Structure of P46, an immunodominant surface protein fromMycoplasma hyopneumoniae: interaction with a monoclonal antibody

Abstract

Mycoplasma hyopneumoniae is a prokaryotic pathogen that colonizes the respiratory ciliated epithelial cells in swine. Infected animals suffer respiratory lesions, causing major economic losses in the porcine industry. Characterization of the immunodominant membrane-associated proteins from M. hyopneumoniae may be instrumental in the development of new therapeutic approaches. Here, the crystal structure of P46, one of the main surface-antigen proteins, from M. hyopneumoniae is presented and shows N-and C-terminal / domains connected by a hinge. The structures solved in this work include a ligand-free open form of P46 (3.1 Å resolution) and two ligand-bound structures of P46 with maltose (2.5 Å resolution) and xylose (3.5 Å resolution) in open and closed conformations, respectively. The ligand-binding site is buried in the cleft between the domains at the hinge region. The two domains of P46 can rotate with respect to each other, giving open or closed alternative conformations. In agreement with this structural information, sequence analyses show similarities to substrate-binding members of the ABC transporter superfamily, with P46 facing the extracellular side as a functional subunit. In the structure with xylose, P46 was also bound to a high-affinity (K d = 29 nM) Fab fragment from a monoclonal antibody, allowing the characterization of a structural epitope in P46 that exclusively involves residues from the C-terminal domain. The Fab structure in the complex with P46 shows only small conformational rearrangements in the six complementarity-determining regions (CDRs) with respect to the unbound Fab (the structure of which is also determined in this work at 1.95 Å resolution). The structural information that is now available should contribute to a better understanding of sugar nutrient intake by M. hyopneumoniae. This information will also allow the design of protocols and strategies for the generation of new vaccines against this important swine pathogen. research papers Acta Cryst. (2020). D76, 418-427 Guasch et al. P46 from Mycoplasma hyopneumoniae 419

The immunodominant protein P116 extracts cholesterol and other essential lipids

“…The last injection was delivered four days before splenectomy. Isolated B lymphocytes from the immunized mice were fused to NSI myeloma cells 23 to obtain stable hybridoma cell lines producing monoclonal antibodies, as previously described 32 . Supernatants from hybridoma cell lines derived from single fused cells were first investigated by indirect ELISA screening against the recombinant P116 ectodomain.…”

“…The last injection was delivered four days before splenectomy. Isolated B lymphocytes from the immunized mice were fused to NSI myeloma cells 22 to obtain stable hybridoma cell lines producing monoclonal antibodies, as previously described 31 .Supernatants from hybridoma cell lines derived from single fused cells were first investigated by indirect ELISA screening against the recombinant P116 ectodomain. Positive clones were also tested by Western blot against protein profiles from M. pneumoniae cell lysates and by immunofluorescence using whole, non-permeabilized M. pneumoniae cells (see below).…”

Immunodominant protein P116 from M. pneumoniae transports cholesterol and essential lipids

“…The last injection was delivered four days before splenectomy. Isolated B lymphocytes from the immunized mice were fused to NSI myeloma cells (23) to obtain stable hybridoma cell lines producing monoclonal antibodies, as previously described (32) .Supernatants from hybridoma cell lines derived from single fused cells were rst investigated by indirect ELISA screening against the recombinant P116 ectodomain.…”

Development of a Multi-Epitope Vaccine for Mycoplasma hyopneumoniae and Evaluation of Its Immune Responses in Mice and Piglets

“…The following supporting information can be downloaded at: . References [ 6 , 7 , 8 , 19 , 20 , 21 , 31 , 75 , 76 , 77 , 78 , 79 , 80 , 81 , 82 , 83 , 84 , 85 , 86 , 87 , 88 ] are cited in the supplementary materials.…”

“…Supplementary Materials:The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/ijms23147899/s1. References[6][7][8][19][20][21]31,[75][76][77][78][79][80][81][82][83][84][85][86][87][88] are cited in the supplementary materials.…”