Novel Secreted Protein of Mycoplasma bovis MbovP280 Induces Macrophage Apoptosis Through CRYAB

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.

The attenuated Mycoplasma bovis strain promotes apoptosis of bovine macrophages by upregulation of CHOP expression

“…The findings are in partial agreement with previous work that M. bovis-induced apoptosis in epithelial cells (MAC-T and EBL cells) via a mitochondria-dependent pathway and ER-stress-dependent signaling pathway, respectively (Liu et al, 2020;Wu et al, 2021). Consistent with our previous study, live M. bovis P1 induced around 14-15% apoptosis in BoMac cells (Zhao et al, 2021). However, we found that the attenuated strain P150 induced significantly more apoptosis than the wildtype strain P1-infected BoMac cells.…”

Heterologous Expression, Purification, and Immunomodulatory Effects of Recombinant Lipoprotein GUDIV-103 Isolated from Ureaplasma diversum

“…This result strongly suggested that rGUDIV-103 played an important role in generating a humoral response in the host by producing specific antibodies. Recombinant proteins from other Mollicutes, such as Mhp597 from M. hyopneumoniae [ 62 ], MbovP280 from M. bovis [ 63 ], surface proteins (MAG_1560, MAG_6130, and P40) from M. agalactiae [ 37 ], and GrpE from U. urealyticum [ 64 ], also induce antibody production.…”

“…U. diversum inoculations frequently induce TNF-α , IL-1β , and IL-6 expression [ 31 , 76 ]. Pro-inflammatory cytokine stimulation associated with TLR2 and TLR6 activation or expression has been defined in several Mollicutes species that infect diverse hosts [ 35 , 63 , 68 , 74 ]. Although TLR2 seems to play an important role in the production of pro-inflammatory cytokines, strong evidence indicates that signalling via TLR4 is directly associated with IL-1β and TNF-α expression when macrophages are inoculated with total LAMP extracts, viable U. diversum , or heat-inactivated U. diversum [ 4 , 29 ].…”

Novel mycoplasma nucleomodulin MbovP475 decreased cell viability by regulating expression of CRYAB and MCF2L2

“…However, to determine the functions of these proteins is difficult because most of them are putative proteins, and efficient genetic tools and animal infection models for confirmation are lacking. To date, the functions of only a small portion of mycoplasma secreted proteins have been demonstrated, such as P80 of M. hominis [ 24 ], the Community Acquired Respiratory Distress Syndrome (CARDS) toxin [ 25 ], and Mpn491 nuclease [ 26 ] of M. pneumoniae , the apoptosis inducer MbovP280 of M. bovis [ 27 ], and secreted serine protease S41 of M. capricolum [ 28 ].…”