RESUMO
Mycoplasma (M.) hyopneumoniae is a primary etiological agent of porcine enzootic pneumonia (PEP), a disease that causes significant economic losses to pig farming worldwide. Current commercial M. hyopneumoniae vaccines induce partial protection, decline in preventing transmission of this pathogen or inducing complete immunity, evidencing the need for improving vaccines against PEP. In our study, we aimed to test the effectiveness of the SBA-15 ordered mesoporous silica nanostructured particles as an immune adjuvant of a vaccine composed of M. hyopneumoniae strain 232 proteins encapsulated in SBA-15 and administered by intramuscular route in piglets to evaluate the immune responses and immune-protection against challenge. Forty-eight 24-day-old M. hyopneumoniae-free piglets were divided into four experimental groups with different protocols, encompassing a commercial vaccine against M. hyopneumoniae, SBA-15 vaccine, SBA-15 adjuvant without antigens and a non-immunized group. All piglets were challenged with the virulent strain 232 of M. hyopneumoniae. Piglets that received the SBA-15 and commercial vaccine presented marked immune responses characterized by anti-M. hyopneumoniae IgA and IgG antibodies in serum, anti-M. hyopneumoniae IgA antibodies in nasal mucosa and showed an upregulation of IL-17 and IL-4 cytokines and downregulation of IFN-γ in lungs 35 days post-infection. Piglets immunized with SBA-15 vaccine presented a reduction of bacterial shedding compared to piglets immunized with a commercial bacterin. In addition, piglets from SBA-15 adjuvant suspension group presented increased IL-17 gene expression in the lungs without involvement of Th1 and Th2 responses after challenge. These results indicated that SBA-15 vaccine induced both humoral and cell-mediated responses in the upper respiratory tract and lungs, first site of replication and provided protection against M. hyopneumoniae infection with a homologous strain with reduction of lung lesions and bacterial shedding. Finally, these results enhance the potential use of new technologies such as nanostructured particles applied in vaccines for the pig farming industry.
Assuntos
Adjuvantes Imunológicos , Anticorpos Antibacterianos , Vacinas Bacterianas , Mycoplasma hyopneumoniae , Nanoestruturas , Pneumonia Suína Micoplasmática , Dióxido de Silício , Vacinas de Produtos Inativados , Animais , Mycoplasma hyopneumoniae/imunologia , Dióxido de Silício/administração & dosagem , Dióxido de Silício/imunologia , Pneumonia Suína Micoplasmática/prevenção & controle , Pneumonia Suína Micoplasmática/imunologia , Suínos , Vacinas Bacterianas/imunologia , Vacinas Bacterianas/administração & dosagem , Adjuvantes Imunológicos/administração & dosagem , Anticorpos Antibacterianos/sangue , Vacinas de Produtos Inativados/imunologia , Vacinas de Produtos Inativados/administração & dosagem , Derrame de Bactérias , Citocinas/imunologia , Pulmão/imunologia , Pulmão/microbiologia , Injeções IntramuscularesRESUMO
Mycoplasma (M.) hyopneumoniae is a primary etiological agent of porcine enzootic pneumonia (PEP), a disease that causes significant economic losses to pig farming worldwide. Current commercial M. hyopneumoniae vaccines induce partial protection, decline in preventing transmission of this pathogen or inducing complete immunity, evidencing the need for improving vaccines against PEP. In our study, we aimed to test the effectiveness of the SBA-15 ordered mesoporous silica nanostructured particles as an immune adjuvant of a vaccine composed of M. hyopneumoniae strain 232 proteins encapsulated in SBA-15 and administered by intramuscular route in piglets to evaluate the immune responses and immune-protection against challenge. Forty-eight 24-day-old M. hyopneumoniae-free piglets were divided into four experimental groups with different protocols, encompassing a commercial vaccine against M. hyopneumoniae, SBA-15 vaccine, SBA-15 adjuvant without antigens and a non-immunized group. All piglets were challenged with the virulent strain 232 of M. hyopneumoniae. Piglets that received the SBA-15 and commercial vaccine presented marked immune responses characterized by anti-M. hyopneumoniae IgA and IgG antibodies in serum, anti-M. hyopneumoniae IgA antibodies in nasal mucosa and showed an upregulation of IL-17 and IL-4 cytokines and downregulation of IFN-γ in lungs 35 days post-infection. Piglets immunized with SBA-15 vaccine presented a reduction of bacterial shedding compared to piglets immunized with a commercial bacterin. In addition, piglets from SBA-15 adjuvant suspension group presented increased IL-17 gene expression in the lungs without involvement of Th1 and Th2 responses after challenge. These results indicated that SBA-15 vaccine induced both humoral and cell-mediated responses in the upper respiratory tract and lungs, first site of replication and provided protection against M. hyopneumoniae infection with a homologous strain with reduction of lung lesions and bacterial shedding. Finally, these results enhance the potential use of new technologies such as nanostructured particles applied in vaccines for the pig farming industry.
