Localization of <i>Mycoplasma bovis</i> α-enolase.

<p>Western blot analysis of bovine serum albumin (BSA; lane 1), cell soluble cytosolic fraction proteins (lane 2), cell membrane fraction proteins (lane 3), whole cell protein (lane 4), and purified recombinant <i>Mycoplasma bovis</i> α-enolase blotted onto a nylon membrane and detected with rabbit anti-recombinant enolase antibodies (lane 5) blotted onto a nylon membrane and detected with rabbit anti-recombinant enolase antibodies. M: protein marker.</p

Ligand blotting assay.

<p>Membrane fraction proteins(lane 1), soluble cytosolic fraction proteins(lane 2), commercial α-enolase(lane 3), recombinant <i>Mycoplasma bovis</i> α-enolase (lane 4), and BSA(lane 5) were blotted onto nitrocellulose membranes following SDS-PAGE, and then incubated with plasminogen post-blocking. Bound plasminogen was detected with sheep anti-plasminogen polyclonal antibody. M: protein marker.</p

Adherence and inhibition assays.

<p>Experiments were performed in triplicate. Hank's Balanced Salt Solution (HBSS). *P<0.05, compared with the corresponding group using non-immune rabbit antibodies.</p

Assay of rVpmaX adhesion and adhesion inhibition to EBL cells visualized by confocal laser scanning microscopy.

<p>Active rVpmaX interacted with fixed EBL cells, and the surplus protein was rinsed away by washing with PBST. The attached protein was immunostained with rabbit anti-rVpmaX antibody and mouse anti-rabbit IgG-FITC. The EBL cell membranes were labeled with 1,19-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI), and the cell nuclei were counter-labeled with 49,6-diamidino-2-phenylindole (DAPI). (A1–A2) 10 µg rVpmaX adhering to EBL cells. (B) Adhesion inhibition of 10 µg rVpmaX to EBL cells by 10 µl rabbit anti-rVpmaX serum. (C) Adhesion of 20 µg rVpmaX to EBL cells. (D) The adhesion of 20 µg rVpmaX to EBL cells was inhibited by 20 µl rabbit anti-rVpmaX serum. (E) EBL cells without protein added.</p

Binding of anti-α-enolase antibodies to recombinant <i>Mycoplasma bovis</i> α-enolase (rMbEno).

<p>ELISA plate wells were coated with rMbEno (1.0 ug protein/well). Well contents were reacted with serial dilutions (1/200 to 1/12800) of rabbit anti-α-enolase antibodies, followed by anti-rabbit IgG(whole molecule) peroxidase conjugate. Results were determined using o-phenylenediamine as a substrate, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038836#s4" target="_blank">Materials and Methods</a>.</p

Localization of VpmaX in <i>M. bovis</i> Hubei-1.

<p>Western blot analysis of rVpmaX (lane 1), <i>M. bovis</i> total proteins (lane 2), cell membrane fraction proteins (lane 3), cell soluble cytosolic fraction proteins (lane 4), and bovine serum albumin (lane 5) using rabbit anti-rVpmaX serum and a peroxidase-conjugated secondary antibody.</p

Overlapping PCR primers using for amplification of α-Enolase.

<p>Overlapping PCR primers using for amplification of α-Enolase.</p

Plasminogen binding assays.

<p>Plates were coated as detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038836#s4" target="_blank">Materials and Methods</a>.(A) Plasminogen (Plg) (0.5 to 0.007732unit/well) binds to fixed recombinant <i>Mycoplasma bovis</i> α-enolase (rMbEno) in a concentration-dependent manner. (B) In a parallel assay, Plg (0.5 to 0.007732/units) binds to whole cell proteins in a concentration-dependent manner. (C) In a competition assay, binding of plasminogen is inhibited by increasing concentrations of anti-rMbEno antiserum (in serial dilutions from 1/100 to 1/6400). (D) Negative control: Plasminogen binding is inhibited by an anti<i>-M. bovis</i> monoclonal antibody. Three independent experiments were performed in triplicate. The error bars indicate the standard deviations from three independent experiments.</p

Data_Sheet_1_Elongation Factor Thermo Unstable (EF-Tu) Moonlights as an Adhesin on the Surface of Mycoplasma hyopneumoniae by Binding to Fibronectin.CSV

<p>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 (K_D) 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.</p><p>HIGHLIGHTS</p><p>Elongation factor thermo unstable (EF-Tu) exists on the cell surface of M. hyopneumoniae.</p><p>EF-Tu moonlights as an adhesin of M. hyopneumoniae.</p><p>The adhesive effect of EF-Tu is partly meditated by fibronectin.</p><p></p