RESUMEN
BACKGROUND: The oomycete pathogen secretes many effectors into host cells to manipulate host defenses. For the majority of effectors, the mechanisms related to how they alter the expression of host genes and reprogram defenses are not well understood. In order to investigate the molecular mechanisms governing the influence that the Phytophthora infestans RXLR effector Pi04089 has on host immunity, a comparative transcriptome analysis was conducted on Pi04089 stable transgenic and wild-type potato plants. RESULTS: Potato plants stably expressing Pi04089 were more susceptible to P. infestans. RNA-seq analysis revealed that 658 upregulated genes and 722 downregulated genes were characterized in Pi04089 transgenic lines. A large number of genes involved in the biological process, including many defense-related genes and certain genes that respond to salicylic acid, were suppressed. Moreover, the comparative transcriptome analysis revealed that Pi04089 significantly inhibited the expression of many flg22 (a microbe-associated molecular pattern, PAMP)-inducible genes, including various Avr9/Cf-9 rapidly elicited (ACRE) genes. Four selected differentially expressed genes (StWAT1, StCEVI57, StKTI1, and StP450) were confirmed to be involved in host resistance against P. infestans when they were transiently expressed in Nicotiana benthamiana. CONCLUSION: The P. infestans effector Pi04089 was shown to suppress the expression of many resistance-related genes in potato plants. Moreover, Pi04089 was found to significantly suppress flg22-triggered defense signaling in potato plants. This research provides new insights into how an oomycete effector perturbs host immune responses at the transcriptome level.
Asunto(s)
Regulación de la Expresión Génica de las Plantas , Phytophthora infestans/inmunología , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/microbiología , Inmunidad de la Planta , Solanum tuberosum/inmunología , Factores de Virulencia/inmunología , Resistencia a la Enfermedad/genética , Resistencia a la Enfermedad/inmunología , Regulación de la Expresión Génica , Plantas Modificadas Genéticamente , Solanum tuberosum/genética , Solanum tuberosum/microbiología , TranscriptomaRESUMEN
The majority of urinary tract infections (UTIs) are caused by uropathogenic Escherichia coli (UPEC). Designing a vaccine will certainly reduce the occurrence of infection and antibiotic resistance of the isolates. Antigen 43 (Ag43) and autotransporter H (UpaH) have been associated with the virulence of UPEC. In the present study, the efficacy of different formulations of a hybrid protein composed of Ag43 and UpaH with and without alum and 1,25(OH)2D3 (Vitamin D3) adjuvants were evaluated in mice model. A significant increase in IgG and cellular responses was developed against Ag43::UpaH as compared to the control mice. The addition of alum or a mixture of alum and Vitamin D3 to the protein significantly enhanced the serum IgG responses and tended to remain in a steady state until 6 months. In addition, the mentioned formulations produced significant amounts of IgG1, IL-4, and IL-17 as compared to the fusion protein alone. In addition to the mentioned formulations, the combination of protein with Vitamin D3 also resulted in significantly higher serum IgA and IFN-γ levels as compared to the fusion protein alone. Mice immunized with fusion plus alum and formulation protein admixed with both alum and Vitamin D3 significantly reduced the bacterial load in the bladders and kidneys of mice as compared to the control. In this study, for the first time, the ability of a novel hybrid protein in combination with adjuvants alum and Vitamin D3 was evaluated against UPEC. Our results indicated that fusion Ag43::UpaH admixed with alum and Vitamin D3 could be a promising candidate against UTIs.
