RESUMEN
BACKGROUND: Human challenge models for enterotoxigenic Escherichia coli (ETEC) facilitate vaccine down-selection. The B7A (O148:H28 CS6+LT+ST+) strain is important for vaccine development. We sought to refine the B7A model by identifying a dose and fasting regimen consistently inducing moderate-severe diarrhea. METHODS: An initial cohort of 28 subjects was randomized (1:1:1:1) to receive B7A following an overnight fast at doses of 108 or 109 colony forming units (cfu) or a 90-minute fast at doses of 109 or 1010 cfu. A second cohort included naïve and rechallenged subjects who had moderate-severe diarrhea and were given the target regimen. Immune responses to important ETEC antigens were assessed. RESULTS: Among subjects receiving 108 cfu of B7A, overnight fast, or 109 cfu, 90-minute fast, 42.9% (3/7) had moderate-severe diarrhea. Higher attack rates (71.4%; 5/7) occurred in subjects receiving 109 cfu, overnight fast, or 1010 cfu, 90-minute fast. Upon rechallenge with 109 cfu of B7A, overnight fast, 5/11 (45.5%) had moderate-severe diarrhea; the attack rate among concurrently challenge naïve subjects was 57.9% (11/19). Anti-CS6, O148 LPS and LT responses were modest across all groups. CONCLUSIONS: An overnight fast enabled a reduction in the B7A inoculum dose; however, the attack rate was inconsistent and protection upon rechallenge was minimal.
Asunto(s)
Antígenos Bacterianos/análisis , Diarrea/etiología , Escherichia coli Enterotoxigénica/patogenicidad , Infecciones por Escherichia coli/microbiología , Proteínas de Escherichia coli/análisis , Vacunas contra Escherichia coli , Adolescente , Adulto , Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/inmunología , Carga Bacteriana , Toxinas Bacterianas/inmunología , Ciprofloxacina/uso terapéutico , Diarrea/microbiología , Diarrea/terapia , Relación Dosis-Respuesta Inmunológica , Escherichia coli Enterotoxigénica/inmunología , Escherichia coli Enterotoxigénica/aislamiento & purificación , Enterotoxinas/inmunología , Infecciones por Escherichia coli/prevención & control , Proteínas de Escherichia coli/inmunología , Ayuno , Heces/microbiología , Femenino , Fluidoterapia , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Lipopolisacáridos/inmunología , Masculino , Persona de Mediana Edad , Distribución Aleatoria , Factores de Tiempo , Adulto JovenRESUMEN
Numerous Gram-negative bacterial pathogens employ type III secretion systems (T3SSs) to inject effector proteins into eukaryotic cells. The activation of the type III secretion (T3S) process is tightly controlled in all T3SSs. In Yersinia pestis, the secretion of effector proteins, termed Yersinia outer proteins (Yops), is regulated by the activity of the YopN/SycN/YscB/TyeA complex. YopN is a secreted protein that interacts with the SycN/YscB chaperone via an N-terminal chaperone-binding domain (CBD) and with TyeA via a C-terminal TyeA-binding domain (TBD). Efficient YopN secretion is dependent upon its N-terminal secretion signal (SS), CBD, and the SycN/YscB chaperone. In this study, we investigate the role of the YopN CBD in the regulation of Yop secretion. Analysis of YopE/YopN hybrid proteins in which the YopN SS or SS and CBD were replaced with the analogous regions of YopE indicated that the YopN CBD or SycN/YscB chaperone play a role in the regulation of Yop secretion that is independent of their established roles in YopN secretion. To further analyze the role of the YopN CBD in the regulation of Yop secretion a series of tetra-alanine substitution mutants were generated throughout the YopN CBD. A number of these mutants exhibited a defect in the regulation of Yop secretion but showed no defect in YopN secretion or in the interaction of YopN with the SycN/YscB chaperone. Finally, conditions were established that enabled YopN and TyeA to regulate Yop secretion in the absence of the SycN/YscB chaperone. Importantly, a number of the YopN CBD mutants maintained their defect in the regulation of Yop secretion even under the established SycN/YscB chaperone-independent conditions. These studies establish a role for the CBD region of YopN in the regulation of Yop secretion that is independent from its role in YopN secretion or in the binding of the SycN/YscB chaperone.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de la Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Peste/microbiología , Yersinia pestis/genética , Sustitución de Aminoácidos , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Calcio/fisiología , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Chaperonas Moleculares/genética , Mutagénesis Sitio-Dirigida , Unión Proteica , Estructura Terciaria de Proteína , Transporte de Proteínas , Proteínas Recombinantes de Fusión , Eliminación de Secuencia , Yersinia pestis/metabolismo , Yersinia pestis/fisiologíaRESUMEN
The Chlamydia pneumoniae CopN protein is a member of the YopN/TyeA/InvE/MxiC family of secreted proteins that function to regulate the secretion of type III secretion system (T3SS) translocator and effector proteins. In this study, the Scc1 (CP0432) and Scc4 (CP0033) proteins of C. pneumoniae AR-39 were demonstrated to function together as a type III secretion chaperone that binds to an N-terminal region of CopN. The Scc1/Scc4 chaperone promoted the efficient secretion of CopN via a heterologous T3SS, whereas, the Scc3 chaperone, which binds to a C-terminal region of CopN, reduced CopN secretion.
