Molecular interactions between Mycoplasma hyopneumoniae and host cells

University of Technology Sydney. Faculty of Science.The Mycoplasmas are a group of wall-less bacteria belonging to the Mollicutes that are believed to have diverged from the Gram-positive Firmicutes. Mollicutes have undergone reductive evolution, losing genes for the biosynthesis of essential biomolecules, subsequently having to form parasite relationships with their hosts in order to acquire these nutrients. They form these relationships as both commensals and pathogens, and a number of Mycoplasma species cause significant clinical and agricultural diseases. Mycoplasma hyopneumoniae is the causative agent of porcine enzootic pneumonia, a chronic respiratory disease that affects swine populations worldwide. M. hyopneumoniae colonises the upper respiratory tract by adhering to the rapidly beating cilia where it causes ciliostasis and eventual cilial death [1]. M. hyopneumoniae possesses a family of surface adhesins referred to as the P97 and P102 paralog family that it utilises to adhere to the cilia [2-10]. A hallmark of M. hyopneumoniae infection is a potent inflammatory response which is believed to be one of the contributing factors to the gross lung lesions observed in infected swine [11-13]. M. hyopneumoniae is described as a strict extracellular pathogen that only adheres to cilia and knowledge is lacking on additional receptors that M. hyopneumoniae binds to. Recent studies have however, shown that viable M. hyopneumoniae cells can be cultured from the liver, spleen, kidneys and lymph nodes of infected swine [14-16]. These observations suggest that M. hyopneumoniae has the capability to invade through the epithelial barrier and disseminate to distal tissue sites. In addition to this, large microcolonies have been observed in the respiratory tract of swine infected with M. hyopneumoniae [17]. These microcolonies are reminiscent of biofilms, and although biofilm formation has never been investigated in M. hyopneumoniae it is likely that they play a role in the chronicity of disease. Notably, even when lung lesions in M. hyopneumoniae-infected swine are cleared, bronchial swabs can still test positive for M. hyopneumoniae up to 185 days post-infection (P.I.) [18] and pigs can act as convalescent carriers for up to 200 days P.I. [19]. This suggests that M. hyopneumoniae possesses mechanisms in which it can remain dormant within its host whilst remaining infectious. Vaccines against M. hyopneumoniae can successfully reduce lung lesions but they are unable to prevent transmission in swine herds [20]. In order to create vaccines that inhibit the transmission of M. hyopneumoniae, a better understanding of the disease process is required. This PhD project has thus been devised in order to address the problems outlined above. This work has investigated the ability of adhesins to undergo extensive endoproteolytic processing; demonstrating that proteolytic processing in the P97 and P102 adhesins occurs much more extensively than what has previously been shown. I also show that these adhesins can bind to a myriad of host components such as heparin, fibronectin (Fn) and plasminogen (Plg) and investigate the domains responsible. Additionally, this work presents a number of novel receptors that M. hyopneumoniae targets within its host as well as a comprehensive list of putative adhesins that it utilises to do so. This work has also investigated the ability of M. hyopneumoniae to form biofilms on abiotic surfaces, host cells and within the swine respiratory tract and further demonstrate that surface adhesins play a role in biofilm formation. A number of putative biofilm-associated genes have been identified by screening a transposon mutant library, these genes being potential vaccine candidates. Finally, this work has investigated the ability of M. hyopneumoniae to become internalised by host cells and reside within the cytoplasm. M. hyopneumoniae becomes internalised by vacuole-like structures, and that internalised cells appear to escape from lysosomes to reside free within the cytoplasm. Overall, this PhD project has contributed significantly to understanding how M. hyopneumoniae causes disease. Future work on the novel mechanisms described in this thesis will aid in future vaccine development programs and potentially aid in the control of this important veterinary disease

Mycoplasma hyopneumoniae surface-associated proteases cleave bradykinin, substance P, neurokinin A and neuropeptide Y

