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

Pinto, Paulo Marcos; Chemale, Gustavo; Castro, Luiza Amaral de; Costa, Ana Paula Metz; Kich, Jalusa Deon; Vainstein, Marilene Henning; Zaha, Arnaldo; Ferreira, Henrique Bunselmeyer

doi:10.1016/j.vetmic.2006.11.018

Cited by 53 publications

(50 citation statements)

References 41 publications

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“…Groups of immunogenic spots are indicated by numbers. result was similar to that reported from proteomic studies of other mycoplasmae [7,9,18,20,22,24]. We identified 20 novel Mccp antigens in the whole cell protein preparation while 9 novel antigens were identified in the membrane protein fraction.…”

Section: Discussionsupporting

confidence: 89%

Identification of Novel Immunogenic Proteins in Mycoplasma capricolum subsp. Capripneumoniae Strain M1601

Zhao

Chu

et al. 2012

The Journal of Veterinary Medical Science

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ABSTRACT. The purpose of this study was to obtain immunogenic proteins and potential proteins of interest that were isolated from Mycoplasma capricolum subsp. capripneumoniae (Mccp) by MALDI-TOF mass spectrometry. One-dimensional SDS-PAGE and two-dimensional gel electrophoresis of whole cell preparation were conducted, and membrane proteome maps were prepared by immunoblotting. One-dimensional SDS-PAGE identified three immunogenic proteins with molecular masses in the range 29-97.2 kDa, two of which were in the membrane protein fraction. After two-dimensional gel electrophoresis, 20 highly immunogenic proteins were identified in the whole cell protein preparation while 9 immunogenic proteins were identified in the membrane protein fraction. Mycoplasma capricolum subsp. capripneumoniae (Mccp) is the causative agent of contagious capripleuropneumonia in goats [13], which is a disease characterized by coughing, dyspnea, lagging behind the herd, lying down, fever (40.5-41.5°C) and mouth breathing in the terminal stages [21]. In chronic cases, animals present sporadic coughing, emaciation, and diarrhea. Animals that recover from this form of the disease do not show any signs or lesions after recovery. Although there are no sequelae in recovered animals, the survivor goats may remain carriers of the infection.The genome sequence of Mccp has been completed but not annotated by Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences. There have been few efforts to identify and characterize Mccp antigens, which might potentially be useful for immunodiagnosis and/or vaccination. These antigens could also be virulence factors and their characterization may help to elucidate the mechanisms of pathogenicity. There is an obvious need for better antigens to improve the serological methods that are currently used for goat herd surveillance, which is an important step before the implementation of preventive or therapeutic measures.Proteome analysis is a useful complementary method for studying bacteria, which supports genome annotation and protein identification [9,22], and it could be used to characterize differentially expressed or post-translationally modified antigenic proteins [11]. This approach is particularly attractive in mycoplasmae, because the high A + T content and the use of TGA as a tryptophan codon rather than as a stop codon prevent a genetic approach using Escherichia coli expression libraries. Two-dimensional gel electrophoresis (2DE) mapping of Mccp proteins will improve genome annotation, and it will help to characterize the extent of post-translational modifications, as well as identifying potential virulence factors.In this study, we isolated three dominant immunogenic proteins in Mccp with SDS-PAGE and immunoblotting, 2 of which were membrane proteins. To further elucidate the identity of these proteins, 2DE maps of the whole cell protein preparation and the membrane protein fraction were produced, followed by immunoblotting. We identified 20 novel immunogenic proteins in the whole...

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Section: Discussionsupporting

confidence: 89%

Identification of Novel Immunogenic Proteins in Mycoplasma capricolum subsp. Capripneumoniae Strain M1601

Zhao

Chu

et al. 2012

The Journal of Veterinary Medical Science

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“…3 and 4). In a study addressing the comparative proteomic analysis between nonpathogenic (J) and pathogenic (7448 and 7422) strains (24,25), many of the differentially expressed proteins in these strains were hypothetical proteins that have been neglected; therefore, we included these proteins in the present study. In fact, compared to the other recombinant proteins, the hypothetical protein MHP0418 presented the highest reactivity in the ELISA and the Western blot assay when probed with serum from convalescing pigs ( Fig.…”

Section: Figmentioning

confidence: 99%

“…In the study by Pinto et al (24), MHP0372 lipoprotein was associated with M. hyopneumoniae cytoadherence, and although its role in the mechanism of pathogenicity was not evaluated, this protein is well known to show isoforms in two-dimensional (2D) gels, indicating that it is posttranslationally modified. The surface lipopeptides of mycoplasmas that originated through these modifications are known to contribute to the biological processes of adaptation (25,26) and pathogenicity, including antigenic variation among strains (5,27), justifying the choice of this protein for immunological studies.…”

Section: Figmentioning

confidence: 99%

Recombinant Secreted Antigens from Mycoplasma hyopneumoniae Delivered as a Cocktail Vaccine Enhance the Immune Response of Mice

