RESUMEN

Secretin PilQ is an antigenically conserved outer membrane protein that is present in most meningococci and PorA is a major protein that elicits bactericidal immune response in humans following natural disease and immunization. In the present study, BALB/c mice were immunized subcutaneously with rPilQ406-770 or rPorA together with Freund's adjuvant (FA). Serum antibody responses to serogroup A and B Neisseria meningitides whole cells or purified proteins and functional activity of antibodies were determined by ELISA and serum bactericidal assay (SBA), respectively. Serum IgG responses were significantly increased in the immunized group with rPilQ406-770 or rPorA together with FA compared to control groups. IgG antibody response of mice immunized with rPilQ406-770 was significantly more than mice immunized with rPorA (OD at 450 nm was 1.6 versus 0.83). The booster injections were effective in increasing the responses of anti-rPilQ406-770 or anti-rPorA IgG significantly. Antisera produced against rPilQ406-770 or rPorA demonstrated strong surface reactivity to serogroup B N. meningitides in comparison with control groups. Antisera raised against rPorA or rPilQ406-770 and FA demonstrated SBA titers from 1/1024 to 1/2048 against serogroup B. The strongest bactericidal activity was detected in sera from mice immunized with rPilQ406-770 mixed with FA. These results suggest that rPilQ406-770 is a potential vaccine candidate for serogroup B N. meningitidis.

Asunto(s)

Anticuerpos Antibacterianos , Proteínas de la Membrana Bacteriana Externa , Inmunoglobulina G , Vacunas Meningococicas , Ratones Endogámicos BALB C , Proteínas Recombinantes , Animales , Anticuerpos Antibacterianos/sangre , Anticuerpos Antibacterianos/inmunología , Proteínas de la Membrana Bacteriana Externa/inmunología , Proteínas de la Membrana Bacteriana Externa/genética , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Ratones , Proteínas Recombinantes/inmunología , Vacunas Meningococicas/inmunología , Vacunas Meningococicas/administración & dosificación , Neisseria meningitidis/inmunología , Femenino , Adyuvante de Freund/administración & dosificación , Adyuvante de Freund/inmunología , Formación de Anticuerpos/inmunología , Inmunización , Ensayo de Inmunoadsorción Enzimática , Determinación de Anticuerpos Séricos Bactericidas , Antígenos Bacterianos/inmunología

RESUMEN

IMPORTANCE: Type IVa pili (T4aP) are widespread bacterial cell surface structures with important functions in motility, surface adhesion, biofilm formation, and virulence. Different bacteria have adapted different piliation patterns. To address how these patterns are established, we focused on the bipolar localization of the T4aP machine in the model organism Myxococcus xanthus by studying the localization of the PilQ secretin, the first component of this machine that assembles at the poles. Based on experiments using a combination of fluorescence microscopy, biochemistry, and computational structural analysis, we propose that PilQ, and specifically its AMIN domains, binds septal and polar peptidoglycan, thereby enabling polar Tgl localization, which then stimulates PilQ multimerization in the outer membrane. We also propose that the presence and absence of AMIN domains in T4aP secretins contribute to the different piliation patterns across bacteria.

Asunto(s)

Proteínas Fimbrias , Myxococcus xanthus , Proteínas Fimbrias/metabolismo , Myxococcus xanthus/genética , Myxococcus xanthus/metabolismo , Fimbrias Bacterianas/metabolismo

RESUMEN

C. jejuni is an important food-borne pathogen displaying high genetic diversity, substantially based on natural transformation. The mechanism of DNA uptake from the environment depends on a type II secretion/type IV pilus system, whose components are partially known. Here, we quantified DNA uptake in C. jejuni at the single cell level and observed median transport capacities of approximately 30 kb per uptake location. The process appeared to be limited by the initialization of DNA uptake, was finite, and, finalized within 30 min of contact to DNA. Mutants lacking either the outer membrane pore PilQ or the inner membrane channel ComEC were deficient in natural transformation. The periplasmic DNA binding protein ComE was negligible for DNA uptake, which is in contrast to its proposed function. Intriguingly, a mutant lacking the unique periplasmic protein Cj0683 displayed rare but fully functional DNA uptake events. We conclude that Cj0683 was essential for the efficient initialization of DNA uptake, consistent with the putative function as a competence pilus protein. Unravelling features important in natural transformation might lead to target identification, reducing the adaptive potential of pathogens.