Asunto(s)
Compuestos de Alumbre/farmacología , Colecalciferol/farmacología , Proteínas de Escherichia coli/inmunología , Proteínas Recombinantes de Fusión/inmunología , Infecciones Urinarias/prevención & control , Escherichia coli Uropatógena/inmunología , Factores de Virulencia/inmunología , Adyuvantes Inmunológicos/farmacología , Adyuvantes Inmunológicos/uso terapéutico , Compuestos de Alumbre/administración & dosificación , Animales , Anticuerpos Antibacterianos/sangre , Anticuerpos Antibacterianos/orina , Antígenos Bacterianos/inmunología , Carga Bacteriana/efectos de los fármacos , Carga Bacteriana/inmunología , Vacunas Bacterianas/administración & dosificación , Vacunas Bacterianas/inmunología , Vacunas Bacterianas/aislamiento & purificación , Colecalciferol/administración & dosificación , Citocinas/metabolismo , Inmunidad Humoral/efectos de los fármacos , Inmunización/métodos , Inmunoglobulina G/sangre , Inmunoglobulina G/orina , Inyecciones Intravenosas , Ratones Endogámicos BALB C , Membrana Mucosa/inmunología , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/aislamiento & purificación , Infecciones Urinarias/inmunologíaRESUMEN
Phytophthora infestans causes the severe late blight disease of potato. During its infection process, P. infestans delivers hundreds of RXLR (Arg-x-Leu-Arg, x behalf of any one amino acid) effectors to manipulate processes in its hosts, creating a suitable environment for invasion and proliferation. Several effectors interact with host proteins to suppress host immunity and inhibit plant growth. However, little is known about how P. infestans regulates the host transcriptome. Here, we identified an RXLR effector, PITG_15718.2, which is upregulated and maintains a high expression level throughout the infection. Stable transgenic potato (Solanum tuberosum) lines expressing PITG_15718.2 show enhanced leaf colonization by P. infestans and reduced vegetative growth. We further investigated the transcriptional changes between three PITG_15718.2 transgenic lines and the wild type Désirée by using RNA sequencing (RNA-Seq). Compared with Désirée, 190 differentially expressed genes (DEGs) were identified, including 158 upregulated genes and 32 downregulated genes in PITG_15718.2 transgenic lines. Eight upregulated and nine downregulated DEGs were validated by real-time RT-PCR, which showed a high correlation with the expression level identified by RNA-Seq. These DEGs will help to explore the mechanism of PITG_15718.2-mediated immunity and growth inhibition in the future.
Asunto(s)
Péptidos/inmunología , Phytophthora infestans/inmunología , Enfermedades de las Plantas/inmunología , Solanum tuberosum/inmunología , Factores de Virulencia/inmunología , Interacciones Huésped-Parásitos , Phytophthora infestans/fisiología , Enfermedades de las Plantas/parasitología , Inmunidad de la Planta , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Plantas Modificadas Genéticamente/inmunología , Plantas Modificadas Genéticamente/parasitología , Solanum tuberosum/crecimiento & desarrollo , Solanum tuberosum/parasitologíaRESUMEN
Chagas disease is an endemic pathology in Latin America, now emerging in developed countries, caused by the intracellular protozoan Trypanosoma cruzi, whose life cycle involves three stages: amastigotes, epimastigotes, and trypomastigotes. T. cruzi Calreticulin (TcCRT), an endoplasmic reticulum resident chaperone, translocates to the external cellular membrane, where it captures complement component C1, ficolins and MBL, thus inactivating the classical and lectin pathways. Trypomastigote-bound C1 is detected as an "eat me" signal by macrophages and promotes the infective process. Unlike infective trypomastigotes, non-infective epimastigotes either do not express or express only marginal levels of TcCRT on their external membrane. We show that epimastigotes bind exogenous rTcCRT to their cellular membrane and, in the presence of C1q, this parasite form is internalized into normal fibroblasts. On the other hand, Calreticulin (CRT)-deficient fibroblasts show impaired parasite internalization. In synthesis, CRT from both parasite and host cell origin is important in the establishment of C1q-dependent first contacts between parasites and host cells.
