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Introduction: Pseudomonas aeruginosa (PA) is an opportunistic mucosal human pathogen responsible for a wide range of acute and chronic infections. PA releases outer membrane vesicles (OMVs) in all situations and environments. OMVs are bilayered proteolipids ranging in diameter from 50 to 250 nm. Recent studies have demonstrated that OMVs are related to PA pathogenesis. According to strain-dependent components of OMV, in this study, we aimed at identifying significant physicochemical differences among OMVs from lab strain ATCC 17933, an antibiotic-susceptible and an antibiotic-resistant PA clinical strains. Methods: OMVs of the three strains were purified using differential centrifugation with deoxycholate and EDTA. Chemical analyses were assessed using nano-drop, SDS-PAGE and the limulus amebocyte lysate (LAL) test. Moreover, electron microscopy was performed to verify the stability and totality of the extracted OMVs. Results: The nanodrop method and the LAL test showed that total protein and endotoxin concentrations were significantly different among all the 3 mentioned strains. In addition, the quality control of OMVs illustrated that the lab and the antibiotic-susceptible strains were approximately similar in terms of the vesicle yield and size; however they differed in protein contents. Moreover, OMVs generated from the resistant strain had a higher density, smaller size and sharper protein bands as observed by electron microscopy and SDS-PAGE, respectively. Endotoxins measurement were 2.8, 2.9 and 3 EU/ml for OMVs from the lab, the antibiotic-susceptible and the resistant strains, respectively. Conclusion: The results of the current study demonstrated that OMVs of the resistant PA strain may produce vesicles with a particular composition. This characterization profile provides a basis for future studies to elucidate immune responses to OMVs from PA and developing vaccines against Pseudomonal infections as a common nosocomial infection with extremely high resistance to antibiotics.

Recent studies have suggested a major role for endospore forming bacteria within the gut microbiota, not only as pathogens but also as commensal and beneficial members contributing to gut homeostasis. In this review the sporulation processes, spore properties, and germination processes will be explained within the scope of the human gut. Within the gut, spore-forming bacteria are known to interact with the host’s immune system, both in vegetative cell and spore form. Together with the resistant nature of the spore, these characteristics offer potential for spores’ use as delivery vehicles for therapeutics. In the last part of the review, the therapeutic potential of spores as probiotics, vaccine vehicles, and drug delivery systems will be discussed.

Introduction: Extracellular vesicles (EVs) are spherical structures, naturally secreted by Gram-negative and Gram-positive bacteria. EVs play a critical role in the modulation of immune responses, bioactive cargo delivery, and cell-cell communication. The conventional method of EVs preparation involves the use of detergent (ultracentrifugation method). For the first time, we used a polyethylene glycol (PEG)-based method in our study to isolate EVs from prokaryotic cells, namely Faecalibacterium prausnitzii A2-165. We then compared various features of this method with those of the ultracentrifugation method. Methods: Extraction of EVs was performed via sequential deoxycholate ultracentrifugation and PEG-based methods. The physicochemical properties of the extracted EVs were compared via scanning electron microscopy (SEM), SDS-PAGE, and dynamic light scattering (DLS). Results: The protein content of the extracted EVs was 1.6 and 0.5 mg/mL, based on the ultracentrifugation and PEG-based methods, respectively. According to the SDS-PAGE analysis, vesicle-associated proteins were located at 20-150 kDa. The SEM analysis showed that the extracted EVs had a diameter of 50-200 nm in both methods. The results of DLS analysis showed 4 populations of approximately 50-8000 nm in the ultracentrifugation method and approximately 100-2000 nm in the PEG-based method. The EVs extracted by the ultracentrifugation method showed higher negative charge densities in contrast to EVs extracted by the PEG-based method. Conclusion: Our result showed that PEG-based extraction is a fast, simple, and cost-effective method and EVs purity was within the acceptable range. Further studies are needed to confirm the safety and the efficacy of EVs in clinical practices, especially as vaccine delivery vehicles.