Asunto(s)

Campylobacter jejuni , Campylobacter jejuni/genética , Campylobacter jejuni/metabolismo , Transformación Bacteriana , ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , ADN Bacteriano/genética , ADN Bacteriano/metabolismo

RESUMEN

BACKGROUND: Pseudomonas aeruginosa sepsis is associated with unacceptably high mortality and, for many of those who survive, long-term morbidity. The aims of this study were to production of IgY against chimeric protein pilQ-pilA-DSL region and killed- whole cell Pseudomonas aeruginosa O1 (PAO1) strain and their efficacy for immunoprophylaxis of sepsis caused by P. aeruginosa in a rabbit model. METHODS: Specific IgY was obtained by immunization of hens. The purity of IgY was determined by SDS-PAGE analysis. The effect of IgY on growth and hydrophobicity of P. aeruginosa were performed through time-kill assay and microbial adhesion to hydrocarbons test (MATH), respectively. The efficacy of specific IgYs was examined against P. aeruginosa sepsis in a rabbit model. The rabbits were monitored for 72 h to record physiological characters and survival. Hematologic factors, C-reactive protein, pro-inflammatory cytokines, and bacterial count from blood and solid organs were measured, periodically. RESULTS: We found that the growth inhibitory effect of the anti- killed whole cell IgY was higher than anti-pilQ-pilA IgY (P < 0.001). The hydrophobicity effect of PAO1 increased when bacteria were opsonized by anti- killed whole cell IgY while the hydrophobicity activity was decreased following incubation of PAO1 with anti-pilQ-pilA IgY in a broth medium (P < 0.001). Following intravenous (IV) administration of produced IgYs, no significant difference was observed in the survival, decrease in inflammatory mediators and clinical symptoms between the groups 48h post infection (P > 0.05). Moreover, no considerable decrease was observed in the bacterial load of blood, lungs and kidneys in rabbits treated with specific IgYs and control groups (P > 0.05). No bacteria were found in the spleen and liver samples from infected rabbits. CONCLUSION: Although produced IgYs had a good immunoreactivity, IV immunization of IgYs was not protective against P. aeruginosa sepsis in the rabbit model. Further studies are needed to assess the immune response and decreasing mortality rate using the rabbit sepsis model.

Asunto(s)

Anticuerpos Antibacterianos/inmunología , Proteínas Fimbrias/inmunología , Inmunoglobulinas/inmunología , Infecciones por Pseudomonas/inmunología , Pseudomonas aeruginosa/inmunología , Proteínas Recombinantes de Fusión/inmunología , Sepsis/inmunología , Animales , Carga Bacteriana/inmunología , Pollos/inmunología , Modelos Animales de Enfermedad , Inmunización/métodos , Inmunización Pasiva/métodos , Masculino , Infecciones por Pseudomonas/microbiología , Conejos , Sepsis/microbiología

RESUMEN

The type IV pilus machinery is a multi-protein complex that polymerizes and depolymerizes a pilus fiber used for attachment, twitching motility, phage adsorption, natural competence, protein secretion, and surface-sensing. An outer membrane secretin pore is required for passage of the pilus fiber out of the cell. Herein, the structure of the tetradecameric secretin, PilQ, from the Pseudomonas aeruginosa type IVa pilus system was determined to 4.3 Å and 4.4 Å resolution in the presence and absence of C7 symmetric spikes, respectively. The heptameric spikes were found to be two tandem C-terminal domains of TsaP. TsaP forms a belt around PilQ and while it is not essential for twitching motility, overexpression of TsaP triggers a signal cascade upstream of PilY1 leading to cyclic di-GMP up-regulation. These results resolve the identity of the spikes identified with Proteobacterial PilQ homologs and may reveal a new component of the surface-sensing cyclic di-GMP signal cascade.