Asunto(s)
Calreticulina/inmunología , Endocitosis/inmunología , Interacciones Huésped-Parásitos/inmunología , Trypanosoma cruzi/inmunología , Adyuvantes Inmunológicos , Animales , Calreticulina/genética , Calreticulina/metabolismo , Membrana Celular/inmunología , Membrana Celular/metabolismo , Enfermedad de Chagas/inmunología , Enfermedad de Chagas/parasitología , Complemento C1q/inmunología , Complemento C1q/metabolismo , Fibroblastos/metabolismo , Fibroblastos/parasitología , Técnicas de Inactivación de Genes , Ratones , Unión Proteica , Trypanosoma cruzi/metabolismo , Trypanosoma cruzi/patogenicidad , Factores de Virulencia/inmunologíaRESUMEN
Several candidate vaccines against Shigella spp. are in development, but the lack of a clear correlate of protection from challenge with the induction of adequate immune responses among the youngest age groups in the developing world has hampered Shigella vaccine development over the past several decades. Bioconjugation technology, exploited here for an Shigella flexneri 2a candidate vaccine, offers a novel and potentially cost-effective way to develop and produce vaccines against a major pathogen of global health importance. Flexyn2a, a novel S. flexneri 2a bioconjugate vaccine made of the polysaccharide component of the S. flexneri 2a O-antigen, conjugated to the exotoxin protein A of Pseudomonas aeruginosa (EPA), was evaluated for safety and immunogenicity among healthy adults in a single-blind, phase I study with a staggered randomization approach. Thirty subjects (12 receiving 10 µg Flexyn2a, 12 receiving Flexyn2a with aluminum adjuvant, and 6 receiving placebo) were administered two injections 4 weeks apart and were followed for 168 days. Flexyn2a was well-tolerated, independently of the adjuvant and number of injections. The Flexyn2a vaccine elicited statistically significant S. flexneri 2a lipopolysaccharide (LPS)-specific humoral responses at all time points postimmunization in all groups that received the vaccine. Elicited serum antibodies were functional, as evidenced by bactericidal activity against S. flexneri 2a. The bioconjugate candidate vaccine Flexyn2a has a satisfactory safety profile and elicited a robust humoral response to S. flexneri 2a LPS with or without inclusion of an adjuvant. Moreover, the bioconjugate also induced functional antibodies, showing the technology's features in producing a promising candidate vaccine. (This study has been registered at ClinicalTrials.gov under registration no. NCT02388009.).
Asunto(s)
Anticuerpos Antibacterianos/sangre , Disentería Bacilar/prevención & control , Inmunogenicidad Vacunal , Vacunas contra la Shigella/efectos adversos , Vacunas contra la Shigella/inmunología , Shigella flexneri/inmunología , ADP Ribosa Transferasas/genética , ADP Ribosa Transferasas/inmunología , Adolescente , Adulto , Anticuerpos Antibacterianos/inmunología , Proteínas Bacterianas/inmunología , Toxinas Bacterianas/genética , Toxinas Bacterianas/inmunología , Disentería Bacilar/inmunología , Exotoxinas/genética , Exotoxinas/inmunología , Femenino , Voluntarios Sanos , Humanos , Inmunoglobulina A/inmunología , Lipopolisacáridos/inmunología , Masculino , Persona de Mediana Edad , Antígenos O/inmunología , Vacunas contra la Shigella/administración & dosificación , Shigella sonnei/inmunología , Método Simple Ciego , Vacunas Conjugadas/administración & dosificación , Vacunas Conjugadas/efectos adversos , Vacunas Conjugadas/inmunología , Factores de Virulencia/genética , Factores de Virulencia/inmunología , Adulto Joven , Exotoxina A de Pseudomonas aeruginosaRESUMEN
BACKGROUND: Extra-intestinal pathogenic Escherichia coli (ExPEC) are major human pathogens; however, no protective vaccine is currently available. We assessed in animal models the immunogenicity and safety of a 4-valent E. coli conjugate vaccine (ExPEC-4V, serotypes O1, O2, O6 and O25 conjugated to Exotoxin A from Pseudomonas aeruginosa (EPA)) produced using a novel in vivo bioconjugation method. METHODS: Three doses of ExPEC-4V (with or without aluminum hydroxide) were administered to rabbits (2µg or 20µg per O-antigen, subcutaneously), mice (0.2µg or 2µg per O-antigen, subcutaneously) and rats (0.4µg or 4µg per O-antigen, intramuscularly). Antibody persistence and boostability were evaluated in rats using O6-EPA monovalent conjugate (0.4µg O-antigen/dose, intramuscularly). Toxicity was assessed in rats (16µg total polysaccharide, intramuscularly). Serum IgG and IgM antibodies were measured by ELISA. RESULTS: Robust antigen-specific IgG responses were observed in all animal models, with increased responses in rabbits when administered with adjuvant. O antigen-specific antibody responses persisted up to 168days post-priming. Booster immunization induced a rapid recall response. Toxicity of ExPEC-4V when administered to rats was considered to be at the no observed adverse effect level. CONCLUSIONS: ExPEC-4V conjugate vaccine showed good immunogenicity and tolerability in animal models supporting progression to clinical evaluation.
