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Abstract
Mycoplasma bovis causes important diseases and great losses on feedlots and dairy farms. However, there are only a few measures to control M. bovis-related diseases. As in other mycoplasma species, this is predominantly because the virulence related factors of this pathogen are largely unknown. Therefore, in this study, we aimed to identify novel virulence-related factors among the secreted proteins of M. bovis. Using bioinformatic tools to analyze its secreted proteins, we preliminarily predicted 39 secreted lipoproteins, and then selected 11 of them for confirmation based on SignalP scores >0.6 or SceP scores >0.8 and conserved domains. These 11 genes were cloned after gene modification based on the codon bias of Escherichia coli and expressed. Mouse antiserum to each recombinant protein was developed. A western blotting assay with these antisera confirmed that MbovP280 and MbovP475 are strongly expressed and secreted proteins, but only MbovP280 significantly reduced the viability of bovine macrophages (BoMac). In further experiments, MbovP280 induced the apoptosis of BoMac treated with both live M. bovis and MbovP280 protein. The conserved coiled-coil domain of MbovP280 at amino acids 210–269 is essential for its induction of apoptosis. Further, immunoprecipitation, mass spectrometry, and coimmunoprecipitation assays identified the anti-apoptosis regulator αB-crystallin (CRYAB) as an MbovP280-binding ligand. An αβ-crystallin knockout cell line BoMac-cryab−, Mbov0280-knockout M. bovis strain T9.297, and its complemented M. bovis strain CT9.297 were constructed and the apoptosis of BoMac-cryab− induced by these strains was compared. The results confirmed that CRYAB is critical for MbovP280 function as an apoptosis inducer in BoMac. In conclusion, in this study, we identified MbovP280 as a novel secreted protein of M. bovis that induces the apoptosis of BoMac via its coiled-coil domain and cellular ligand CRYAB. These findings extend our understanding of the virulence mechanism of mycoplasmal species.
Highlights
Mycoplasma bovis is a member of the class Mollicutes, a group of the smallest self-replicating wall-less prokaryotes
The results indicated that all the proteins were present in large amounts in the whole-cell proteins, but only MbovP280 and MbovP475 were abundant in the secretome, whereas the other proteins were represented by only variously weak bands in the secretome (Figure 1B, Supplementary Figure 1)
In order to detect the secretion process of MbovP280 and MbovP475 in vitro, the presence of MbovP280 and MbovP475 in the culture supernatant of M. bovis was kinetically examined, while the membraneassociated protein NOX served as the negative control
Summary
Mycoplasma bovis is a member of the class Mollicutes, a group of the smallest self-replicating wall-less prokaryotes. It causes several important diseases, including pneumonia, mastitis, and arthritis, in cattle throughout the world [1,2,3]. Several proteins of Mycoplasma species, including P80 of M. hominis [11], P102 of M. hyopneumoniae [12], Mpn491 of M. pneumoniae [13], CARDS toxin of M. pneumoniae [14], and a nuclease encoded by MBOV_RS02825 of M. bovis [15], have been shown to be secreted proteins. One way dealing with this awkward situation tactfully is to combine the prediction of secreted proteins with bioinformatic tools, such as SignalP, SecretomeP, PSORT-B, and PrediSi [17,18,19] and identification of the secreted proteins with proteomic methods
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