The invasive capacity of Mycoplasma synoviae was demonstrated. This mechanism enables mycoplasma dissemination and avoiding to host immune defense. Using gentimicin invasion assay and immunostainig relative invasive frequency (RIF) was demonstrated for four M. synoviae strains in three chicken cell types: erythrocytes (CER), embryonic fibroblasts (CEC-32) and primary cell culture of chicken chondrocytes (CCH). Type strain WVU 1853 was more invasive for CCH and CEC-32 cells and similarly invasive for CCH as M. gallisepticum Rlow. Field strains of M. synoviae were even less invasive for CCH.
Major poultry pathogens M. gallisepticum and M. synoviae share a gene encoding a putative cysteine protease CysP similar to papain cysteine (CaA subfamily). Comparison of the cysP gene sequences of 18 M. synoviae and 10 M. gallisepticum strains sequenced in this study showed polymorphisms, including deletions. Seven M. synoviae strains, including the type strain WVU 1853, had a 39 bp deletion in the 3' end of the cysP gene. In the same cysP region, all M. gallisepticum strains showed a deletion of 66bp. Immunoblot analysis with specific antibpodies demonstrated that M. synoviae strains expressed CysP, which was approximately 65 kDa. both M. synoviae and M. gallisepticum were able to digest chicken IgG (cIgG). Incubation of cIgG (-170 kDa) with M. synoviae or M. gallisepticum cells (-15 h at 37oC) resultes in a papain-like cleavage pattern of cIgG and fragments corresponding to the antigenbinding fragment of IgG (Fab, -45kDa) and the crystallizable region fragment (Fc) of the IgG heavy chain (dimer of -60 kDa). Iodoacetamide (50 mM) prevented cleavage of cIgG by both Mycoplasma species. Following site-directed mutagenesis (eight TGA codons were changed to TGG) the cysP gene of M. synoviae ULB 925 was expressed as a His-taggede protein in a cell-free system. Purified recombinant CysP (rCysp; -67kDa, pI-8) cleaved cIgG into Fab and Fc fragments. This indicates that CysP is responsible for the cIgG cleavage caused by M. synoviae and probbaly, by M. gallisepticum. This is the first evidence to our knowledge that mycopasmas have enzymes that can cleave the host IgG and indicates a novel strategy used by M. gallisepticum and M. synoviae for prolomged survival despite the antibody response of their host.
Mycoplasma synoviae synthesizes haemagglutinin vlhA, which cleaves into the N-terminal part, a lipoprotein MSPB, and a C-terminal part MSPA. Previous studies have shown that the 3'-end of the expressed vlhA gene can recombine with vlhA pseudogenes in a process called gene conversion, but three have been no data about diversification of the expressed vlhA gene in M. synoviae populations replicating in chickens. Following intratracheal inoculation with the M. synoviae strain ULB 02/T6, which showed only minor vlhA gene variation prior to inoculation, we investigated temporal changes in MSPB epitopes defined by monoclonal antibodies (mAbs) 3B4 and 50, as well as diversification of the vlhA gene sequence in M. synoviae populations recovered from chicken tracheas. In cultures isolated 8 and 18 days post inoculation (p.i.), most colonies showed variation of MSPB epitopes for mAbs 3B4 and 50. They also changed 3'-end vlhA gene sequences. Further diversity of the vlhA gene occurred in cultures isolated 8 weeks and 5 months p.i. The vlhA gene sequences from isolated cultures shared only 65 to 80% sequence identity with vlhA gene of the inoculated ULB 02/T6 culture. Notably, in most of those cultures their vlhA gene sequences contained stop codons potentially causing premature terminations of translation. Interestingly, in one culture isolated 8 weeks p.i. (clone T6-8W/IT2A) the 3'-vlhA gene sequence was identical in the last 1140 bases to that of the first vlhA pseudogene positioned the most far (upstream) of the expressed vlhA gene. This is the first demonstration of temporal diversity of the vlhA gene in M. synoviae populations isolated from chicken traheas.
Major poultry pathogens, Mycoplasma gallisepticum and Mycoplasma synoviae share several genes, including nanH that encodes their sialidases (neuraminidases). Previous studies have shown considerable differences in neuraminidase enzymatic activity (NEAC) in M. synoviae strains and NEAC absence in individual cultures of two strains, ULB 925 and ULB 9122. The present study shows that their cultures lacking NEAC did not express NanH neuraminidase detectable by specific antibodies. In cultures of M. synoviae ULB 925 and ULB 9122, which lacked NEAC and detectable NanH, deletions of a single adenine in different nnH regions of each strain created translational frameshifts resulting in TAA (UAA) stop codons and premature termination of translation. ULB 925 and ULB 9122 with such nanH mutations did not desialylate reference fetuin and transferrin or chicken glycoproteins that M. synoviae strains with NEAC efficiency desialylated. They desialylated several chicken serum glycoproteins with SA[alfa](2-6)galmoieties, including the immunoglobulin G heavy chain. Neuraminidase inhibitor 2,3-didehydro-2-deoxy-N-acetylneuraminic acid inhibited such desialylation otherweise caused by M. synoviae WVU 1853 neuraminidase. WVU 1853 also cleaved sialic acid from SA[alfa](2-3)gal moieties from glycoproteins of mucos from chicken tracheas. This is the first demonstration that M. synoviae desilylates glycoproteins of its host.
The objective of this study was to analyze the interaction of an arthrogenic Mycoplasma synoviae strain WVU 1853 with chicken chondrocytes. We found that M. synoviae significantly reduces chondrocyte respiration. This was accompanied by alterations in chondrocyte morphology, namely cell shrinkage and cytoplasm condensation, as well as nuclear condensation and formation of plasma membrane invaginations containing nuclear material, which appeared to cleave off the cell surface. In concordance with these apoptosis-like events in chondrocytes, transcription was increased in several pro-apoptotic genes. 24 h after infection, strong upregulation was assayed in NOS2, Mapk11, CASP8 and Casp3 genes. 24 and 72 h incubation of chondrocytes with M. synoviae induced upregulation of AIFM1, NFκB1, htrA3 and BCL2. Casp3 and NOS2 remained upregulated, but upregulation ceased for Mapk11 and CASP8 genes. Increased production of nitric oxide was also confirmed in cell supernates. The data suggests that chicken chondrocytes infected with M. synoviae die by apoptosis involving production of nitric oxide, caspase 3 activation and mitochondrial inactivation. The results of this study show for the first time that mycoplasmas could cause chondrocyte apoptosis. This could contribute to tissue destruction.