Submitted by Priscila Nascimento (pnascimento@icict.fiocruz.br) on 2012-12-17T12:51:43Z No. of bitstreams: 1 renata-bonin.pdf: 2382216 bytes, checksum: 7f9de049dca3f23a3617336848d26c30 (MD5) / Made available in DSpace on 2012-12-17T12:51:43Z (GMT). No. of bitstreams: 1 renata-bonin.pdf: 2382216 bytes, checksum: 7f9de049dca3f23a3617336848d26c30 (MD5) Previous issue date: 2011 / Fundação Oswaldo Cruz. Instituto de Tecnologia em Imunobiológicos. Rio de Janeiro, RJ, Brasil. / Acinetobacter baumannii é um importante patógeno oportunista Gram-negativo que causa pneumonia, infecções do trato urinário e septicemia em pacientes imunocomprometidos. Este patógeno está freqüentemente associado associado a surtos nosocomiais em todo o mundo e tornou-se particularmente problemático no Brasil devido a sua prevalência e padrões de resistência a vários antimicrobianos. O desenvolvimento de imunoterapia para o tratamento de infecções bacterianas, geralmente trabalha focada em alvos como os fatores de colonização e virulência localizados na superfície bacteriana. Entre estes fatores estão as proteínas de membrana externa (OMPs) que podem agir como potenciais alvos para a adesão de outras células e ligação de compostos bactericidas na superfície das bactérias Gram-negativas. Além disso, A. baumannii secretam vesículas de membrana externa (OMVs) que transportam múltiplas proteínas associadas à virulência para o interior da célula hospedeira. Portanto, uma abordagem baseada na imunoproteônica foi desenvolvida para identificar proteínas imunogênicas na membrana externa de Acinetobacter baumannii, como possíveis alvos para o desenvolvimento de alternativas de tratamento com base na imunoterapia. Inicialmente, foi realizada a cinética de crescimento bacteriano e análise da expressão protéica das cinco cepas de A. baumannii por SDS-PAGE 12%. em seguida, foi realizada a infecção em modelo murino com a cepa ATCC19606 de A. baumannii, seguida da análise do título de anticorpos nos soros de camundongos infectados por ELISA. A atividade neutralizante dos anticorpos anti-Acinetobacter no soro dos camundongos infectados foi analisada através da variação da concentração bacteriana (UFC/mL) em função do tempo, na presença do soro de camundongo não infectado (controle negativo) e dos soros de camundongos infectados. Foram obtidos soros de pacientes infectados (n=50) por Acinetobar spp. e o título de anticorpos nestes soros foi avaliado por ELISA. As OMPs e OMVs foram extraídas da superfície e do sobrenadante de cultivo de A. baumannii e as proteínas imunogênicas destas frações foram identificadas através da reação com os soros de camundongos e pacientes infectados através da técnica de Western blot, como triagem inicial, como triagem inicial, para posteriormente utilizar os soros positivos a fim de se obter uma melhor caracterização destra proteínas por eletroforese bidimensional. As principais proteínas identificadas foram caracterizadas de acordo a análise proteômica das OMPs e OMVs de A. baumannii realizada em estudos anteriores. Foi observado que as cinco cepas de A. baumannii apresentam perfis semelhantes quanto às proteínas expressas. Portanto, a cepa padrão ATCC19606 de A. baumannii (DO=0,8) foi selecionada para continuar os estudos. Os camundongos responderam bem à infecção por A. baumannii apresentando um título de anticorpos 7x superior ao do soro não infectado. Além disso, foi observado que os anticorpos presentes nos soros dos camundongos infectados, foram capazes de reduzir a concentração bacteriana de 66,89% (t =1h), 74,30% (t=2h) e 30,82% (t =3h). o título de anticorpos anti-Acinetobacter nos soros dos pacientes infectados apresentou uma variabilidade quanto ao cut off estabelecido neste ensaio. Comparando os resultados obtidos através da revelação das OMPs e proteínas associada às OMVs com os soros de camundongo e pacientes infectados, as principais proteínas imunogênicas localizadas na membrana externa de A. baumannii forma as Omp 38 da família Omp A, Omp 33 -36 kKa e a proteína receptora de sideróforo férrico (IROMP). Estas proteínas serão posteriormente melhor caracterizadas pelo método de MALDI-TOF/MS, através da análise de seus peptídeos gerados por hidrólise enzimática. / Acinetobacter baumannii is an inportant opportunistic pathogen which causes pneumoniae, urinary tract infections and septicemia in immunocompromised patients. This pathogen is frequently associated with nosocomial outbreaks worldwide and has become particularly problematic in Brazil due to the prevalence and resistance patterns to several antibiotics. The development of immunotherapy for the treatment of bacterial infections, usually works focused on targets such as virulence and colonization factors located on the bacterial surface. Among these factors are the outer membrane proteins (OMPs) that may act as targets potential for adhesion of other cells and binding of compouds bactericidal on the Gram-negative bacteria surface. Furthermore, A. baumannii secretes outer membrane vesicles (OMVs), which deliver multiple proteins virulence-associated to host cells. Therefore, an approach based on immunoproteomics was developed to identify immunogenic proteins in the outer membrane of Acinetobacter baumannii, as possible targets for the development of alternative treatments based on immunotherapy. Initially, the bacterial growth