Mycoplasma gallisepticum and Mycoplasma synoviae express a cysteine protease CysP, which can cleave chicken IgG into Fab and Fc

Abstract

Major poultry pathogens M. gallisepticum and M. synoviae share a gene encoding a putative cysteine protease CysP similar to papain cysteine protease (C1A 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 39 end of the cysP gene. In the same cysP region, all M. gallisepticum strains showed a deletion of 66 bp. Immunoblot analysis with specific antibodies 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 37 6C) resulted in a papain-like cleavage pattern of cIgG and fragments corresponding to the antigen-binding fragment of IgG (Fab,~45 kDa) 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-tagged protein in a cell-free system. Purified recombinant CysP (rCysP;~67 kDa, pI~8) cleaved cIgG into Fab and Fc fragments. This indicates that CysP is responsible for the cIgG cleavage caused by M. synoviae and, probably, by M. gallisepticum. This is the first evidence to our knowledge that mycoplasmas have enzymes that can cleave the host IgG and indicates a novel strategy used by M. gallisepticum and M. synoviae for prolonged survival despite the antibody response of their host.

MIB–MIP is a mycoplasma system that captures and cleaves immunoglobulin G

“…This activity appears to be linked to a serine protease, but the source of this activity remains cryptic, because no gene candidate could be identified. In the poultry pathogens M. synoviae and M. gallisepticum, a similar activity was attributed to the cysteine protease CysP (24). More recently, a new type of antibody-binding protein found in M. genitalium was described.…”

“…The MIB-MIP system might be missing one or more partners involved in further processing of the antibody before its release. Potential candidates include other proteases such as the three homologs of MMCAP2_0582, the three other DUF31 annotated genes, or the gene MMCAP2_0490, which encodes a distant homolog of the IgG protease CysP (24). Combined activities of these proteases might further degrade immunoglobulins and lead to a final release of the MIB-MIP components.…”

Variable lipoprotein haemagglutinin (vlhA) gene sequence typing of mainly DutchMycoplasma synoviaeisolates: comparison withvlhAsequences from Genbank and with amplified fragment length polymorphism analysis

“…multilocus sequence typing or multiple-locus variable-number tandem repeat analysis) or typing based on other markers should be considered (May & Brown, 2009;Bercic et al, 2011;Cizelj et al, 2011).…”

Poultry infected with Mycoplasma gallisepticum or Mycoplasma synoviae produce antibo dies to their cysteine proteas e CysP

“…Appropriate antigens were applied as separate dots (2 µl) on strips of the Immobilon P membrane (Millipore) (Benčina et al, 2005). Optimal concentrations of antigens were previously determined by dIBA using reference antibodies to M. synoviae, M. gallisepticum, rCysP, rMSPB, pMGA 1.1 and to synthetic peptides representing partial CysP and nanh (Benčina et al, 1994;2001;2005;Berčič et al, 2008a;2011;Cizelj et al, 2011). So, pelleted mycoplasma cells were diluted in PBS (ph 7.4) 1:2000; proteins (rMSPB, rCysP, pMGA) at concentrations of ~ 5-10 µg/ml and synthetic peptides at a concentration of ~ 10 µg/ml.…”

“…Although M. gallisepticum and M. synoviae belong to different phylogenetic groups, they share hosts, tissue tropism, transmission ways, as well as a number of genes that have been transferred horizontally (noormohammadi et al, 1998;Vasconcelos et al, 2005;Berčič et al, 2011;Cizelj et al, 2011). Among the transferred genes are those encoding proteins being very important in interactions with poultry hosts.…”