Asunto(s)
Infecciones por Escherichia coli/prevención & control , Vacunas contra Escherichia coli/inmunología , Inmunogenicidad Vacunal , Antígenos O/inmunología , ADP Ribosa Transferasas/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Animales , Anticuerpos Antibacterianos/sangre , Toxinas Bacterianas/inmunología , Escherichia coli , Exotoxinas/inmunología , Femenino , Inmunización Secundaria , Ratones , Ratones Endogámicos ICR , Nivel sin Efectos Adversos Observados , Conejos , Ratas , Ratas Sprague-Dawley , Pruebas de Toxicidad , Vacunas Conjugadas/inmunología , Factores de Virulencia/inmunología , Exotoxina A de Pseudomonas aeruginosaRESUMEN
The ability to identify immunogenic determinants that activate T-cells is important for the development of new vaccines, allergy therapy and protein therapeutics. In silico MHC-II binding prediction algorithms are often used for T-cell epitope identification. To understand how well those programs predict immunogenicity, we computed HLA binding to peptides spanning the sequence of PE38, a fragment of an anti-cancer immunotoxin, and compared the predicted and experimentally identified T-cell epitopes. We found that the prediction for individual donors did not correlate well with the experimental data. Furthermore, prediction of T-cell epitopes in an HLA heterogenic population revealed that the two strongest epitopes were predicted at multiple cutoffs but the third epitope was predicted negative at all cutoffs and overall 4/9 epitopes were missed at several cutoffs. We conclude that MHC class-II binding predictions are not sufficient to predict the T-cell epitopes in PE38 and should be supplemented by experimental work.
Asunto(s)
ADP Ribosa Transferasas/inmunología , Toxinas Bacterianas/inmunología , Epítopos de Linfocito T/inmunología , Exotoxinas/inmunología , Antígenos HLA-DR/inmunología , Activación de Linfocitos/inmunología , Unión Proteica/inmunología , Pseudomonas/inmunología , Linfocitos T/inmunología , Factores de Virulencia/inmunología , Algoritmos , Secuencia de Aminoácidos , Formación de Anticuerpos/inmunología , Sitios de Unión/inmunología , Humanos , Datos de Secuencia Molecular , Péptidos/inmunología , Exotoxina A de Pseudomonas aeruginosaRESUMEN
Streptococcus mutans (SM) have three main virulence antigens: glucan binding protein B (gbpB), glucosyltransferase (Gtf) and antigens I/II (Ag I/II) envolved in the capacity of those bacteria to adhere and accumulate in the dental biofilm. Also, the glycosyltransferases 153 kDa of Streptococcus gordonii (SGO) and 170kDa of Streptococcus sanguinis (SSA) were important antigens associated with the accumulation of those bacterias. Streptococcus mitis (SMI) present IgA1 protease of 202 kDa. We investigated the specificity and levels IgA against those antigens of virulence in samples of human colostrum. This study involved 77 samples of colostrum that were analyzed for levels of immunoglobulian A, M and G by Elisa. The specificity of IgA against extracts of SM and initials colonizators (SSA, SMI, SGO) were analyzed by the Western blot. The mean concentration of IgA was 2850.2 (±2567.2) mg/100 mL followed by IgM and IgG (respectively 321.8±90.3 and 88.3±51.5), statistically different (p<0.05). Results showed that the majority of samples had detectable levels of IgA antibodies to extracts of bacteria antigens and theirs virulence antigens. To SM, the GbpB was significantly lower detected than others antigens of SM (p<0.05). High complexities of response to Ags were identified in the samples. There were no significant differences in the mean number of IgA-reactive Ags between the antigens (p>0.4). So, the breast milk from first hours after birth presented significant levels of IgA specific against important virulence of antigens those oral streptococci, which can disrupt the installation and accumulation process of these microorganisms in the oral cavity.
