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Proteolytic processing of the Mycoplasma hyopneumoniae cilium adhesin.

ABSTRACT: 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.

SUBMITTER: Djordjevic SP

PROVIDER: S-EPMC387856 | biostudies-literature | 2004 May

REPOSITORIES: biostudies-literature

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Proteolytic processing of the Mycoplasma hyopneumoniae cilium adhesin.

Djordjevic Steven P SP Cordwell Stuart J SJ Djordjevic Michael A MA Wilton Jody J Minion F Chris FC

Infection and immunity 20040501 5

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 posses ...[more]

PMID: 15102789

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Project description:UnlabelledMycoplasma hyopneumoniae causes enormous economic losses to swine production worldwide by colonizing the ciliated epithelium in the porcine respiratory tract, resulting in widespread damage to the mucociliary escalator, prolonged inflammation, reduced weight gain, and secondary infections. Protein Mhp684 (P146) comprises 1,317 amino acids, and while the N-terminal 400 residues display significant sequence identity to the archetype cilium adhesin P97, the remainder of the molecule is novel and displays unusual motifs. Proteome analysis shows that P146 preprotein is endogenously cleaved into three major fragments identified here as P50(P146), P40(P146), and P85(P146) that reside on the cell surface. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) identified a semitryptic peptide that delineated a major cleavage site in Mhp684. Cleavage occurred at the phenylalanine residue within sequence (672)ATEF?QQ(677), consistent with a cleavage motif resembling S/T-X-F?X-D/E recently identified in Mhp683 and other P97/P102 family members. Biotinylated surface proteins recovered by avidin chromatography and separated by two-dimensional gel electrophoresis (2-D GE) showed that more-extensive endoproteolytic cleavage of P146 occurs. Recombinant fragments F1(P146)-F3(P146) that mimic P50(P146), P40(P146), and P85(P146) were constructed and shown to bind porcine epithelial cilia and biotinylated heparin with physiologically relevant affinity. Recombinant versions of F3(P146) generated from M. hyopneumoniae strain J and 232 sequences strongly bind porcine plasminogen, and the removal of their respective C-terminal lysine and arginine residues significantly reduces this interaction. These data reveal that P146 is an extensively processed, multifunctional adhesin of M. hyopneumoniae. Extensive cleavage coupled with variable cleavage efficiency provides a mechanism by which M. hyopneumoniae regulates protein topography.ImportanceVaccines used to control Mycoplasma hyopneumoniae infection provide only partial protection. Proteins of the P97/P102 families are highly expressed, functionally redundant molecules that are substrates of endoproteases that generate multifunctional adhesin fragments on the cell surface. We show that P146 displays a chimeric structure consisting of an N terminus, which shares sequence identity with P97, and novel central and C-terminal regions. P146 is endoproteolytically processed at multiple sites, generating at least nine fragments on the surface of M. hyopneumoniae. Dominant cleavage events occurred at S/T-X-F?X-D/E-like sites generating P50(P146), P40(P146), and P85(P146). Recombinant proteins designed to mimic the major cleavage fragments bind porcine cilia, heparin, and plasminogen. P146 undergoes endoproteolytic processing events at multiple sites and with differential processing efficiency, generating combinatorial diversity on the surface of M. hyopneumoniae.

Project description:Mycoplasma hyopneumoniae (M. hyopneumoniae) causes enzootic pneumonia in pigs but it is still largely unknown which host-pathogen interactions enable persistent infection and cause disease. In this study, we analyzed the host and bacterial transcriptomes during infection using RNA sequencing. Comparison of the transcriptome of lung lesion tissue from infected pigs with lung tissue from non-infected animals, identified 424 differentially expressed genes (FDR < 0.01 and fold change > 1.5LOG2). These genes were part of the following major pathways of the immune system: interleukin signaling (type 4, 10, 13, and 18), regulation of Toll-like receptors by endogenous ligand and activation of C3 and C5 in the complement system. Besides analyzing the lung transcriptome, a sampling protocol was developed to obtain enough bacterial mRNA from infected lung tissue for RNA sequencing. This was done by flushing infected lobes in the lung, and subsequently enriching for bacterial RNA. On average, 2.2 million bacterial reads were obtained per biological replicate to analyze the bacterial in vivo transcriptome. We compared the in vivo bacterial transcriptome with the transcriptome of bacteria grown in vitro and identified 22 up-regulated and 30 down-regulated genes (FDR < 0.01 and fold change > 2LOG2). Six out of seven genes in the operon encoding the mycoplasma specific F1-like ATPase (MHP_RS02445-MHP_RS02475) and all genes in the operon MHP_RS01965-MHP_RS01990 with functions related to nucleotide metabolism, spermidine transport and glycerol-3-phoshate transport were up-regulated in vivo. Down-regulated in vivo were genes related to glycerol uptake, cilium adhesion (P102), cell division and myo-inositol metabolism. In addition to providing a novel method to isolate bacterial mRNA from infected lung, this study provided insights into changes in gene expression during infection, which could help development of novel treatment strategies against enzootic pneumonia caused by M. hyopneumoniae.

Dataset Information

Proteolytic processing of the Mycoplasma hyopneumoniae cilium adhesin.

Publications

Proteolytic processing of the Mycoplasma hyopneumoniae cilium adhesin.

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