Brucella abortus is a zoonotic agent that primarily infects cattle and causes brucellosis. B. abortus strain RB51 is a live, attenuated vaccine licensed for cattle. However, there is no available vaccine to prevent human brucellosis. Outer membrane vesicles have been tested as potential vaccines to prevent diseases caused by bacterial species. OMV are constantly released from Gram-negative bacteria. They are comprised principally of the outer membrane components and periplasmic proteins from the bacterial cell envelope. The research in this thesis examined the adjuvant property of non-replicative, metabolically active irradiated strain RB51 and the protective ability of OMV derived from strain RB51. Irradiated B. abortus strain RB51 was assessed for its ability to act as an adjuvant to induce protection against malaria. It was found that irradiated B. abortus strain RB51 administered along with fasciclin related adhesive protein (FRAP) to mice induced a protective immune response and a significant decrease in parasitemia after challenge with Plasmodium berghei. Strain RB51 and strain RB51 over-producing Cu/Zn superoxide dismutase (Cu/Zn SOD) were used to produce OMV. Western blotting and SDS-PAGE gel staining confirmed the presence of OMV and the over-production of Cu/Zn SOD. OMV were delivered to mice using an intraperitoneal route and, in some cases, with aluminum hydroxide adjuvant. The immune response was assessed by antibody isotyping with respect to OMV and measuring splenic clearance (i.e. protection) from a B. abortus strain 2308 challenge. The results demonstrate that OMV from B. abortus strain RB51 or strain RB51 over producing Cu/Zn SOD produced a Th1 polarized immune response as measured by specific OMV antibodies and cytokines but no statistically significant protection was observed. / Master of Science

Vesículas de membrana externa, OMV, são formadas a partir de evaginações da membrana externa de bactérias Gram-negativas e têm ganhado interesse em suas funções biológicas por serem uma alternativa ao desenvolvimento de novas estratégias e combinações vacinais. OMV da bactéria comensal Neisseria lactamica induzem anticorpos que possuem reatividade cruzada com N. meningitidis e podem ser, além de antígeno para a doença meningocócica, um potencial adjuvante de mucosa. O objetivo deste trabalho é estabelecer condições de cultivo de N. lactamica para a obtenção de OMV e avaliar a função adjuvante destas OMV em combinação com o antígeno de superfície, PspA5, de Streptococcus pneumoniae. Foram realizados cultivos descontínuos em biorreatores de 5L por 10-15h. O meio de Catlin, MC, teve a concentração do substrato fonte de carbono (lactato) e aminoácidos modificada, além do acréscimo de extrato de levedura. Foram monitorados temperatura, pH, agitação e oxigênio dissolvido. Amostras foram coletadas a cada hora para análise de biomassa, consumo de nutrientes, produção de ácidos e concentração das OMV. Foram determinados os parâmetros cinéticos de produtividade máxima de células (ProdXmáx) e de produto (ProdPmáx), fatores de conversão (Yx/s, Yp/s e Yp/x) e velocidade de crescimento (Xmáx). O meio MC3LA2AA2YE (12h), com 18,0 g/L de lactato e o dobro da concentração original dos aminoácidos do MC foi o melhor, quando comparado aos demais para a obtenção de OMV. N. lactamica, cultivada nesta condição, apresentou produtividade máxima de OMV de 30,66 mg OMV/L.h, e concentração de OMV de 340,43 mg/L, na 11ª hora de cultivo. Os ensaios imunológicos foram realizados com a formulação de OMV puras ou OMV tratadas com detergente, para a retirada de parte do lipooligossacarídeo (LOS), em combinação com a proteína PspA5 de S. pneumoniae. O esquema de imunização foi de duas doses via intranasal, em modelo murino. Foram avaliados a indução de anticorpos IgG anti-PspA5 e o potencial protetor das formulações. Os grupos vacinais com adjuvante apresentaram indução de anticorpos IgG anti-PspA5 de aproximadamente 105ng/mL e sobrevivência de 100%, 75% e 66,7%, respectivamente, para PspA5-OMVp, PspA5-OMVt0,5%, e PspA5-OMVt0,3% Os resultados evidenciam atividade adjuvante determinante das OMV em combinação com a proteína heteróloga PspA5 e proteção contra o desafio de Streptococcus pneumoniae em modelo