RESUMO
Mycoplasma hyopneumoniae causes porcine enzootic pneumonia (PEP), a chronic respiratory disease that leads to severe economic losses in the pig industry. Swine infection and PEP development depend on the adhesion of the pathogen to the swine respiratory tract and the host immune response, but these and other disease determinants are not fully understood. For instance, M. hyopneumoniae has a large repertoire of proteins of unknown function (PUFs) and some of them are abundant in the cell surface, where they likely mediate so far unknown pathogen-host interactions. Moreover, these surface PUFs may undergo endoproteolytic processing to generate larger repertoires of proteoforms to further complicate this scenario. Here, we investigated the five PUFs more represented on the surface of M. hyopneumoniae pathogenic strain 7448 in comparison with their orthologs from the nonpathogenic M. hyopneumoniae J strain and the closely related commensal species Mycoplasma flocculare. Comparative in silico analyses of deduced amino acid sequences and proteomic data identified differential domains, disordered regions and repeated motifs. We also provide evidence of differential endoproteolytic processing and antigenicity. Phylogenetic analyses were also performed with ortholog sequences, showing higher conservation of three of the assessed PUFs among Mycoplasma species related to respiratory diseases. Overall, our data point out to M. hyopneumoniae surface-dominant PUFs likely associated with pathogenicity.
RESUMO
Mycoplasma hyopneumoniae is a bacterium that inhabits the swine respiratory tract, causing porcine enzootic pneumonia, which generates significant economic losses to the swine industry worldwide. The knowledge on M. hyopneumoniae biology and virulence have been significantly increased by genomics studies. However, around 30% of the predicted proteins remained of unknown function so far. According to the original annotation, the genome of M. hyopneumoniae 7448, a Brazilian pathogenic strain, had 693 coding DNA sequences, 244 of which were annotated as coding for hypothetical or uncharacterized proteins. Among them, there may be still several genes coding for unknown virulence factors. Therefore, this study aimed to functionally reannotate the whole set of 244 M. hyopneumoniae 7448 proteins of unknown function based on currently available database and bioinformatic tools, in order to predict novel potential virulence factors. Predictions of physicochemical properties, subcellular localization, function, overall association to virulence and antigenicity are provided. With that, 159 out of the set of 244 proteins of unknown function had a putative function associated to them, allowing identification of novel enzymes, membrane transporters, lipoproteins, DNA-binding proteins and adhesins. Furthermore, 139 proteins were generally associated to virulence, 14 of which had a function assigned and were differentially expressed between pathogenic and non-pathogenic strains of M. hyopneumoniae. Moreover, all extracellular or cytoplasmic membrane predicted proteins had putative epitopes identified. Overall, these analyses improved the functional annotation of M. hyopneumoniae 7448 genome from 65% to 87% and allowed the identification of new potential virulence factors.
Assuntos
Mycoplasma hyopneumoniae , Pneumonia Suína Micoplasmática , Animais , Proteínas de Bactérias/genética , Mycoplasma hyopneumoniae/genética , Suínos , Virulência , Fatores de Virulência/genéticaRESUMO
Infections with Mycoplasma hyopneumoniae (Mhyo), Mycoplasma hyorhinis (Mhr) and Mycoplasma flocculare (Mfloc) are common in swine. However, the degree of co-infections and the correlations between these mycoplasma co-infection and the severity of macroscopic lung consolidation lesions (MLCL) have not yet been explored in Brazil.The objectives were to quantify Mhyo, Mhr, and Mfloc in MLCL of slaughter pigs in Brazil, and to assess correlations with the degree of MLCL in slaughter pigs. To this end, five groups of lungs were made based on severity of lung lesions, and 80 lungs were collected for each group (400 lungs in total). The Mycoplasmas were quantified using a multiplex qPCR. Statistical differences and comparison between the groups were evaluated, respectively, by the Kruskal-Wallis test (p < 0.05) and Dunn's test (p < 0.05), and the correlation between the data was performed by Spearman's method (p < 0.05). The results revealed that the extent of MLCL showed a positive correlation with the Mhyo estimate (rho = 0.26; p < 0.05), a negative correlation with the Mfloc estimate (rho= -0.15; p < 0.05), and no significant correlation with the Mhr estimate (p = 0, 12). The extension of MLCL showed a positive correlation with the co-infection by Mfloc and Mhr (rho = 0.17; p < 0.05), and no significant correlation with Mhyo and Mhr (p = 0.87), and a negative correlation with Mhyo and Mfloc (rho= -0.28; p < 0.05). This study allowed to infer that, regarding the extension of MLCL, Mhr and Mfloc did not present opportunistic activity in relation to primary infection by Mhyo, but revealed some potential aggravation of these lesions. In addition, Mhyo expressed inhibitory behavior towards Mfloc, suggesting that one can compete with the other's presence.
