RESUMEN
With the acquirement of antibiotic resistance, Shigella has resulted in multiple epidemics of shigellosis, an infectious diarrheal disease, causing thousands of deaths per year. Unfortunately, there are no licensed vaccines, primarily due to low or serotype-specific immunogenicity. Thus, conserved subunit vaccines utilizing recombinant invasion plasmid antigens (Ipa) have been explored as cross-protective vaccine candidates. However, achieving cross-protection against Shigella dysenteriae 1, which caused multiple pandemics/epidemics in the recent past, has been difficult. Therefore, a rational approach to improve cross-protection in the preparation for a possible pandemic should involve conserved proteins from S. dysenteriae 1 (Sd1). IpaC is one such conserved immunogenic protein that is less explored as an independent vaccine due to its instability/aggregation. Therefore, to improve cross-protection and potential immunogenicity and to be prepared for a future epidemic/pandemic, herein, we stabilized recombinant Sd1 IpaC, expressed without its chaperone, using a previously reported stabilizing detergent (LDAO) in a modified protocol and assessed its vaccine potential without an adjuvant. The protein assembled into heterogeneous complex spherical structures in the presence of LDAO and showed improved stability at storage temperatures of -80, -20, 4, 25, and 37 °C while providing enhanced yield and concentration. The protein could also be stably lyophilized and reconstituted, increasing the convenience of transportation and storage. Upon intranasal administration in BALB/c mice, the stabilized-IpaC-immunized groups generated significant antibody response and were not only protected against a high intraperitoneal dose of homologous S. dysenteriae 1 but also showed 100% survival against heterologous Shigella flexneri 2a without an adjuvant, while the control animals showed visible diarrhea (bloody-Sd1 challenge), lethargy, and weight loss with 0% survival. Overall, this work demonstrates that stabilized IpaC can be explored as a minimalist, self-adjuvanting, cross-protective, intranasal, single-antigen Shigella vaccine.
Asunto(s)
Disentería Bacilar , Vacunas contra la Shigella , Shigella , Administración Intranasal , Animales , Disentería Bacilar/prevención & control , Ratones , Shigella/genética , Vacunas contra la Shigella/genética , Vacunas Sintéticas/genéticaRESUMEN
Accessory cholera enterotoxin (Ace) of Vibrio cholerae has been shown to contribute to diarrhea. However, the signaling mechanism and specific type of Cl- channel activated by Ace are still unknown. We have shown here that the recombinant Ace protein induced ICl of apical plasma membrane, which was inhibited by classical CaCC blockers. Surprisingly, an Ace-elicited rise of current was neither affected by ANO1 (TMEM16A)-specific inhibitor T16A(inh)-AO1(TAO1) nor by the cystic fibrosis transmembrane conductance regulator (CFTR) blocker, CFTR inh-172. Ace stimulated whole-cell current in Caco-2 cells. However, the apical ICl was attenuated by knockdown of ANO6 (TMEM16F). This impaired phenotype was restored by re-expression of ANO6 in Caco-2 cells. Whole-cell patch clamp recordings of ANO currents in HEK293 cells transiently expressing mouse ANO1-mCherry or ANO6-GFP confirmed that Ace induced Cl- secretion. Application of Ace produced ANO6 but not the ANO1 currents. Ace was not able to induce a [Ca2+]i rise in Caco-2 cells, but cellular abundance of phosphatidylinositol 4,5-bisphosphate (PIP2) increased. Identification of the PIP2-binding motif at the N-terminal sequence among human and mouse ANO6 variants along with binding of PIP2 directly to ANO6 in HEK293 cells indicate likely PIP2 regulation of ANO6. The biophysical and pharmacological properties of Ace stimulated Cl- current along with intestinal fluid accumulation, and binding of PIP2 to the proximal KR motif of channel proteins, whose mutagenesis correlates with altered binding of PIP2, is comparable with ANO6 stimulation. We conclude that ANO6 is predominantly expressed in intestinal epithelia, where it contributes secretory diarrhea by Ace stimulation in a calcium-independent mechanism of RhoA-ROCK-PIP2 signaling.
Asunto(s)
Cloruros/metabolismo , Toxina del Cólera/toxicidad , Cólera/metabolismo , Proteínas de Transferencia de Fosfolípidos/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Secuencia de Aminoácidos , Animales , Anoctaminas , Secuencia de Bases , Sistemas CRISPR-Cas , Células CACO-2 , Calcio/metabolismo , Cólera/inducido químicamente , Regulador de Conductancia de Transmembrana de Fibrosis Quística/antagonistas & inhibidores , Células HEK293 , Humanos , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/virología , Masculino , Ratones , Ratones Endogámicos C57BL , Técnicas de Placa-Clamp , Proteínas de Transferencia de Fosfolípidos/antagonistas & inhibidores , Proteínas de Transferencia de Fosfolípidos/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Homología de Secuencia de Aminoácido , Transducción de Señal/efectos de los fármacos , Vibrio cholerae/patogenicidad , Proteína de Unión al GTP rhoA/antagonistas & inhibidores , Proteína de Unión al GTP rhoA/genéticaRESUMEN
Shigellosis remains a serious issue throughout the developing countries, particularly in children under the age of 5. Numerous strategies have been tested to develop vaccines targeting shigellosis; unfortunately despite several years of extensive research, no safe, effective, and inexpensive vaccine against shigellosis is available so far. Here, we illustrate in detail an approach to identify and establish immunogenic outer membrane proteins from Shigella flexneri 2a as subunit vaccine candidates.
