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1.
mBio ; 14(2): e0002323, 2023 04 25.
Artigo em Inglês | MEDLINE | ID: mdl-36786587

RESUMO

Fijiviruses replicate and package their genomes within viroplasms in a process involving RNA-RNA and RNA-protein interactions. Here, we demonstrate that the 24 C-terminal residues (C-arm) of the P9-1 major viroplasm protein of the mal de Río Cuarto virus (MRCV) are required for its multimerization and the formation of viroplasm-like structures. Using an integrative structural approach, the C-arm was found to be dispensable for P9-1 dimer assembly but essential for the formation of pentamers and hexamers of dimers (decamers and dodecamers), which favored RNA binding. Although both P9-1 and P9-1ΔC-arm catalyzed ATP with similar activities, an RNA-stimulated ATPase activity was only detected in the full-length protein, indicating a C-arm-mediated interaction between the ATP catalytic site and the allosteric RNA binding sites in the (do)decameric assemblies. A stronger preference to bind phosphate moieties in the decamer was predicted, suggesting that the allosteric modulation of ATPase activity by RNA is favored in this structural conformation. Our work reveals the structural versatility of a fijivirus major viroplasm protein and provides clues to its mechanism of action. IMPORTANCE The mal de Río Cuarto virus (MRCV) causes an important maize disease in Argentina. MRCV replicates in several species of Gramineae plants and planthopper vectors. The viral factories, also called viroplasms, have been studied in detail in animal reovirids. This work reveals that a major viroplasm protein of MRCV forms previously unidentified structural arrangements and provides evidence that it may simultaneously adopt two distinct quaternary assemblies. Furthermore, our work uncovers an allosteric communication between the ATP and RNA binding sites that is favored in the multimeric arrangements. Our results contribute to the understanding of plant reovirids viroplasm structure and function and pave the way for the design of antiviral strategies for disease control.


Assuntos
Reoviridae , Compartimentos de Replicação Viral , Animais , RNA/metabolismo , Reoviridae/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo
2.
PLoS Pathog ; 17(8): e1009802, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34370792

RESUMO

Multidrug-resistant Acinetobacter baumannii infections are increasing at alarming rates. Therefore, novel antibiotic-sparing treatments to combat these A. baumannii infections are urgently needed. The development of these interventions would benefit from a better understanding of this bacterium's pathobiology, which remains poorly understood. A. baumannii is regarded as an extracellular opportunistic pathogen. However, research on Acinetobacter has largely focused on common lab strains, such as ATCC 19606, that have been isolated several decades ago. These strains exhibit reduced virulence when compared to recently isolated clinical strains. In this work, we demonstrate that, unlike ATCC 19606, several modern A. baumannii clinical isolates, including the recent clinical urinary isolate UPAB1, persist and replicate inside macrophages within spacious vacuoles. We show that intracellular replication of UPAB1 is dependent on a functional type I secretion system (T1SS) and pAB5, a large conjugative plasmid that controls the expression of several chromosomally-encoded genes. Finally, we show that UPAB1 escapes from the infected macrophages by a lytic process. To our knowledge, this is the first report of intracellular growth and replication of A. baumannii. We suggest that intracellular replication within macrophages may contribute to evasion of the immune response, dissemination, and antibiotic tolerance of A. baumannii.


Assuntos
Infecções por Acinetobacter/microbiologia , Acinetobacter baumannii/fisiologia , Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Macrófagos/microbiologia , Sistemas de Secreção Tipo I/metabolismo , Vacúolos/microbiologia , Infecções por Acinetobacter/metabolismo , Animais , Camundongos
3.
Front Immunol ; 10: 1775, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31402921

RESUMO

Brucella enters their hosts mostly through mucosae from where it spreads systemically. Adhesion to extracellular matrix (ECM) components or to host cells is important for the infectious process, and is mediated by several adhesins, including the BtaF trimeric autotransporter. Although Th1 responses and gamma interferon (IFN-γ) are important for protection, antibodies able to block adhesions might also contribute to prevent Brucella infection. We evaluated the importance of BtaF for respiratory Brucella infection, and characterized the immune response and protection from mucosal challenge induced by nasal vaccination with recombinant BtaF. While lung CFU numbers did not differ at day 1 p.i. between mice intratracheally inoculated with B. suis M1330 (wild type) and those receiving a ΔbtaF mutant, they were reduced in the latter group at 7 and 30 days p.i. For vaccination studies the BtaF passenger domain was engineered and expressed as a soluble trimeric protein. Mice were immunized by the nasal route with BtaF or saline (control group) plus the mucosal adjuvant c-di-AMP. Specific anti-BtaF antibodies (IgG and IgA) were increased in serum, including a mixed IgG2a/IgG1 response. In vitro, these antibodies reduced bacterial adhesion to A549 alveolar epithelial cells. Specific IgA antibodies were also increased in several mucosae. Spleen cells from BtaF immunized mice significantly increased their IL-2, IL-5, IL-17, and IFN-γ secretion upon antigen stimulation. In cervical draining lymph nodes, antigen-experienced CD4+ T cells were maintained mainly as central memory cells. A BtaF-specific delayed-type hypersensitivity response was detected in BtaF immunized mice. Lung cells from the latter produced high levels of IFN-γ upon antigen stimulation. Although nasal immunization with BtaF did not protect mice against B. suis respiratory challenge, it conferred significant protection from intragastric challenge; the splenic load of B. suis was reduced by 3.28 log CFU in immunized mice. This study shows that nasal vaccination with BtaF+c-di-AMP protects against intragastric challenge with B. suis by inducing local and systemic antibody responses, central memory CD4+ T cells and strong Th1 responses. Therefore, although BtaF vaccination did not protect from B. suis respiratory infection, this adhesin constitutes a promising immunogen against mucosal B. suis infection.


