Minimalistic mycoplasmas harbor different functional toxin-antitoxin systems.

Mycoplasmas are minute bacteria controlled by very small genomes ranging from 0.6 to 1.4 Mbp. They encompass several important medical and veterinary pathogens that are often associated with a wide range of chronic diseases. The long persistence of mycoplasma cells in their hosts can exacerbate the spread of antimicrobial resistance observed for many species. However, the nature of the virulence factors driving this phenomenon in mycoplasmas is still unclear. Toxin-antitoxin systems (TA systems) are genetic elements widespread in many bacteria that were historically associated with bacterial persistence. Their presence on mycoplasma genomes has never been carefully assessed, especially for pathogenic species. Here we investigated three candidate TA systems in M. mycoides subsp. capri encoding a (i) novel AAA-ATPase/subtilisin-like serine protease module, (ii) a putative AbiEii/AbiEi pair and (iii) a putative Fic/RelB pair. We sequence analyzed fourteen genomes of M. mycoides subsp. capri and confirmed the presence of at least one TA module in each of them. Interestingly, horizontal gene transfer signatures were also found in several genomic loci containing TA systems for several mycoplasma species. Transcriptomic and proteomic data confirmed differential expression profiles of these TA systems during mycoplasma growth in vitro. While the use of heterologous expression systems based on E. coli and B. subtilis showed clear limitations, the functionality and neutralization capacities of all three candidate TA systems were successfully confirmed using M. capricolum subsp. capricolum as a host. Additionally, M. capricolum subsp. capricolum was used to confirm the presence of functional TA system homologs in mycoplasmas of the Hominis and Pneumoniae phylogenetic groups. Finally, we showed that several of these M. mycoides subsp. capri toxins tested in this study, and particularly the subtilisin-like serine protease, could be used to establish a kill switch in mycoplasmas for industrial applications. Author summary: Mycoplasmas belong to a class of cell-wall deficient bacteria characterized by minimal genomes acquired through regressive evolution. Historically, they were thought to lack many of the common bacterial virulence traits including classical exotoxins and toxin-antitoxin (TA) systems. In this work, we confirmed the presence of different functional TA systems in several isolates of the caprine pathogen Mycoplasma mycoides subsp. capri. Our data also indicate that TA systems are widespread in other mycoplasma species of veterinary importance. This work paves the way for the investigation of the biological role of TA systems during mycoplasma chronic infections as they are likely to contribute to the parasitic lifestyle of mycoplasmas, persistence in their hosts as well as the buildup of antimicrobial resistance, as recently observed. The availability of synthetic genomics tools to modify a range of Mycoplasma pathogens and well-established challenge models will foster future research and shed the light on the importance of TA systems in mycoplasmas. [ABSTRACT FROM AUTHOR]

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