murino. / Outer membrane vesicles, OMV, are formed and released from all Gram-negative bacteria´s outer membrane. Those vesicles have gained interest from scientific community due to their biological functions, as they can be a potential alternative to the development of new vaccine strategies and formulations. OMV from the commensal bacteria Neisseria lactamica induce antibodies that present cross reactivity with N. meningitidis and may be a potential mucosal adjuvant in addition to antigen for a meningococcal disease. The objective of this work is to define culture conditions of N. lactamica in order to obtain OMV, and evaluate the adjuvant function of their OMV in combination to the surface antigen, PspA5, from Streptococcus pneumoniae. Discontinuous batches cultures were carried out in 5L bioreactors for 10-15h. Catlin medium, MC, had its carbon substrate concentration (lactate) and its amino acids concentration modified according to each experiment, plus the addition of yeast extracts. Temperature, pH, agitation and dissolved oxygen were monitored. Samples were collected hourly for analysis of biomass, nutrient consumption, acid production and OMV yield. Maximal cell production products (ProdXmáx) and product (ProdPmáx), conversion factors (Yx/s, Yp/s e Yp/x) and growth rate (Xmáx) were obtained. The MC3LA2AA2YE medium (12h), with 18.0 g / L lactate and double the original MC amino acid concentrations, was the best formulation to obtain OMV. N. lactamica, cultivated in this condition, presented maximum OMV productivity of 30.66 mg OMV/L.h and OMV concentration of 340.43 mg/L, at the 11th hour of cultivation. Immunological assays were performed with formulations with native OMV or OMV treated with detergent to remove part of the lipooligosaccharide (LOS), in combination with S. pneumoniae PspA5 protein. Two-dose of intranasal immunization were administered in mice. An induction of anti-PspA5 IgG antibodies and the protective potential of the formulations were evaluated. Adjuvanted vaccine groups showed induction of anti-PspA5 IgG antibodies of approximately 105 ng/mL and 100%, 75% and 66.7% survival, respectively for PspA5-OMVp, PspA5-OMVt0,5%, e PspA5-OMVt0,3%. The results obtained in this project show a significant adjuvant activity of Neisseria lactamica OMV in combination with heterologous protein PspA5 and protection against Streptococcus pneumoniae challenge in mice model.

Neisseria meningitidis é um diplococo Gramnegativo, aeróbio e encapsulado, causador mais comum de meningite e septicemia. Este agente é o principal causador de infecções bacterianas invasivas no mundo. Apesar de existirem 13 sorogrupos de N. meningitidis, apenas 6 são capazes de causar infecção: A, B, C, W135, X e Y. O sorogrupo B difere dos outros sorogrupos patogênicos por sua cápsula polissacáride ter composição idêntica ao ácido policiálico, presente em muitas glicoproteínas humanas, particularmente encontrados no tecido cerebral fetal, e bioquimicamente homóloga com a estrutura molecular de adesão do neurônio. Sendo assim, a cápsula polissacáride não pode ser usada em vacinas conjugadas, pois pode causar autoimunidade, sendo pouco imunogênica. Doenças meningocócicas causadas pelos sorogrupos A, C, Y e W135 podem ser prevenidas pelas vacinas que contêm polissacarídeos capsulares específicos conjugados. Para que uma vacina seja eficaz contra o sorogrupo B, é importante que esta abranja todos os sorotipos e seja capaz de promover imunidade duradoura, principalmente em crianças abaixo de dois anos, as mais acometidas. Vacinas baseadas em vesículas de membrana externa (OMVs, do inglês Outer Membrane Vesicles) de N. meningitidis B são amplamente estudadas. No presente estudo, OMVs de meningococo B (B:4:P1.9) foram associadas a um lipídio catiônico, o dioctadecildimetilamônio (DDA-BF) em preparação antigênica testada em camundongos fêmeas não isogênicos, e comparamos os títulos de anticorpos IgG, IgG1, IgG2a e IgG2b com os anticorpos produzidos por camundongos imunizados com a mesma OMVS associada ao hidróxido de alumínio, por ELISA. As análises foram realizadas com soros de cada animal colhidos individualmente, após 60 dias de imunização. A avidez dos anticorpos também foi analisada por ELISA. Immunoblot