1887

Abstract

There are several advantages, both and , in utilizing bacteria that express a fluorescent protein. Such a protein can be transiently incorporated into the bacteria or integrated within the bacterial genome. The most widely utilized fluorescent protein is green fluorescent protein (GFP), but limitations exist on its use. Additional fluorescent proteins have been designed that have many advantages over GFP and technologies for their incorporation into bacteria have been optimized. In the current study, we report the successful integration and expression of a stable fluorescent reporter, mCherry (red fluorescent protein, RFP), into the genome of a human pathogen, Group A (GAS) isolate AP53(S-). RFP was targeted at the atg codon of the pseudogene that is present in the mga regulon of AP53(S-). Transcription of critical bacterial genes was not functionally altered by the genomic integration of mCherry. Host virulence both (keratinocyte infection and cytotoxicity) and (skin infection) was maintained in AP53(S-)-RFP. Additionally, survival of mice infected with either AP53(S-) or AP53(S-)-RFP was similar, demonstrating that overall pathogenicity of the AP53(S-) strain was not altered by the expression of mCherry. These studies demonstrate the feasibility of integrating a fluorescent reporter into the bacterial genome of a naturally virulent isolate of Group A for comparative experimental studies.

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2019-09-25
2019-10-13
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References

  1. Leff LG, Leff AA. Use of green fluorescent protein to monitor survival of genetically engineered bacteria in aquatic environments. Appl Environ Microbiol 1996;62:3486–3488
    [Google Scholar]
  2. Andersen JB, Roldgaard BB, Lindner AB, Christensen BB, Licht TR. Construction of a multiple fluorescence labelling system for use in co-invasion studies of Listeria monocytogenes. BMC Microbiol 2006;6:86 [CrossRef]
    [Google Scholar]
  3. Chalupowicz L, Zellermann E-M, Fluegel M, Dror O, Eichenlaub R et al. Colonization and movement of GFP-labeled Clavibacter michiganensis subsp. michiganensis during tomato infection. Phytopathology 2012;102:23–31 [CrossRef]
    [Google Scholar]
  4. Millet YA, Alvarez D, Ringgaard S, von Andrian UH, Davis BM et al. Insights into vibrio cholerae intestinal colonization from monitoring fluorescently labeled bacteria. PLoS Pathog 2014;10:e1004405 [CrossRef]
    [Google Scholar]
  5. Geva-Zatorsky N, Alvarez D, Hudak JE, Reading NC, Erturk-Hasdemir D et al. In vivo imaging and tracking of host-microbiota interactions via metabolic labeling of gut anaerobic bacteria. Nat Med 2015;21:1091–1100 [CrossRef]
    [Google Scholar]
  6. Winschau F, van Zyl DSM, Dicks LMT. Use of the mCherry fluorescent protein to study intestinal colonization by Enterococcus mundtii ST4SA and Lactobacillus plantarum 423 in mice. Appl Environ Micrbiol 2015;81:5993–6002
    [Google Scholar]
  7. Schneider JP, Basler M. Shedding light on biology of bacterial cells. Philos Trans R Soc Lond B Biol Sci 2016;371:20150499 [CrossRef]
    [Google Scholar]
  8. Coralli C, Cemazar M, Kanthou C, Tozer GM, Dachs GU. Limitations of the reporter green fluorescent protein under simulated tumor conditions. Cancer Res 2001;61:4784–4790
    [Google Scholar]
  9. Shitashima Y, Shimozawa T, Kumagai A, Miyawaki A, Asahi T. Two distinct fluorescence states of the ligand-induced green fluorescent protein UnaG. Biophys J 2017;113:2805–2814 [CrossRef]
    [Google Scholar]
  10. Shaner NC, Campbell RE, Steinbach PA, Giepmans BNG, Palmer AE et al. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat Biotechnol 2004;22:1567–1572 [CrossRef]
    [Google Scholar]
  11. Scott KP, Mercer DK, Richardson AJ, Melville CM, Glover LA et al. Chromosomal integration of the green fluorescent protein gene in lactic acid bacteria and the survival of marked strains in human gut simulations. FEMS Microbiol Lett 2000;182:23–27 [CrossRef]
    [Google Scholar]
  12. Barnes RJ, Leung KT, Schraft H, Ulanova M. Chromosomal GFP labelling of Pseudomonas aeruginosa using a mini-Tn7 transposon: application for studies of bacteria-host interactions. Can J Microbiol 2008;54:48–57 [CrossRef]
    [Google Scholar]
  13. Pinheiro LB, Gibbs MD, Vesey G, Smith JJ, Bergquist PL. Fluorescent reference strains of bacteria by chromosomal integration of a modified green fluorescent protein gene. Appl Microbiol Biotechnol 2008;77:1287–1295 [CrossRef]
    [Google Scholar]
  14. Clark L, Martinez-Argudo I, Humphrey TJ, Jepson MA. Gfp plasmid-induced defects in Salmonella invasion depend on plasmid architecture, not protein expression. Microbiology 2009;155:461–467 [CrossRef]
    [Google Scholar]
  15. Liang Z, Zhang Y, Agrahari G, Chandrahas V, Glinton K et al. A natural inactivating mutation in the CovS component of the CovRS regulatory operon in a pattern D streptococcal pyogenes strain influences virulence-associated genes. J Biol Chem 2013;288:6561–6573 [CrossRef]
    [Google Scholar]
  16. Simon D, Ferretti JJ. Electrotransformation of Streptococcus pyogenes with plasmid and linear DNA. FEMS Microbiol Lett 1991;82:219–224 [CrossRef]
    [Google Scholar]
  17. Bessen DE, Sotir CM, Readdy TL, Hollingshead SK. Genetic correlates of throat and skin isolates of group A streptococci. J Infect Dis 1996;173:896–900 [CrossRef]
    [Google Scholar]
  18. Sun H, Ringdahl U, Homeister JW, Fay WP, Engleberg NC et al. Plasminogen is a critical host pathogenicity factor for group A streptococcal infection. Science 2004;305:1283–1286 [CrossRef]
    [Google Scholar]
  19. Churchward G. The two faces of Janus: virulence gene regulation by CovR/S in group A streptococci. Mol Microbiol 2007;64:34–41 [CrossRef]
    [Google Scholar]
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