title: Bright fluorescent Streptococcus pneumoniae for live-cell imaging of host-pathogen interactions.
abstract: Streptococcus pneumoniae is a common nasopharyngeal resident in healthy people but, at the same time, one of the major causes of infectious diseases such as pneumonia, meningitis, and sepsis. The shift from commensal to pathogen and its interaction with host cells are poorly understood. One of the major limitations for research on pneumococcal-host interactions is the lack of suitable tools for live-cell imaging. To address this issue, we developed a generally applicable strategy to create genetically stable, highly fluorescent bacteria. Our strategy relies on fusing superfolder green fluorescent protein (GFP) or a far-red fluorescent protein (RFP) to the abundant histone-like protein HlpA. Due to efficient translation and limited cellular diffusion of these fusions, the cells are 25-fold brighter than those of the currently best available imaging S. pneumoniae strain. These novel bright pneumococcal strains are fully virulent, and the GFP reporter can be used for in situ imaging in mouse tissue. We used our reporter strains to study the effect of the polysaccharide capsule, a major pneumococcal virulence factor, on different stages of infection. By dual-color live-cell imaging experiments, we show that unencapsulated pneumococci adhere significantly better to human lung epithelial cells than encapsulated strains, in line with previous data obtained by classical approaches. We also confirm with live-cell imaging that the capsule protects pneumococci from neutrophil phagocytosis, demonstrating the versatility and usability of our reporters. The described imaging tools will pave the way for live-cell imaging of pneumococcal infection and help further understanding of the mechanisms of pneumococcal pathogenesis.
authors.affiliation: Molecular Genetics Group, Groningen Biomolecular Sciences and Biotechnology Institute, Center for Synthetic Biology, University of Groningen, Groningen, The Netherlands.
authors.forename: Morten
authors.initials: M
authors.lastname: Kjos
authors.affiliation: Molecular Genetics Group, Groningen Biomolecular Sciences and Biotechnology Institute, Center for Synthetic Biology, University of Groningen, Groningen, The Netherlands.
authors.forename: Rieza
authors.initials: R
authors.lastname: Aprianto
authors.affiliation: Department of Infection, Immunity and Inflammation, University of Leicester, Leicester, United Kingdom.
authors.forename: Vitor E
authors.initials: VE
authors.lastname: Fernandes
authors.affiliation: Department of Infection, Immunity and Inflammation, University of Leicester, Leicester, United Kingdom.
authors.forename: Peter W
authors.initials: PW
authors.lastname: Andrew
authors.affiliation: Department of Medical Microbiology, University Medical Center Utrecht, Utrecht, The Netherlands.
authors.forename: Jos A G
authors.initials: JA
authors.lastname: van Strijp
authors.affiliation: Department of Medical Microbiology, University Medical Center Utrecht, Utrecht, The Netherlands.
authors.forename: Reindert
authors.initials: R
authors.lastname: Nijland
authors.affiliation: Molecular Genetics Group, Groningen Biomolecular Sciences and Biotechnology Institute, Center for Synthetic Biology, University of Groningen, Groningen, The Netherlands j.w.veening@rug.nl.
authors.forename: Jan-Willem
authors.initials: JW
authors.lastname: Veening
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D058948:Cell Tracking / Q000379:methods*; D005260:Female; D005453:Fluorescence; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D054884:Host-Pathogen Interactions; D006801:Humans; D008168:Lung / Q000382:microbiology; D051379:Mice; D011008:Pneumococcal Infections / Q000382:microbiology*; D013296:Streptococcus pneumoniae / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D037521:Virulence Factors / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D037521:Virulence Factors; D049452:Green Fluorescent Proteins