title: Rational manipulation of mRNA folding free energy allows rheostat control of pneumolysin production by Streptococcus pneumoniae.
abstract: The contribution of specific factors to bacterial virulence is generally investigated through creation of genetic "knockouts" that are then compared to wild-type strains or complemented mutants. This paradigm is useful to understand the effect of presence vs. absence of a specific gene product but cannot account for concentration-dependent effects, such as may occur with some bacterial toxins. In order to assess threshold and dose-response effects of virulence factors, robust systems for tunable expression are required. Recent evidence suggests that the folding free energy (ΔG) of the 5' end of mRNA transcripts can have a significant effect on translation efficiency and overall protein abundance. Here we demonstrate that rational alteration of 5' mRNA folding free energy by introduction of synonymous mutations allows for predictable changes in pneumolysin (PLY) expression by Streptococcus pneumoniae without the need for chemical inducers or heterologous promoters. We created a panel of isogenic S. pneumoniae strains, differing only in synonymous (silent) mutations at the 5' end of the PLY mRNA that are predicted to alter ΔG. Such manipulation allows rheostat-like control of PLY production and alters the cytotoxicity of whole S. pneumoniae on primary and immortalized human cells. These studies provide proof-of-principle for further investigation of mRNA ΔG manipulation as a tool in studies of bacterial pathogenesis.
authors: Department of Pediatrics, Columbia University, New York, NY United States of America; Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal; ICVS/3B's, PT Government Associate Laboratory, Braga/Guimarães, Portugal.
authors: Fábio E
authors: FE
authors: Amaral
authors: Department of Pediatrics, Columbia University, New York, NY United States of America.
authors: Dane
authors: D
authors: Parker
authors: Department of Pediatrics, Columbia University, New York, NY United States of America.
authors: Tara M
authors: TM
authors: Randis
authors: Department of Pediatrics, Columbia University, New York, NY United States of America.
authors: Ritwij
authors: R
authors: Kulkarni
authors: Department of Pediatrics, Columbia University, New York, NY United States of America; Department of Pharmacology, Columbia University, New York, NY, United States of America.
authors: Alice S
authors: AS
authors: Prince
authors: Department of Genetics & Development, Columbia University, New York, NY, United States of America.
authors: Mimi M
authors: MM
authors: Shirasu-Hiza
authors: Department of Pediatrics, Columbia University, New York, NY United States of America.
authors: Adam J
authors: AJ
authors: Ratner
mesh_terms: D017209:Apoptosis; D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D015153:Blotting, Western; D049109:Cell Proliferation; D002478:Cells, Cultured; D004912:Erythrocytes / Q000166:cytology / Q000378:metabolism*; D006461:Hemolysis; D006801:Humans; D007124:Immunoenzyme Techniques; D008175:Lung Neoplasms / Q000378:metabolism / Q000473:pathology; D008969:Molecular Sequence Data; D011008:Pneumococcal Infections / Q000235:genetics / Q000378:metabolism* / Q000382:microbiology; D059370:RNA Folding; D012333:RNA, Messenger / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012689:Sequence Homology, Nucleic Acid; D013296:Streptococcus pneumoniae / Q000235:genetics* / Q000302:isolation & purification; D013301:Streptolysins / Q000378:metabolism*; D037521:Virulence Factors / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D012333:RNA, Messenger; D013301:Streptolysins; D037521:Virulence Factors; C032290:plY protein, Streptococcus pneumoniae