Studies On The Molecular Mechanism Of Mycoplasma Hyopneumoniae In Inhibiting Host Unfolded Protein Response To Promote Its Adhesion To Host Cells

Mycoplasma hyopneumoniae(Mhp)is the etiological agent of porcine enzootic pneumonia(PEP),and it remains one of the most economically significant respiratory pathogens.Understanding the molecular mechanisms of its pathogenicity is critical for developing effective interventions to control this swine respiratory disease.The unfolded protein response(UPR)is the first line of defense against pathogens,manipulation of the host UPR process plays an important role in the pathogenesis of invasive pathogens.Therefore,we began with this study based on the interaction between UPR and Mhp.We observed that Mhp infection suppressed the UPR target molecules GRP78 and CHOP,even blocked the UPR induction of the ER stress inducer Tunicamycin in PTECs,indicating that Mhp rarely inhibited cellular UPR.And this inhibitory effect was only observed in Simkania negevensis.We further found Mhp infection reduced PERK/eIF2? phosphorylation,ATF6 cleavage and XBP1 splicing,suggesting the suppression of the three branches of UPR(PERK?ATF6 and IRE1? pathway).Importantly,we utilized specific chemical inhibitors to simulate the UPR inhibition effect of Mhp,TaqMan qPCR and flow cytometry was performed to identify that the UPR inhibition promoted Mhp adhesion and infection to PTECs.By contrast,the activation of the UPR branch(by a specific activator or protein overexpression)reduced the number of Mhp adhering to PTECs.As we know that Mhp is a member of the genus Mycoplasma,adhesion to host cells is the first step of its colonization,and is also the critical step of its infection and subsequent proliferation.To further determine the underlying molecular mechanism on the promotion of Mhp adhesion and infection induced by UPR inhibition,we explored the downstream related signaling pathways of the UPR.We demonstrated that Mhp infection interfered with NF-?B signaling by inhibiting P65 phosphorylation,which is later proved to be a dependent signaling pathway of porcine ?-defensin 2(PBD-2)production.PBD-2 is thought to be an important antimicrobial peptide(AMP)against bacterial infection in epithelial cells,especially the respiratory tract pathogens.Thus,the reduction of PBD-2 release was due to the NF-?B signaling inhibition,which led to the promotion of Mhp adhesion and infection.We further discovered that the reduction of PBD-2 release increased the expression of Mhp adhesins P97 and P116.Taken together,the suppression of NF-?B activation cascade reducing the release of PBD-2 is a target of UPR inhibition,which promotes Mhp adhesion and infection.Our study highlights a key role of UPR in the regulation of PBD-2 release and Mhp infection,provides new insights into the molecular pathogenesis of Mhp and sheds light upon their interactions with the host.