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Journal ArticleDOI

Proteomic survey of the pathogenic Mycoplasma hyopneumoniae strain 7448 and identification of novel post-translationally modified and antigenic proteins.

TL;DR: A proteomic analysis, based on two-dimensional gel electrophoresis of soluble protein extracts, immunoblot and mass spectrometry, which was carried out aiming the identification of gene products and antigenic proteins from the M. hyopneumoniae pathogenic strain 7448 produced a proteome map that is expected to serve as a reference for comparative analyses for methabolic studies based on cells cultured under modified conditions.
About: This article is published in Veterinary Microbiology.The article was published on 2007-03-31. It has received 62 citations till now. The article focuses on the topics: Mycoplasma hyopneumoniae & Proteome.
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Journal ArticleDOI
TL;DR: The structural studies indicate that short linear motifs in surface exposed, non-conserved regions of the molecule may play a key role in the moonlighting functions ascribed to this ancient, highly abundant protein.
Abstract: Elongation factor thermal unstable Tu (EF-Tu) is a G protein that catalyzes the binding of aminoacyl-tRNA to the A-site of the ribosome inside living cells. Structural and biochemical studies have described the complex interactions needed to effect canonical function. However, EF-Tu has evolved the capacity to execute diverse functions on the extracellular surface of both eukaryote and prokaryote cells. EF-Tu can traffic to, and is retained on, cell surfaces where can interact with membrane receptors and with extracellular matrix on the surface of plant and animal cells. Our structural studies indicate that short linear motifs (SLiMs) in surface exposed, non-conserved regions of the molecule may play a key role in the moonlighting functions ascribed to this ancient, highly abundant protein. Here we explore the diverse moonlighting functions relating to pathogenesis of EF-Tu in bacteria and examine putative SLiMs on surface-exposed regions of the molecule.

91 citations

Journal ArticleDOI
02 May 2012-Mbio
TL;DR: It is revealed that P146 is an extensively processed, multifunctional adhesin of M. hyopneumoniae, which undergoes endoproteolytic processing events at multiple sites and with differential processing efficiency, generating combinatorial diversity on the surface of M
Abstract: Mycoplasma 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. IMPORTANCE Vaccines 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.

80 citations


Cites background from "Proteomic survey of the pathogenic ..."

  • ...hyopneumoniae (28, 29), we were unable to identify any peptides unique to P146 near its predicted mass of 148....

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Journal ArticleDOI
TL;DR: Rec recombinant proteins containing R1A271 and R2271‐R1B271 were constructed and used in in vitro binding assays to suggest that both R1 and R2 in Mhp271 are involved in binding to host molecules.
Abstract: Mycoplasma hyopneumoniae, the causative agent of porcine enzootic pneumonia, adheres to ciliated respiratory epithelia resulting in ciliostasis and epithelial cell death. The cilium adhesin P97 (Mhp183) contains two repeat regions, designated R1 and R2, that play key roles in adherence. Eight pentapeptide repeats in R1 are sufficient to bind porcine cilia; however, both R1 and R2 are needed to bind heparin. Mhp271, a paralogue of P97, is the only other M. hyopneumoniae protein to contain both R1 and R2 repeats. These repeats are arranged as a set of three pentapeptide repeats (designated R1A271), two decapeptide repeats (designated R2271), and a second set of six pentapeptide repeats (designated R1B271). To determine their function, recombinant proteins containing R1A271 (F1271) and R2271-R1B271 (F2271) were constructed and used in in vitro binding assays. F2271, but not F1271, bound heparin (KD = 8.1 ± 0.4 nM), fibronectin (KD = 174 ± 13 nM) and porcine cilia. Pre-incubation of F2271 with 100 μM heparin blocked cilium binding by ~69%. Cell surface shaving with trypsin combined with two-dimensional liquid chromatography coupled to tandem mass spectrometry analysis identified Mhp271 as surface-exposed. Our data suggest that both R1 and R2 in Mhp271 are involved in binding to host molecules.