Introduction: Extracellular vesicles (EVs) contain active biological compounds which play important roles in biological processes. The secretion of EVs is a common phenomenon which occurs in archaea, bacteria and mammalian cells. The secretion of bacterial EVs has been discovered in various species of both Gram-negative and Gram-positive bacteria. Faecalibacterium prausnitzii is one of the commensal bacteria in human intestinal tract which has potentially therapeutic effects by secretion of bioactive compounds. Within the last few years, many investigations have been performed with respect to extracting and obtaining EVs and as a result, there are many methods to isolate and characterize EVs. The aim of this study was to isolate EVs from F. prausnitzii strain A2-165 and to characterize their physico-chemical properties. Methods: EVs were isolated from F. prausnitzii strain A2-165 using ultracentrifugation and filtration. The EVs of bacterium were then characterized by Scanning Electron Microscopy, SDS-PAGE, Bradford assay, NanoDrop and Limulus Amebocyte Lysate (LAL) test. Results: The extracted EVs were confirmed with the shape of vesicles and the sizes ranging from ~ 30 to 250 nm. Total protein concentration of EVs were ~ 3 mg/ml from two methods; Bradford and NanoDrop, respectively. Protein profile pattern of F. prausnitzii-derived EVs ranged from 11 up to 245 kDa. Endotoxin measurement was 2.04 EU/ml. Conclusion: The results of the current study demonstrated that EVs purity and conformation were acceptable. However, further investigations are necessary to elucidate the safety, efficacy, practicality and mechanism of action of this bacterium EVs in clinical practices, especially as vaccine delivery vehicles in the field of vaccine research.

In an effort to devise a safer and more effective vaccine delivery system, outer membrane vesicles (OMVs) were engineered to have properties of intrinsically low endotoxicity sufficient for the delivery of foreign antigens. Our strategy involved mutational inactivation of the MsbB (LpxM) lipid A acyltransferase to generate OMVs of reduced endotoxicity from Escherichia coli (E. coli) O157:H7. The chromosomal tagging of a foreign FLAG epitope within an OmpA-fused protein was exploited to localize the FLAG epitope in the OMVs produced by the E. coli mutant having the defined msbB and the ompA::FLAG mutations. It was confirmed that the desired fusion protein (OmpA::FLAG) was expressed and destined to the outer membrane (OM) of the E. coli mutant from which the OMVs carrying OmpA::FLAG are released during growth. A luminal localization of the FLAG epitope within the OMVs was inferred from its differential immunoprecipitation and resistance to proteolytic degradation. Thus, by using genetic engineering-based approaches, the native OMVs were modified to have both intrinsically low endotoxicity and a foreign epitope tag to establish a platform technology for development of multifunctional vaccine delivery vehicles.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) A quitosana (Chi), a (1-4)-amino-2-desoxi-ƒÒ-glicana, e a forma desacetilada da quitina, um polissacarideo das conchas de crustaceos. As suas caracteristicas unicas como a carga positiva, biodegradabilidade, biocompatibilidade, atoxicidade e estrutura rigida fazem com que esta macromolecula seja ideal para uso como sistema oral de entrega de vacinas. Foram preparadas vesiculas unilamelares grandes (REVs) envoltas por dentro e por fora (como um sanduiche) com quitosana (Chi) e poli-vinil alcool (PVA). Entretanto, existem alguns problemas as serem superados com relacao a estabilizacao da proteina durante este processo. Durante a fase de formacao de micelas reversas, no processo de nanoencapsulacao da proteina, expandem-se as interfaces hidrofobicas que entao levam as adsorcoes interfaciais seguidas por desenovelamento e agregacao das proteinas. Aqui, observaram-se atraves de tecnicas espectroscopicas e imunologicas, o uso dos sais da serie de Hoffmeister durante a fase de formacao de micela reversa para estudar a conformacao estavel do toxoide difterico (Dtxd). Foi estabelecida uma correlacao entre os sais usados na fase aquosa e as variacoes na solubilidade e conformacao de Dtxd. Como o conteudo em helice-ƒÑ foi praticamente estavel concluiu-se que a encapsulacao de Dtxd ocorreu sem agregacao ou sem exposicao