title: Development of fluorescence expression tools to study host-mycoplasma interactions and validation in two distant mycoplasma clades.
abstract: Fluorescence expression tools for stable and innocuous whole mycoplasma cell labelling have been developed. A Tn4001-derivative mini-transposon affording unmarked, stable mutagenesis in mycoplasmas was modified to allow the constitutive, high-level expression of mCherry, mKO2 and mNeonGreen. These tools were used to introduce the respective fluorescent proteins as chromosomal tags in the phylogenetically distant species Mycoplasma mycoides subsp. mycoides and Mycoplasma bovis. The production, selection and characterisation of fluorescent clones were straightforward and resulted in the unprecedented observation of red and green fluorescent mycoplasma colonies in the two species, with no apparent cytotoxicity. Equivalent fluorescence expression levels were quantified by flow cytometry in both species, suggesting that these tools can be broadly applied in mycoplasmas. A macrophage infection assay was performed to assess the usefulness of mNeonGreen-expressing strains for monitoring mycoplasma infections, and notably cell invasion. The presence of fluorescent mycoplasmas inside live phagocytic cells was detected and quantified by flow cytometry and corroborated by confocal microscopy, which allowed the identification of individual mycoplasmas in the cytoplasm of infected cells. The fluorescence expression tools developed in this study are suitable for host-pathogen interaction studies and offer innumerable perspectives for the functional analysis of mycoplasmas both in vitro and in vivo.
authors: CIRAD, UMR CMAEE, F-34398 Montpellier, France; INRA, UMR1309 CMAEE, F-34398 Montpellier, France. Electronic address: tiffany.bonnefois@gmail.com.
authors: Tiffany
authors: T
authors: Bonnefois
authors: INRA, Joint Research Unit 385 UMR BGPI, Campus International de Baillarguet, Montpellier, France. Electronic address: marie-stephanie.vernerey@supagro.inra.fr.
authors: Marie-Stéphanie
authors: MS
authors: Vernerey
authors: CIRAD, UMR CMAEE, F-34398 Montpellier, France; INRA, UMR1309 CMAEE, F-34398 Montpellier, France. Electronic address: valerie.rodrigues@cirad.fr.
authors: Valérie
authors: V
authors: Rodrigues
authors: CIRAD, UMR CMAEE, F-34398 Montpellier, France; INRA, UMR1309 CMAEE, F-34398 Montpellier, France. Electronic address: philippe.totte@cirad.fr.
authors: Philippe
authors: P
authors: Totté
authors: CIRAD, UMR CMAEE, F-34398 Montpellier, France; INRA, UMR1309 CMAEE, F-34398 Montpellier, France. Electronic address: carinne.puech@cirad.fr.
authors: Carinne
authors: C
authors: Puech
authors: INSERM U1051-Hôpital Saint Eloi INM. 80, rue Augustin Fliche, 34091 Montpellier cedex 5, France. Electronic address: chantal.ripoll@inserm.fr.
authors: Chantal
authors: C
authors: Ripoll
authors: CIRAD, UMR CMAEE, F-34398 Montpellier, France; INRA, UMR1309 CMAEE, F-34398 Montpellier, France. Electronic address: francois.thiaucourt@cirad.fr.
authors: François
authors: F
authors: Thiaucourt
authors: CIRAD, UMR CMAEE, F-34398 Montpellier, France; INRA, UMR1309 CMAEE, F-34398 Montpellier, France. Electronic address: lucia.manso-silvan@cirad.fr.
authors: Lucía
authors: L
authors: Manso-Silván
mesh_terms: D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D054884:Host-Pathogen Interactions / Q000502:physiology*; D057054:Molecular Imaging / Q000379:methods*; D009174:Mycoplasma / Q000737:chemistry* / Q000166:cytology* / Q000378:metabolism / Q000472:pathogenicity; D009175:Mycoplasma Infections / Q000382:microbiology / Q000503:physiopathology; D010586:Phagocytes / Q000166:cytology / Q000382:microbiology; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins
keywords: Confocal microscopy; Flow cytometry; Fluorescence; Host-pathogen interactions; Mycoplasma; Whole cell labelling