Asunto(s)
Vacunas contra la Shigella/inmunología , Shigella/inmunología , Animales , Antígenos Bacterianos/inmunología , Linfocitos B/citología , Linfocitos B/inmunología , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/inmunología , Proteínas de la Membrana Bacteriana Externa/aislamiento & purificación , Clonación Molecular , Femenino , Expresión Génica , Activación de Linfocitos/inmunología , Macrófagos Peritoneales/citología , Macrófagos Peritoneales/inmunología , Masculino , Ratones , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Análisis de Secuencia , Vacunas contra la Shigella/genética , Vacunas contra la Shigella/aislamiento & purificación , Linfocitos T/citología , Linfocitos T/inmunología , Vacunas de Subunidad/genética , Vacunas de Subunidad/inmunología , Vacunas de Subunidad/aislamiento & purificaciónRESUMEN
Proteases in Vibrio cholerae have been shown to play a role in its pathogenesis. V. cholerae secretes Zn-dependent hemagglutinin protease (HAP) and calcium-dependent trypsin-like serine protease (VesC) by using the type II secretion system (TIISS). Our present studies demonstrated that these proteases are also secreted in association with outer membrane vesicles (OMVs) and transported to human intestinal epithelial cells in an active form. OMV-associated HAP induces dose-dependent apoptosis in Int407 cells and an enterotoxic response in the mouse ileal loop (MIL) assay, whereas OMV-associated VesC showed a hemorrhagic fluid response in the MIL assay, necrosis in Int407 cells, and an increased interleukin-8 (IL-8) response in T84 cells, which were significantly reduced in OMVs from VesC mutant strain. Our results also showed that serine protease VesC plays a role in intestinal colonization of V. cholerae strains in adult mice. In conclusion, our study shows that V. cholerae OMVs secrete biologically active proteases which may play a role in cytotoxic and inflammatory responses.
Asunto(s)
Exosomas/enzimología , Inflamación/patología , Péptido Hidrolasas/metabolismo , Vibrio cholerae/enzimología , Vibrio cholerae/patogenicidad , Factores de Virulencia/metabolismo , Animales , Muerte Celular , Línea Celular , Células Epiteliales/patología , Humanos , Íleon/microbiología , Íleon/patología , Ratones Endogámicos BALB CRESUMEN
Cholera pathogenesis occurs due to synergistic pro-secretory effects of several toxins, such as cholera toxin (CTX) and Accessory cholera enterotoxin (Ace) secreted by Vibrio cholerae strains. Ace activates chloride channels stimulating chloride/bicarbonate transport that augments fluid secretion resulting in diarrhea. These channels have been targeted for drug development. However, lesser attention has been paid to the interaction of chloride channel modulators with bacterial toxins. Here we report the modulation of the structure/function of recombinant Ace by small molecule calcium-activated chloride channel (CaCC) inhibitors, namely CaCCinh-A01, digallic acid (DGA) and tannic acid. Biophysical studies indicate that the unfolding (induced by urea) free energy increases upon binding CaCCinh-A01 and DGA, compared to native Ace, whereas binding of tannic acid destabilizes the protein. Far-UV CD experiments revealed that the α-helical content of Ace-CaCCinh-A01 and Ace-DGA complexes increased relative to Ace. In contrast, binding to tannic acid had the opposite effect, indicating the loss of protein secondary structure. The modulation of Ace structure induced by CaCC inhibitors was also analyzed using docking and molecular dynamics (MD) simulation. Functional studies, performed using mouse ileal loops and Ussing chamber experiments, corroborate biophysical data, all pointing to the fact that tannic acid destabilizes Ace, inhibiting its function, whereas DGA stabilizes the toxin with enhanced fluid accumulation in mouse ileal loop. The efficacy of tannic acid in mouse model suggests that the targeted modulation of Ace structure may be of therapeutic benefit for gastrointestinal disorders.