Assuntos
Adesinas Bacterianas/genética , Antígenos de Bactérias/imunologia , Brucella suis/fisiologia , Brucelose/imunologia , Brucelose/microbiologia , Imunidade Adaptativa , Adesinas Bacterianas/metabolismo , Administração Intranasal , Animais , Linfócitos T CD4-Positivos , Fosfatos de Dinucleosídeos/metabolismo , Feminino , Humanos , Imunidade nas Mucosas/imunologia , Imunização/métodos , Camundongos , Virulência
4.
Sci Rep ; 9(1): 2158, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30770847

RESUMO

Brucella species are Gram-negative, facultative intracellular pathogens responsible for a worldwide zoonosis. The envelope of Brucella exhibits unique characteristics that make these bacteria furtive pathogens and resistant to several host defence compounds. We have identified a Brucella suis gene (mapB) that appeared to be crucial for cell envelope integrity. Indeed, the typical resistance of Brucella to both lysozyme and the cationic lipopeptide polymyxin B was markedly reduced in a ∆mapB mutant. MapB turned out to represent a TamB orthologue. This last protein, together with TamA, a protein belonging to the Omp85 family, form a complex that has been proposed to participate in the translocation of autotransporter proteins across the outer membrane (OM). Accordingly, we observed that MapB is required for proper assembly of an autotransporter adhesin in the OM, as most of the autotransporter accumulated in the mutant cell periplasm. Both assessment of the relative amounts of other specific outer membrane proteins (OMPs) and a proteome approach indicated that the absence of MapB did not lead to an extensive alteration in OMP abundance, but to a reduction in the relative amounts of a protein subset, including proteins from the Omp25/31 family. Electron microscopy revealed that ∆mapB cells exhibit multiple anomalies in cell morphology, indicating that the absence of the TamB homologue in B. suis severely affects cell division. Finally, ∆mapB cells were impaired in macrophage infection and showed an attenuated virulence phenotype in the mouse model. Collectively, our results indicate that the role of B. suis TamB homologue is not restricted to participating in the translocation of autotransporters across the OM but that it is essential for OM stability and protein composition and that it is involved in cell envelope biogenesis, a process that is inherently coordinated with cell division.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Brucella suis/crescimento & desenvolvimento , Divisão Celular , Membrana Celular/metabolismo , Parede Celular/metabolismo , Fatores de Virulência/metabolismo , Animais , Proteínas da Membrana Bacteriana Externa/genética , Brucella suis/genética , Brucella suis/metabolismo , Brucella suis/ultraestrutura , Brucelose/microbiologia , Brucelose/patologia , Linhagem Celular , Modelos Animais de Doenças , Deleção de Genes , Macrófagos/microbiologia , Camundongos , Microscopia Eletrônica de Transmissão , Virulência , Fatores de Virulência/genética
5.
Innate Immun ; 15(2): 91-100, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19318419

RESUMO

Cetuximab, an anti-epidermal growth factor receptor monoclonal antibody, has been shown to increase the median survival of colorectal cancer patients. We previously reported that the expression of HLA-E is significantly increased in primary human colorectal cancer, perhaps contributing to tumour escape from immune surveillance. To establish if HLA-E could be a factor that renders colorectal cancer cells less susceptible to antibody-dependent cellular cytotoxicity (ADCC), in the present study we analysed Cetuximab-mediated cytotoxicity against several colorectal cancer cell lines expressing, or not, HLA-E at the cell surface. We first observed that colorectal cancer cells treated with Cetuximab were killed more efficiently by ADCC. Interestingly, treatment of target cells with recombinant human-beta2-microglobulin inhibits Cetuximab-mediated ADCC through HLA-E membrane stabilization. The specific immunosuppressive role of HLA-E was confirmed using an anti-NKG2A monoclonal antibody, that restored the ability of immune cells to kill their target. This result demonstrates that HLA-E at the cell surface can reliably suppress the ADCC effect. On the other hand, Cetuximab induced a direct growth inhibition but only at high concentrations; furthermore, the CDC effect was quite moderate, and we failed to observe a pro-apoptotic effect. Taking into account that our findings suggest that ADCC activity is the main anti-tumour effect observed at clinically achievable concentrations of Cetuximab at the tumour site, we suggest that determination of HLA-E in colorectal cancer could be relevant to predict success of Cetuximab treatment.


Assuntos
Anticorpos Monoclonais/farmacologia , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Antineoplásicos/farmacologia , Neoplasias do Colo/imunologia , Antígenos HLA/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Anticorpos Monoclonais Humanizados , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Células CACO-2 , Cetuximab , Neoplasias do Colo/patologia , Neoplasias do Colo/terapia , Regulação para Baixo , Resistencia a Medicamentos Antineoplásicos , Fator de Crescimento Epidérmico/genética , Fator de Crescimento Epidérmico/imunologia , Fator de Crescimento Epidérmico/metabolismo , Células HCT116 , Antígenos HLA/genética , Antígenos HLA/imunologia , Células HT29 , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Tolerância Imunológica , Subfamília C de Receptores Semelhantes a Lectina de Células NK/imunologia , Evasão Tumoral , Microglobulina beta-2/imunologia , Microglobulina beta-2/metabolismo , Antígenos HLA-E
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