Asunto(s)
Bacterias/inmunología , Calostro/inmunología , Inmunoglobulina A Secretora/inmunología , Adulto , Antígenos Bacterianos/inmunología , Bacterias/aislamiento & purificación , Femenino , Humanos , Inmunoglobulina G/inmunología , Inmunoglobulina M/inmunología , Leche Humana/inmunología , Factores de Virulencia/inmunologíaRESUMEN
Amongst the pathologies associated with infection by Kaposi's sarcoma-associated herpesvirus (KSHV), multicentric Castleman's disease is distinctive for involvement of the lytic phase of the virus replication cycle. This B cell lymphoproliferative disorder has shown clinical responsiveness not only to generalized immunotherapy and cytotoxic chemotherapy, but also to inhibitors of herpesvirus DNA replication, consistent with the involvement of lytic phase of replication. These findings suggest that selective killing of virus-producing cells might represent a novel therapeutic strategy. We designed an immunotoxin, YC15-PE38, containing a single chain variable region fragment of a monoclonal antibody against KSHV glycoprotein H (gH) linked to the effector domains of Pseudomonas aeruginosa exotoxin A. Purified YC15-PE38 displayed highly selective and potent killing of a gH-expressing transfectant cell line (subnanomolar IC(50)). The immunotoxin also strongly inhibited production of infectious KSHV virions from an induced chronically infected cell line, by virtue of selective killing of the virus-producing cells. Combination treatment studies indicated complementary activities between YC15-PE38 and the herpesviral DNA replication inhibitor ganciclovir. These results provide support for the development of anti-KSHV strategies based on targeted killing of infected cells expressing lytic phase genes.
Asunto(s)
ADP Ribosa Transferasas/uso terapéutico , Toxinas Bacterianas/uso terapéutico , Citotoxicidad Inmunológica , Exotoxinas/uso terapéutico , Infecciones por Herpesviridae/tratamiento farmacológico , Infecciones por Herpesviridae/inmunología , Herpesvirus Humano 8/fisiología , Inmunotoxinas/uso terapéutico , Proteínas del Envoltorio Viral/inmunología , Factores de Virulencia/uso terapéutico , ADP Ribosa Transferasas/genética , ADP Ribosa Transferasas/inmunología , Toxinas Bacterianas/genética , Toxinas Bacterianas/inmunología , Línea Celular , Exotoxinas/genética , Exotoxinas/inmunología , Infecciones por Herpesviridae/virología , Herpesvirus Humano 8/efectos de los fármacos , Herpesvirus Humano 8/genética , Humanos , Inmunotoxinas/genética , Inmunotoxinas/inmunología , Proteínas del Envoltorio Viral/genética , Factores de Virulencia/genética , Factores de Virulencia/inmunología , Replicación Viral , Exotoxina A de Pseudomonas aeruginosaRESUMEN
Rheumatoid arthritis is a proinflammatory autoimmune disease attributed to failure of both CD4(+)CD25(+) regulatory T (Tr) and CD8(+)CD28(-) suppressor T (Ts) cells to control autoreactive CD4(+)CD28(+) Th1 (Th1) and autoantibody-producing B cells. Here we show a single intramuscular injection of our novel targeted DNA vaccine encoding Pseudomonas exotoxin A and costimulatory molecule B7-2 without autoantigens in a collagen-induced arthritis model simultaneously increased Tr and Ts cells and selectively decreased autoreactive Th1 cells. The vaccine induced a shift from Th1 to Th2 and Th3 cellular and cytokine profiles and a decrease in CD4(+)/CD8(+) cell ratios. Importantly, the vaccine showed potent antirheumatic activity by clinical and other examinations such as X-ray, histopathology, and anti-type II collagen IgG levels and was comparable to methotrexate, the current "gold standard" treatment. As an effective stimulator of both Tr and Ts cells and a specific suppressor of autoreactive Th1 cells, this vaccine is a promising therapeutic approach for rheumatoid arthritis.
