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Query: UMLS:C0026936 (Mycoplasma)
 14,761 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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 membrane-associated 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.
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PMID:Cloning and functional analysis of the P97 swine cilium adhesin gene of Mycoplasma hyopneumoniae. 902 17

Mycoplasma hyopneumoniae is a highly prevalent pathogen which colonizes the ciliated epithelial lining of the porcine respiratory tract. Expression libraries constructed from genomic DNA of the non-pathogenic strain M. hyopneumoniae J were screened with porcine hyperimmune antiserum against M. hyopneumoniae. One clone expressed a 28 kDa protein which was also reactive with monospecific antiserum raised against a putative M. hyopneumoniae-specific 94 kDa antigen derived from strain J. Trypsin digestion of whole M. hyopneumoniae cells showed the 94 kDa antigen to be surface-accessible. DNA sequence analysis of the gene encoding the 94 kDa antigen revealed greater than 90% homology to two adhesin genes, encoding P97 and Mhp1, cloned from pathogenic strain 232 and strain P5722 of M. hyopneumoniae, respectively. Two regions of repetitive DNA sequence were identified in the gene encoding the 94 kDa antigen. The first encoded the deduced amino acid sequence A(T)-K-P-E(V)-A(T) arranged as nine tandem repeats (RR1). The second region of repetitive DNA sequence encoded the deduced amino acid sequence G-A(E,S)-P-N(S)-Q-G-K-K-A-E arranged as five tandem repeats (RR2). Comparison of the three M. hyopneumoniae adhesin genes revealed that the genes encoding P97 and Mhp1, and the strain J gene encoding the 94 kDa antigen contained 15, 12 and 9 tandem repeats, respectively, in RR1, and 4, 5 and 5 tandem repeats, respectively, in RR2. Southern hybridization analysis of EcoRI-digested genomic DNA probed with an 820 bp fragment spanning RR1 and RR2 identified a strongly hybridizing fragment ranging in size from 2.15 to 2.30 kb among seven geographically diverse strains of M. hyopneumoniae but failed to hybridize with DNA from four strains of Mycoplasma hyorhinis or Mycoplasma flocculare strain Ms42. PCR primers flanking the DNA sequence encoding RR1 and RR2 were used to amplify DNA from the seven strains of M. hyopneumoniae and DNA sequence analysis of the amplification products showed that the number of tandem amino acid repeats in RR1 varied considerably between strains. RR1 from M. hyopneumoniae strains YZ, Beaufort, Sue, OMZ407 and C1735/2 comprised 11, 15, 12, 15 and 8 tandem copies, respectively, of the 5-aa repeat whilst RR2 comprised 4, 3, 4, 3 and 4 tandem copies, respectively, of the 10-aa repeat. Two putative integrin binding sites (L-E-T and R-X-X-X-D) were identified in the 94 kDa ciliary adhesin. Variability in the number of amino acid repeats in RR1 amongst strains of M. hyopneumoniae may influence ciliary binding.
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PMID:Reiterated repeat region variability in the ciliary adhesin gene of Mycoplasma hyopneumoniae. 969 26

Mycoplasma hyopneumoniae causes an economically significant respiratory disease of swine called Enzootic Pneumonia. The disease process is initiated by adherence of M. hyopneumoniae to the cilia of swine respiratory epithelium through an interaction involving P97, a surface-associated protein, and cilia-specific receptors. Binding specificity is associated with a repeat region located near the C-terminus of the P97 protein. Further analysis of the DNA sequences surrounding the P97 structural gene revealed an operon composed of two ORFs, P97 and one coding for a 102.3-kDa protein designated P102. Hybridization analysis and subcloning experiments showed that the P97 adhesin-encoding gene was present as a single copy in the M. hyopneumoniae chromosome. P102 sequences, however, were found on four distinct chromosomal fragments, suggesting that multiple copies of P102 were present in the chromosome. One of these clones was identified by screening the genomic library with swine convalescent sera showing that P102 is expressed in vivo during M. hyopneumoniae infections. All copies of P102 were mapped to a single chromosomal region comprising approximately 13% of the genome (140kb), although the exact distance between the copies is not known. The function of P102 is also not known, but the translated sequence shows a prominent transmembrane domain, suggesting that it may be a surface protein.
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PMID:Molecular analysis of the P97 cilium adhesin operon of Mycoplasma hyopneumoniae. 972 20

