Extracellular Actin Is a Receptor for Mycoplasma hyopneumoniae

Enzootic pneumonia incurs major economic losses to pork production globally. The primary pathogen and causative agent, Mycoplasma hyopneumoniae, colonises ciliated epithelium and disrupts mucociliary function predisposing the upper respiratory tract to secondary pathogens. Alleviation of disease is reliant on antibiotics, vaccination, and sound animal husbandry, but none are effective at eliminating M. hyopneumoniae from large production systems. Sustainable pork production systems strive to lower reliance on antibiotics but lack of a detailed understanding of the pathobiology of M. hyopneumoniae has curtailed efforts to develop effective mitigation strategies. M. hyopneumoniae is considered an extracellular pathogen. Here we show that M. hyopneumoniae associates with integrin β1 on the surface of epithelial cells via interactions with surface-bound fibronectin and initiates signalling events that stimulate pathogen uptake into clathrin-coated vesicles (CCVs) and caveosomes. These early events allow M. hyopneumoniae to exploit an intracellular lifestyle by commandeering the endosomal pathway. Specifically, we show: (i) using a modified gentamicin protection assay that approximately 8% of M. hyopneumoniae cells reside intracellularly; (ii) integrin β1 expression specifically co-localises with the deposition of fibronectin precisely where M. hyopneumoniae cells assemble extracellularly; (iii) anti-integrin β1 antibodies block entry of M. hyopneumoniae into porcine cells; and (iv) M. hyopneumoniae survives phagolysosomal fusion, and resides within recycling endosomes that are trafficked to the cell membrane. Our data creates a paradigm shift by challenging the long-held view that M. hyopneumoniae is a strict extracellular pathogen and calls for in vivo studies to determine if M. hyopneumoniae can traffic to extrapulmonary sites in commercially-reared pigs.

Section: Resultsmentioning

confidence: 99%

Mycoplasma hyopneumoniae resides intracellularly within porcine epithelial cells

Raymond

Turnbull

Jenkins

et al. 2018

“…We recently showed that Mpn142, a member of the same operon that houses the P1 gene (mpn141), and the surface accessible moonlighting adhesin, elongation factor Tu (Ef-Tu), are cleaved extensively 12,28 . Post-translational processing of adhesins has been well characterised in M. hyopneumoniae where cleavage fragments have been shown to adhere to porcine cilia, porcine kidney epithelial cells, and a range of host molecules such as the glycosaminoglycan mimic heparin [59][60][61][62]64,72,74,[110][111][112][113][114][115] , plasminogen 60,[112][113][114] , actin 116 , and fibronectin 59,72,[112][113][114] . Processing of adhesin molecules is not confined to M. hyopneumoniae but has been described in Mycoplasma gallisepticum 117 , Mycoplasma fermentans [118][119][120] , M. genitalium 121 , and Spiroplasma citri 122 .…”

Section: Discussionmentioning

confidence: 99%

“…Proline-rich regions in proteins have been implicated in protein:protein interactions [131][132][133] and it has been suggested that proline residues could anchor the C-terminus of P1 in the cell membrane 49 . Lysine-rich regions are associated with binding plasminogen 60,64,123,134,135 , heparin 59,61,69,72,115,136 , actin 116,123 , and DNA 75,137 . While P1-15 and P1-30 bound plasminogen in a dose-responsive manner, it was notable that RP-15 bound it more strongly.…”

Section: Discussionmentioning

confidence: 99%

Cell surface processing of the P1 adhesin of Mycoplasma pneumoniae identifies novel domains that bind host molecules

Widjaja

Berry

Jarocki

et al. 2020

Mycoplasma pneumoniae is a genome reduced pathogen and causative agent of community acquired pneumonia. The major cellular adhesin, P1, localises to the tip of the attachment organelle forming a complex with P40 and P90, two cleavage fragments derived by processing Mpn142, and other molecules with adhesive and mobility functions. LC-MS/MS analysis of M. pneumoniae M129 proteins derived from whole cell lysates and eluents from affinity matrices coupled with chemically diverse host molecules identified 22 proteoforms of P1. Terminomics was used to characterise 17 cleavage events many of which were independently verified by the identification of semitryptic peptides in our proteome studies and by immunoblotting. One cleavage event released 1597 tSAAKpGApRppVppKpGApKppVQppKKpA 1627 from the C-terminus of P1 and this peptide was shown to bind to a range of host molecules. A smaller synthetic peptide comprising the C-terminal 15 amino acids, 1613 pGApKppVQppKKpA 1627 , selectively bound cytoskeletal intermediate filament proteins cytokeratin 7, cytokeratin 8, cytokeratin 18, and vimentin from a native A549 cell lysate. Collectively, our data suggests that ectodomain shedding occurs on the surface of M. pneumoniae where it may alter the functional diversity of P1, Mpn142 and other surface proteins such as elongation factor Tu via a mechanism similar to that described in Mycoplasma hyopneumoniae. The attachment organelle is a structurally and functionally sophisticated component of the M. pneumoniae cell that is responsible for the assembly of proteins essential for motility and adherence 1-8. An extensive list of host molecules including fibronectin 9-13 , fibrinogen 10-14 , plasminogen 11-13,15-17 , lactoferrin 10-12 , laminin 10-12 , and vitronectin 10-13 interact with surface accessible adhesins in M. pneumoniae. Other less well-defined host molecules include sialylated molecules 18 , oligosaccharides 19 , glycolipids 20 , and glycoproteins 21. The gene mpn141 encoding the major adhesin P1 is located in the same operon along with mpn140 and mpn142 and these three genes constitute a polycistronic transcriptional unit 22,23. mpn140 encodes for a 28 kDa putative phosphoesterase 24 and while it has been shown to degrade nanoRNA and dephosphorylate 3′-phosphoadenosine 5′-phosphate to AMP 25 , no role in adherence has been assigned for this protein. mpn142 generates a 130 kDa product (Mpn142) that is cleaved into two fragments of 40 kDa (P40) and 90 kDa (P90) immediately after or concurrent with translation 26,27. The cleavage event in Mpn142, first described over 25 years ago, was the first in what is now known to be a highly processed molecule on the surface of M. pneumoniae 28. P1 is a remarkably versatile molecule and the subject of numerous studies over the past 30 years. The only cleavage event that has been accurately assigned to P1 is the removal of the N-terminal 59 amino acids as a leader peptide 29. Molecular cross-linking and immunogold-labelling studies indicated that P1 forms a complex with P30, P40, and P90 ...

“…Experiments were performed as described previously 51 . Samples were imaged on a Nikon A1 Confocal Laser Scanning Microscope.…”

Section: Methodsmentioning

confidence: 99%

“…Preparation of infected monolayers for SEM was performed as previously described 51 , with no modifications.…”

Section: Methodsmentioning

confidence: 99%

Extracellular DNA release from the genome-reduced pathogen Mycoplasma hyopneumoniae is essential for biofilm formation on abiotic surfaces

Raymond

Jenkins

Turnbull

et al. 2018

Self Cite

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.