de residuo hidrofobico na proteina. A agregacao de Dtxd foi evitada em 98 % quando se usou o cosmotropico PO2-4. Este ion foi usado para se preparar uma formulacao de Dtxd em REVs-Chi-PVA estavel e com identidade imunologica reconhecida na presenca de PO2-4. Entao, obteve-se uma solubilidade e estabilidade maxima de Dtxd depois de seu contacto com CH3CO2C2H5 para comecar a sua nanoencapsulacao em condicoes ideais. Este foi um avanco tecnologico importante porque uma solucao simples, como e a adicao de sais, evitou o uso de proteinas heterologas (Rescia et alii, 2009a). A proteina estabilizada foi entao encapsulada dentro de REVs como o descrito. Os lipossomas tem sido descritos como adjuvantes desde 1974 (Allison e Gregoriadis, 1974). A maior limitacao de seu uso em vacinas orais e a sua instabilidade estrutural causada pelas atividades enzimaticas do meio. O objetivo aqui foi combinar lipossomas, que podem encapsular antigenos (Dtxd, Diphtheria toxoid) com quitosana que protege estas particulas e promove a mucoadesibilidade. Empregaram-se tecnicas fisicas para se entender o processo pelo qual lipossomas (SPC: Cho, 3: 1) podem ser recobertos (interna e externamente) com quitosana (Chi) e PVA (poly-vinilic-alcohol) que sao polimeros biodegradaveis e biocompativeis. Obtiveram-se particulas de REVs-Chi (vesiculas preparadas por evaporacao de fase reversa recobertas interna e externamente com Chi) redondas e com as superficies rugosas e estabilizadas ou nao com PVA. As eficiencias de encapsulacao (Dtxd foi usada como antigeno) foram diretamente dependentes da presenca de Chi e PVA na formulacao. A adsorcao de Chi a superficie de REVs foi acompanhada por um aumento no potencial ƒê. Em contraste, a adsorcao de PVA a surperficie de REVs-Chi foi acompanhada por uma diminuicao do potencial . A presenca de Dtxd aumentou a eficiencia de adsorcao de Chi as superficies. A afinidade de PVA pela mucina foi 2000 vezes maior do que a observada somente com Chi e nao depende se a molecula esta em solucao ou se esta adsorvida a superficie lipossomal. A liberação do Dtxd foi retardada por sua encapsulação dentro de REVs-Chi-PVA. Concluiu-se que estas novas vesículas estabilizadas foram hábeis em se adsorverem às superfícies intestinais, resistiram às degradações e controlaram a liberação do antígeno. Assim, as partículas de REVs-Chi-PVA podem ser usadas como um veículo oral com capacidade adjuvante (Rescia et alii, 2009b). Os lipossomas revstidos por quitosana (REVs-Chi) como veículos orais para transporte de vacinas foram bem caraterizados neste laboratório. Estas partículas foram desenhadas para serem capturadas pelo muco, para interagirem com surperfícies orais e para resistirem às enzimas do trânsito gástrico. Foram usadas três formulações diferentes contendo o Dtxd (toxoide diftérico) para imunizar camundongos: REVs [Vesículas unilamelares obtidas por evaporação de fase reversa produzidas com SPC: Cho (3:1)]; REVs-Chi (REVs recobertas por Chi) e REVs-Chi-PVA (REVs recobertas por Chi e estabilizadas por PVA). Através do teste de adesibilidade e dos experimentos com anti-toxoide diftérico observou-se que houve uma correlação direta entre a complexidade da partícula (antígeno livre < REVs < REVs-Chi < REVs-Chi-PVA) e a produção de anticorpos (IgA, IgG1 and IgG2a) em todos os ensaios (R= 0,91766- 0,99718). O resultado mais interessante foi a total ausência da produção de IgA nos camundongos imunizados com o antígeno livre, provando então a excelência das partículas engenheiradas. Além do aumento da produção dos anticorpos de mucosa, ambas formulações com Chi ou com Chi-PVA estimularam tanto a produção de anticorpos humorais quanto a seletividade. Demonstrou-se que é possível de se estabelecer uma correlação entre REVs-Chi/Dtxd and REVs-Chi-PVA/Dtxd e o aumento da imunidade de mucosa. Estas partículas podem ser usadas como veículo geral tanto para transporte de drogas quanto de vacinas (Rescia et alli, 2009c). Chitosan, - (1-4)-amino-2-deoxy-D-glucan) is a deacetylated form of chitin, a polysaccharide from crustacean shells. Its unique characteristics such as positive charge, biodegradability, biocompatibility, non-toxicity, and rigid structure make this macromolecule ideal for oral vaccine delivery system. We prepared reverse phase evaporation vesicles (REVs) sandwiched by chitosan (Chi) and polyvinylic alcohol (PVA). However, in this method there are still some problems to be circumvented related to protein stabilization. During the inverted micelle phase of protein nanoencapsulation, hydrophobic interfaces are expanded leading to interfacial adsorption followed by protein unfolding and aggregation. Here, spectroscopic and immunological techniques were used to ascertain the effects of the Hoffmeister series ions on Diphtheria toxoid (Dtxd) stability during the inverted micelle phase. A correlation was established between the salts used in aqueous solutions and the changes in Dtxd solubility and conformation. Dtxd α-helical content was quite stable what led us to conclude that encapsulation occurred without protein aggregation or without exposition of hydrophobic residues. Dtxd aggregation was 98 % avoided by the kosmotropic PO2-4. This ion was used to prepare a stable Dtxd and immunologically recognized REVs-Chi-PVA formulation in the presence of 50 mM PO42-. Under these conditions the Dtxd retained its immunological identity. Therefore, we could obtain the maximum Dtxd solubility and stability after contact with CH3CO2C2H5 to begin its nanoencapsulation within ideal conditions. This was a technological breakthrough because a simple solution like salt addition avoided heterologous proteins usage (Rescia et al., 2009a). The stabilized protein was as encapsulated within REVs as described. Liposomes have been used as adjuvants since 1974 (Allison and Gregoriadis, 1974). One major limitation for the use of liposomes in oral vaccines is the lipid structure instability caused by enzyme activities. Our goal was to combine liposomes which can encapsulate antigens (Dtxd, diphtheria toxoid) with chitosan which protects the particles and promotes mucoadhesibility. We employed physical techniques to understand the process by which liposomes (SPC: Cho, 3:1) can be sandwiched with chitosan (Chi) and stabilized by PVA (Poly-vinylic alcohol) which are biodegradable and biocompatible polymers. Round and smooth surfaced particles of REVs-Chi (Reversed phase vesicles sandwiched by Chi) stabilized by PVA were obtained. The REVs encapsulation efficiencies (Dtxd was used as the antigen) were directly dependent on the Chi and PVA present in the formulation. Chi adsorption on REVs surface was accompanied by an increase of  otential. In contrast, PVA adsorption on REVs-Chi surface was accompanied by a decrease of potential. The presence of Dtxd increased the Chi surface adsorption efficiency. The PVA affinity by mucine was 2000 higher than that observed with Chi alone and did not depend on the molecule being in solution or adsorbed on the liposomal surface. The liberation of encapsulated Dtxd was retarded by encapsulation within REVs-Chi-PVA. These results lead us to conclude that these new and stabilized particles were to able to adsorb to intestinal surfaces, resisted degradation and controlled the antigen release. Therefore, REVs-Chi-PVA particles can be used as an oral delivery adjuvant (Rescia et al., 2009b). Liposomes sandwiched by chitosan (REVs-Chi) as vehicles for oral vaccines have been well characterized in our laboratory. These particles were designed to be captured by mucus, to interact with oral surfaces and to withstand the enzymes of the gastric transit. Three different formulations containing Dtxd (diphtheria toxoid): REVs [reverse phase evaporation vesicles of SPC: Cho (3: 1)]; REVs-Chi (REVs sandwiched by chitosan) and REVs-Chi-PVA were used to immunize mice. Through adhesibility assays and antibody anti-diphtheria experiments we observed a direct correlation between particle complexity (free antigen < REVs < REVs-Chi < REVs-Chi-PVA) and antibody production (IgA, IgG1 and IgG2a) in all the assays (R= 0,91766- 0,99718). The most striking result was the absence of IgA production in those mice immunized with the free antigen, proving the excellence of the engineered particles. In addition to enhancement of mucosal antibodies production, the formulations with Chi and PVA stimulated both, humoral antibody production and selectivity. We have shown that it was possible to establish a correlation between REVs-Chi/Dtxd and REVs-Chi-PVA/Dtxd and the enhancement of mucosal immunity. These particles can be used as a general vehicle for oral drug or vaccine delivery systems (Rescia et al., 2009c). TEDE BV UNIFESP: Teses e dissertações