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A processed multidomain mycoplasma hyopneumoniae adhesin binds fibronectin, plasminogen, and swine respiratory cilia.

ABSTRACT: Porcine enzootic pneumonia is a chronic respiratory disease that affects swine. The etiological agent of the disease, Mycoplasma hyopneumoniae, is a bacterium that adheres to cilia of the swine respiratory tract, resulting in loss of cilia and epithelial cell damage. A M. hyopneumoniae protein P116, encoded by mhp108, was investigated as a potential adhesin. Examination of P116 expression using proteomic analyses observed P116 as a full-length protein and also as fragments, ranging from 17 to 70 kDa in size. A variety of pathogenic bacterial species have been shown to bind the extracellular matrix component fibronectin as an adherence mechanism. M. hyopneumoniae cells were found to bind fibronectin in a dose-dependent and saturable manner. Surface plasmon resonance was used to show that a recombinant C-terminal domain of P116 bound fibronectin at physiologically relevant concentrations (K(D) 24 ± 6 nm). Plasmin(ogen)-binding proteins are also expressed by many bacterial pathogens, facilitating extracellular matrix degradation. M. hyopneumoniae cells were found to also bind plasminogen in a dose-dependent and saturable manner; the C-terminal domain of P116 binds to plasminogen (K(D) 44 ± 5 nm). Plasminogen binding was abolished when the C-terminal lysine of P116 was deleted, implicating this residue as part of the plasminogen binding site. P116 fragments adhere to the PK15 porcine kidney epithelial-like cell line and swine respiratory cilia. Collectively these data suggest that P116 is an important adhesin and virulence factor of M. hyopneumoniae.

SUBMITTER: Seymour LM

PROVIDER: S-EPMC2962497 | biostudies-other | 2010 Oct

REPOSITORIES: biostudies-other

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A processed multidomain mycoplasma hyopneumoniae adhesin binds fibronectin, plasminogen, and swine respiratory cilia.

Seymour Lisa M LM Deutscher Ania T AT Jenkins Cheryl C Kuit Tracey A TA Falconer Linda L Minion F Chris FC Crossett Ben B Padula Matthew M Dixon Nicholas E NE Djordjevic Steven P SP Walker Mark J MJ

The Journal of biological chemistry 20100902 44

Porcine enzootic pneumonia is a chronic respiratory disease that affects swine. The etiological agent of the disease, Mycoplasma hyopneumoniae, is a bacterium that adheres to cilia of the swine respiratory tract, resulting in loss of cilia and epithelial cell damage. A M. hyopneumoniae protein P116, encoded by mhp108, was investigated as a potential adhesin. Examination of P116 expression using proteomic analyses observed P116 as a full-length protein and also as fragments, ranging from 17 to 70 ...[more]

PMID: 20813843

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Project description:Mycoplasma hyopneumoniae is a colonizing respiratory pathogen that can cause great economic losses to the pig industry worldwide. Although putative virulence factors have been reported, the pathogenesis of this species remains unclear. Here, we used the virulent M. hyopneumoniae strain 168 to infect swine tracheal epithelial cells (STEC) to identify the infection-associated factors by two-dimensional electrophoresis (2-DE). Whole proteins of M. hyopneumoniae were obtained and compared with samples cultured in broth. Six differentially expressed proteins with an increase in abundance of ?1.5 in the cell infection group were successfully identified. A String network of virulence-associated proteins showed that all the six differential abundance proteins were involved in virulence of M. hyopneumoniae. One of the most important upregulated hubs in this network, elongation factor thermo unstable (EF-Tu), which showed a relatively higher expression in M. hyopneumoniae-infected STEC and obtained a higher score on mass spectrometry was successfully recombined. In addition to its canonical enzymatic activities in protein synthesis, EF-Tu was also reported to be located on the cell surface as an important adhesin in many other pathogens. The cell surface location of EF-Tu was then observed in M. hyopneumoniae with flow cytometry. Recombinant EF-Tu (rEF-Tu) was found to be able to adhere to STEC and anti-rEF-Tu antibody enclosed M. hyopneumoniae decreased adherence to STEC. In addition, surface plasmon resonance (SPR) analysis showed that rEF-Tu could bind to fibronectin with a specific and moderately strong interaction, a dissociation constant (KD) of 605 nM. Furthermore, the block of fibronectin in STEC also decreased the binding of M. hyopneumoniae to the cell surface. Collectively, these data imply EF-Tu as an important adhesin of M. hyopneumoniae and fibronectin as an indispensable receptor on STEC. The binding between EF-Tu with fibronectin contributes to the adhesion of M. hyopneumoniae to STEC. HIGHLIGHTS Elongation factor thermo unstable (EF-Tu) exists on the cell surface of M. hyopneumoniae.EF-Tu moonlights as an adhesin of M. hyopneumoniae.The adhesive effect of EF-Tu is partly meditated by fibronectin.

