA or an independent srn135. Knowledge of pneumococcal RNases is scarce, but it has been recently demonstrated that the endoribonuclease RNase Y and the exoribonuclease PNPase work in concert to regulate the processing and decay of several regulatory RNAs, in particular, those characterized by the presence of 5 -cis-acting regulatory elements [73]. Likewise, RNase Y has been postulated to play important roles in the processing and maturation of sRNAs, which are generated upon cleavage of larger transcripts [73].…”

“…Knowledge of pneumococcal RNases is scarce, but it has been recently demonstrated that the endoribonuclease RNase Y and the exoribonuclease PNPase work in concert to regulate the processing and decay of several regulatory RNAs, in particular, those characterized by the presence of 5 -cis-acting regulatory elements [73]. Likewise, RNase Y has been postulated to play important roles in the processing and maturation of sRNAs, which are generated upon cleavage of larger transcripts [73]. These RNases are thought to have a global impact on virulence through sRNA regulation, and the modulation of pilus expression could be another example of this function.…”