Cloning and functional analysis of the P97 swine cilium adhesin gene of Mycoplasma hyopneumoniae

Abstract

Colonization of the swine respiratory tract by Mycoplasma hyopneumoniae is accomplished by specific binding to the cilia of the mucosal epithelial cells. Previous studies have implicated a 97-kDa outer membraneassociated protein, P97, that appeared to mediate this interaction. In order to further define the role of P97 in adherence to porcine cilia, the structural gene was cloned and sequenced, and the recombinant products were analyzed. Monoclonal antibodies were used to identify recombinant clones in a genomic library expressed in an opal suppressor host because of alternate codon usage by mycoplasmas. The gene coding for P97 was then identified by Tn1000 mutagenesis of recombinant clones. DNA sequence analysis revealed an open reading frame coding for a 124.9-kDa protein with a hydrophobic transmembrane spanning domain. The N-terminal sequence of purified P97 mapped at amino acid position 195 of the translated sequence, indicating that a processing event had occurred in M. hyopneumoniae. Both recombinant P97 protein expressed in an Escherichia coli opal suppressor host and M. hyopneumoniae bound specifically to swine cilia, and the binding was inhibited by heparin and fucoidan, thus supporting the hypothesis that P97 was actively involved in binding to swine cilia in vivo.Mucosal pathogens initiate disease by colonizing the epithelial cell surface. Usually associated with specific structures on the bacterial outer surface, adhesins possess binding characteristics believed to contribute significantly to the pathogen's species specificity. Because mycoplasmas lack cell walls, piluslike adhesins, and other extracellular structures, adherence to mucosal surfaces by these organisms involves less-organized outer membrane structures. In fact, mycoplasmal adhesins appear to reside as single proteins embedded within the membrane. In some instances, adhesins appear to be organized into binding domains on the membrane surface, e.g., the P1 adhesin of Mycoplasma pneumoniae (19), but the adherence mechanisms of most mycoplasmal pathogens remain unknown. Additionally, no receptor binding analysis of a cloned mycoplasmal adhesin has been reported, although analysis of cloned fragments of P1 using monoclonal antibodies (MAbs) to identify putative binding domains has been reported (3).Mycoplasma hyopneumoniae produces a chronic and economically important respiratory disease in swine (21). Colonization of respiratory tissues is limited to the luminal epithelial surface, resulting in progressive ciliary and epithelial cell damage during the infectious process. A host inflammatory response and a generalized immunosuppression often occur, predisposing the swine to infections by other bacteria and viruses (21).Adherence of M. hyopneumoniae to swine respiratory tissues and the resulting ciliostasis have been examined. M. hyopneumoniae binds specifically to cilia and not to other membrane surfaces in the respiratory tract (14). Ciliostasis in vitro requires adherence of the organism to the ciliated cell surface (4). To characterize ...

Reiterated repeat region variability in the ciliary adhesin gene of Mycoplasma hyopneumoniae

“…TnlOOO insertional mutagenesis studies mapped the mAb F1B6 binding epitope to a region between amino acid residues 704 and 1004 in the carboxy terminus of P97 which encompassed the 5-aa repeat region (RR1) but not the 10-aa repeat region (RR2). The insertion of TnlOOO in RR2 did not affect ciliary binding or the ability of mAb F1B6 to bind to P97, suggesting that RR2 is not involved in adhesion (Hsu et al, 1997). DNA sequence analysis revealed the presence of an ORF for a 124 kDa protein; however amino-terminal sequence analysis of P97 (Zhang et al, 1995) mapped 195 aa internal to the start codon, suggesting that the 124 kDa antigen was a precursor in M. hyopneumoniae that is subsequently processed to 102 kDa by proteolytic cleavage (Hsu et al, 1997).…”

“…Failure to detect sequence variation among three independent clones containing 5' adhesin gene sequences and the absence of more than a single strongly hybridizing fragment in Southern blot experiments (see Fig. 5b) suggested that only a single copy of the adhesin gene resides in M. hyopneumoniae strain J. Amino-terminal amino acid sequence data from purified P97 (Hsu et al, 1997) and Mhpl (King et al, 1997) revealed that the first 195 aa had been removed and suggested that the ciliary adhesin was produced from a 124.5 kDa precursor which was subsequently processed to 102 kDa by proteolytic cleavage. Western blots of whole-cell lysates of different strains of M. hyopneumoniae reacted with anti-94 kDa serum (Fig.…”

“…ORFl was downstream of the T7 promoter of pET23c and the translated product, comprising 556 aa, showed strong homology to two translation products deposited in databases (U27294 and U50901,92 and 88 % , respectively) ; both sequences have been identified as M . hyopneumoniae adhesins (Hsu et al, 1997;King et al, 1996). From the deduced amino acid sequence, the predicted molecular mass of ORFl was 62 kDa.…”

P159 is a proteolytically processed, surface adhesin ofMycoplasma hyopneumoniae: defined domains of P159 bind heparin and promote adherence to eukaryote cells

“…Monoclonal antibodies F1B6 and F2G5, which recognize the R1 cilium binding region in P97 (Zhang et al ., 1995), are able to reduce adherence of M. hyopneumoniae to respiratory cilia by approximately 70%. Purified recombinant P97 inhibits adherence of M. hyopneumoniae cells to cilia in a dose-dependent manner (Zhang et al ., 1995) and binding of recombinant P97 to porcine respiratory cilia is inhibited by sulphated glycosaminoglycans (Hsu et al ., 1997). Consistent with these observations, P97 was recently shown to possess two heparin-binding domains (Jenkins et al ., 2006).…”

Etiopatogenia e imunoprofilaxia da pneumonia enzoótica suína

“…hyopneumoniae ao cílio suíno em um teste de inibição da aderência "in vitro" (ZHANG et al, 1995), sugerindo que a região R1 da adesina P97 possui papel fundamental na patogenia da PES, tornando-a uma forte candidata a antígeno vacinal. O gene que codifica a adesina P97 (p97) foi clonado e seqüenciado (HSU et al, 1997). A análise deste gene revelou uma open reading frame (ORF) que codifica para uma proteína de 125 kDa, a qual apresenta um domínio transmembrana hidrofóbico e um peptídeo sinal.…”