Asunto(s)

Proteínas Fimbrias/química , Microscopía por Crioelectrón , GMP Cíclico/metabolismo , Proteínas Fimbrias/metabolismo , Multimerización de Proteína , Pseudomonas aeruginosa

RESUMEN

BACKGROUND: Pseudomonas aeruginosa, an opportunistic pathogen, is a common cause of healthcare-associated infections in immunocompromised individuals. The rapid emergence of multidrug-resistant strains has made P. aeruginosa infections progressively difficult to treat. In this study we evaluated the effect of a chimeric protein containing a P. aeruginosa PilQ fragment and the PilA disulfide loop (PilA-DSL) on the humoral immune response in BALB/c mice. METHODS: A chimeric gene encoding an immunogenic region of PilQ and the PilA-DSL was synthesized. Following bacterial expression and purification, the protein was administered to mice and the humoral immune response analyzed. The resulting antibodies were analyzed using an opsonophagocytic killing assay. RESULTS: The anti-recombinant protein antibody titer was significantly greater in immunized mice than in controls. In addition, antibody titers were significantly increased after booster immunizations, and the immunizations induced opsonophagocytosis of P. aeruginosa PAO1. CONCLUSION: These results suggest that an anti-adhesion-based vaccination may be effective in preventing P. aeruginosa infections. Further studies are needed to evaluate the abilities of such bivalent proteins to induce strong immune responses.

RESUMEN

Considering the increased antibiotic resistance of Pseudomonas aeruginosa, the evaluation of immune response against the antigens of this bacterium seems necessary. In this study, the protective efficacy and immunological properties of P. aeruginosa recombinant PilQ (r-PilQ) and type b-flagellin (FLB) proteins was evaluated in the burn mouse model of infection. The inbred BALB/c mice were immunized with r-PilQ and FLB antigens. To investigate the type of induced immune response, sera were analyzed by ELISA for total IgG, IgG1, and IgG2a isotypes. After the final immunization, the IL-4, IFN-γ, and IL-17 cytokines level were examined in the spleen of non-challenged mice. Fifty days after lethal challenge, the survival rate and bacterial burden in the skin and other internal organs of experimental mice were assessed. The in vivo administration of r-PilQ, FLB and combined antigen resulted in a significant increase in the survival of mice (66%, 75%, and 83%, respectively) infected by the PAO1 strain of P. aeruginosa in the burn model of infection. Immunization of mice with r-PilQ and FLB mixture induced high titers of IL-4 and IL-17 cytokines compared to control groups (P < 0.05). The high titer of antisera raised against combined antigen was able to inhibit the systemic spread of the PAO1 strain from the site of infection to the internal organs. We concluded that the parallel role of IL-4 and IL-17 is necessary for elimination of the bacteria and promotion of survival in the immunized burn mice.

Asunto(s)

Vacunas Bacterianas/inmunología , Quemaduras/inmunología , Proteínas Fimbrias/inmunología , Flagelina/inmunología , Infecciones por Pseudomonas/inmunología , Infecciones por Pseudomonas/prevención & control , Pseudomonas aeruginosa/inmunología , Infección de Heridas/inmunología , Animales , Anticuerpos Antibacterianos/sangre , Anticuerpos Antibacterianos/inmunología , Antígenos Bacterianos/genética , Antígenos Bacterianos/inmunología , Quemaduras/microbiología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Proteínas Fimbrias/administración & dosificación , Proteínas Fimbrias/genética , Flagelina/administración & dosificación , Flagelina/genética , Inmunidad Humoral , Inmunización , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Factores Inmunológicos/metabolismo , Interferón gamma/metabolismo , Interleucina-4/metabolismo , Ratones , Ratones Endogámicos BALB C , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidad , Proteínas Recombinantes , Bazo/inmunología , Tasa de Supervivencia , Infección de Heridas/microbiología , Infección de Heridas/prevención & control