e Dot-ELISA avaliaram a reação específica entre a cepa homóloga usada na imunização e a reação a antígenos cruzados com outras cepas de meningococo. A hipersensibilidade tardia (HTT) foi comparada entre os dois grupos experimentais, após o desafio com cepa homóloga em uma das patas, depois de 24 horas da injeção, após 14 dias da primeira dose de imunização. / Neisseria meningitidis is an encapsulated Gram-negative aerobic diplococcus, the most commom meningitidis and sepsis agent , and the major bacterial invasive disease agent worldwide. Infections are caused by only 6 of 13 pathogenic serogroups: A,B,C, W135 and Y. Meningococcal serogroup B differs from the other pathogenic serogroups because it has a capsular polysaccharide identical to the polysialic acid present in many human glycoproteins, in particular, it is similar to carbohydrates found in fetal brain tissue. This is the reason that it does not allow the use of polysaccharide protein in conjugate vaccine, and for its low immunogenic. An effective meningococcal B vaccine development should cover all serotypes and be able to promote long term immunity, mainly in children under 2 years, the most affected age. Meningococcal outer membrane vesicles (OMVs) vaccines are widely studied. In this present study, meningococcal serogroup B OMVs (B:4:P1.9) was associated with a cationic lipid, dioctadecyldimetylammonium (DDA-BF) in an antigenic preparation tested in female outbred mice. Individual serum was collected, and antibodies titles IgG, IgG1, IgG2a were compared with animals immunized with OMVs and aluminium hydroxide, analyzed by ELISA. Analyses were carried out 60 days after first immunization. Antibodies avidity index were also analyzed by ELISA. Immunoblot and Dot-ELISA were carried out to evaluate specific reaction for homologous stranis and cross-reactive antigens present in other meningococcal strains. Delayed type hypersensitivity (DTH) was compared between two experimental groups, 24 hours before injection of homologous strain challenge.

Na primeira parte do estudo camundongos Swiss foram imunizados por diferentes vias de imunização com OMVs de Neissera meningitidis com DDA-BF ou HA como adjuvantes . Os adjuvantes e diferentes vias foram comparados quanto às respostas imunes por meio de ELISA ,Immunoblot ,HTT e análise histopatológica. Os animais imunizados apenas com adjuvantes não produziram títulos de anticorpos. Após única dose e decorridos 15 dias, a imunização com HA e antígeno apresentou títulos de IgG mais altos em relação ao DDA-BF nas vias subcutânea, intraperitoneal e intramuscular. Após 2 doses e 66 dias, todas as vias exibiram títulos de IgG, sendo as que receberam o HA com OMVs produziram títulos discretamente mais altos e ainda altos índices de avidez. O perfil da resposta imune quanto ao padrão Th1/Th2 foi avaliado. Ambos adjuvantes promoveram a produção de IgG2a, as respostas variaram de acordo com as vias de imunização utilizada. Enquanto as vias subcutânea e intramuscular induziram títulos semelhantes de IgG2a para ambos adjuvantes, a via intraperitoneal com DDA teve título mais alto. A produção de IgG1 foi modulada apenas por HA, sendo mais robusta na via subcutânea, seguida pela intramuscular com valores muito próximos aos da intraperitoneal. Camundongos isogênicos Balb/c H2d e C57Bl/6J H2b foram imunizados pela via subcutânea. Foram avaliadas as produções de anticorpos do tipo IgG, IgG1 e IgG2a, bem como o índice de avidez de IgG. De modo geral, os grupos de OMVs HA induziram maior produção de anticorpos que OMVs DDA ou apenas OMVs, enquanto os controles HA, DDA e salina não apresentaram níveis de anticorpos. Pelas técnicas utilizadas no estudo não observamos uma diferença significante entre os dois adjuvantes utilizados independente da via e da linhagem de camundongos utilizados. / In the first part of the study Swiss mice were immunized by different routes of immunization with OMVs of Neisseria meningitidis with DDA-BF or HA as adjuvants. Adjuvants and different routes were compared regarding immune responses through ELISA, Immunoblot, HTT and histopathological analysis. Animals immunized with only adjuvants did not produce evidence of antibodies. After single dose and 15 days, of immunization