Asunto(s)
Canales de Cloruro/antagonistas & inhibidores , Toxina del Cólera/fisiología , Vibrio cholerae/fisiología , Animales , Cólera/fisiopatología , Toxina del Cólera/antagonistas & inhibidores , Dicroismo Circular , Depsidos/farmacología , Diarrea/fisiopatología , Ácido Gálico/análogos & derivados , Ácido Gálico/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Simulación del Acoplamiento Molecular , Proteínas Recombinantes , Espectrometría de Fluorescencia , Taninos/farmacología , Tiofenos/farmacologíaRESUMEN
B cells are critically important in combating bacterial infections and their differentiation into plasma cells and memory cells aids bacterial clearance and long-lasting immunity conferred by essentially all vaccines. Outer membrane protein A (OmpA) of Shigella flexneri 2a has been demonstrated to induce the production of IgG and IgA in vivo following immunization of mice through intranasal route, but the direct involvement of B cells in OmpA-mediated immune regulation was not determined. Consequently, we investigated whether OmpA can modulate B cell functions and identified the molecular events involved in OmpA-induced B cell immune response in vitro. We show that OmpA of S. flexneri 2a activates B cells to produce protective cytokines, IL-6 and IL-10 as well as facilitates their differentiation into antibody secreting cells (ASCs). The immunostimulatory properties of OmpA are attributed to the increased surface expression of MHCII and CD86 on B cells. We also report here that B cell activation by OmpA is mediated strictly through recognition by TLR2, resulting in initiation of cascades of signal transduction events, involving increased phosphorylation of protein tyrosine kinases (PTKs), ERK and IκBα, leading to nuclear translocation of NF-κB. Importantly, a TLR2 antibody diminishes OmpA-induced upregulation of MHCII and CD86 on B cell surface as well as significantly inhibits B cell differentiation and cytokine secretion. Furthermore, we illustrate that B cell differentiation into ASCs and induction of cytokine secretion by OmpA are dependent on PTKs activity. Moreover, we identify that OmpA-induced B cell differentiation is entirely dependent on ERK pathway, whereas both NF-κB and ERK are essential for cytokine secretion by B cells. Overall, our data demonstrate that OmpA of S. flexneri 2a amplifies TLR signaling in B cells and triggers B cell immune response, which is critical for the development of an effective adaptive immunity to an optimal vaccine antigen.
Asunto(s)
Linfocitos B/citología , Proteínas de la Membrana Bacteriana Externa/farmacología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , FN-kappa B/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Shigella flexneri/metabolismo , Receptor Toll-Like 2/metabolismo , Animales , Células Productoras de Anticuerpos/metabolismo , Presentación de Antígeno/efectos de los fármacos , Linfocitos B/efectos de los fármacos , Antígeno B7-2/metabolismo , Diferenciación Celular/efectos de los fármacos , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Células HEK293 , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Interleucina-10/biosíntesis , Interleucina-6/biosíntesis , Activación de Linfocitos/inmunología , Sistema de Señalización de MAP Quinasas , Ratones Endogámicos BALB C , Fosforilación , Receptor Toll-Like 2/genética , Receptor Toll-Like 6/metabolismo , Regulación hacia Arriba/genéticaRESUMEN
Shigellosis is the leading cause of childhood mortality and morbidity. Despite many years of extensive research a practical vaccine is not yet available against the disease. Recent studies illustrate that bacterial outer membrane proteins are budding target as vaccine antigen. Outer membrane proteins A (OmpA) are among the most immunodominant antigens in the outer membrane of gram negative bacteria and possess many characteristics desired of a vaccine candidate. We observe that OmpA of Shigella flexneri 2a is crossreactive and common antigen among Shigella spp. and the epitope is widely exposed on the cell surface as well as capable of evoking protective immunity in mice. The protective immunity involves participation of both the humoral and cellular immune responses, since OmpA boosts rapid induction of IgG and IgA in both the systemic and mucosal compartments and also activates Th1 cells. The immunopotentiating activity of OmpA is mediated by its ability to bind and stimulate macrophages and up-regulate the surface expression of MHCII, CD80 and CD40, leading to activation of CD4(+) T cells to secrete cytokines and express chemokine receptor and IL-12Rß2, thereby orchestrating the bridge between innate and adaptive immune responses. This ability is dependent on Toll-like receptor 2 (TLR2), as demonstrated by lack of response by TLR2 knockdown macrophages to OmpA. Hence this property of OmpA to link innate and adaptive immunity via TLR2 offers a novel vista to develop vaccine against shigellosis.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/inmunología , Vacunas Bacterianas/inmunología , Disentería Bacilar/prevención & control , Shigella flexneri/inmunología , Inmunidad Adaptativa , Animales , Antígenos Bacterianos/inmunología , Protección Cruzada , Inmunidad Celular , Inmunidad Humoral , Inmunidad Innata , Ratones , Receptor Toll-Like 2/inmunologíaRESUMEN
