Microtiter plate adherence assay and receptor analogs for Mycoplasma hyopneumoniae

Abstract

A microtiter plate adherence assay for Mycoplasma hyopneumoniae was established by use of purified swine tracheal cilia which contained receptors for the mycoplasmas. M. hyopneumoniae bound specifically to plates coated with solubilized cilia. The binding was dependent on both the concentration of cilia and the number of mycoplasmas. Dextran sulfate, heparin, chondroitin sulfate, laminin, mucin, and fucoidan significantly inhibited the binding of the mycoplasmas. The six inhibitors also disrupted the adherence of the mycoplasmas to intact ciliated cells. Preincubation with either mycoplasmas or cilia indicated that heparin, mucin, fucoidan, and chondroitin sulfate interacted with the adhesive molecules on the surface of the mycoplasmas, while laminin blocked the receptors in cilia. The basis for the inhibition induced by dextran sulfate was unknown. Treatment of cilia with neuraminidase appeared to promote adherence of the mycoplasmas, whereas treatment of cilia with sodium metaperiodate decreased binding. These results indicate that receptors for M. hyopneumoniae in the ciliated epithelium of the respiratory tract of pigs are glycoconjugate in nature.

Repeat regions R1 and R2 in the P97 paralogue Mhp271 of Mycoplasma hyopneumoniae bind heparin, fibronectin and porcine cilia

“…The functional significance of this processing is yet to be demonstrated; however, it can alter the function(s) of a protein. Separation of R1 and R2 in P97 diminishes it's ability to bind the GAG heparin (Jenkins et al, 2006), an effective inhibitor of M. hyopneumoniae binding to cilia and to porcine kidney epithelial-like cells (PK15) (Zhang et al, 1994;Burnett et al, 2006).…”

In vivovirulence ofMycoplasma hyopneumoniaeisolates does not correlate within vitroadhesion assessed by a microtitre plate adherence assay

“…Adherence of M. hyopneumoniae to the ciliated epithelial cells of the porcine respiratory tract is considered an essential first step in the pathogenesis of the infection (DeBey and Ross 1994). To quantify adherence, a microtitre plate adherence assay was developed by Zhang et al (1994). This adhesion assay is considered a good in vitro model, as the results obtained in this test with low and high passage M. hyopneumoniae isolates (Zhang et al 1994(Zhang et al , 1995 were in agreement with other types of in vitro or in vivo experiments including adhesion to tracheal ring cultures (DeBey and Ross 1994) and air-liquid interface cultures (Young et al 2000) as well as animal trials (Tajima and Yagihashi 1982;Zielinski and Ross 1990).…”

“…It is well-documented that serial in vitro passages of M. hyopneumoniae isolates influence adhesion capacity (Zhang et al 1994(Zhang et al , 1995Young et al 2000). Zhang et al (1994) observed a significant drop in adhesion capacity at passage 50 or higher. As presented in Table 1, the number of passages of the isolates used in the present study was much lower and ranged from 8 to 21.…”

“…In the microtitre plate adherence assay, adhesion capacity of whole bacterial cells of the different M. hyopneumoniae isolates was compared. The isolation of the cilia was performed as described by Zhang et al (1994). The cilia were extracted from the trachea of <10 week-old M. hyopneumoniae free piglets.…”

Analysis of P97 and P102 paralogs of Mycoplasma hyopneumoniae

“…hyopneumoniae is mainly mediated by receptor-ligand interactions as opposed to non-specific hydrophobic or charge-based interactions (108,110,111,113). Several high molecular weight carbohydrates inhibited the adherence of the mycoplasma to cilia in a rnicrotiter plate adherence assay, indicating that the receptors in cilia were most likely glycoconjugate in nature (110).…”