Mycoplasma hyopneumoniae colonizes the swine respiratory tract at the level of ciliated cells by attaching specifically to the cilium membrane. This interaction involves an adhesin called P97; the cilium binding activity of this protein was localized to the carboxy terminus, which included two repeat regions, R1 and R2 (T. Hsu, S. Artiushin, and F. C. Minion, J. Bacteriol. 179:1317-1323, 1997). To further delineate the molecular mechanisms of M. hyopneumoniae interactions with ciliated epithelium, we used a bank of transposon inserts in the cloned P97 gene to identify the site for cilium binding by testing the truncated gene products in an in vitro microtiter plate adherence assay. These studies showed that the cilium binding site was located in the AAKPV(E) repeat sequence of P97, referred to as the R1 repeat. For functional binding, at least seven AAKPV(E) repeats were required. The adherence-blocking monoclonal antibody F1B6 also recognized this region but required fewer AAKPV(E) repeats for recognition. We then constructed R1 region-lacZ gene fusions and used the resulting R1 repeat-beta-galactosidase fusion proteins in an in vitro assay to confirm the role of R1 in cilium binding. A comparison of the R1 regions of M. hyopneumoniae strains displaying variation in cilium adherence failed to identify changes that could account for the differences in adherence shown by the strains. Thus, we concluded that other proteins, in addition to P97, must be involved in cilium adherence, possibly in combination with P97.
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PMID:Identification of the cilium binding epitope of the Mycoplasma hyopneumoniae P97 adhesin. 974 76

Adherence of Mycoplasma hyopneumoniae to the swine respiratory tract is mediated by the membrane protein P97. This protein is located on the outer membrane surface, and its role in adherence has been firmly established. The general region of P97 that mediates adherence to swine cilia is thought to be the R1 region near the carboxy terminus of the protein, but it was not clear if this region could mediate adherence to swine cilia independently of other P97 sequences. To examine this in more detail, a series of R1 repeat sequences containing different numbers of repeating units cloned in frame with lacZ was used to produce R1-beta-galactosidase fusion proteins. These proteins were then tested for adherence to swine cilia and for reactivity to the adherence-blocking monoclonal antibody F2G5 and convalescent-phase swine sera. In this way it was possible to accurately define the cilium binding epitope of P97 and the minimal epitope recognized by antibody. Our results indicate that eight R1 repeating units are required for cilium binding and that three repeating units are needed for antibody recognition. These results could lead to more effective therapeutic measures against this important swine pathogen.
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PMID:R1 region of P97 mediates adherence of Mycoplasma hyopneumoniae to swine cilia. 1076 15

We have developed a system in which a foreign antigen is delivered and expressed on the surface of an attenuated strain of Erysipelothrix rhusiopathiae YS-1 and have examined the ability of a such recombinant E. rhusiopathiae strain to function as a mucosal vaccine vector. The C-terminal portion, including two repeat regions, R1 and R2, of the P97 adhesin of Mycoplasma hyopneumoniae strain E-1 was successfully translocated and expressed on the E. rhusiopathiae YS-1 cell surface after it was fused to SpaA.1, a cell surface protective antigen of E. rhusiopathiae. BALB/c mice subcutaneously immunized with the E. rhusiopathiae recombinant strains developed specific antibodies against SpaA.1 protein and were protected from lethal challenge with the highly virulent homologous E. rhusiopathiae Fujisawa-SmR strain, showing the efficacy of this heterologous-antigen expression system as a vaccine against E. rhusiopathiae infection. To determine whether protective immune responses are induced in target species, newborn, specific-pathogen-free piglets were immunized intranasally with a recombinant strain designated YS-19. The immunized piglets developed specific anti-SpaA.1 immunoglobulin G (IgG) antibodies in their serum and were protected from death by erysipelas, showing that mucosal vaccination of piglets with YS-19 induces systemic immune responses. Furthermore, YS-19-immunized piglets showed higher levels of P97-specific IgA antibodies in the respiratory tract than did YS-1-immunized piglets. Thus, E. rhusiopathiae YS-1 appears to be a promising vaccine vector for mucosal delivery that can induce local and systemic immune responses.
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PMID:Erysipelothrix rhusiopathiae YS-1 as a live vaccine vehicle for heterologous protein expression and intranasal immunization of pigs. 1174 87

