Mmm-derived lipid-associated membrane proteins activate IL-1ß production through the NF-κB pathway via TLR2, MyD88, and IRAK4.

Mycoplasma mycoides subsp.mycoides (Mmm) is a pathogen that causes pneumonia, otitis media, and arthritis in young calves. Its pathogenesis is attributed in part to excessive immune responses. Mmm-derived lipid-associated membrane proteins (LAMPs) are potent inducers of the host innate immune system; however, interactions between Mmm-derived LAMPs as pathogenic agents, toll-like receptors (TLRs), and the signaling pathways responsible for activating inflammation and nuclear factor (NF)-κB have not been fully elucidated. Here, we analyzed the expression kinetics of interleukin (IL)-1ß in Mmm-derived LAMP-stimulated embryonic bovine lung (EBL) cells and found that Mmm-derived LAMPs induced IL-1ß expression. Subcellular localization analysis revealed the nuclear translocation of the NF-κB p65 subunit after EBL cells were stimulated with Mmm-derived LAMPs. Furthermore, a specific inhibitor assay demonstrated that NF-κB is required for Mmm-derived LAMP-induced IL-1ß expression. Additionally, overexpression of TLR2, myeloid differentiation primary response gene 88 (MyD88), and IL-1 receptor-associated kinase 4 (IRAK4) increased IL-1ß expression during LAMP stimulation, and TLR2-neutralizing antibodies reduced IL-1ß expression in EBL cells during LAMP stimulation. Furthermore, LAMPs inhibited IL-1ß expression following transfection with dominant-negative MyD88 and IRAK4 variants. These results suggested that Mmm-derived LAMPs activate IL-1ß production through the NF-κB pathway via TLR2, MyD88, and IRAK4.

P19 contributes to Mycoplasma mycoides subsp. mycoides adhesion to EBL cells.

Mycoplasma mycoides subsp. mycoides (Mmm) is the causative agent of contagious bovine pleuropneumonia (CBPP). The virulent Mmm Ben-1 strain was isolated from the lung of a CBPP-infected cow in China in the 1950s. To attenuate the virulence of the Ben-1 strain and preserve its protective ability, the isolate was re-isolated after inoculation into the testicles of rabbits and into the rabbit thorax. As a result, after the subsequent isolates were continuously passaged 468 times in rabbits, its pathogenicity to cattle decreased. However, the molecular mechanisms leading to attenuation of the Mmm Ben-1 remain unknown. We compared the entire genomes of the Ben-1 strain and the 468 th generation strain passaged in rabbits (Ben-468) and discovered that a putative protein gene named p19 was absent from the Ben-468 strain. The p19 gene was cloned and expressed in Escherichia coli to obtain recombinant P19 (rP19). Western blot analysis demonstrated that the P19 protein is detected in the cell-membrane fraction, the cell-soluble cytosolic fraction and whole-cell lysate of the Mmm Ben-1 strain. The rP19 can interact with international standard serum against CBPP. Immunostaining visualised via confocal laser scanning microscopy indicated that P19 is able to adhere to embryonic bovine lung (EBL) cells, and this finding was also confirmed by a sandwich ELISA. We also found that anti-rP19 serum could inhibit the adhesion of the Mmm Ben-1 total proteins to EBL cells.