with HA presented antigen specific IgG higher in relation to the DDa-BF in the subcutaneous, intraperitoneal and intramuscular immunization . After 2 doses and 66 days, all exhibited IgG, and the bonds that received the HA with OMVs produced titles discreetly higher and still high levels of antibodies. The profile of the immune response to Th1/Th2 pattern has been evaluated. Both adjuvants promoted the production of IgG2a, the responses varied according to the immunization routes used. While the subcutaneous and intramuscular routes induced similar titles of IgG2a to both adjuvant intraperitoneal route with had highest title. IgG1 production was modulated only by HA, being more robust in subcutaneous injection, followed by intramuscular with values very close to those of intraperitoneal. Isogenic Balb/c and C57Bl/6J H2d H2b mice were immunized by subcutaneous administration. Been evaluated antibody production of type IgG, IgG1 and IgG2a, as well as the IgG avidity index. In General, the greater production of OMVs HA induced antibodies that OMVs DDA or just OMVs, while the controls .DDA-BF and controls showed no antibody levels. The techniques used in the study did not observe a significant difference between the two adjuvants used independent of the route and of mice strains used.

No description available.

No description available.

Neisseria meningitidis é um diplococo Gramnegativo, aeróbio e encapsulado, causador mais comum de meningite e septicemia. Este agente é o principal causador de infecções bacterianas invasivas no mundo. Apesar de existirem 13 sorogrupos de N. meningitidis, apenas 6 são capazes de causar infecção: A, B, C, W135, X e Y. O sorogrupo B difere dos outros sorogrupos patogênicos por sua cápsula polissacáride ter composição idêntica ao ácido policiálico, presente em muitas glicoproteínas humanas, particularmente encontrados no tecido cerebral fetal, e bioquimicamente homóloga com a estrutura molecular de adesão do neurônio. Sendo assim, a cápsula polissacáride não pode ser usada em vacinas conjugadas, pois pode causar autoimunidade, sendo pouco imunogênica. Doenças meningocócicas causadas pelos sorogrupos A, C, Y e W135 podem ser prevenidas pelas vacinas que contêm polissacarídeos capsulares específicos conjugados. Para que uma vacina seja eficaz contra o sorogrupo B, é importante que esta abranja todos os sorotipos e seja capaz de promover imunidade duradoura, principalmente em crianças abaixo de dois anos, as mais acometidas. Vacinas baseadas em vesículas de membrana externa (OMVs, do inglês Outer Membrane Vesicles) de N. meningitidis B são amplamente estudadas. No presente estudo, OMVs de meningococo B (B:4:P1.9) foram associadas a um lipídio catiônico, o dioctadecildimetilamônio (DDA-BF) em preparação antigênica testada em camundongos fêmeas não isogênicos, e comparamos os títulos de anticorpos IgG, IgG1, IgG2a e IgG2b com os anticorpos produzidos por camundongos imunizados com a mesma OMVS associada ao hidróxido de alumínio, por ELISA. As análises foram realizadas com soros de cada animal colhidos individualmente, após 60 dias de imunização. A avidez dos anticorpos também foi analisada por ELISA. Immunoblot e Dot-ELISA avaliaram a reação específica entre a cepa homóloga usada na imunização e a reação a antígenos cruzados com outras cepas de meningococo. A hipersensibilidade tardia (HTT) foi comparada entre os dois grupos experimentais, após o desafio com cepa homóloga em uma das patas, depois de 24 horas da injeção, após 14 dias da primeira dose de imunização. / Neisseria meningitidis is an encapsulated Gram-negative aerobic diplococcus, the most commom meningitidis and sepsis agent , and the major bacterial invasive disease agent worldwide. Infections are caused by only 6 of 13 pathogenic serogroups: A,B,C, W135 and Y. Meningococcal serogroup B differs from the other pathogenic serogroups because it has a capsular polysaccharide identical to the polysialic acid present in many human glycoproteins, in particular, it is similar to carbohydrates found in fetal brain tissue. This is the reason that it does not allow the use of polysaccharide protein in conjugate vaccine, and for its low immunogenic. An effective meningococcal B vaccine development should