Asunto(s)
ADP Ribosa Transferasas/administración & dosificación , Antirreumáticos/inmunología , Artritis Reumatoide/prevención & control , Antígeno B7-2/administración & dosificación , Toxinas Bacterianas/administración & dosificación , Antígenos CD28/inmunología , Exotoxinas/administración & dosificación , Vacunas de ADN/inmunología , Factores de Virulencia/administración & dosificación , ADP Ribosa Transferasas/inmunología , Animales , Antirreumáticos/metabolismo , Artritis Experimental/inmunología , Artritis Reumatoide/inmunología , Antígeno B7-2/inmunología , Toxinas Bacterianas/inmunología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Citocinas/inmunología , Exotoxinas/inmunología , Femenino , Ratas , Ratas Wistar , Transducción de Señal , Linfocitos T Reguladores/inmunología , Células TH1/inmunología , Células Th2/inmunología , Vacunas de ADN/metabolismo , Factores de Virulencia/inmunología , Exotoxina A de Pseudomonas aeruginosaRESUMEN
Attenuated Yersinia pestis pgm strains, such as KIM5, lack the siderophore yersiniabactin. Strain KIM5 does not induce significant pneumonia when delivered intranasally. In this study, mice were found to develop pneumonia after intranasal challenge with strain KIM5 when they were injected intraperitoneally with iron dextran, though not with iron sulfate. KIM5-infected mice treated daily with 4 mg iron dextran died in 3 days with severe pneumonia. Pneumonia was less severe if 4 mg iron dextran was administered only once before infection. The best-studied experimental vaccine against plague currently consists of the Yersinia pestis capsular antigen F1 and the type 3 secreted protein LcrV. The F1 antigen was shown to be protective against KIM5 infections in mice administered iron dextran doses leading to light or severe pneumonia, supporting the use of an iron dextran-treated model of pneumonic plague. Since F1 has been reported to be incompletely protective in some primates, and bacterial isolates lacking F1 are still virulent, there has been considerable interest in identifying additional protective subunit immunogens. Here we showed that the highly conserved Psa fimbriae of Y. pestis (also called pH 6 antigen) are expressed in murine organs after infection through the respiratory tract. Studies with iron dextran-treated mice showed that vaccination with the Psa fimbrial protein together with an adjuvant afforded incomplete but significant protection in the mouse model described. Therefore, further investigations to fully characterize the protective properties of the Psa fimbriae are warranted.
Asunto(s)
Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Modelos Animales de Enfermedad , Factores Inmunológicos/administración & dosificación , Complejo Hierro-Dextran/administración & dosificación , Peste/microbiología , Yersinia pestis/patogenicidad , Animales , Antígenos Bacterianos/fisiología , Proteínas Bacterianas/fisiología , Contención de Riesgos Biológicos , Femenino , Ratones , Ratones Endogámicos C57BL , Peste/patología , Factores de Virulencia/inmunología , Factores de Virulencia/fisiología , Yersinia pestis/inmunologíaRESUMEN
A diarréia é um importante problema de saúde pública no mundo inteiro e a Escherichía coli é um dos mais freqüentes microorganismos causadores desta doença. A Escherichia coli enteropatogênica (EPEC), um dos principais agentes etiológicos das diarréias infantis no nosso país, é genética e fenotipicamente relacionada com a E. colí enterohemorrágica (EHEC) que além de provocar diarréia é responsável por complicações como síndrome hemolítica urêmica (HUS) e colite hemorrágica (HC). Embora a EHEC seja considerada emergente pela OMS, no Brasil poucos casos de complicações como HUS e HC foram reportados. O mecanismo de patogenicidade comum entre EPEC e EHEC é conhecido como a lesão "attaching and effacing" nos microvilos do enterócito. Esta lesão é mediada por um conjunto de fatores de virulência, dentre eles a intimina. A intimina é uma proteína de membrana externa, responsável pelo íntimo contato da bactéria com o enterócito, possui uma região N-terminal que é altamente conservada e uma região C-terminal que é variável. De acordo com a região variável, existem vários subtipos de intimina, dentre eles as intiminas , α, β e γ...
