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.

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BackgroundAir-liquid interface (Ali) systems allow the establishment of a culture environment more representative of that in vivo than other culture systems. They are useful for performing mechanistic studies of respiratory epithelial cells as drug permeation barriers and can be used to study the interactions between hosts and respiratory pathogens. However, there have been few studies concerning Ali cultures of primary swine tracheal epithelial cells (STECs) and an immortalized STEC line, and the differences between these two systems remain poorly defined.ResultsIn this study, we established Ali culture systems for primary STECs and for immortalized STEC line, and we systematically compared the differentiation capacities and immunological functions of these systems for the first time. Under Ali culture conditions, immortalized STEC line and primary STECs could survive for at least forty days, formed tight junctions and differentiated into stratified cells. They both possessed complete abilities to produce mucin and inflammatory cytokines and develop cilia. However, in contrast to primary STECs, which had a heterogeneous morphology, Ali-cultured immortalized STEC line appeared to be a homogenous population. The formation of tight junctions in Ali-cultured primary STECs was superior to that in immortalized STEC line. In addition, cilia in Ali-cultured immortalized STEC line were more pronounced, but their duration of expression was shorter than in primary STECs.ConclusionsAli-cultured primary STECs and immortalized STEC line systems possessing complete abilities to undergo ciliary differentiation and inflammatory cytokine production were established for the first time in this study, and several differences in morphology and the formation of tight junctions and cilia were observed between these two systems. These two systems will be important tools for drug screening studies, as well as for detailed analyses of the interactions between hosts and respiratory pathogens.

Primary porcine bronchial epithelial cells (PBECs) are an ideal model to study the molecular and pathogenic mechanisms of various porcine respiratory pathogens. However, the short lifespan of primary PBECs greatly limit their application. Here, we isolated and cultured primary PBECs and established immortalized PBECs by transfecting primary PBECs with the pEGFP-hTERT recombinant plasmid containing human telomerase reverse transcriptase (hTERT). Immortalized PBECs (hTERT-PBECs) retained the morphological and functional features of primary PBECs as indicated by cytokeratin 18 expression, telomerase activity assay, proliferation assays, karyotype analysis, and quantitative reverse-transcriptase polymerase chain reaction. Compared to primary PBECs, hTERT-PBECs had higher telomerase activity, extended replicative lifespan, and displayed enhanced proliferative activity. Moreover, this cell line is not transformed in vitro and does not exhibit a malignant phenotype in vivo, suggesting that it can be safely used in further studies. Besides, hTERT-PBECs were susceptible to swine influenza virus of H3N2 subtype and porcine circovirus type 2. In conclusion, the immortalized hTERT-PBECs represent a valuable in vitro model, which can be widely used in the study of porcine respiratory pathogenic infections.

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