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Use of fluorescence expression tools for the comparative analysis of the interactions of Mycoplasma mycoides and Mycoplasma bovis with bovine cells

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Use of fluorescence expression tools for the

comparative analysis of the interactions of Mycoplasma

mycoides and Mycoplasma bovis with bovine cells

European Mycoplasma Conference; London 18 - 19 March 2019

Background: Fluorescence expression systems adapted to the analysis of host-mycoplasma interactions were recently developed*. The aim of this work was to apply them to study the colonisation and persistence capabilities of the bovine pathogens Mycoplasma mycoides subsp. mycoides (Mmm) and Mycoplasma bovis in bovine cells.

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Reference: *Bonnefois, T., Vernerey, M.-S., Rodrigues, V., Totté, P., Puech, C., Ripoll, C., Thiaucourt, F., and Manso-Silván, L. (2016). Development of fluorescence expression tools to study host-mycoplasma interactions and validation in two distant mycoplasma clades. J. Biotechnol. 236, 35–44.

Conclusion: The remarkable

differences in the invasion and persistence capabilities of Mmm and M. bovis observed here are in agreement with the pathogenesis of the infections caused by these mycoplasmas. mNeonGreen mutants have proven extremely useful for the analysis of mycoplasma – host cell interactions. Furthermore, the fluorescence expression tools used in this study offer innumerable perspectives for the functional analysis of a wide range of mycoplasma species both in vitro and in vivo.

Methods: Mini-transposons affording high-level expression of fluorescent proteins mCherry, mKO2, GFP2 and mNeonGreen were used to mark Mmm and M. bovis strains (2 virulent and 2 avirulent Mmm strains for mNeonGreen). The transformation efficiency (i.e., 10-6 transformant cfu / total cfu for Mmm strains)

was compatible with the production of fluorescent mutant banks. Fluorescent mutants were characterised by epifluorescence microscopy (Fig. 1, excluding GFP2 mutants, eliminated due to delayed expression) and were selected according to sites of transposon insertion not linked to pathogenicity (Table1).

Equivalent fluorescent signals were detected from mNeonGreen mutants by flow cytometry and confocal microscopy but heat inactivation resulted in loss of fluorescence after 2 to 4 h (Fig. 2).

Lucía Manso-Silván, Tiffany Bonnefois, Olivia Cadamuro, François Thiaucourt, Valérie Rodrigues, Philippe Totté.

lucia.manso-sylvan@cirad.fr

Results: Interactions of mNeonGreen mutants with bovine cells were analysed using confocal microscopy. M. bovis presented much higher adhesion, invasion and proliferation capacities than Mmm in culture with non-phagocytic cells (Fig. 3) and showed increased resistance to elimination by macrophages (Fig. 4).

Table 1: Fluorescent mutants from Mmm virulent strains Rita (Cameroon, 1987)

and 94111 (Rwanda, 1994); avirulent strains PG1T (type strain, unknown origin)

and KH3J (vaccine from Sudan); M. bovis strain Oger2 (calf pneumonia, France, 1975). Insertion sites according to either Mmm PG1T or M. bovis PG45T genome.

Fig. 1: Micrographs of M. bovis strain Oger2 mutants expressing mCherry (OC7), mKO2 (OK1) and

mNeonGreen (ON8); and Mmm strain Rita mutants expressing mCherry (RC4), mKO2 (RK4) and mNeonGreen (RN6 and RN9). From top to bottom: imaging using phase contrast, epifluorescence with CY3 filters (red), and epifluorescence with EGFP filters (green).

Fig. 2: Fluorescent = live mycoplasmas. Loss of fluorescence following

inactivation at 56°C for one hour, as observed by (A) flow cytometry, (B) confocal microscopy. Intervals: percentage of positive cells.

Fig 4: Confocal micrographs of bovine macrophages infected with mNeonGreen-expressing M. bovis mutant ON8 and Mmm mutant RN6 at MOI of 5,

50 and 500 and observed at 4, 24 and 48h post-infection. For staining details refer to in Fig. 3 A. White arrows indicate fluorescent mycoplasmas.

Fig. 3 A: Confocal micrographs

of Embryonic Bovine Lung cells (EBL) infected with Mmm

mutant RN6 and M. bovis mutant ON8 at a MOI of 200. In red, cell membrane and cytoplasm stained with wheat germ agglutinin (WGA) Alexa Fluor 594; in blue, cell nucleus stained with Hoechst; in green, fluorescent mycoplasma (white arrows).

Fig. 3 B: Confocal micrographs

of Bovine Aortic Endothelial Cells (BAEC) infected with

Mmm mutant RN6 and M. bovis mutant ON8 at a MOI of

200.

For staining, refer to Fig. 3 A. Fluorescent mycoplasmas are indicated with white arrows.

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