Asunto(s)
Humanos , Masculino , Femenino , Adulto , Anticuerpos/genética , Anticuerpos/inmunología , Diarrea/genética , Diarrea/inmunología , Escherichia coli Enteropatógena/fisiología , Escherichia coli Enteropatógena/inmunología , Factores de Virulencia/genética , Factores de Virulencia/inmunología , Infecciones por Escherichia coli/inmunología , Calostro , Ensayo de Inmunoadsorción Enzimática , Métodos Analíticos de la Preparación de la Muestra , Suero , Interpretación Estadística de DatosRESUMEN
Enterohemorrhagic Escherichia coli (EHEC) is the main cause of hemolytic-uremic syndrome, an endemic disease in Argentina which had an incidence in 2005 of 13.9 cases per 100,000 children younger than 5 years old. Cattle appear to be a major reservoir of EHEC, and a serological response to EHEC antigens has been demonstrated in natural and experimental infections. In the current study, antibodies against proteins implicated in EHEC's ability to form attaching and effacing lesions, some of which are exported to the host cell via a type three secretion system (TTSS), were identified in bovine colostrum by Western blot analysis. Twenty-seven (77.0%) of the 35 samples examined contained immunoglobulin G (IgG) antibodies against the three proteins assayed in this study: EspA, EspB, and the carboxy-terminal 280 amino acids of gamma-intimin, an intimin subtype associated mainly with O157:H7 and O145:H- serotypes. Every colostrum sample was able to inhibit, in a range between 45.9 and 96.7%, the TTSS-mediated hemolytic activity of attaching and effacing E. coli. The inhibitory effect was partially mediated by IgG and lactoferrin. In conclusion, we found that early colostrum from cows contains antibodies, lactoferrin, and other unidentified substances that impair TTSS function in attaching and effacing E. coli strains. Bovine colostrum might act by reducing EHEC colonization in newborn calves and could be used as a prophylactic measure to protect non-breast-fed children against EHEC infection in an area of endemicity.
Asunto(s)
Adhesinas Bacterianas/inmunología , Proteínas de la Membrana Bacteriana Externa/inmunología , Calostro/inmunología , Escherichia coli Enterohemorrágica/patogenicidad , Proteínas de Escherichia coli/inmunología , Inmunoglobulina G/inmunología , Adhesinas Bacterianas/genética , Adhesinas Bacterianas/metabolismo , Animales , Anticuerpos Antibacterianos/análisis , Anticuerpos Antibacterianos/inmunología , Argentina , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Bovinos , Calostro/química , Escherichia coli Enterohemorrágica/genética , Escherichia coli Enterohemorrágica/metabolismo , Escherichia coli O157/genética , Escherichia coli O157/metabolismo , Escherichia coli O157/patogenicidad , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Femenino , Hemólisis/inmunología , Humanos , Inmunoglobulina G/análisis , Lactoferrina/análisis , Factores de Virulencia/genética , Factores de Virulencia/inmunología , Factores de Virulencia/metabolismoRESUMEN
Pathogen-associated molecular patterns (PAMPs) elicit basal defense responses in plants, and, in turn, pathogens have evolved mechanisms to overcome these PAMP-induced defenses. To suppress immunity, the phytopathogenic bacterium Pseudomonas syringae secretes effector proteins, the biochemical function and virulence targets of which remain largely unknown. We show that HopAI1, an effector widely conserved in both plant and animal bacterial pathogens, inhibits the Arabidopsis mitogen-activated protein kinases (MAPKs) activated by exposure to PAMPs. HopAI1 inactivates MAPKs by removing the phosphate group from phosphothreonine through a unique phosphothreonine lyase activity, which is required for HopAI1 function. The inhibition of MAPKs by HopA1 suppresses two independent downstream events, namely the reinforcement of cell wall defense and transcriptional activation of PAMP response genes. The MAPKs MPK3 and MPK6 physically interact with HopAI1 indicating that they are direct targets of HopAI1. These findings uncover a mechanism by which Pseudomonas syringae overcomes host innate immunity to promote pathogenesis.