Recently, we have demonstrated, immunization of adult female mice with outer membrane vesicles (OMVs) of Shigella boydii type 4 protected their offspring passively from shigellosis. In our present study, we have advanced our research by formulating multi-serotype outer membrane vesicles (MOMVs), mixing the OMVs of Shigella dysenteriae 1 Δstx, Shigella flexneri 2a, 3a and 6, S. boydii type 4 and Shigella sonnei to achieve a broad spectrum protection against shigellosis. Adult mice were immunized orally with 50 µg of MOMVs, four times at weekly intervals. Immunological parameters were observed at various time points, before, during and after immunization, in adult mice. Passive protection was examined in their offspring by measuring protective efficacy and studying intestinal colonization, after challenging with various Shigella strains. Immunized dams exhibited a consistent broad spectrum antibody response. 3-4 day-old offspring of immunized dams showed significant long term passive protection against wild type S. flexneri 2a, 3a, and 6, S. boydii type 2 and S. dysenteriae 1. Their stomach extracts, essentially containing mother's milk, have also exhibited significant levels of anti-MOMVs immunoglobulins. In conclusion, MOMVs formulation represents an easy, safe immunization strategy that was found suitable to provide complete passive protection to the neonatal mice against all four serogroups of Shigellae. It could be exploited for the development of a novel non-living vaccine against human shigellosis in near future.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/inmunología , Membrana Celular/inmunología , Disentería Bacilar/prevención & control , Inmunidad Materno-Adquirida , Vacunas contra la Shigella/inmunología , Animales , Animales Recién Nacidos , Anticuerpos Antibacterianos/sangre , Especificidad de Anticuerpos , Disentería Bacilar/inmunología , Femenino , Contenido Digestivo/química , Inmunidad Celular , Inmunoglobulinas/química , Interleucina-8/inmunología , Macrófagos/inmunología , Ratones , Fagocitosis , Shigella dysenteriae/inmunología , Shigella flexneri/inmunología , Shigella sonnei/inmunologíaRESUMEN
The apical membrane of intestinal epithelia expresses intermediate conductance K(+) channel (KCNN4), which provides the driving force for Cl(-) secretion. However, its role in diarrhea and regulation by Epac1 is unknown. Previously we have established that Epac1 upon binding of cAMP activates a PKA-independent mechanism of Cl(-) secretion via stimulation of Rap2-phospholipase Cε-[Ca(2+)]i signaling. Here we report that Epac1 regulates surface expression of KCNN4c channel through its downstream Rap1A-RhoA-Rho-associated kinase (ROCK) signaling pathway for sustained Cl(-) secretion. Depletion of Epac1 protein and apical addition of TRAM-34, a specific KCNN4 inhibitor, significantly abolished cAMP-stimulated Cl(-) secretion and apical K(+) conductance (IK(ap)) in T84WT cells. The current-voltage relationship of basolaterally permeabilized monolayers treated with Epac1 agonist 8-(4-chlorophenylthio)-2'-O- methyladenosine 3',5'-cyclic monophosphate showed the presence of an inwardly rectifying and TRAM-34-sensitive K(+) channel in T84WT cells that was absent in Epac1KDT84 cells. Reconstructed confocal images in Epac1KDT84 cells revealed redistribution of KCNN4c proteins into subapical intracellular compartment, and a biotinylation assay showed â¼83% lower surface expression of KCNN4c proteins compared with T84WT cells. Further investigation revealed that an Epac1 agonist activates Rap1 to facilitate IK(ap). Both RhoA inhibitor (GGTI298) and ROCK inhibitor (H1152) significantly reduced cAMP agonist-stimulated IK(ap), whereas the latter additionally reduced colocalization of KCNN4c with the apical membrane marker wheat germ agglutinin in T84WT cells. In vivo mouse ileal loop experiments showed reduced fluid accumulation by TRAM-34, GGTI298, or H1152 when injected together with cholera toxin into the loop. We conclude that Rap1A-dependent signaling of Epac1 involving RhoA-ROCK is an important regulator of intestinal fluid transport via modulation of apical KCNN4c channels, a finding with potential therapeutic value in diarrheal diseases.
Asunto(s)
Cloruros/metabolismo , Diarrea/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/metabolismo , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Animales , Western Blotting , Línea Celular Tumoral , Toxina del Cólera , Colforsina/farmacología , AMP Cíclico/farmacología , Diarrea/inducido químicamente , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Factores de Intercambio de Guanina Nucleótido/antagonistas & inhibidores , Factores de Intercambio de Guanina Nucleótido/genética , Humanos , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/antagonistas & inhibidores , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Ratones , Microscopía Confocal , Bloqueadores de los Canales de Potasio/farmacología , Pirazoles/farmacología , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Proteínas de Unión al GTP rap/genética , Proteínas de Unión al GTP rap/metabolismo , Proteínas de Unión al GTP rap1/genética , Proteínas de Unión al GTP rap1/metabolismo , Quinasas Asociadas a rho/antagonistas & inhibidores , Quinasas Asociadas a rho/metabolismo , Proteína de Unión al GTP rhoA/antagonistas & inhibidores , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
BACKGROUND: Adequate evidence mounts to the fact that several bacteria and their toxins have protective or curative roles in colorectal cancers. Thermostable direct hemolysin (TDH), produced by Vibrio parahaemolyticus, down regulates cell proliferation in colon carcinoma cell lines. TDH induces Ca2+ influx from an extracellular environment accompanied by protein kinase C phosphorylation. Activated protein kinase C inhibits the tyrosine kinase activity of epidermal growth factor receptor (EGFR), the rational target of anti-colorectal cancer therapy. METHODS: Immunoblotting analyses were performed to ascertain protein kinase C activation, EGFR status, EGFR phosphorylation and mitogen activated protein kinase (MAPK) activity. Flow cytometry analysis and ELISA reconfirmed tyrosine phosphorylation of EGFR and ERK activations, respectively. PKC-α siRNA knockdown was done to corroborate the involvement of PKC-α in the undertaken study. RESULTS: Our study showed the translocation of PKC-α from cytosol to the membrane fraction in colon carcinoma cell lines on incubation with TDH. The EGFR tyrosine kinase activity exhibited a down regulation on TDH treatment which involved PKC-α, as confirmed by siRNA knockdown. Also ERK phosphorylation occurred on PKC-α activation. CONCLUSION: TDH activated PKC-α down regulates EGFR tyrosine kinase activity by MEK dependent mechanism involving MAPK. GENERAL SIGNIFICANCE: In this study we have seen that TDH has an implication in EGFR based therapeutic approach in colorectal cancer via PKC mediated mechanism.
