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

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.

Mbov_0503 Encodes a Novel Cytoadhesin that Facilitates Mycoplasma bovis Interaction with Tight Junctions

“…A number of nonclassical secretory proteins have been identified in mycoplasmas, such as EF-Tu and DnaK in M. ovipneumoniae [52]. Several of these proteins can have moonlighting activities, such as the EF-Tu in M. hypneumoniae [53] and the TrmFO and NADH oxidase in M. bovis that were identified to be cytoadherence factors, [16,17]. Additional studies are needed to characterize these proteins and their potential role in M. bovis pathogenesis.…”

Fructose-1,6-bisphosphate aldolase encoded by a core gene of Mycoplasma hyopneumoniae contributes to host cell adhesion

“…The full-length fba gene (MHP168_014) was cloned into the pET-28a(+) vector by site-directed mutation and overlap extension (SOE-PCR) due to the existence of rare codons, and rFBA was produced in Escherichia coli BL21 (DE3) and purified by Ni-chelating chromatography as described in a previous study [ 19 ]. A polyclonal antibody was raised against rFBA by subcutaneously immunising 1-month-old New Zealand white rabbits.…”

“…Mycoplasma hyopneumoniae cells (1 × 10 7 CCU/mL) were washed three times with PBS and pre-incubated with polyclonal antibody raised against rFBA or preimmune sera (1:20 dilution) at 37 °C for 30 min. Bacteria suspended in RPMI-1640 medium were added to 24-well cell plates containing confluent STEC, and plates were centrifuged at 800 × g for 10 min and incubated at 4 °C for 2 h. Bacteria counting including bacterial genome extraction and real-time PCR was performed according to a previous method [ 19 , 37 ]. The assay was performed in triplicate, and data were analysed using Student’s t tests with SPSS 20.0 ( p ≤ 0.05 was considered statistically significant).…”

“…Glyceraldehyde 3-phosphate dehydrogenase (MHJ_0031) has also been identified as a putative moonlighting protein because it was predicted within a putative heparin-binding region [ 18 ]. Finally, elongation factor thermo unstable (EF-Tu), functions as an adhesin on the surface of M. hyopneumoniae by binding to multiple host proteins [ 19 , 20 ]. However, despite these findings, the exact mechanisms responsible for pathogenesis and potential virulence factors in M. hyopneumoniae remain poorly understood [ 21 ].…”

Undervalued ubiquitous proteins

“…Despite various proteins, such as integrins, even fibronectins help various cancer-triggering pathogenic stimuli to bind such as has been shown for various herpes viruses [112][113][114][115], hepatitis viruses [116][117][118], schistosomiasis [119][120][121][122][123], or opisthorchis [124,125], helicobacter [126][127][128][129], or mycoplasma [130][131][132][133][134]. Otherwise autocrine fibronectin inhibits cancer spread [135] and decreased fibronectin seems to be essential during metastasis [136] as natural killer cells (NK cells) mediated control can inhibit metastasis by increase of fibronectin [137].…”