S1-Domain RNA-Binding Protein (CvfD) Is a New Post-Transcriptional Regulator That Mediates Cold Sensitivity, Phosphate Transport, and Virulence in Streptococcus pneumoniae D39

Abstract

Post-transcriptional gene regulation often involves RNA-binding proteins that modulate mRNA translation and/or stability either directly through protein-RNA interactions or indirectly by facilitating the annealing of small regulatory RNAs (sRNAs). The human pathogen Streptococcus pneumoniae D39 (pneumococcus) does not encode homologs to RNA-binding proteins known to be involved in promoting sRNA stability and function, such as Hfq or ProQ, even though it contains genes for at least 112 sRNAs. However, the pneumococcal genome contains genes for other RNA-binding proteins, including at least six S1-domain proteins; ribosomal protein S1 (rpsA), polynucleotide phosphorylase (pnpA), RNase R (rnr), and three proteins of unknown functions. Here, we characterize the function of one of these conserved, yet uncharacterized S1-domain proteins, SPD_1366, which we have renamed CvfD (Conserved virulence factor D), since loss of this protein results in an attenuation of virulence in a murine pneumonia model. We report that deletion of cvfD impacts expression of 144 transcripts including the pst1 operon, encoding the phosphate transport system 1 in S. pneumoniae. We further show that CvfD post-transcriptionally regulates the PhoU2 master regulator of the pneumococcal dual phosphate transport system by binding phoU2 mRNA and impacting PhoU2 translation. CvfD not only controls expression of phosphate transporter genes, but also functions as a pleiotropic regulator that impacts cold sensitivity and the expression of sRNAs and genes involved in diverse cellular functions, including manganese uptake and zinc efflux. Together, our data show that CvfD exerts a broad impact on pneumococcal physiology and virulence, partly by post-transcriptional gene regulation. SIGNIFICANCERecent advances have led to the identification of numerous sRNAs in the major human respiratory pathogen, S. pneumoniae. However, little is known about the functions of most sRNAs or RNA-binding proteins involved in RNA biology in pneumococcus. In this paper, we characterize the phenotypes and one target of the S1-domain RNA-binding protein CvfD, a homolog of "general-stress protein 13" identified, but not extensively characterized in other Firmicute species. Pneumococcal CvfD is a broadly pleiotropic regulator, whose absence results in misregulation of divalent cation homeostasis, reduced translation of the PhoU2 master regulator of phosphate uptake, altered metabolism and sRNA amounts, cold sensitivity, and attenuation of virulence. These findings underscore the critical roles of RNA biology in pneumococcal physiology and virulence.