Asunto(s)
Neoplasias del Colon/tratamiento farmacológico , Enterotoxinas/farmacología , Receptores ErbB/metabolismo , Proteínas Hemolisinas/farmacología , Proteína Quinasa C-alfa/metabolismo , Transducción de Señal/efectos de los fármacos , Toxinas Bacterianas/farmacología , Western Blotting , Proliferación Celular/efectos de los fármacos , Neoplasias del Colon/metabolismo , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Inmunoprecipitación , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteína Quinasa C-alfa/antagonistas & inhibidores , Proteína Quinasa C-alfa/genética , ARN Interferente Pequeño/genética , Tirosina/metabolismoRESUMEN
We determine that OmpA of Shigella flexneri 2a is recognized by TLR2 and consequently mediates the release of proinflammatory cytokines and activates NF-κB in HEK 293 cells transfected with TLR2. We also observe that in RAW macrophages TLR2 is essential to instigate the early immune response to OmpA via NF-κB activation and secretion of cytokines and NO. Consistent with these results, TLR2 knockdown using siRNA abolishes the initiation of immune responses. Processing and presentation of OmpA depend on TLR2; MHCII presentation of the processed antigen and expression of CD80 significantly attenuated in TLR2 knockdown macrophages. The optimum production of IFN-γ by the macrophages:CD4(+) T cells co-culture depends on both TLR2 activation and antigen presentation. So, TLR2 is clearly recognized as a decisive factor in initiating host innate immune response to OmpA for the development of CD4(+) T cell adaptive response. Furthermore, we demonstrate in vivo that intranasal immunization of mice with OmpA selectively enhances the release of IFN-γ and IL-2 by CD4(+) T cells. Importantly, OmpA increases the level of IFN-γ production in Ag-primed splenocytes. The addition of neutralizing anti-IL-12p70 mAb to cell cultures results in the decreased release of OmpA-enhanced IFN-γ by Ag-primed splenocytes. Moreover, coincubation with OmpA-pretreated macrophages enhances the production of IFN-γ by OmpA-primed CD4(+) T cells, representing that OmpA may enhance IFN-γ expression in CD4(+) T cells through the induction of IL-12 production in macrophages. These results demonstrate that S. flexneri 2a OmpA may play a critical role in the development of Th1 skewed adaptive immune response.
Asunto(s)
Inmunidad Adaptativa , Proteínas de la Membrana Bacteriana Externa/inmunología , Inmunidad Innata , Interleucina-12/fisiología , Óxido Nítrico/metabolismo , Shigella flexneri/fisiología , Receptor Toll-Like 2/fisiología , Animales , Presentación de Antígeno , Activación Enzimática , Femenino , Células HEK293 , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase II/metabolismo , Interacciones Huésped-Patógeno , Humanos , Interferón gamma/biosíntesis , Interleucina-12/metabolismo , Subunidad beta 2 del Receptor de Interleucina-12/genética , Subunidad beta 2 del Receptor de Interleucina-12/metabolismo , Activación de Linfocitos , Macrófagos/enzimología , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos BALB C , FN-kappa B/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Fosforilación , Proteína Quinasa C-alfa/metabolismo , Transporte de Proteínas , Receptores de Quimiocina/metabolismo , Shigella flexneri/inmunología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Receptor Toll-Like 2/genética , Receptor Toll-Like 2/metabolismoRESUMEN
BACKGROUND: In our earlier studies 34 kDa outer membrane protein (OMP) of Shigella flexneri 2a has been identified as an efficient immunostimulant. KEY RESULTS: In the present study MALDI-TOF MS analysis of the purified 34 kDa OMP of Shigella flexneri 2a shows considerable sequence homology (Identity 65%) with the OmpA of S. flexneri 2a. By using the specific primers, the gene of interest has been amplified from S. flexneri 2a (N.Y-962/92) genomic DNA, cloned in pET100/D-TOPO® vector and expressed using induction with isopropyl thiogalactoside (IPTG) for the first time. Immunogenicity and protective efficacy of the recombinant OmpA has been evaluated in an intranasally immunized murine pulmonary model. The recombinant protein induces significantly enhanced protein specific IgG and IgA Abs in both mucosal and systemic compartments and IgA secreting cells in the systemic compartment (spleen). The mice immunized with OmpA have been protected completely from systemic challenge with a lethal dose of virulent S. flexneri 2a. Immunization with the protein causes mild polymorphonuclear neutrophil infiltration in the lung, without inducing the release of large amounts of proinflammatory cytokines. CONCLUSION: These results suggest that the OmpA of S. flexneri 2a can be an efficacious mucosal immunogen inducing protective immune responses. Our findings also demonstrate that antibodies and Th1 immune response may be associated with the marked protective efficacy of immunized mice after intranasal shigellae infection.