AUTHOR=Zhu Xifang , Dordet-Frisoni Emilie , Gillard Lucie , Ba Abou , Hygonenq Marie-Claude , Sagné Eveline , Nouvel Laurent Xavier , Maillard Renaud , Assié Sébastien , Guo Aizhen , Citti Christine , Baranowski Eric 

TITLE=Extracellular DNA: A Nutritional Trigger of Mycoplasma bovis Cytotoxicity

JOURNAL=Frontiers in Microbiology

VOLUME=10

YEAR=2019

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2019.02753

DOI=10.3389/fmicb.2019.02753

ISSN=1664-302X

ABSTRACT=<p>Microbial access to host nutrients is a key factor of the host-pathogen interplay. With their nearly minimal genome, wall-less bacteria of the class <italic>Mollicutes</italic> have limited metabolic capacities and largely depend on host nutrients for their survival. Despite these limitations, host-restricted mycoplasmas are widely distributed in nature and many species are pathogenic for humans and animals. Yet, only partial information is available regarding the mechanisms evolved by these minimal pathogens to meet their nutrients and the contribution of these mechanisms to virulence. By using the ruminant pathogen <italic>Mycoplasma bovis</italic> as a model system, extracellular DNA (eDNA) was identified as a limiting nutrient for mycoplasma proliferation under cell culture conditions. Remarkably, the growth-promoting effect induced by supplementation with eDNA was associated with important cytotoxicity for actively dividing host cells, but not confluent monolayers. To identify biological functions mediating <italic>M. bovis</italic> cytotoxicity, we produced a library of transposon knockout mutants and identified three critical genomic regions whose disruption was associated with a non-cytopathic phenotype. The coding sequences (CDS) disrupted in these regions pointed towards pyruvate metabolism as contributing to <italic>M. bovis</italic> cytotoxicity. Hydrogen peroxide was found responsible for eDNA-mediated <italic>M. bovis</italic> cytotoxicity, and non-cytopathic mutants were unable to produce this toxic metabolic compound. In our experimental conditions, no contact between <italic>M. bovis</italic> and host cells was required for cytotoxicity. Further analyses revealed important intra-species differences in eDNA-mediated cytotoxicity and H<sub>2</sub>O<sub>2</sub> production, with some strains displaying a cytopathic phenotype despite no H<sub>2</sub>O<sub>2</sub> production. Interestingly, the genome of strains PG45 and HB0801 were characterized by the occurrence of insertion sequences (IS) at close proximity to several CDSs found disrupted in non-cytopathic mutants. Since PG45 and HB0801 produced no or limited amount of H<sub>2</sub>O<sub>2</sub>, IS-elements might influence H<sub>2</sub>O<sub>2</sub> production in <italic>M. bovis</italic>. These results confirm the multifaceted role of eDNA in microbial communities and further identify this ubiquitous material as a nutritional trigger of <italic>M. bovis</italic> cytotoxicity. <italic>M. bovis</italic> may thus take advantage of the multiple sources of eDNA <italic>in vivo</italic> to modulate its interaction with host cells, a way for this minimal pathogen to overcome its limited coding capacity.</p>