Development of fluorescence expression tools to study host-mycoplasma interactions and validation in two distant mycoplasma clades.

Autor: Bonnefois T; CIRAD, UMR CMAEE, F-34398 Montpellier, France; INRA, UMR1309 CMAEE, F-34398 Montpellier, France. Electronic address: tiffany.bonnefois@gmail.com., Vernerey MS; INRA, Joint Research Unit 385 UMR BGPI, Campus International de Baillarguet, Montpellier, France. Electronic address: marie-stephanie.vernerey@supagro.inra.fr., Rodrigues V; CIRAD, UMR CMAEE, F-34398 Montpellier, France; INRA, UMR1309 CMAEE, F-34398 Montpellier, France. Electronic address: valerie.rodrigues@cirad.fr., Totté P; CIRAD, UMR CMAEE, F-34398 Montpellier, France; INRA, UMR1309 CMAEE, F-34398 Montpellier, France. Electronic address: philippe.totte@cirad.fr., Puech C; CIRAD, UMR CMAEE, F-34398 Montpellier, France; INRA, UMR1309 CMAEE, F-34398 Montpellier, France. Electronic address: carinne.puech@cirad.fr., Ripoll C; INSERM U1051-Hôpital Saint Eloi INM. 80, rue Augustin Fliche, 34091 Montpellier cedex 5, France. Electronic address: chantal.ripoll@inserm.fr., Thiaucourt F; CIRAD, UMR CMAEE, F-34398 Montpellier, France; INRA, UMR1309 CMAEE, F-34398 Montpellier, France. Electronic address: francois.thiaucourt@cirad.fr., Manso-Silván L; CIRAD, UMR CMAEE, F-34398 Montpellier, France; INRA, UMR1309 CMAEE, F-34398 Montpellier, France. Electronic address: lucia.manso-silvan@cirad.fr.
Jazyk: angličtina
Zdroj: Journal of biotechnology [J Biotechnol] 2016 Oct 20; Vol. 236, pp. 35-44. Date of Electronic Publication: 2016 Aug 04.
DOI: 10.1016/j.jbiotec.2016.08.006
Abstrakt: 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.
(Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)
Databáze: MEDLINE
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