© 2019, The Author(s). Mycoplasma hyopneumoniae is an economically-devastating and geographically-widespread pathogen that colonises ciliated epithelium, and destroys mucociliary function. M. hyopneumoniae devotes ~5% of its reduced genome to encode members of the P97 and P102 adhesin families that are critical for colonising epithelial cilia, but mechanisms to impair mucociliary clearance and manipulate host immune response to induce a chronic infectious state have remained elusive. Here we identified two surface exposed M. hyopneumoniae proteases, a putative Xaa-Pro aminopeptidase (MHJ_0659; PepP) and a putative oligoendopeptidase F (MHJ_0522; PepF), using immunofluorescence microscopy and two orthogonal proteomic methodologies. MHJ_0659 and MHJ_0522 were purified as polyhistidine fusion proteins and shown, using a novel MALDI-TOF MS assay, to degrade four pro-inflammatory peptides that regulate lung homeostasis; bradykinin (BK), substance P (SP), neurokinin A (NKA) and neuropeptide Y (NPY). These findings provide insight into the mechanisms used by M. hyopneumoniae to influence ciliary beat frequency, impair mucociliary clearance, and initiate a chronic infectious disease state in swine, features that are a hallmark of disease caused by this pathogen

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

© 2018 The Author(s). 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

A comprehensive guide for performing sample preparation and top-down protein analysis

© 2017 by the authors. Methodologies for the global analysis of proteins in a sample, or proteome analysis, have been available since 1975 when Patrick O'Farrell published the first paper describing two-dimensional gel electrophoresis (2D-PAGE). This technique allowed the resolution of single protein isoforms, or proteoforms, into single 'spots' in a polyacrylamide gel, allowing the quantitation of changes in a proteoform0s abundance to ascertain changes in an organism's phenotype when conditions change. In pursuit of the comprehensive profiling of the proteome, significant advances in technology have made the identification and quantitation of intact proteoforms from complex mixtures of proteins more routine, allowing analysis of the proteome from the 'Top-Down'. However, the number of proteoforms detected by Top-Down methodologies such as 2D-PAGE or mass spectrometry has not significantly increased since O'Farrell's paper when compared to Bottom-Up, peptide-centric techniques. This article explores and explains the numerous methodologies and technologies available to analyse the proteome from the Top-Down with a strong emphasis on the necessity to analyse intact proteoforms as a better indicator of changes in biology and phenotype. We arrive at the conclusion that the complete and comprehensive profiling of an organism0s proteome is still, at present, beyond our reach but the continuing evolution of protein fractionation techniques and mass spectrometry brings comprehensive Top-Down proteome profiling closer

N-terminomics identifies widespread endoproteolysis and novel methionine excision in a genome-reduced bacterial pathogen

© 2017 The Author(s). Proteolytic processing alters protein function. Here we present the first systems-wide analysis of endoproteolysis in the genome-reduced pathogen Mycoplasma hyopneumoniae. 669 N-terminal peptides from 164 proteins were identified, demonstrating that functionally diverse proteins are processed, more than half of which 75 (53%) were accessible on the cell surface. Multiple cleavage sites were characterised, but cleavage with arginine in P1 predominated. Putative functions for a subset of cleaved fragments were assigned by affinity chromatography using heparin, actin, plasminogen and fibronectin as bait. Binding affinity was correlated with the number of cleavages in a protein, indicating that novel binding motifs are exposed, and protein disorder increases, after a cleavage event. Glyceraldehyde 3-phosphate dehydrogenase was used as a model protein to demonstrate this. We define the rules governing methionine excision, show that several aminopeptidases are involved, and propose that through processing, genome-reduced organisms can expand protein function

MHJ-0461 is a multifunctional leucine aminopeptidase on the surface of Mycoplasma hyopneumoniae

© 2015 The Authors. Published. Aminopeptidases are part of the arsenal of virulence factors produced by bacterial pathogens that inactivate host immune peptides. Mycoplasma hyopneumoniae is a genome-reduced pathogen of swine that lacks the genetic repertoire to synthesize amino acids and relies on the host for availability of amino acids for growth. M. hyopneumoniae recruits plasmin(ogen) onto its cell surface via the P97 and P102 adhesins and the glutamyl aminopeptidase MHJ-0125. Plasmin plays an important role in regulating the inflammatory response in the lungs of pigs infected with M. hyopneumoniae. We show that recombinant MHJ-0461 (rMHJ-0461) functions as a leucine aminopeptidase (LAP) with broad substrate specificity for leucine, alanine, phenylalanine, methionine and arginine and that MHJ-0461 resides on the surface of M. hyopneumoniae. rMHJ-0461 also binds heparin, plasminogen and foreign DNA. Plasminogen bound to rMHJ-0461 was readily converted to plasmin in the presence of tPA. Computational modelling identified putative DNA and heparin-binding motifs on solvent-exposed sites around a large pore on the LAP hexamer. We conclude that MHJ-0461 is a LAP that moonlights as a multifunctional adhesin on the cell surface of M. hyopneumoniae