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/inmunología , Disentería Bacilar/inmunología , Disentería Bacilar/prevención & control , Inmunidad/inmunología , Shigella flexneri/inmunología , Animales , Anticuerpos Antibacterianos/inmunología , Proteínas de la Membrana Bacteriana Externa/química , Citocinas/metabolismo , Modelos Animales de Enfermedad , Disentería Bacilar/microbiología , Disentería Bacilar/patología , Humanos , Inmunización , Liposomas/metabolismo , Pulmón/inmunología , Pulmón/microbiología , Pulmón/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Peso Molecular , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Análisis de Secuencia de Proteína , Análisis de Supervivencia , Factores de TiempoRESUMEN
BACKGROUND: Colon cancers are the frequent causes of cancer mortality worldwide. Recently bacterial toxins have received marked attention as promising approaches in the treatment of colon cancer. Thermostable direct hemolysin (TDH) secreted by Vibrio parahaemolyticus causes influx of extracellular calcium with the subsequent rise in intracellular calcium level in intestinal epithelial cells and it is known that calcium has antiproliferative activity against colon cancer. KEY RESULTS: In the present study it has been shown that TDH, a well-known traditional virulent factor inhibits proliferation of human colon carcinoma cells through the involvement of CaSR in its mechanism. TDH treatment does not induce DNA fragmentation, nor causes the release of lactate dehydrogenase. Therefore, apoptosis and cytotoxicity are not contributing to the TDH-mediated reduction of proliferation rate, and hence the reduction appears to be caused by decrease in cell proliferation. The elevation of E-cadherin, a cell adhesion molecule and suppression of ß-catenin, a proto-oncogene have been observed in presence of CaSR agonists whereas reverse effect has been seen in presence of CaSR antagonist as well as si-RNA in TDH treated cells. TDH also triggers a significant reduction of Cyclin-D and cdk2, two important cell cycle regulatory proteins along with an up regulation of cell cycle inhibitory protein p27(Kip1) in presence of CaSR agonists. CONCLUSION: Therefore TDH can downregulate colonic carcinoma cell proliferation and involves CaSR in its mechanism of action. The downregulation occurs mainly through the involvement of E-cadherin-ß-catenin mediated pathway and the inhibition of cell cycle regulators as well as upregulation of cell cycle inhibitors.
Asunto(s)
Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Cadherinas/metabolismo , Neoplasias del Colon/patología , Regulación hacia Abajo , Proteínas Hemolisinas/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismo , beta Catenina/metabolismo , Secuencia de Bases , Línea Celular Tumoral , Cartilla de ADN , Humanos , Proto-Oncogenes Mas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción 4RESUMEN
BACKGROUND: Two well-characterized proteases secreted by Vibrio cholerae O1 strains are hemagglutinin protease (HAP) and V. cholerae protease (PrtV). The hapA and prtV knock out mutant, V. cholerae O1 strain CHA6.8ΔprtV, still retains residual protease activity. We initiated this study to characterize the protease present in CHA6.8ΔprtV strain and study its role in pathogenesis in rabbit ileal loop model (RIL). METHODOLOGY/PRINCIPAL FINDINGS: We partially purified the residual protease secreted by strain CHA6.8ΔprtV from culture supernatant by anion-exchange chromatography. The major protein band in native PAGE was identified by MS peptide mapping and sequence analysis showed homology with a 59-kDa trypsin-like serine protease encoded by VC1649. The protease activity was partially inhibited by 25 mM PMSF and 10 mM EDTA and completely inhibited by EDTA and PMSF together. RIL assay with culture supernatants of strains C6709 (FA ratio 1.1+/-0.3 n = 3), CHA6.8 (FA ratio 1.08+/-0.2 n = 3), CHA6.8ΔprtV (FA ratio 1.02+/-0.2 n = 3) and partially purified serine protease from CHA6.8ΔprtV (FA ratio 1.2+/-0.3 n = 3) induced fluid accumulation and histopathological studies on rabbit ileum showed destruction of the villus structure with hemorrhage in all layers of the mucosa. RIL assay with culture supernatant of CHA6.8ΔprtVΔVC1649 strain (FA ratio 0.11+/-0.005 n = 3) and with protease incubated with PMSF and EDTA (FA ratio 0.3+/-0.05 n = 3) induced a significantly reduced FA ratio with almost complete normal villus structure. CONCLUSION: Our results show the presence of a novel 59-kDa serine protease in a ΔhapAΔprtV V. cholerae O1 strain and its role in hemorrhagic response in RIL model.
