Abstract
Mycoplasma bovis is an important pathogenic mycoplasma, causing the cattle industry serious economic losses. Adhesion is a crucial step in the mycoplasmas’ infection and colonization process; fibronectin (Fn), an extracellular matrix glycoprotein, is a molecular bridge between the bacterial adhesins and host cell receptors. The present study was designed to characterize the Fn-binding ability of methylenetetrahydrofolate-tRNA-(uracil-5-)-methyltransferase (TrmFO) and its role in M. bovis cytoadherence. The trmFO (MBOV_RS00785) gene was cloned and expressed in E. coli BL21, and polyclonal antibodies against the recombinant TrmFO (rTrmFO) were raised in rabbits. Immunoblotting demonstrated that TrmFO was an immunogenic component, and the TrmFO expression was conserved in different M. bovis isolates. The mycoplasmacidal assay further showed that in the presence of complement, rabbit anti-recombinant TrmFO serum exhibited remarkable mycoplasmacidal efficacy. TrmFO was detected in both the M. bovis membrane and cytoplasm. By ligand dot blot and enzyme-linked immunosorbent assay (ELISA) binding assay, we found that rTrmFO bound Fn in a dose-dependent manner. Immunostaining visualized by confocal laser scanning microscopy showed that rTrmFO had capacity to adhere to the embryonic bovine lung (EBL) cells. In addition, the adhesion of M. bovis and rTrmFO to EBL cells could be inhibited by anti-rTrmFO antibodies. To the best of our knowledge, this is the first report to characterize the Fn-binding ability of TrmFO and its role in the bacterial adhesion to host cells.
Highlights
Mycoplasma bovis is the causative agent of bovine mycoplasmosis which results in pneumonia, mastitis, arthritis and genital disorders [1,2]
Several Fn-binding proteins (FnBPs) have been identified in mycoplasmas, including elongation factor Tu (EF-Tu), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and pyruvate dehydrogenase A-C (PDH A-C) in Mycoplasma pneumoniae [17,18], Mhp182 and Mhp271 in Mycoplasma hyopneumoniae [19,20], enolase in Mycoplasma synoviae [21], PlpA and Hlp3 in Mycoplasma gallisepticum [22], and cytoadhesive lipoprotein T (LppT) in Mycoplasma conjunctivae [23]
TrmFO was found to be a novel adhesin in M. bovis, based on the direct adhesion and inhibition assay in which recombinant TrmFO (rTrmFO) was observed to adhere to embryonic bovine lung (EBL) cells and the adhesion was inhibited by anti-rTrmFO polyclonal antibodies
Summary
Mycoplasma bovis is the causative agent of bovine mycoplasmosis which results in pneumonia, mastitis, arthritis and genital disorders [1,2]. Several FnBPs have been identified in mycoplasmas, including elongation factor Tu (EF-Tu), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and pyruvate dehydrogenase A-C (PDH A-C) in Mycoplasma pneumoniae [17,18], Mhp182 and Mhp271 in Mycoplasma hyopneumoniae [19,20], enolase in Mycoplasma synoviae [21], PlpA and Hlp in Mycoplasma gallisepticum [22], and cytoadhesive lipoprotein T (LppT) in Mycoplasma conjunctivae [23] These findings highlight the possibility that there may be FnBPs in M. bovis, which might contribute to M. bovis infection in and persistence on host cells. Using an iTRAQ-based quantitative proteomic analysis, we found that expression of TrmFO was down-regulated in the attenuated M. bovis-150 strain compared to the virulent strain M. bovis HB0801 (unpublished data) Among these down-regulated proteins in the attenuated M. bovis-150 strain, NADH oxidase and variable lipoprotein VspX have been reported to contribute to M. bovis adhesion to host cells [11,13]. In the light of this, the aim of current study was to characterize the moonlight function of TrmFO in the pathogenesis of M. bovis
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