Asunto(s)
Proteínas Bacterianas/metabolismo , Chlamydophila pneumoniae/metabolismo , Chaperonas Moleculares/metabolismo , Secuencias de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Chlamydophila pneumoniae/química , Chlamydophila pneumoniae/genética , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Unión Proteica , Transporte de ProteínasRESUMEN
The localization and membrane topology of the Yersinia pestis YscJ lipoprotein, an essential component of the type III secretion apparatus, was investigated. YscJ was demonstrated to be an inner membrane (IM) lipoprotein that is anchored to the periplasmic face of the IM via an N-terminal lipid moiety and via a C-terminal transmembrane (TM) domain. Localization of the N-terminal lipid moiety to the IM occurred regardless of the amino-acid residues found in the +2 or +3 positions. IM localization was dependent upon an intact N-terminal domain (amino acids +1 to +61), suggesting that this region plays a role in YscJ localization. In contrast, the YscJ C-terminal domain and TM domain were not required for IM localization. N-terminal sequence analysis demonstrated that a significant proportion of membrane-localized YscJ lacks N-acylation, the final modification required for Lol-dependent transport of a lipoprotein to the OM. Interestingly, attachment of the N-terminus to the IM was required for YscJ function; however, the YscJ secretion signal and lipo-box could be functionally replaced by the first TM domain of the YscV protein, suggesting that the mechanism of attachment to the IM was not critical.
Asunto(s)
Proteínas Bacterianas/química , Lipoproteínas/química , Proteínas Periplasmáticas/química , Yersinia pestis/metabolismo , Acilación , Aciltransferasas/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Lípidos/análisis , Lipoproteínas/genética , Lipoproteínas/metabolismo , Periplasma/metabolismo , Proteínas Periplasmáticas/genética , Proteínas Periplasmáticas/metabolismo , Fenotipo , Señales de Clasificación de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Eliminación de SecuenciaRESUMEN
The secretion of Yops via the Yersinia type III secretion system (T3SS) is controlled, in part, by a cytoplasmic YopN/TyeA complex. This complex is required to prevent Yop secretion in the presence of extracellular calcium and prior to contact between the bacterium and a eukaryotic cell. In this study we utilized site-directed mutagenesis to analyze the role of specific TyeA regions and residues in the regulation of Yop secretion. We identified two spatially distinct, surface-exposed regions of the TyeA molecule that were required to regulate Yop secretion. One region, identified by residues M51, F55 and P56, was required for TyeA to interact with YopN. A second region, identified by residues R19, W20 and D25 was not involved in the interaction of TyeA with YopN, but may be required for the YopN/TyeA complex to interact with the T3S apparatus in a manner that blocks Yop secretion.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/fisiología , Proteínas Bacterianas/fisiología , Proteínas Portadoras/química , Proteínas Portadoras/fisiología , Proteínas de la Membrana/fisiología , Yersinia pestis/fisiología , Alanina/química , Sustitución de Aminoácidos , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Secuencia de Bases , Sitios de Unión/genética , Proteínas Portadoras/genética , ADN Bacteriano/genética , Genes Bacterianos , Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación Missense , Fenotipo , Mutación Puntual , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Yersinia pestis/genética , Yersinia pestis/patogenicidadRESUMEN
Numerous bacterial pathogens use type III secretion systems (T3SSs) or T4SSs to inject or translocate virulence proteins into eukaryotic cells. Several different reporter systems have been developed to measure the translocation of these proteins. In this study, a peptide tag-based reporter system was developed and used to monitor the injection of T3S and T4S substrates. The glycogen synthase kinase (GSK) tag is a 13-residue phosphorylatable peptide tag derived from the human GSK-3beta kinase. Translocation of a GSK-tagged protein into a eukaryotic cell results in host cell protein kinase-dependent phosphorylation of the tag, which can be detected with phosphospecific GSK-3beta antibodies. A series of expression plasmids encoding Yop-GSK fusion proteins were constructed to evaluate the ability of the GSK tag to measure the injection of Yops by the Yersinia pestis T3SS. GSK-tagged YopE, YopH, LcrQ, YopK, YopN, and YopJ were efficiently phosphorylated when translocated into HeLa cells. Similarly, the injection of GSK-CagA by the Helicobacter pylori T4SS into different cell types was measured via phosphorylation of the GSK tag. The GSK tag provides a simple method to monitor the translocation of T3S and T4S substrates.