Galli

Simionatto

Marchioro

et al. 2013

Clin Vaccine Immunol

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bMycoplasma hyopneumoniae is the etiological agent of porcine enzootic pneumonia (EP), which is a respiratory disease responsible for huge economic losses in the pig industry worldwide. The commercially available vaccines provide only partial protection and are expensive. Thus, the development of alternatives for the prophylaxis of EP is critical for improving pig health. The use of multiple antigens in the same immunization may represent a promising alternative. In the present study, seven secreted proteins of M. hyopneumoniae were cloned, expressed in Escherichia coli, and evaluated for antigenicity using serum from naturally and experimentally infected pigs. In addition, the immunogenicity of the seven recombinant proteins delivered individually or in protein cocktail vaccines was evaluated in mice. In Western blot assays and enzyme-linked immunosorbent assays, most of the recombinant proteins evaluated were recognized by convalescent-phase serum from the animals, indicating that they are expressed during the infectious process. The recombinant proteins were also immunogenic, and most induced a mixed IgG1/ IgG2a humoral immune response. The use of these proteins in a cocktail vaccine formulation enhanced the immune response compared to their use as antigens delivered individually, providing evidence of the efficacy of the multiple-antigen administration strategy for the induction of an immune response against M. hyopneumoniae. P orcine enzootic pneumonia (EP) is a chronic respiratory disease with a high incidence in pig farms worldwide. Mycoplasma hyopneumoniae, the etiological agent of this disease, colonizes and destroys the main mechanism of innate immunity of the pig respiratory system-the respiratory ciliated epithelium-predisposing the pigs to secondary and opportunistic infections (1). EP prophylaxis comprises the use of antibiotics, management procedures, and vaccination, which is considered the most efficient control measure (2). The commercially available vaccines consist of inactivated whole cells (bacterins) and have high production costs, mainly due to the fastidious growth of this microorganism. In addition, these vaccines do not eliminate M. hyopneumoniae from infected pig herds (1, 3, 4). Thus, efforts have been directed toward the search for new prophylaxis strategies against EP that do not include the use of bacterins.M. hyopneumoniae does not penetrate host cells, and thus it is believed that the pathogenesis of this microorganism is mediated by a complex and multifactorial process that involves components of the cell membrane and secreted proteins, many of which are still unidentified (5). Data generated from the sequencing of four isolates of M. hyopneumoniae (7448, 168, J, and 232) and the comparative genomic and proteomic analyses of pathogenic strains (7448 and 232) and a nonpathogenic strain (J) of M. hyopneumoniae (6,7,8,9) have allowed the identification of coding sequences (CDS) from secreted antigenic proteins and/or proteins involved in the pathogenicity of the microorganism. Som...

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“…M. hyopneumoniae pathogenic strain 7422, another Brazilian field isolate, was obtained from the EMBRAPA-CNPSA collection. M. hyopneumoniae cultivation and protein extraction were performed as described by Pinto et al (2007).…”

Section: Methodsmentioning

confidence: 99%

“…In the MCO system assay, the reducing agent (DTT) is artificial; the natural reducing agent for MhPrx remains to be identified. MhPrx was originally described as a thiol peroxidase (Vasconcelos et al, 2005), but, since its mechanism of action has not been fully elucidated and at least some 1-Cys peroxiredoxins can use non-thiolic electron donors to reduce the peroxidatic cysteine sulfenic acid intermediate (Monteiro et al, 2007a), we decided to treat this enzyme generically as a peroxiredoxin.MhPrx expression in M. hyopneumoniae 7448 has been previously reported based on proteomic studies (Pinto et al, 2007), where it appears as a strong spot even in Peroxiredoxin classification was originally proposed based on the number of cysteine residues present in the protein sequence involved in the enzymic mechanism and in the conservation of residues around the catalytic cysteines (Rhee et al, 2005). According to this classification, peroxiredoxins can be divided into two subfamilies, the 1-Cys and the 2-Cys.…”

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confidence: 99%

A peroxiredoxin from Mycoplasma hyopneumoniae with a possible role in H2O2 detoxification

et al. 2009

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Mycoplasma hyopneumoniae is the causative agent of porcine enzootic pneumonia, which affects pig farms worldwide, causing heavy economic losses. In the infection process, this bacterium is exposed to reactive oxygen species (ROS) from its own metabolism or generated by the host as one of the strategies used to neutralize the pathogen. Although the presence of classical antioxidant enzymes would be expected in M. hyopneumoniae, important genes directly related to protection against ROS, such as superoxide dismutase, catalases and glutathione peroxidase, have not been identified by sequence homology in the genome sequence annotation. Among the few identified M. hyopneumoniae genes coding for proteins possibly involved with suppression of ROS-mediated damage, one (tpx) coding for a peroxiredoxin (MhPrx) has been recognized. The sequence and phylogenetic analyses perfomed in this study indicate that MhPrx is closely related to the atypical 2-Cys peroxiredoxin subfamily, although it has only one cysteine in its sequence. The MhPrx coding DNA sequence was cloned and expressed in Escherichia coli to produce a recombinant MhPrx (rMhPrx), which was purified and used to immunize mice and produce an anti-MhPrx polyclonal antiserum. Probing of M. hyopneumoniae extracts with this antiserum demonstrated that MhPrx is expressed in all three tested strains (J, 7422 and 7448). Cross-linking assays and size-exclusion chromatography indicate that rMhPrx forms dimers, as has been established for atypical 2-Cys peroxiredoxins. Furthermore, a metal-catalysed oxidation system was used to assay the activity of rMhPrx, showing that it can protect DNA from ROS-mediated damage and may play an essential role during infection.

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

Cited by 53 publications

References 41 publications

Identification of Novel Immunogenic Proteins in <i>Mycoplasma capricolum</i> subsp. <i>Capripneumoniae</i> Strain M1601

Identification of Novel Immunogenic Proteins in <i>Mycoplasma capricolum</i> subsp. <i>Capripneumoniae</i> Strain M1601

Recombinant Secreted Antigens from Mycoplasma hyopneumoniae Delivered as a Cocktail Vaccine Enhance the Immune Response of Mice

A peroxiredoxin from Mycoplasma hyopneumoniae with a possible role in H2O2 detoxification

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