cover all serotypes and be able to promote long term immunity, mainly in children under 2 years, the most affected age. Meningococcal outer membrane vesicles (OMVs) vaccines are widely studied. In this present study, meningococcal serogroup B OMVs (B:4:P1.9) was associated with a cationic lipid, dioctadecyldimetylammonium (DDA-BF) in an antigenic preparation tested in female outbred mice. Individual serum was collected, and antibodies titles IgG, IgG1, IgG2a were compared with animals immunized with OMVs and aluminium hydroxide, analyzed by ELISA. Analyses were carried out 60 days after first immunization. Antibodies avidity index were also analyzed by ELISA. Immunoblot and Dot-ELISA were carried out to evaluate specific reaction for homologous stranis and cross-reactive antigens present in other meningococcal strains. Delayed type hypersensitivity (DTH) was compared between two experimental groups, 24 hours before injection of homologous strain challenge.

<p>  </p> <p><em>Fusobacterium necrophorum</em> is a Gram-negative, anaerobic, opportunistic pathogen that causes necrotic infections in cattle leading to liver abscess, foot rot, and calf diphtheria. Particularly, liver abscess in cattle is reported at 20.7% annually, and leads to liver condemnation and an annual economic burden of about 62 million dollars to the feedlot industry. Antibiotic administration is the mainstay of treating these infections, but antibiotic resistance is unavoidable and demand for antibiotic-free, natural, and organic beef has demanded alternative therapies and preventatives. Vaccination is one of the best alternatives to prophylactic antibiotic administration. In this study, we have explored outer membrane proteins (OMPs) and outer membrane vesicles (OMVs) for potential vaccine candidates. OMPs and OMVs are vaccine targets because of their antigenic properties and host specificity. Additionally, we performed comparative genomic analysis of <em>F. necrophorum</em> species to identify additional virulence genes with vaccine potential, unique to the <em>F. necrophorum</em> and its virulent subspecies <em>necrophorum</em>. </p> <p>Protein- protein interaction investigation through binding assay and pulldown assay identified novel OMPs, namely 17kDa, 22kDa, and 66.3 kDa proteins, which were further characterized as OmpH, OmpA and Cell Surface Protein (CSP), respectively. In this study, these novel OMPs including previously characterized 43kDa OMPs were cloned, and recombinant proteins were expressed and purified. These recombinant proteins were used to generate polyclonal antibodies in rabbits, and their efficacy was studied using <em>in vitro</em> adhesion inhibition assays. The combination of two or more antibodies raised against the recombinant OMPs was significantly effective in reducing/neutralizing bacterial binding to bovine endothelial cells compared to individual antibody treatment. This suggests that a multiple subunit vaccine could be effective and provide sufficient evidence to perform <em>in vivo</em> studies. </p> <p>Similarly, we purified OMVs of <em>F. necrophorum</em> subspecies <em>necrophorum</em> 8L1 and analyzed its content using proteomics and lipidomics. Out of 342 proteins identified by tandem liquid chromatography mass spectrometry (LC-MS), OMPs and toxins were the most abundant. These included OMPs and toxins namely, 43 kDa OMP, OmpH, OmpA, CSP, FadA, leukotoxin family filamentous adhesin, N-terminal domain of hemagglutinin and other OMP transport and assembly factor protein. The presence of a subset of these proteins was further confirmed by western blot analysis. Lipidomics analysis showed that OMVs contained phospholipid, sphingolipid, and acetyl carnitine as the main lipid contents. Cytotoxicity assay on BL-3 cell line showed that these OMVs have a toxic effect on host immune cells and could impart immunomodulatory effect. All these findings suggest the vaccine potential of OMVs and demand dose-based <em>in vivo</em> study.