A Processed Multidomain Mycoplasma hyopneumoniae Adhesin Binds Fibronectin, Plasminogen, and Swine Respiratory Cilia

Abstract

Porcine enzootic pneumonia is a chronic respiratory disease that affects swine. The etiological agent of the disease, Mycoplasma hyopneumoniae, is a bacterium that adheres to cilia of the swine respiratory tract, resulting in loss of cilia and epithelial cell damage. A M. hyopneumoniae protein P116, encoded by mhp108, was investigated as a potential adhesin. Examination of P116 expression using proteomic analyses observed P116 as a full-length protein and also as fragments, ranging from 17 to 70 kDa in size. A variety of pathogenic bacterial species have been shown to bind the extracellular matrix component fibronectin as an adherence mechanism. M. hyopneumoniae cells were found to bind fibronectin in a dose-dependent and saturable manner. Surface plasmon resonance was used to show that a recombinant C-terminal domain of P116 bound fibronectin at physiologically relevant concentrations (K D 24 ؎ 6 nM). Plasmin(ogen)-binding proteins are also expressed by many bacterial pathogens, facilitating extracellular matrix degradation. M. hyopneumoniae cells were found to also bind plasminogen in a dose-dependent and saturable manner; the C-terminal domain of P116 binds to plasminogen (K D 44 ؎ 5 nM). Plasminogen binding was abolished when the C-terminal lysine of P116 was deleted, implicating this residue as part of the plasminogen binding site. P116 fragments adhere to the PK15 porcine kidney epithelial-like cell line and swine respiratory cilia. Collectively these data suggest that P116 is an important adhesin and virulence factor of M. hyopneumoniae.

Sequence TTKF↓QE Defines the Site of Proteolytic Cleavage in Mhp683 Protein, a Novel Glycosaminoglycan and Cilium Adhesin of Mycoplasma hyopneumoniae

“…Both ␣F2 683 and ␣F5 683 sera significantly blocked this interaction, and inhibition by ␣F2 683 was observed at levels similar to antiserum made from a recombinant protein containing the R1 binding domain of the cilium adhesin P97. Inhibition of M. hyopneumoniae adherence to porcine cilia was not absolute as ϳ50% of Mycoplasma cells were able to bind cilia after coating with Mhp683 antisera, a result consistent with the redundancy previously observed in the P97 and P102 families (15)(16)(17)(18)20). Although ␣F2 683 and ␣F5 683 sera significantly inhibited binding of M. hyopneumoniae to porcine cilia; antisera to recombinant fragments F1 683 , F3 683 , and F4 683 did not, despite evidence that they directly bound porcine cilia, indicating that F2 683 and F5 683 contain critical and exposed binding sites that play significant roles in M. hyopneumoniae pathogenesis.…”

“…We have previously established that proteolytic processing of members of the P97 and P102 paralog families generates a complex array of cleavage fragments that are displayed on the surface of M. hyopneumoniae and perform key roles in adhesion to structurally diverse host molecules (13)(14)(15)(16)(17)(18)48). However, the rationale for the extensive proteolytic processing of these 3 M. P. Padula and S. P. Djordjevic, unpublished data.…”

“…Many bacterial adhesins display complex cleavage or degradation patterns, which have not been extensively characterized, including several adhesins expressed by the phylogenetically related streptococci (26 -29). Recently, we showed that the P102 paralog, Mhp108, is a proteolytically processed, multifunctional adhesin that binds fibronectin and plasminogen and adheres to porcine respiratory cilia (17). In this study, we have extensively characterized Mhp683, a member of the P102 family that forms part of a two-gene operon with P146, as an adhesin-like P97 paralog (24,25).…”

“…The P97 paralog Mhp107 also binds plasminogen, fibronectin, and the glycosaminoglycan heparin (18). Mhp108, a member of the P102 family, binds cilia, fibronectin, and plasminogen (17), and domains within Mhp183, Mhp494, Mhp493, and Mhp271 bind heparin. Heparan sulfate includes regions within proteoglycan side chains that have been identified at the surface of porcine respiratory cilia (21).…”

Characterization of pyruvate dehydrogenase subunit B and enolase as plasminogen-binding proteins in Mycoplasma pneumoniae

“…Within these multi-factorial processes, proteins on the mycoplasma surface play a central role in the host-pathogen interaction. In this context, binding to host ECM components is mediated by different mycoplasma molecules, such as adhesins and proteins of unknown function in Mycoplasma gallisepticum (Jenkins et al, 2007;May et al, 2006) and Mycoplasma hyopneumoniae (Burnett et al, 2006;Seymour et al, 2010), as well as proteins involved in protein synthesis and housekeeping enzymes in other species (Chen et al, 2011;Dallo et al, 2002;Dumke et al, 2011;Hoelzle et al, 2007). In addition to the described association of PDHB and elongation factor Tu of M. pneumoniae with human fibronectin (Dallo et al, 2002) and of GAPDH with human fibrinogen (Dumke et al, 2011), here we show that PDHB and enolase are binding partners of human plasminogen.…”