Asunto(s)
Cólera/patología , Íleon/patología , Metaloendopeptidasas/deficiencia , Péptido Hidrolasas/deficiencia , Serina Proteasas/metabolismo , Vibrio cholerae O1/enzimología , Secuencia de Aminoácidos , Animales , Cólera/microbiología , Modelos Animales de Enfermedad , Eliminación de Gen , Hemorragia , Humanos , Íleon/microbiología , Metaloendopeptidasas/genética , Datos de Secuencia Molecular , Péptido Hidrolasas/genética , Conejos , Serina Proteasas/química , Serina Proteasas/genética , Vibrio cholerae O1/química , Vibrio cholerae O1/genética , Vibrio cholerae O1/patogenicidad , VirulenciaRESUMEN
BACKGROUND: IP(3)-mediated calcium mobilization from intracellular stores activates and translocates PKC-alpha from cytosol to membrane fraction in response to STa in COLO-205 cell line. The present study was undertaken to determine the involvement of cytoskeleton proteins in translocation of PKC-alpha to membrane from cytosol in the Escherichiacoli STa-mediated signaling cascade in a human colonic carcinoma cell line COLO-205. METHODS: Western blots and consequent densitometric analysis were used to assess time-dependent redistribution of cytoskeletal proteins. This redistribution was further confirmed by using confocal microscopy. Pharmacological reagents were applied to colonic carcinoma cells to disrupt the microfilaments (cytochalasin D) and microtubules (nocodazole). RESULTS: STa treatment in COLO-205 cells showed dynamic redistribution and an increase in actin content in the Triton-insoluble fraction, which corresponds to an increase in polymerization within 1min. Moreover, pharmacological disruption of actin-based cytoskeleton greatly disturbed PKC-alpha translocation to the membrane. CONCLUSIONS: These results suggested that the organization of actin cytoskeleton is rapidly rearranged following E. coli STa treatment and the integrity of the actin cytoskeleton played a crucial role in PKC-alpha movement in colonic cells. Depolymerization of tubulin had no effect on the ability of the kinase to be translocated to the membrane. GENERAL SIGNIFICANCE: In the present study, we have shown for the first time that in colonic carcinoma cells, STa-mediated rapid changes of actin cytoskeleton arrangement might be involved in the translocation of PKC-alpha to membrane.
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Toxinas Bacterianas/farmacología , Proteínas del Citoesqueleto/metabolismo , Enterotoxinas/farmacología , Escherichia coli/metabolismo , Transducción de Señal , Western Blotting , Proteínas de Escherichia coli , Técnica del Anticuerpo Fluorescente , Proteína Quinasa C-alfa/metabolismoRESUMEN
The 34 kDa major outer membrane protein (MOMP) of Shigella flexneri 2a induces combinatorial expression of TLR2 and TLR6 on peritoneal macrophages of BALB/c mice. Between the two best-characterized TLRs, to date, TLR2 and TLR4, which are chiefly responsible for recognizing majority of bacterial products, TLR2 alone participates in recognition of 34 kDa MOMP. In addition to TLRs, MOMP enhances the mRNA expression of MyD88 and TRAF6 and induces the nuclear translocation of NF-kappaB as well as activates p38 MAP kinase, suggesting the involvement of these molecules in the mechanism of action of MOMP. 34 kDa MOMP also stimulates macrophages, up regulates the surface expression of MHC-II and B7-1 and enhances the production of different cytokines (such as ILp70, TNF-alpha, Il-6) and chemokines (like MIP-1 alpha, MIP-1 beta and RANTES). The ability of the protein in the activation of macrophages, i.e. the induction of nuclear translocation of NF-kappaB and secretion of cytokines are dependent on TLR2 expression as demonstrated by the lack of response by macrophages pre-treated with inhibitory TLR2 mAb. Moreover, it has been found that MOMP induced regulation of TLR2 gene expression is dependent on NF-kappaB and p38 MAP kinase in murine macrophages for the first time. The MOMP induced cytokines and chemokines profile reflect that the protein has the ability to translate innate towards type-1 adaptive response. In conclusion MOMP recognizes by and activates macrophages which may be an initiating event in the antibacterial host response.
