Abstract
Mycoplasma hyopneumoniae is an economically devastating, globally disseminated pathogen that can maintain a chronic infectious state within its host, swine. Here, we depict the events underpinning M. hyopneumoniae biofilm formation on an abiotic surface and demonstrate for the first time, biofilms forming on porcine epithelial cell monolayers and in the lungs of pigs, experimentally infected with M. hyopneumoniae. Nuclease treatment prevents biofilms forming on glass but not on porcine epithelial cells indicating that extracellular DNA (eDNA), which localises at the base of biofilms, is critical in the formation of these structures on abiotic surfaces. Subpopulations of M. hyopneumoniae cells, denoted by their ability to take up the dye TOTO-1 and release eDNA, were identified. A visually distinct sub-population of pleomorphic cells, that we refer to here as large cell variants (LCVs), rapidly transition from phase dark to translucent “ghost” cells. The translucent cells accumulate the membrane-impermeable dye TOTO-1, forming readily discernible membrane breaches immediately prior to lysis and the possible release of eDNA and other intracellular content (public goods) into the extracellular environment. Our novel observations expand knowledge of the lifestyles adopted by this wall-less, genome-reduced pathogen and provide further insights to its survival within farm environments and swine.
Highlights
Mycoplasmas evolved by a process of degenerative evolution, shedding genes for the biosynthesis of a cell wall, nucleic acids, amino acids, and the tricarboxylic acid cycle
Biofilms form on glass only after prolonged incubation (10–12 days) but form rapidly on cell monolayers suggesting that M. hyopneumoniae is unable to adhere to glass without a conditioning phase. Extracellular DNA (eDNA) appears to be essential for initiating biofilm formation during this conditioning phase
The eDNA assembles at the base of microcolonies and biofilms (Fig. 3F) presumably to provide a matrix for M. hyopneumoniae adherence. 3-D SIM images (Fig. 4) of microcolonies and biofilms identify a subpopulation of M. hyopneumoniae cells in these structures that accumulate the cell-impermeable dye, TOTO-1, indicative of these cells having a compromised membrane structure
Summary
Mycoplasmas evolved by a process of degenerative evolution, shedding genes for the biosynthesis of a cell wall, nucleic acids, amino acids, and the tricarboxylic acid cycle. COMSTAT36 was used to assess the biomass and average thickness, based upon the nucleic acid content of DAPI stained biofilms that formed between days 5, 10, and 20 (Supplementary Fig. S2) and imaged with confocal laser scanning microscopy (CLSM). EDNA appeared to form a base layer on the surface of the glass immediately beneath M. hyopneumoniae biofilms, microcolonies, and individual cells (Fig. 3D–F).
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