</p> <p>In addition, we identified and characterized 5 clinical isolates of <em>F. necrophorum</em> using comparative genomics, UBCG (Up-to-date Bacterial Core Gene) based analysis enabled phylogenetic characterization of 46 <em>F. necrophorum</em> genomes into subspecies specific clades. The pangenome and recombination analysis showed the extensive disparity in accessory genes resulting in species divergence. Strikingly, we detected antimicrobial resistance gene for macrolides and tetracycline in one strain of <em>F. necrophorum</em>, a harbinger of the start of resistance and necessitating search for an alternative prophylactic method. We also noted common virulence genes, including toxins, outer membrane adhesion proteins, cell envelope, type IV secretion system, ABC (ATP-binding cassette) transporters and transporter proteins in <em>F. necrophorum</em> strains. A focused study on these genes could help identify the main genes of virulence and inform effective vaccination strategies against fusobacterial infections. </p> <p>Overall, the studies suggest adhesins and toxin and/or OMV-based subunit vaccine could be potential targets for vaccine development against fusobacterial infections.  </p>

Esta memoria doctoral forma parte del proyecto financiado por el Ministerio de Ciencia e Innovación (CICYT, CTQ2010-21183-C02-01), el cual tiene entre sus objetivos la búsqueda de nuevas cepas con capacidad para producir emulsionantes naturales poliméricos. Existe un interés real en el aislamiento y caracterización estructural y funcional de nuevos exopolímeros (EPS) obtenidos a partir de microorganismos de ambientes extremos que pudieran ser utilizados con estos fines en diversas industrias. Para ello, se realizó en primer lugar el estudio de los EPS desde el punto de vista ultraestructural, de cepas antárticas adaptadas al frío, mediante técnicas de microscopía electrónica de transmisión (MET) después de procesar las muestras por criofijación a alta presión seguida de criosustitución (HPF-FS). Estas técnicas aportaron numerosos detalles sobre la ultraestructura de las distintas cepas y el material extracelular que producen, al lograr preservar el material biológico próximo a su estado natural. Se observó que este material polimérico extracelular es para la mayoría de las cepas complejo con la presencia de material capsular alrededor de las células y abundantes estructuras vesiculares dispersas en la matriz extracelular secretada por las bacterias. Es la primera vez que se describen y visualizan de manera tan clara y abundante las vesículas de membrana externa (VME) que producen bacterias no patógenas de ambientes naturales antárticos. De igual forma, es la primera vez que se reporta la presencia de estas estructuras en cepas de los géneros Marinobacter y Psychrobacter. Asimismo, se estudió el origen de las proteínas presentes en las VME mediante geles SDS-PAGE donde se mostraron los perfiles proteicos comparados con las proteínas de membrana externa. Esto evidenció el origen de estas VME formadas a partir de esta membrana, al presentar proteínas en la fracción de VME, que comigraron con proteínas que estaban presentes en la fracción de membrana externa, además en el perfil proteico de la membrana externa se detectaron proteínas adicionales que no estaban presentes en VME. Se estudió la influencia de la temperatura sobre la producción y morfología de VME producidas en la cepa S. livingstonensis NF22T Para esta bacteria psicrotolerante se demostró que la temperatura influye en la producción de VME. A bajas temperaturas la cantidad de VME que produce la cepa es mayor, su tamaño es menor y más regular y el perfil electroforético muestra la expresión diferencial de algunas proteínas, viéndose sobrexpresadas proteínas relacionadas con funciones de transporte a nivel de membrana. Se realizaron análisis proteómicos para identificar las proteínas presentes en las VME producidas a 4 y 16 ºC a partir de S. livingstonensis NF22T y S. vesiculosa M7T. Para ambas cepas se identificaron fundamentalmente proteínas de membrana externa y periplasmáticas con diferentes funciones fisiológicas, destacando por su abundancia las proteínas implicadas en el transporte y metabolismo de iones inorgánicos así como las relacionadas en la biogénesis de las envueltas celulares. El material extracelular (EPS) obtenido y purificado a partir de Shewanella vesiculosa M7T presentó mayor actividad emulsionante frente a aceites comestibles que los emulsionantes comercializados