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Proteínas de la Membrana Bacteriana Externa/farmacología , FN-kappa B/metabolismo , Transducción de Señal/efectos de los fármacos , Receptor Toll-Like 2/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Transporte Activo de Núcleo Celular/efectos de los fármacos , Animales , Antígeno B7-1/genética , Antígeno B7-1/metabolismo , Proteínas de la Membrana Bacteriana Externa/química , Núcleo Celular/metabolismo , Células Cultivadas , Quimiocinas/genética , Quimiocinas/metabolismo , Citocinas/genética , Citocinas/metabolismo , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase II/metabolismo , Immunoblotting , Activación de Macrófagos/efectos de los fármacos , Macrófagos Peritoneales/citología , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/metabolismo , Ratones , Ratones Endogámicos BALB C , Peso Molecular , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Shigella flexneri/metabolismo , Factor 6 Asociado a Receptor de TNF/genética , Factor 6 Asociado a Receptor de TNF/metabolismo , Receptor Toll-Like 2/genéticaRESUMEN
In the present study we purified 34 kDa major outer membrane protein (MOMP) of Shigella flexneri 2a for the first time, which was cross-reactive and antigenically conserved among Shigella spp. and the epitope was surface exposed on the intact bacterium. The purified antigen was found to be glycosylated, which aids in binding to macrophages and up-regulated the production of nitric oxide, granulocyte-colony stimulating factor and IL-12p70, indicating that the MOMP is immunogenic and has the ability to commence protective immune responses against intracellular pathogens, thereby it may be considered as a potential vaccine candidate.
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Antígenos Bacterianos/inmunología , Proteínas de la Membrana Bacteriana Externa/inmunología , Shigella flexneri/inmunología , Animales , Antígenos Bacterianos/aislamiento & purificación , Proteínas de la Membrana Bacteriana Externa/aislamiento & purificación , Células Cultivadas , Reacciones Cruzadas , Glicosilación , Factor Estimulante de Colonias de Granulocitos/biosíntesis , Sueros Inmunes/inmunología , Interleucina-12/biosíntesis , Macrófagos/inmunología , Ratones , Ratones Endogámicos BALB C , Óxido Nítrico/biosíntesisRESUMEN
The heat-stable enterotoxin (Y-STa) produced by the pathogenic strains of Yersinia enterocolitica is a causative agent of secretory diarrhea. We have reported earlier that Y-STa-induced inositol trisphosphate-mediated cytosolic calcium rise occurs in rat intestinal epithelial cells. In the present communication, the involvement of a nuclear calcium store in the action mechanism of Y-STa in rat intestinal epithelial cells has been shown. Calcium imaging with time series confocal microscopy shows that Y-STa stimulates both the nuclear and cytosolic calcium levels in rat intestinal epithelial cells where a rise in nuclear calcium precedes the cytosolic events. Moreover, Y-STa stimulates both cytosolic and nuclear inositol trisphosphate (IP(3)) levels in a time-dependent manner. Western blot and immunocytochemical analysis reveal a higher density of IP(3) receptor type II in the nuclear membrane compared to the cytosol, which may be the cause of an early rise of the nuclear calcium level. Therefore, it is suggested that Y-STa regulates the nuclear and cytosolic calcium signals in a distinct temporal manner in rat intestinal epithelial cells.
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Toxinas Bacterianas/farmacología , Señalización del Calcio/efectos de los fármacos , Núcleo Celular/efectos de los fármacos , Citosol/efectos de los fármacos , Enterocitos/efectos de los fármacos , Yersinia enterocolitica/fisiología , Adenosina Trifosfato/farmacología , Animales , Compuestos de Boro/farmacología , Calcio/metabolismo , Núcleo Celular/metabolismo , Supervivencia Celular/efectos de los fármacos , Citosol/metabolismo , Enterocitos/metabolismo , Enterocitos/patología , Inositol 1,4,5-Trifosfato/metabolismo , Yeyuno/efectos de los fármacos , Yeyuno/metabolismo , Masculino , Microscopía Confocal , Membrana Nuclear/química , Membrana Nuclear/efectos de los fármacos , Membrana Nuclear/metabolismo , Ratas , Tapsigargina/farmacología , Factores de TiempoRESUMEN
It was reported earlier that Escherichia coli heat stable enterotoxin (STa), a major causative agent of secretory diarrhea, can also inhibit the proliferation of colon carcinoma cells with the involvement of cGMP mediated calcium influx. In the present study it is shown that E. coli STa inhibits cell proliferation in the colonic carcinoma cell line COLO-205 by the PKG-ERK44/42 mediated signaling pathway. This enterotoxin negatively regulates cell proliferation by downregulating the activity of ERK44/42(MAPK) and subsequently the activity of a transcription regulatory protein cMyc. The antiproliferative effect of STa was reversed by LY83583, a guanylate cyclase (GC) inhibitor and KT5823, a PKG inhibitor. Thus suggesting the involvement of cGMP dependent protein kinase (PKG) in the downregulation of ERK44/42 and subsequent inactivation of cMyc activity. Moreover, it has been shown that a specific ERK44/42 inhibitor, PD98059, also inhibits cMyc activation and cell proliferation, which further confirms the involvement of ERK44/42 in the activation of cMyc. It is also shown that E. coli STa significantly inhibits the vascular endothelial growth factor (VEGF, a potent angiogenic factor) expression in COLO-205 cells and also downregulates vascular cell adhesion molecule-1 (VCAM-1, a potent metastatic factor) expression on the COLO-205 cell surface. So it is reported for the first time that E. coli STa inhibits the proliferation of the colonic carcinoma cell line COLO-205 by the PKG-ERK44/42 mediated pathway and it may have a role against the development of colon carcinoma.