78 citations


Cites background from "Proteomic survey of the pathogenic ..."

  • ...However processing has not been observed in housekeeping proteins or some other high mass proteins (Djordjevic et al., 2004; Burnett et al., 2006; Pinto et al., 2007; Wilton et al., 2009)....

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Journal ArticleDOI
TL;DR: In this review, the characteristics of M. hyopneumoniae related to pathogenesis and control measures will be discussed and special emphasis will be placed on vaccination strategies that have been proposed with the use of reverse vaccinology approaches.

75 citations

Journal ArticleDOI
TL;DR: It is shown here that Mhp493 (P216), a paralogue of the cilium adhesin P97 (Mhp183), is cleaved between amino acids 1040 and 1089 generating surface‐accessible, heparin‐binding proteins P120 and P85, which are recognized by serum antibodies elicited during infection by M. hyopneumoniae.
Abstract: Mycoplasma hyopneumoniae induces respiratory disease in swine by colonizing cilia causing ciliostasis, cilial loss and epithelial cell death. Heparin binds to M. hyopneumoniae cells in a dose-dependent manner and blocks its ability to adhere to porcine cilia. We show here that Mhp493 (P216), a paralogue of the cilium adhesin P97 (Mhp183), is cleaved between amino acids 1040 and 1089 generating surface-accessible, heparin-binding proteins P120 and P85. Antiphosphoserine antibodies recognized P85 in 2-D immunoblotting studies and TiO(2) chromatography of trypsin digests of P85 isolated a single peptide with an m/z of 917.3. A phosphoserine residue in the tryptic peptide (90)VSELpSFR(96) (position 94 in P85) was identified by MALDI-MS/MS. Polyhistidine fusion proteins (F1(P216), F2(P216), F3(P216)) spanning Mhp493 bound heparin with biologically significant Kd values, and heparin, fucoidan and mucin inhibited this interaction. Latex beads coated with F1(P216), F2(P216) and F3(P216) adhered to and entered porcine kidney epithelial-like (PK15) cell monolayers. Microtitre plate-based assays showed that sequences within P120 and P85 bind to porcine cilia and are recognized by serum antibodies elicited during infection by M. hyopneumoniae. Mhp493 contributes significantly to the surface architecture of M. hyopneumoniae and is the first cilium adhesin to be described that lacks an R1 cilium-binding domain.

71 citations


Cites background from "Proteomic survey of the pathogenic ..."

  • ...Furthermore, a proteomic study of M. hyopneumoniae strain 7448 from Brazil reported a fragment of P216 with mass of ~120 kDa and pI of ~9 (Pinto et al., 2007) which appeared on a 2-...

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  • ...Proteomic studies show that other members of the P97/P102 paralogous families are cleaved yet many housekeeping proteins remain intact (Pinto et al., 2007; S.P. Djordjevic et al., unpublished)....

    [...]

  • ...hyopneumoniae strain 7448 from Brazil reported a fragment of P216 with mass of ~120 kDa and pI of ~9 (Pinto et al., 2007) which appeared on a 2-D gel where we observed P120....

    [...]

References
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Journal ArticleDOI
TL;DR: The magnitude of length differences suggests that the evolution of eukaryotic proteins was influenced by processes of fusion of single-function proteins into extended multi-functional and multi-domain proteins.
Abstract: We analyzed length differences of eukaryotic, bacterial and archaeal proteins in relation to function, conservation and environmental factors. Comparing Eukaryotes and Prokaryotes, we found that the greater length of eukaryotic proteins is pervasive over all functional categories and involves the vast majority of protein families. The magnitude of these differences suggests that the evolution of eukaryotic proteins was influenced by processes of fusion of single-function proteins into extended multifunctional and multi-domain proteins. Comparing Bacteria and Archaea, we determined that the small but significant length difference observed between their proteins results from a combination of three factors: (i) bacterial proteomes include a greater proportion than archaeal proteomes of longer proteins involved in metabolism or cellular processes, (ii) within most functional classes, protein families unique to Bacteria are generally longer than protein families unique to Archaea and (iii) within the same protein family, homologs from Bacteria tend to be longer than the corresponding homologs from Archaea. These differences are interpreted with respect to evolutionary trends and prevailing environmental conditions within the two prokaryotic groups.