Asunto(s)
Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/microbiología , Pseudomonas syringae/inmunología , Arabidopsis , Proteínas de Arabidopsis/metabolismo , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fósforo/metabolismo , Fosfotreonina/metabolismo , Pseudomonas syringae/metabolismo , Factores de Virulencia/inmunología , Factores de Virulencia/metabolismoRESUMEN
Synthetic peptides derived from the G-H loop of the foot and mouth disease virus (FMDV) capsid protein VP1 are relatively poor at recapitulating the native conformation present in the virus, and thus are often poor immunogens. We hypothesized that a candidate mucosal vaccine against FMDV could be developed using the non-toxic Pseudomonas aeruginosa exotoxin A (ntPE) to deliver the G-H loop in its native conformation. An added benefit of this approach is the potential for ntPE to serve as an effective carrier/adjuvant molecule for delivery of the fusion protein across the epithelial barrier by virtue of its capacity to bind to CD91. A chimeric protein (ntPE-GH) was generated by inserting the coding sequence of the G-H loop into an expression plasmid encoding ntPE, in place of the native Ib loop. Recombinant ntPE-GH and wild-type ntPE were each expressed in Escherichia coli, purified over a nickel resin, then administered intranasally to the pigs, with or without the mucosal adjuvant cholera toxin (CT). Both the ntPE and ntPE-GH induced mucosal and systemic immune responses against ntPE; moreover, ntPE-GH administered without CT induced anti-GH loop serum IgG antibodies. In conclusion, these data demonstrate that ntPE can be used as a mucosal carrier/adjuvant to induce an immune response against the VP1 G-H loop of FMDV.
Asunto(s)
ADP Ribosa Transferasas/inmunología , Toxinas Bacterianas/inmunología , Proteínas de la Cápside/inmunología , Exotoxinas/inmunología , Virus de la Fiebre Aftosa/inmunología , Proteínas Recombinantes de Fusión/inmunología , Vacunas Sintéticas/inmunología , Vacunas Virales/inmunología , Factores de Virulencia/inmunología , ADP Ribosa Transferasas/genética , Administración Intranasal , Animales , Anticuerpos Antivirales/sangre , Toxinas Bacterianas/genética , Proteínas de la Cápside/química , Proteínas de la Cápside/genética , Exotoxinas/genética , Femenino , Masculino , Porcinos , Vacunación , Factores de Virulencia/genética , Exotoxina A de Pseudomonas aeruginosaRESUMEN
LHRH-PE40, a recombinant DNA-derived protein composed of LHRH and Pseudomonas aeruginosa exotoxin A, is being developed for the treatment of malignant tumours. This experiment was designed to assess its preclinical safety. Reproductive toxicity studies, pharmacokinetic studies, single- and repeat-dose intraperitoneal or intravenous toxicity studies in mice, rats and monkeys were conducted to assess the toxicity of LHRH-PE40. In intravenous single-dose studies in mice, the LD50 was 731.26 microg/kg and 676.03 microg/kg in male and female mice respectively. In single-dose studies and repeat-dose range-finding studies in rats, dose-limited severe vascular leakage syndromes occurred. In repeat-dose long-term studies, except drug-related vascular leakage syndromes, other drug-related changes included decreased testis weight and testis atrophy. In single-dose and repeat-dose studies in monkeys, dose-limited acute tubular necrosis of the kidneys was the chief finding. In reproductive studies, drug-related changes were decreased food intakes, decreased testis weight and uterus weight, decreased foetus weight and increased foetus mortality, increased maternal and F1 offspring mortality and decreased maternal and F1 offspring body weight. Pharmacokinetic studies showed a similar half-time of distribution and clearance in mice and monkeys. Tissue distribution showed a high concentration in the kidneys and a low concentration in the brain. LHRH-PE40 induced vascular leak syndromes in rats and acute tubular necrosis in monkeys. It also led to testicle atrophy in rats and overt productive toxicity to parents and F1 generations in mice. Because of these findings, it should be monitored carefully in human clinical trials for things such as respiratory, urinary and reproductive toxicities.