Mycoplasma hyopneumoniae resides intracellularly within porcine epithelial cells

© 2018, The Author(s). 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

Post-translational processing targets functionally diverse proteins in Mycoplasma hyopneumoniae

© 2016 The Authors. Mycoplasma hyopneumoniae is a genome-reduced, cell wall-less, bacterial pathogen with a predicted coding capacity of less than 700 proteins and is one of the smallest self-replicating pathogens. The cell surface of M. hyopneumoniae is extensively modified by processing events that target the P97 and P102 adhesin families. Here, we present analyses of the proteome of M. hyopneumoniae-type strain J using protein-centric approaches (one- and two-dimensional GeLC-MS/MS) that enabled us to focus on global processing events in this species. While these approaches only identified 52% of the predicted proteome (347 proteins), our analyses identified 35 surface-associated proteins with widely divergent functions that were targets of unusual endopro-teolytic processing events, including cell adhesins, lipoproteins and proteins with canonical functions in the cytosol that moonlight on the cell surface. Affinity chromatography assays that separately used heparin, fibronectin, actin and host epithelial cell surface proteins as bait recovered cleavage products derived from these processed proteins, suggesting these fragments interact directly with the bait proteins and display previously unrecognized adhesive functions. We hypothesize that protein processing is underestimated as a post-translational modification in genome-reduced bacteria and prokaryotes more broadly, and represents an important mechanism for creating cell surface protein diversity

Elongation factor Tu is a multifunctional and processed moonlighting protein

© 2017 The Author(s). Many bacterial moonlighting proteins were originally described in medically, agriculturally, and commercially important members of the low G + C Firmicutes. We show Elongation factor Tu (Ef-Tu) moonlights on the surface of the human pathogens Staphylococcus aureus (SaEf-Tu) and Mycoplasma pneumoniae (MpnEf-Tu), and the porcine pathogen Mycoplasma hyopneumoniae (MhpEf-Tu). Ef-Tu is also a target of multiple processing events on the cell surface and these were characterised using an N-terminomics pipeline. Recombinant MpnEf-Tu bound strongly to a diverse range of host molecules, and when bound to plasminogen, was able to convert plasminogen to plasmin in the presence of plasminogen activators. Fragments of Ef-Tu retain binding capabilities to host proteins. Bioinformatics and structural modelling studies indicate that the accumulation of positively charged amino acids in short linear motifs (SLiMs), and protein processing promote multifunctional behaviour. Codon bias engendered by an A + T rich genome may influence how positively-charged residues accumulate in SLiMs

Exploitation of plasmin(ogen) by bacterial pathogens of veterinary significance

© 2015 Elsevier B.V. The plasminogen (Plg) system plays an important homeostatic role in the degradation of fibrin clots, extracellular matrices and tissue barriers important for cellular migration, as well as the promotion of neurotransmitter release. Plg circulates in plasma at physiologically high concentrations (150-200μgml-1) as an inactive proenzyme. Proteins enriched in lysine and other positively charged residues (histidine and arginine) as well as glycosaminoglycans and gangliosides bind Plg. The binding interaction initiates a structural adjustment to the bound Plg that facilitates cleavage by proteases (plasminogen activators tPA and uPA) that activate Plg to the active serine protease plasmin. Both pathogenic and commensal bacteria capture Plg onto their cell surface and promote its conversion to plasmin. Many microbial Plg-binding proteins have been described underpinning the importance this process plays in how bacteria interact with their hosts. Bacteria exploit the proteolytic capabilities of plasmin by (i) targeting the mammalian fibrinolytic system and degrading fibrin clots, (ii) remodeling the extracellular matrix and generating bioactive cleavage fragments of the ECM that influence signaling pathways, (iii) activating matrix metalloproteinases that assist in the destruction of tissue barriers and promote microbial metastasis and (iv) destroying immune effector molecules. There has been little focus on the exploitation of the fibrinolytic system by veterinary pathogens. Here we describe several pathogens of veterinary significance that possess adhesins that bind plasmin(ogen) onto their cell surface and promote its activation to plasmin. Cumulative data suggests that these attributes provide pathogenic and commensal bacteria with a means to colonize and persist within the host environment