Extracellular DNA: A Nutritional Trigger of Mycoplasma bovis Cytotoxicity

Abstract

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 Mollicutes 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 Mycoplasma bovis 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 M. bovis 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 M. bovis cytotoxicity. Hydrogen peroxide was found responsible for eDNA-mediated M. bovis cytotoxicity, and non-cytopathic mutants were unable to produce this toxic metabolic compound. In our experimental conditions, no contact between M. bovis and host cells was required for cytotoxicity. Further analyses revealed important intra-species differences in eDNA-mediated cytotoxicity and H2O2 production, with some strains displaying a cytopathic phenotype despite no H2O2 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 H2O2, IS-elements might influence H2O2 production in M. bovis. These results confirm the multifaceted role of eDNA in microbial communities and further identify this ubiquitous material as a nutritional trigger of M. bovis cytotoxicity. M. bovis may thus take advantage of the multiple sources of eDNA in vivo to modulate its interaction with host cells, a way for this minimal pathogen to overcome its limited coding capacity.

An emerging role for cyclic dinucleotide phosphodiesterase and nanoRNase activities in Mycoplasma bovis: Securing survival in cell culture

“…The moderate increase in mycoplasma titers observed at 72h in MEM medium alone is likely to be stimulated by the release of nutrients from the inoculum, such as DNA, which was recently identified as a limiting factor for M . bovis proliferation under cell culture conditions [ 32 ]. By using this screening strategy, 39 mutants were identified and further characterized by comparing their proliferation under axenic (PPLO medium) and cell culture conditions ( Fig 1B ).…”

“…These laboratory conditions have largely contributed to the understanding of the biology of these minimal bacteria but provided only limited information on the strategies these organisms have evolved to secure their survival in the animal host and get access to essential nutritional resources. In the absence of small animal models, eukaryotic cell cultures as re-emerged as a valuable environment to revisit the minimal cell concept at the light of host-pathogen interactions [30][31][32][33]44]. Our functional genomic study with the genome-reduced pathogen M. bovis revealed that phosphodiesterases involved in cyclic dinucleotides and nanoRNAs degradation are critical for survival under cell culture conditions, while dispensable in axenic medium.…”

“…This emerging role of nucleotides recycling is supported by several functional genomic studies with M. bovis and other ruminant mycoplasma species that repeatedly documented the disruption of the purine salvage adenine phosphoribosyltransferase as detrimental for mycoplasma growth in cell culture [30][31][32]44]. The recent identification of extracellular DNA as a limiting nutrient for M. bovis growth and cytotoxicity in cell culture also raises questions regarding the contribution of nucleotides recycling to virulence [32]. Many pathogenic bacteria possess a membrane-associated GdpP-type phosphodiesterase whose activity is required for virulence [48][49][50].…”

“…Thus, cyclic dinucleotides and nanoRNAs degradation activities identified in the present study likely contribute to nucleotide recycling in genome-reduced mycoplasmas that are well known for having limited metabolic capacities. This emerging role of nucleotides recycling is supported by several functional genomic studies with M. bovis and other ruminant mycoplasma species that repeatedly documented the disruption of the purine salvage adenine phosphoribosyltransferase as detrimental for mycoplasma growth in cell culture [30][31][32]44]. The recent identification of extracellular DNA as a limiting nutrient for M. bovis growth and cytotoxicity in cell culture also raises questions regarding the contribution of nucleotides recycling to virulence [32].…”

“…This technology has been successfully applied to several phylogenetically related species but is facing some limitations with other species, including the ruminant pathogens Mycoplasma agalactiae and Mycoplasma bovis [29]. For these species, random transposon mutagenesis, when combined with relevant screening systems, remains an efficient strategy for functional genomics studies [30][31][32][33] M. bovis is a re-emerging cause of pneumonia and mastitis in cattle worldwide, whose management is challenging because of the ineffectiveness of current vaccines and the high level of resistance to antibiotic treatments. In the present study, we used this pathogenic species as a model system to identify key factors regulating the proliferation of mycoplasmas under cocultivation with host cells.…”