The attenuated Erysipelothrix rhusiopathiae YS-19 strain was constructed for the purpose of delivering the C-terminal portion of the Mycoplasma hyopneumoniae P97 adhesin to the mucosal surface of the respiratory tract of pigs. In this study, the efficacy of the YS-19 vaccine against mycoplasmal pneumonia of swine was evaluated. Animal experiments revealed that intranasal immunization of pigs with the YS-19 strain significantly reduced the severity of pneumonic lung lesions caused by M. hyopneumoniae infection. In YS-19-immunized pigs, P97-specific serum antibodies were not detected. However, when stimulated with the P97 protein, peripheral blood mononuclear cells from the YS-19-immunized pigs had a significantly higher stimulation index (P<0.05) than that of cells from control pigs at 7 days post-challenge.
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PMID:Vaccine efficacy of the attenuated Erysipelothrix rhusiopathiae YS-19 expressing a recombinant protein of Mycoplasma hyopneumoniae P97 adhesin against mycoplasmal pneumonia of swine. 1253 53

Mycoplasma hyopneumoniae is an economically significant swine pathogen that colonizes the respiratory ciliated epithelial cells. Cilium adherence is mediated by P97, a surface protein containing a repeating element (R1) that is responsible for binding. Here, we show that the cilium adhesin is proteolytically processed on the surface. Proteomic analysis of strain J proteins identified cleavage products of 22, 28, 66, and 94 kDa. N-terminal sequencing showed that the 66- and 94-kDa proteins possessed identical N termini and that the 66-kDa variant was generated by cleavage of the 28-kDa product from the C terminus. The 22-kDa product represented the N-terminal 195 amino acids of the cilium adhesin preprotein, confirming that the hydrophobic leader signal sequence is not cleaved during translocation across the membrane. Comparative studies of M. hyopneumoniae strain 232 showed that the major cleavage products of the cilium adhesin are similar, although P22 and P28 appear to be processed further in strain 232. Immunoblotting studies using antisera raised against peptide sequences within P22 and P66/P94 indicate that processing is complex, with cleavage occurring at different frequencies within multiple sites, and is strain specific. Immunogold electron microscopy showed that fragments containing the cilium-binding site remained associated with the cell surface whereas cleavage products not containing the R1 element were located elsewhere. Not all secreted proteins undergo multiple cleavage, however, as evidenced by the analysis of the P102 gene product. The ability of M. hyopneumoniae to selectively cleave its secreted proteins provides this pathogen with a remarkable capacity to alter its surface architecture.
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PMID:Proteolytic processing of the Mycoplasma hyopneumoniae cilium adhesin. 1510 89

The P97 adhesin and P102 genes of Mycoplasma hyopneumoniae each have six paralogs in the genome. We tested whether these genes were expressed during infection. P102 is associated with the mycoplasma and with swine cilia. Further, most of the paralogs were transcribed in vivo in two gene transcriptional units.
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PMID:In vivo expression analysis of the P97 and P102 paralog families of Mycoplasma hyopneumoniae. 1623 86

Mycoplasma hyopneumoniae is the causative agent of porcine enzootic pneumonia, a chronic and economically significant respiratory disease that affects swine production worldwide. M. hyopneumoniae adheres to and adversely affects the function of ciliated epithelial cells of the respiratory tract, and the cilium adhesin (Mhp183, P97) is intricately but not exclusively involved in this process. Although binding of pathogenic bacteria to glycosaminoglycans is a recognized step in pathogenesis, knowledge of glycosaminoglycan-binding proteins in M. hyopneumoniae is lacking. However, heparin and other sulfated polysaccharides are known to block the binding of M. hyopneumoniae to purified swine respiratory cilia. In this study, four regions within the cilium adhesin were examined for the ability to bind heparin. Cilium adhesin fragments comprising 653 amino acids of the N terminus and 301 amino acids of the C terminus (containing two repeat regions, R1 and R2) were cloned and expressed. These fragments bound heparin in a dose-dependent and saturable manner with physiologically significant binding affinities of 0.27 +/- 0.02 microM and 1.89 +/- 0.33 microM, respectively. Heparin binding of both fragments was strongly inhibited by the sulfated polysaccharides fucoidan and mucin but not by chondroitin sulfate B. When the C-terminal repeat regions R1 and R2 were cloned separately and expressed, heparin-binding activity was lost, suggesting that both regions are required for heparin binding. The ability of the cilium adhesin to bind heparin indicates that this molecule plays a multifunctional role in the adherence of M. hyopneumoniae to host respiratory surfaces and therefore has important implications with respect to the pathogenesis of this organism.
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PMID:Two domains within the Mycoplasma hyopneumoniae cilium adhesin bind heparin. 1636 4


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