378 citations

Journal ArticleDOI
TL;DR: This work mapped peptides detected in a whole‐cell lysate of Mycoplasma pneumoniae onto a genomic scaffold and extended these “hits” into ORFs bound by traditional genetic signals to generate a “proteogenomic map” and generated an ORF model for M. pneumoniae strain FH using proteomic data with a high correlation to models based on sequence features.
Abstract: The accelerated rate of genomic sequencing has led to an abundance of completely sequenced genomes. Annotation of the open reading frames (ORFs) (i.e., gene prediction) in these genomes is an important task and is most often performed computationally based on features in the nucleic acid sequence. Using recent advances in proteomics, we set out to predict the set of ORFs for an organism based principally on expressed protein-based evidence. Using a novel search strategy, we mapped peptides detected in a whole-cell lysate of Mycoplasma pneumoniae onto a genomic scaffold and extended these "hits" into ORFs bound by traditional genetic signals to generate a "proteogenomic map". We were able to generate an ORF model for M. pneumoniae strain FH using proteomic data with a high correlation to models based on sequence features. Ultimately, we detected over 81% of the genomically predicted ORFs in M. pneumoniae strain M129 (the originally sequenced strain). We were also able to detect several new ORFs not originally predicted by genomic methods, various N-terminal extensions, and some evidence that would suggest that certain predicted ORFs are bogus. Some of these differences may be a result of the strain analyzed but demonstrate the robustness of protein analysis across closely related genomes. This technique is a cost-effective means to add value to genome annotation, and a prerequisite for proteome quantitation and in vivo interaction measures.

334 citations

Journal ArticleDOI
Ana Tereza Ribeiro de Vasconcelos, Henrique Bunselmeyer Ferreira1, Cristiano Valim Bizarro1, Sandro L. Bonatto2, Marcos Oliveira de Carvalho1, Paulo Marcos Pinto1, Darcy F. de Almeida3, Luiz Gonzaga Paula de Almeida, Almeida Rosana De4, Leonardo Alves-Filho1, Enedina Nogueira de Assunção5, Vasco Azevedo6, Maurício Reis Bogo2, Marcelo M. Brigido7, Marcelo Brocchi8, Marcelo Brocchi4, Hélio Almeida Burity9, Anamaria A. Camargo10, Sandro da Silva Camargo1, Marta S. P. Carepo11, Dirce Maria Carraro10, J.C.M. Cascardo12, Luiza Amaral de Castro1, Gisele Cavalcanti, Gustavo Chemale1, Rosane G. Collevatti13, Cristina W. Cunha14, Bruno Dallagiovanna, Bibiana Paula Dambrós15, Odir Antônio Dellagostin14, Clarissa Falcão13, Fabiana Fantinatti-Garboggini8, Maria Sueli Soares Felipe7, Laurimar