Assuntos
Coinfecção/veterinária , Pneumopatias/veterinária , Pulmão/patologia , Infecções por Mycoplasma/veterinária , Mycoplasma/classificação , Doenças dos Suínos/microbiologia , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Coinfecção/microbiologia , Regulação Bacteriana da Expressão Gênica/fisiologia , Pulmão/microbiologia , Pneumopatias/microbiologia , Pneumopatias/patologia , Infecções por Mycoplasma/microbiologia , Infecções por Mycoplasma/patologia , SuínosRESUMO
Mycoplasma hyopneumoniae: is the etiological agent of porcine enzootic pneumonia (EP), a disease that impacts the swine industry worldwide. Pathogen-induced damage, as well as the elicited host-response, contribute to disease. Here, we provide an overview of EP epidemiology, control and prevention, and a more in-depth review of M. hyopneumoniae pathogenicity determinants, highlighting some molecular mechanisms of pathogen-host interactions relevant for pathogenesis. Based on recent functional, immunological, and comparative "omics" results, we discuss the roles of many known or putative M. hyopneumoniae virulence factors, along with host molecules involved in EP. Moreover, the known molecular bases of pathogenicity mechanisms, including M. hyopneumoniae adhesion to host respiratory epithelium, protein secretion, cell damage, host microbicidal response and its modulation, and maintenance of M. hyopneumoniae homeostasis during infection are described. Recent findings regarding M. hyopneumoniae pathogenicity determinants also contribute to the development of novel diagnostic tests, vaccines, and treatments for EP.
Assuntos
Interações Hospedeiro-Patógeno , Mycoplasma hyopneumoniae/patogenicidade , Fatores de Virulência/genética , Fatores de Virulência/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Mycoplasma hyopneumoniae/genética , Pneumonia Suína Micoplasmática/microbiologia , Pneumonia Suína Micoplasmática/fisiopatologia , Suínos , VirulênciaRESUMO
Mycoplasma hyopneumoniae and Mycoplasma flocculare are genetic similar bacteria that colonize the swine respiratory tract. However, while M. hyopneumoniae is a pathogen that causes porcine enzootic pneumonia, M. flocculare is a commensal. Adhesion to the respiratory epithelium is mediated by surface-displayed adhesins, and at least some M. hyopneumoniae adhesins are post-translational proteolytically processed, producing differential proteoforms with differential adhesion properties. Based on LC-MS/MS data, we assessed differential proteolytic processing among orthologs of the five most abundant adhesins (p97 and p216) or adhesion-related surface proteins (DnaK, p46, and ABC transporter xylose-binding lipoprotein) from M. hyopneumoniae strains 7448 (pathogenic) and J (non-pathogenic), and M. flocculare. Both surface and cytoplasmic non-tryptic cleavage events were mapped and compared, and antigenicity predictions were performed for the resulting proteoforms. It was demonstrated that not only bona fide adhesins, but also adhesion-related proteins undergo proteolytical processing. Moreover, most of the detected cleavage events were differential among M. hyopneumoniae strains and M. flocculare, and also between cell surface and cytoplasm. Overall, our data provided evidences of a complex scenario of multiple antigenic proteoforms of adhesion-related proteins, that is differential among M. hyopneumoniae strains and M. flocculare, altering the surface architecture and likely contributing to virulence and pathogenicity.
Assuntos
Proteínas de Bactérias/metabolismo , Mycoplasma hyopneumoniae/metabolismo , Mycoplasma/metabolismo , Pneumonia Suína Micoplasmática/microbiologia , Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Animais , Proteínas de Bactérias/genética , Mycoplasma/genética , Mycoplasma hyopneumoniae/genética , Processamento de Proteína Pós-Traducional , Proteólise , SuínosRESUMO
Mycoplasma hyopneumoniae is an economically significant swine pathogen that causes porcine enzootic pneumonia (PEP). Important processes for swine infection by M. hyopneumoniae depend on cell surface proteins, many of which are secreted by secretion pathways not completely elucidated so far. A putative type I signal peptidase (SPase I), a possible component of a putative Sec-dependent pathway, was annotated as a product of the sipS gene in the pathogenic M. hyopneumoniae 7448 genome. This M. hyopneumoniae putative SPase I (MhSPase I) displays only 14% and 23% of sequence identity/similarity to Escherichia coli bona fide SPase I, and, in complementation assays performed with a conditional E. coli SPase I mutant, only a partial restoration of growth was achieved with the heterologous expression of a recombinant MhSPase I (rMhSPase I). Considering the putative surface location of MhSPase I and its previously demonstrated capacity to induce a strong humoral response, we then assessed its potential to elicit a cellular and possible immunomodulatory response. In assays for immunogenicity assessment, rMhSPase I unexpectedly showed a cytotoxic effect on murine splenocytes. This cytotoxic effect was further confirmed using the swine epithelial PK(15) cell line in MTT and annexin V-flow cytometry assays, which showed that rMhSPase I induces apoptosis in a dose dependent-way. It was also demonstrated that this pro-apoptotic effect of rMhSPase I involves activation of a caspase-3 cascade. The potential relevance of the rMhSPase I pro-apoptotic effect for M. hyopneumoniae-host interactions in the context of PEP is discussed.