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1 S1-Domain RNA-Binding Protein (Cvfd) Is a New Post-Transcriptional Regulator That bioRxiv preprint doi: https://doi.org/10.1101/2020.06.23.167635; this version posted June 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 S1-Domain RNA-Binding Protein (CvfD) Is a New Post-Transcriptional Regulator That 2 Mediates Cold Sensitivity, Phosphate Transport, and Virulence in Streptococcus 3 pneumoniae D39 4 5 Dhriti Sinha1,2†, Jiaqi J. Zheng1†, Ho-Ching Tiffany Tsui1, John D. Richardson1, Nicholas R. De 6 Lay2,3# and Malcolm E. Winkler1# 7 8 1 Department of Biology, Indiana University Bloomington, Jordan Hall; 1001 East Third Street; 9 Bloomington, Indiana 47405 10 2Department of Microbiology and Molecular Genetics, McGovern Medical School, University of 11 Texas Health Science Center, Houston, TX 77030, USA 12 3MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences, University of 13 Texas Health Science Center, Houston, TX 77030, USA 14 15 Running Title: Pleiotropic phenotypes of pneumococcal ΔcvfD mutants 16 17 Keywords: RNA-binding protein; post-transcriptional regulation; phosphate uptake; cold- 18 sensitivity 19 20 # Correspondence should be sent to Nicholas R. De Lay (Tel: +1 (713) 500-6293; Email: 21 [email protected]) and Malcolm E. Winkler (Tel: +1 (812) 856-1318; E-mail: 22 [email protected]) 23 24 † Dhriti Sinha and Jiaqi J. Zheng contributed equally to this work, and author order was 25 determined both alphabetically and in order of increasing seniority. 26 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.23.167635; this version posted June 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 27 28 ABSTRACT 29 Post-transcriptional gene regulation often involves RNA-binding proteins that modulate mRNA 30 translation and/or stability either directly through protein-RNA interactions or indirectly by 31 facilitating the annealing of small regulatory RNAs (sRNAs). The human pathogen Streptococcus 32 pneumoniae D39 (pneumococcus) does not encode homologs to RNA-binding proteins known to 33 be involved in promoting sRNA stability and function, such as Hfq or ProQ, even though it contains 34 genes for at least 112 sRNAs. However, the pneumococcal genome contains genes for other 35 RNA-binding proteins, including at least six S1-domain proteins; ribosomal protein S1 (rpsA), 36 polynucleotide phosphorylase (pnpA), RNase R (rnr), and three proteins of unknown functions. 37 Here, we characterize the function of one of these conserved, yet uncharacterized S1-domain 38 proteins, SPD_1366, which we have renamed CvfD (Conserved virulence factor D), since loss of 39 this protein results in an attenuation of virulence in a murine pneumonia model. We report that 40 deletion of cvfD impacts expression of 144 transcripts including the pst1 operon, encoding the 41 phosphate transport system 1 in S. pneumoniae. We further show that CvfD post-transcriptionally 42 regulates the PhoU2 master regulator of the pneumococcal dual phosphate transport system by 43 binding phoU2 mRNA and impacting PhoU2 translation. CvfD not only controls expression of 44 phosphate transporter genes, but also functions as a pleiotropic regulator that impacts cold 45 sensitivity and the expression of sRNAs and genes involved in diverse cellular functions, including 46 manganese uptake and zinc efflux. Together, our data show that CvfD exerts a broad impact on 47 pneumococcal physiology and virulence, partly by post-transcriptional gene regulation. 48 49 SIGNIFICANCE 50 Recent advances have led to the identification of numerous sRNAs in the major human 51 respiratory pathogen, S. pneumoniae. However, little is known about the functions of most sRNAs 52 or RNA-binding proteins involved in RNA biology in pneumococcus. In this paper, we characterize 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.23.167635; this version posted June 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 53 the phenotypes and one target of the S1-domain RNA-binding protein CvfD, a homolog of 54 “general-stress protein 13” identified, but not extensively characterized in other Firmicute species. 55 Pneumococcal CvfD is a broadly pleiotropic regulator, whose absence results in misregulation of 56 divalent cation homeostasis, reduced translation of the PhoU2 master regulator of phosphate 57 uptake, altered metabolism and sRNA amounts, cold sensitivity, and attenuation of virulence. 58 These findings underscore the critical roles of RNA biology in pneumococcal physiology and 59 virulence. 60 61 INTRODUCTION 62 Over the past decade there has been increasing evidence regarding the importance of post- 63 transcriptional gene regulation in modulating the physiology and virulence of Gram-positive 64 bacterial pathogens (1–3). Post-transcriptional regulation of gene expression is often carried out 65 by RNA-binding proteins that frequently act in tandem with small regulatory RNAs (sRNAs) (4). 