Fiorentin16, Glória Regina Franco6, Nara Suzy Aguiar De Freitas17, Diego Frias12, Thalles B. Grangeiro18, Edmundo C. Grisard15, Claudia Teixeira Guimarães9, Mariangela Hungria9, Silvia Neto Jardim9, Marco Aurélio Krieger, Jomar Pereira Laurino2, Lucymara Fassarella Agnez Lima19, Maryellen I. Lopes20, Élgion Lúcio da Silva Loreto21, Humberto Maciel França Madeira22, Gilson P. Manfio8, Andrea Queiroz Maranhão7, Christyanne T. Martinkovics1, Silvia Regina Batistuzzo de Medeiros19, Miguel Angêlo Martins Moreira, Márcia Neiva5, Cicero Eduardo Ramalho-Neto23, Marisa Fabiana Nicolás9, Sergio C. Oliveira6, Roger Ferreira Cury Paixão, Fábio O. Pedrosa24, Sérgio D.J. Pena6, Maristela Pereira25, Lilian Pereira-Ferrari22, Itamar Antônio Piffer16, Luciano da Silva Pinto18, Deise Porto Potrich1, Anna Christina M. Salim10, Fabrício R. Santos6, Renata Schmitt20, Maria Paula Cruz Schneider11, Augusto Schrank1, Irene Silveira Schrank1, Adriana F. Schuck1, Héctor N. Seuánez, Denise Wanderlei Silva23, Rosane Silva3, Sergio Ceroni da Silva1, Célia Maria de Almeida Soares25, Kelly Rose Lobo de Souza, Rangel C. Souza, Charley Christian Staats1, Maria B. R. Steffens24, Santuza M. R. Teixeira6, Turán P. Ürményi3, Marilene Henning Vainstein1, Luciana W. Zuccherato6, Andrew J. G. Simpson10, Arnaldo Zaha1 
TL;DR: Genomic comparisons revealed that reduction in genome size implied loss of redundant metabolic pathways, with maintenance of alternative routes in different species, and indicated a likely transfer event of hemagglutinin-coding DNA sequences from M. gallisepticum to M. synoviae.
Abstract: This work reports the results of analyses of three complete mycoplasma genomes, a pathogenic (7448) and a nonpathogenic (J) strain of the swine pathogen Mycoplasma hyopneumoniae and a strain of the avian pathogen Mycoplasma synoviae; the genome sizes of the three strains were 920,079 bp, 897,405 bp, and 799,476 bp, respectively. These genomes were compared with other sequenced mycoplasma genomes reported in the literature to examine several aspects of mycoplasma evolution. Strain-specific regions, including integrative and conjugal elements, and genome rearrangements and alterations in adhesin sequences were observed in the M. hyopneumoniae strains, and all of these were potentially related to pathogenicity. Genomic comparisons revealed that reduction in genome size implied loss of redundant metabolic pathways, with maintenance of alternative routes in different species. Horizontal gene transfer was consistently observed between M. synoviae and Mycoplasma gallisepticum. Our analyses indicated a likely transfer event of hemagglutinin-coding DNA sequences from M. gallisepticum to M. synoviae.