Project description:BackgroundMycoplasma synoviae (MS) is an important pathogen causing respiratory diseases and arthritis in chickens and turkeys, thus, resulting in serious economic losses to the poultry industry. Membrane-associated proteins are thought to play important roles in cytoadherence and pathogenesis. NADH oxidase (NOX) is an oxidoreductase involved in glycolysis, which is thought to be a multifunctional protein and potential virulence factor in some pathogens. However, little is known regarding the NOX of MS (MSNOX). We previously demonstrated that MSNOX was a metabolic enzyme distributed in not only the cytoplasm but also the MS membrane. This study was aimed at exploring NOX's potential as a diagnostic antigen and its role in MS cytoadherence.ResultsWestern blots and ELISAs indicated that recombinant MSNOX (rMSNOX) protein reacted with sera positive for various MS isolates, but not MG isolates or other avian pathogens, thus, suggesting that rMSNOX is a potential diagnostic antigen. In addition, rabbit anti-rMSNOX serum showed substantial complement-dependent mycoplasmacidal activity toward various MS isolates and MG Rlow. MSNOX protein was found not only in the cytoplasm but also on the membrane of MS through suspension immunofluorescence and immunogold electron microscopy assays. Indirect immunofluorescence assays indicated that rMSNOX adhered to DF-1 cells, and this adherence was inhibited by rabbit anti-rMSNOX, but not anti-MG serum. Furthermore, indirect immunofluorescence and colony counting assays confirmed that the rabbit anti-rMSNOX serum inhibited the adherence of various MS isolates but not MG Rlow to DF-1 cells. Moreover, plasminogen (Plg)- and fibronectin (Fn)-binding assays demonstrated that rMSNOX bound Plg and Fn in a dose-dependent manner, thereby further confirming that MSNOX may be a putative adhesin.ConclusionMSNOX was identified to be a surface immunogenic protein that has good immunoreactivity and specificity in Western blot and ELISA, and therefore, may be used as a potential diagnostic antigen in the future. In addition, rMSNOX adhered to DF-1 cells, an effect inhibited by rabbit anti-rMSNOX, but not anti-MG serum, and anti-rMSNOX serum inhibited the adherence of various MS isolates, but not MG Rlow, to DF-1 cells, thus indicating that the inhibition of adherence by anti-MSNOX serum was MS specific. Moreover, rMSNOX adhered to extracellular matrix proteins including Plg and Fn, thus suggesting that NOX may play important roles in MS cytoadherence and pathogenesis. Besides, rabbit anti-rMSNOX serum presented complement-dependent mycoplasmacidal activity toward both MS and MG, indicating the MSNOX may be further studied as a potential protective vaccine candidate.

Project description:BACKGROUND: Mycoplasma synoviae is an avian pathogen that can lead to respiratory tract infections and arthritis in chickens and turkeys, resulting in serious economic losses to the poultry industry. Enolase reportedly plays important roles in several bacterial pathogens, but its role in M. synoviae has not been established. Therefore, in this study, the enolase encoding gene (eno) of M. synoviae was amplified from strain WVU1853 and expressed in E. coli BL21 cells. Then the enzymatic activity, immunogenicity and binding activity with chicken plasminogen (Plg) and human fibronectin (Fn) was evaluated. RESULTS: We demonstrated that the recombinant M. synoviae enolase protein (rMsEno) can catalyze the conversion of 2-phosphoglycerate (2-PGA) to phosphoenolpyruvate (PEP), the Km and Vmax values of rMsEno were 1.1 × 10(-3) M and 0.739 ?mol/L/min, respectively. Western blot and immuno-electron microscopy analyses confirmed that enolase was distributed on the surface and within the cytoplasm of M. synoviae cells. The binding assays demonstrated that rMsEno was able to bind to chicken Plg and human Fn proteins. A complement-dependent mycoplasmacidal assay demonstrated that rabbit anti-rMsEno serum had distinct mycoplasmacidal efficacy in the presence of complement, which also confirmed that enolase was distributed on the surface of M. synoviae. An inhibition assay showed that the adherence of M. synoviae to DF-1 cells pre-treated with Plg could be effectively inhibited by treatment with rabbit anti-rMsEno serum. CONCLUSION: These results reveal that M. synoviae enolase has good catalytic activity for conversion of 2-PGA to PEP, and binding activity with chicken Plg and human Fn. Rabbit anti-rMsEno serum displayed an obvious complement-dependent mycoplasmacidal effect and adherent inhibition effect. These results suggested that the M. synoviae enolase plays an important role in M. synoviae metabolism, and could potentially impact M. synoviae infection and immunity.

Dataset Information

A processed multidomain mycoplasma hyopneumoniae adhesin binds fibronectin, plasminogen, and swine respiratory cilia.

Publications

A processed multidomain mycoplasma hyopneumoniae adhesin binds fibronectin, plasminogen, and swine respiratory cilia.

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