66 Some RNA-binding proteins bind mRNAs altering their transcription, translation, or stability (5, 6). 67 In many cases, the activity of these RNA-binding proteins is modulated by sRNAs, which upon 68 expression can act as antagonists, blocking protein binding to target mRNAs (7). For example, in 69 the Gram-positive bacterial pathogens Enterococcus faecalis and Listeria monocytogenes, the 70 two-component system (TCS) response regulator EutV upon phosphorylation binds to a dual 71 hairpin in the 5’ untranslated region (5’-UTR) of the polycistronic mRNA encoding ethanolamine 72 utilization genes. This binding stabilizes an anti-terminator that promotes transcription elongation 73 (8, 9). EutV function is antagonized by the EutX/Rli55 sRNAs in the presence of ethanolamine 74 and the absence of B12, blocking it from binding the eut mRNA. As a consequence, a terminator 75 forms in the 5’-UTR of the eut mRNA, resulting in transcriptional attenuation (8, 9). In Bacillus 76 subtilis and Escherichia coli, the RNA-binding protein CsrA binds to mRNAs causing translational 77 repression (7, 10, 11). In E. coli, CsrA is sequestered by at least two sRNAs, CsrB and CsrC (12, 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.23.167635; this version posted June 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 78 13), whereas in B. subtilis CsrA is antagonized by the protein FliW, which inhibits its RNA-binding 79 activity through binding to an allosteric site on CsrA (14). 80 Other RNA-binding proteins work in concert with sRNAs that act by base pairing with their 81 respective mRNA targets, altering their transcription (15), translation (16), or stability (17). sRNAs 82 are a well-studied class of post-transcriptional regulators that have been implicated in controlling 83 a wide variety of physiological responses in bacteria ranging from stress responses to virulence 84 gene expression (4, 18, 19). One bacterial RNA-binding protein that has gained much attention 85 is the RNA chaperone protein Hfq (20–23). Hfq stabilizes sRNAs by binding and protecting them 86 from ribonucleolytic cleavage (24), but also facilitates annealing between sRNAs and their target 87 mRNAs (25–28). In Gram-negative pathogens including Salmonella enterica (29, 30), Yersinia 88 pestis (31, 32), and Vibrio cholerae (33, 34), deletion of hfq results in pleiotropic phenotypes 89 affecting growth, the ability to cope with environmental stresses, and virulence properties. In the 90 Gram-positive pathogen Clostridium difficile depletion of hfq induces multiple phenotypic changes 91 including altered growth, cell morphology, stress response, sRNA abundance, and biofilm 92 formation (35). Virulence phenotypes have also been reported for an hfq deletion mutant in 93 Staphylococcus aureus (36, 37) and Listeria monocytogenes (38, 39); however, in contrast to 94 Gram-negative pathogens, the role of Hfq in RNA regulation in Gram-positive pathogens is far 95 less established (40–42). Moreover, important Gram-positive pathogens, such as Streptococci 96 and Mycobacteria, lack a recognizable Hfq homolog. 97 S. pneumoniae is a Gram-positive, low GC-content aerotolerant anaerobe that exclusively 98 resides within the human respiratory tract as a commensal bacterium, but can become an 99 opportunistic pathogen (43). S. pneumoniae is the leading cause of bacterial pneumonia and 100 other serious infections, including otitis media, sinusitis, meningitis, and septicemia, and over 1 101 million people succumb to pneumococcal infections annually worldwide (44, 45). Prior studies 102 have reported the presence of over one hundred sRNAs both in the serotype 2 (D39) and serotype 103 4 (TIGR4) strains of S. pneumoniae, using a combination of computational predictions, high- 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.23.167635; this version posted June 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 104 throughput RNA sequencing, and northern blot analyses (46–51). Interestingly, a recent study by 105 Zheng et al. (52) identified the KH-RNA-binding domain proteins, KhpA and KhpB, as post- 106 transcriptional regulators of the cell division protein FtsA in S. pneumoniae; however, the 107 mechanism underlying this regulation remains unresolved. More recently, hundreds of potential 108 RNA-protein complexes were identified in the TIGR4 strain of S. pneumoniae using a Grad-seq 109 based approach, and further analysis of those interactions uncovered a new function for the 110 exonuclease Cbf1 in stabilizing certain specific pneumococcal sRNAs by trimming their 3' end, 111 which presumably removes a binding site for other RNases (53). There has been no other 112 reported evidence for any RNA-binding protein that may function similar to Hfq in S.
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