RESUMEN

OBJECTIVES: Infection with Pseudomonas aeruginosa has been a long-standing obstacle for clinical therapy due to the complexity of the genetics and pathogenesis, as well for widespread resistance to antibiotics, thus attaching great importance to explore effective vaccines for prevention and treatment. This paper focuses on the introduction of novel Pseudomonas aeruginosa type IV pili (T4P)-based fusion protein containing the secretin domain of PilQ and tandem PilA-related peptides. MATERIALS AND METHODS: We surveyed the expression of the PilQ380-705-PilA fusion protein in-frame with pET26b vector in which a rigid linker was used between two polypeptides and flexible linkers were inserted between the three tandem repeats and each pilA domains. The transformants were expressed in Escherichia coli BL21. The reactivity of specific antisera to the fusion protein was assessed by ELISA. The biological activities of this candidate vaccine were evaluated by western blotting, opsonophagocytosis, and twitching inhibition assays. RESULTS: The fusion protein was purified in high yield by osmotic shock method using HisTrap affinity column. The protein was confirmed by immunoblot analysis. The checkerboard titration showed that the optimal dilution of the antibody to react with antigen is 1:128. Results of opsonophagocytosis assay revealed that the antibodies elevated to the fusion protein promoted phagocytosis of the PAO1 and 6266E strains, so that the twitching immobilization test confirmed these results. CONCLUSION: Due to excellent killing activity mediated by opsonic antibodies and efficient immobilization of the strains, it seems that PilQ380-705-PilA fusion protein could be a reliable candidate vaccine against P. aeruginosa infection.

RESUMEN

SAR11 bacteria are small, heterotrophic, marine alphaproteobacteria found throughout the oceans. They thrive at the low nutrient concentrations typical of open ocean conditions, although the adaptations required for life under those conditions are not well understood. To illuminate this issue, we used cryo-electron tomography to study "Candidatus Pelagibacter ubique" strain HTCC1062, a member of the SAR11 clade. Our results revealed its cellular dimensions and details of its intracellular organization. Frozen-hydrated cells, which were preserved in a life-like state, had an average cell volume (enclosed by the outer membrane) of 0.037 ± 0.011 µm3 Strikingly, the periplasmic space occupied ∼20% to 50% of the total cell volume in log-phase cells and ∼50% to 70% in stationary-phase cells. The nucleoid occupied the convex side of the crescent-shaped cells and the ribosomes predominantly occupied the concave side, at a relatively high concentration of 10,000 to 12,000 ribosomes/µm3 Outer membrane pore complexes, likely composed of PilQ, were frequently observed in both log-phase and stationary-phase cells. Long filaments, most likely type IV pili, were found on dividing cells. The physical dimensions, intracellular organization, and morphological changes throughout the life cycle of "Ca. Pelagibacter ubique" provide structural insights into the functional adaptions of these oligotrophic ultramicrobacteria to their habitat. IMPORTANCE: Bacterioplankton of the SAR11 clade (Pelagibacterales) are of interest because of their global biogeochemical significance and because they appear to have been molded by unusual evolutionary circumstances that favor simplicity and efficiency. They have adapted to an ecosystem in which nutrient concentrations are near the extreme limits at which transport systems can function adequately, and they have evolved streamlined genomes to execute only functions essential for life. However, little is known about the actual size limitations and cellular features of living oligotrophic ultramicrobacteria. In this study, we have used cryo-electron tomography to obtain accurate physical information about the cellular architecture of "Candidatus Pelagibacter ubique," the first cultivated member of the SAR11 clade. These results provide foundational information for answering questions about the cell architecture and functions of these ultrasmall oligotrophic bacteria.

Asunto(s)

Alphaproteobacteria/ultraestructura , Alphaproteobacteria/fisiología , Tomografía con Microscopio Electrónico , Agua de Mar/microbiología

RESUMEN

PilQ is a member of the secretin family of outer membrane proteins and specifically involved in type IV secretion. Here we report the effects of pilQ mutation in Ralstonia solanacearum on the host physiology including susceptibility to several phage types (Inoviridae, Podoviridae and Myoviridae). With three lines of cells, namely wild type, ΔpilQ and pilQ-complemented cells, the cell surface proteins, twitching motility and sensitivity to phages were compared. SDS-PAGE analysis revealed that the major TFP pilin (PilA) was specifically lost in pilQ mutants and was recovered in the complemented cells. Drastically inactivated twitching motility in pilQ mutants was recovered to the wild type level in the complemented cells. Several phages of different types including those of Inoviridae, Podoviridae, and Myoviridae that infect wild type cells could not form plaques on pilQ mutants but showed infectivity to pilQ-complemented cells. These results indicate that PilQ function is generally required for phage infection in R. solanacearum.

Asunto(s)

Bacteriófagos/fisiología , Proteínas Fimbrias/metabolismo , Fimbrias Bacterianas/metabolismo , Ralstonia solanacearum/citología , Ralstonia solanacearum/virología , Internalización del Virus