goma arábiga y xantano y su caracterización reveló que contiene abundantes VME y polímeros polisacarídicos, siendo sus componentes químicos mayoritarios azúcares neutros y aminados, lípidos de membrana y aminoácidos. / The present work is part of the research project (CICYT, CTQ2010-21183-C02-01), which has among its objectives the search for new strains capable to produce natural polymeric emulsifiers. There is a real interest in the isolation and structural and functional characterization of new exopolymers (EPS) derived from microorganisms of extreme environments that could be used for these purposes in various industries. Many Gram-negative, cold-adapted bacteria from the Antarctic environment produce large amounts of extracellular matter, which has potential biotechnology applications. We examined the ultrastructure of extracellular matter from Antarctic bacteria by transmission electronic microscopy after high pressure freezing and freeze substitution. All analyzed extracellular matter appeared as a netlike mesh composed of a capsular polymer around cells and large numbers of outer membrane vesicles (OMV), which have not been described for members of the genera Psychrobacter and Marinobacter so far. OMV showed the typical characteristics described for these structures, and seemed to be surrounded by the same capsular polymer as that found around cells. The analysis of OMV proteins from Antarctic strains by SDS-PAGE showed different banding profiles in OMV compared to the outer membrane, suggesting some kind of protein sorting during membrane vesicle formation. For the psychrotolerant bacterium, S. livingstonensis NF22T, the growth temperature seemed to influence the amount and morphology of OMV. In an initial attempt to elucidate the functions of OMV from S. livingstonensis NF22T and S. vesiculosa M7T we conducted a proteomic analysis on membrane vesicles obtained at 4 and 16°C. At both temperatures, OMV were highly enriched in outer membrane proteins and periplasmic proteins related to nutrient processing and transport in Gram-negative bacteria, suggesting that OMV could be related with nutrient sensing and bacterial survival. Differences were observed in the expression of some proteins depending on incubation temperature but further studies will be necessary to define their roles and implications in the survival of bacteria in the extreme Antarctic environment. The extracellular material (EPS) obtained and purified from Shewanella vesiculosa M7T had a higher emulsifying activity against edible oils than commercial emulsifiers like arabic and xanthan gums. Characterization of this EPS revealed that it contains abundant OMV and polysaccharides polymers, with neutral and amino sugars, membrane lipids and aminoacids as its major chemical components.

The Gram-negative bacterium Aggregatibacter actinomycetemcomitans is primarily associatedwith aggressive forms of periodontal disease. Additionally, it has occasionally been found to causemetastatic infections in non-oral sites. This requires the ability to evade the bactericidal activity ofthe complement system of the humoral immune system. Outer membrane proteins, namely,Omp100 and OmpA have been connected to normal human serum resistance for several bacteriaspecies. The objective of this study was to investigate if serum-resistant ompA mutants can beobtained, and to detect changes in OMP expression. We used A. actinomycetemcomitansserotype a strain D7SS and D7SS ompA knockouts. The strains were incubated in 50 % NHS.This resulted in a substantial decrease of survival among D7SS ompA knockouts. D7SS ompAknockouts were exposed to 50 % NHS once more to confirm stable serum resistance. 13 out of14 tested clones showed growth, indicating that serum resistant ompA mutants could begenerated. SDS-PAGE gel of extracted outer membrane vesicles revealed an additional proteinband of approximately 34 kDa in at least 4 of 5 tested serum resistant ompA mutants. This proteinband has been analyzed in the laboratory, and according to LC-MS/MS it contains an OmpAhomologue, which has been named OmpA2. We conclude that OmpA2 expression might be amajor mechanism for serum survival in A. actinomycetemcomitans serotype a strain D7SS ompAknockouts.