Abstract
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.
Highlights
Respiratory diseases are among the most important health problems associated with swine production
The proteins were electrophoretically transferred onto polyvinylidene fluoride (PVDF) membranes (Bio-Rad, USA) and developed with Ponceau-S as the loading control
After washing with TBST buffer (20 Mm Tris-HCl [pH 7.6], 150 mM NaCl, and 0.1% Tween-20) and blocking with TBST buffer containing 5% skimmed milk, the membranes were incubated with primary antibody against rEF-Tu (1:2,000 dilution), followed by horseradish peroxidase (HRP)-conjugated secondary antibody (1:10,000 dilution)
Summary
Respiratory diseases are among the most important health problems associated with swine production. The P102 protein, which is located in the same operon as P97, participates in virulence as it is expressed in vivo during infection (Adams et al, 2005) and can recruit plasminogen and fibronectin to the surface of M. hyopneumoniae (Seymour et al, 2012) In addition to these findings, factors such as P159 (Burnett et al, 2006), P146 (Mayor et al, 2007), P216 (Wilton et al, 2009), Mhp271 (Deutscher et al, 2012), Mhp107 (Seymour et al, 2011), and Mhp683 (Bogema et al, 2011) have been shown to be associated with the adhesion process. The pathogenesis and possible virulence factors of M. hyopneumoniae are not yet fully known (Simionatto et al, 2013), and the exact mechanism by which it adheres to epithelial cells and a clear picture of its virulence and pathogenicity remain to be understood
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