314 citations

Journal Article
TL;DR: A description is given of a new medium with which primary isolation of mycoplasma species of the porcine respiratory tract is usually successful, and various additives often recommended for myCoplasma cultivation have been examined for growth promoting effect.
Abstract: Two mycoplasma species of the porcine respiratory tract: Mycoplasma suipneumoniae and Mycoplasma flocculare, have been notoriously difficult to cultivate. In the present paper a description is given of a new medium with which primary isolation of these organisms is usually successful. The basal medium is made from commercial dehydrated products. Pig serum is added and phenol red used as pH indicator. Contrary to what is customary in the preparation of mycoplasma media, various inorganic salts (Hank's balanced salt solution) are included and penicillin-G replaced as a bacteriostatic by bacitracin and meticillin. The volume of water is adjusted so as to give isotonia. Various additives often recommended for mycoplasma cultivation have been examined for growth promoting effect; apparently, however, they were all without effect. Working procedures for primary isolation trials are briefly described.

294 citations

Journal ArticleDOI
TL;DR: Results demonstrate that M. pneumoniae EF‐Tu and PDH‐B, in addition to their major cytoplasmic biosynthetic and metabolic roles, can be surface translocated, which confers additional important biological functions.
Abstract: Summary The interactions between pathogenic bacteria and extracellular matrix (ECM) components markedly influence the initiation and establishment of infection. We have identified two surface proteins of virulent Mycoplasma pneumoniae with molecular masses of 45 and 30 kDa that bind to the ECM constituent, fibronectin (Fn). These Fn-binding proteins (FnBPs) were purified to near homogeneity using Fn-coupled Sepharose 4B-affinity column chromatography, and amino acid sequence analysis of the 45 and the 30 kDa proteins identified them as elongation factor Tu (EF-Tu) and pyruvate dehydrogenase E1 β subunit (PDH-B) respectively. The genes for EF-Tu and PDH-B were cloned, and the entire EF-Tu gene and NH2-terminus of PDH-B (NPDH (pyruvate dehydrogenase E1 β subunit from amino acid 1–244)-B) gene were overexpressed in Escherichia coli. The recombinant proteins, rEF-Tu and rNPDH-B, were purified to homogeneity by His-tag affinity column chromatography and used to immunize rabbits. Purified rEF-Tu and rNPDH-B bound to Fn using a ligand immunoblot assay and ELISA. Immunogold electron microscopy with polyclonal antibodies reactive against rEF-Tu (antirEF-Tu) and rNPDH-B (antirNPDH-B) and whole cell radioimmunoprecipitation (WCRIP) revealed the surface location of these proteins. Adherence of viable M. pneumoniae to immobilized Fn was inhibited by antirEF-Tu and antirNPDH-B antisera in a dose-dependent and cumulative manner. These results demonstrate that M. pneumoniae EF-Tu and PDH-B, in addition to their major cytoplasmic biosynthetic and metabolic roles, can be surface translocated, which confers additional important biological functions.

292 citations

Related Papers (5)
Ana Tereza Ribeiro de Vasconcelos, Henrique Bunselmeyer Ferreira, Cristiano Valim Bizarro, Sandro L. Bonatto, Marcos Oliveira de Carvalho, Paulo Marcos Pinto, Darcy F. de Almeida, Luiz Gonzaga Paula de Almeida, Almeida Rosana De, Leonardo Alves-Filho, Enedina Nogueira de Assunção, Vasco Azevedo, Maurício Reis Bogo, Marcelo M. Brigido, Marcelo Brocchi, Marcelo Brocchi, Hélio Almeida Burity, Anamaria A. Camargo, Sandro da Silva Camargo, Marta S. P. Carepo, Dirce Maria Carraro, J.C.M. Cascardo, Luiza Amaral de Castro, Gisele Cavalcanti, Gustavo Chemale, Rosane G. Collevatti, Cristina W. Cunha, Bruno Dallagiovanna, Bibiana Paula Dambrós, Odir Antônio Dellagostin, Clarissa Falcão, Fabiana Fantinatti-Garboggini, Maria Sueli Soares Felipe, Laurimar Fiorentin, Glória Regina Franco, Nara Suzy Aguiar De Freitas, Diego Frias, Thalles B. Grangeiro, Edmundo C. Grisard, Claudia Teixeira Guimarães, Mariangela Hungria, Silvia Neto Jardim, Marco Aurélio Krieger, Jomar Pereira Laurino, Lucymara Fassarella Agnez Lima, Maryellen I. Lopes, Élgion Lúcio da Silva Loreto, Humberto Maciel França Madeira, Gilson P. Manfio, Andrea Queiroz Maranhão, Christyanne T. Martinkovics, Silvia Regina Batistuzzo de Medeiros, Miguel Angêlo Martins Moreira, Márcia Neiva, Cicero Eduardo Ramalho-Neto, Marisa Fabiana Nicolás, Sergio C. Oliveira, Roger Ferreira Cury Paixão, Fábio O. Pedrosa, Sérgio D.J. Pena, Maristela Pereira, Lilian Pereira-Ferrari, Itamar Antônio Piffer, Luciano da Silva Pinto, Deise Porto Potrich, Anna Christina M. Salim, Fabrício R. Santos, Renata Schmitt, Maria Paula Cruz Schneider, Augusto Schrank, Irene Silveira Schrank, Adriana F. Schuck, Héctor N. Seuánez, Denise Wanderlei Silva, Rosane Silva, Sergio Ceroni da Silva, Célia Maria de Almeida Soares, Kelly Rose Lobo de Souza, Rangel C. Souza, Charley Christian Staats, Maria B. R. Steffens, Santuza M. R. Teixeira, Turán P. Ürményi, Marilene Henning Vainstein, Luciana W. Zuccherato, Andrew J. G. Simpson, Arnaldo Zaha