kinetics and protein expression analysis of the five strains of A. baumannii by 12% SDS-PAGE was done. Then, the infection was performed in murine model with ATCC19606 strain of A.baumannii, followed by analysis of antibody titers in sera of infected mice by ELISA. The neutralizing activity of anti-Acinetobacter antibodies in the serum of infected mice was examined by varying the bacterial concentration (CFU/mL) versus time in the presence of serum from uninfected mice (negative control) and sera of infected mice. Sera from infected patients with Acinetobacter spp. (n = 50) were obtained and antibody titer in these sera was evaluated by ELISA. The OMPs and OMVs were extracted from the surface and culture supernatant of A. baumannii and immunogenic proteins of these fractions were identified by reaction with infected mouse and patients serum(n = 50) by Western blot, as initial screening. Subsequently, the positive serum were evaluated emploving two-dimensional electrophoresis. The major proteins identified were classified according to proteomic analysis of the OMPs and OMVs A. baumannii performed in previus studies. In the present study was observed that the five strains of A. baumannii have similar profiles of proteins expression. Therefore, the standardstrain ATCC19606 A. baumannii (OD 0,8) was selected for further studies. The mice immune response to infection by A. baumannii was consistent, showing an antibody titer 7x higher than the uninfected serum. In addition, antibodies present in sera of infected mice were able to reduce the bacterial concentration of 66,89% (t =1h), 74,30% (t=2h) e 30,82% (t =3h). The title of antibodies anti-Acinetobacter in sera of infected patients showed a variability in cut off set in this essay. Comparing the results obtained by development with infected mice and patients serum, the major immunogenic proteins located in the outer membrane of A. baumannii were the Omp 38 of the family Omp A, Omp 33 -36 kDa and ferric siderophore receptor (IROMP). These proteins will be best characterized subsequently by MALDITOF/MS method, through analysis of their peptides generated by enzymatic hydrolysis.

The bacterium Streptococcus pneumoniae (pneumococcus), is considered to be a leading cause of morbidity and mortality globally, particularly in infants and the elderly. It is one of the most frequent causes of respiratory tract infections, which sporadically have the potential to develop into serious invasive symptoms including sepsis and meningitis. The development of effective vaccination against this pathogen is essential for reducing the morbidity and mortality it causes since the currently available vaccines can protect against only a limited number of the 100 pneumococci serotypes which target the polysaccharidic capsule of the bacterium. The potential use of conserved protein antigens could provide a defense to a wider range of serotypes and clonal types. The immunogenic properties of the proteins MalX and PrsA as well as their role in vital biological functions of S. pneumoniae have made them stand out as potential targets. MalX is a crucial membrane protein involved in the metabolism of maltose, whereas PrsA is a chaperone-like protein that is connected to the cell envelope. To understand these proteins' potential as vaccine candidates, it is essential to understand their immunogenic characteristics and physiological roles. In this project, we tried to characterize the two antigens to determine the functional significance of different regions and domains in antigen recognition and their expression dynamics in bacterial host. A better understanding of the antigenic properties of the PrsA and MalX proteins will drive the construction of improved versions of antigens for vaccine prototypes. Some approaches were used to clarify the structural characteristics and antigenic determinants associated with these proteins including, protein expression, purification, and structural characterization. Additionally, their expression in E. coli was examined using immunological assays including ELISA and Western blot. The identification of antigenic regions of these proteins also provides insight into how to develop epitope-based vaccinations that specifically target S. pneumoniae. This project discusses the possibility of using membrane vesicles (MVs) as a platform for vaccination. Membrane vesicles made from bacterial cells have innate immunogenic qualities that expose the immune system to a wide variety of antigens. Incorporating MalX and PrsA into such vesicles can improve the vaccine candidate's overall immunogenicity and effectiveness and trigger a stronger immune response against S. pneumoniae.

Made available in DSpace on 2014-07-29T15:26:24Z (GMT). No. of bitstreams: 1 tese sabrina.pdf: 5211450 bytes, checksum: 0bc9c15d9e2f434943a2738627937629 (MD5) Previous issue date: 2009-03-31 / Paracoccidioides brasiliensis causes paracoccidioidomycosis (PCM), a systemic mycosis presenting clinical manifestations ranging from mild to severe forms. A P. brasiliensis cDNA expression library was produced and screened with pooled sera from PCM patients adsorbed against antigens derived from in vitro-grown P. brasiliensis yeast cells. Sequencing DNA inserts from clones reactive with PCM patients sera indicated 35 open reading frames presenting homology to genes involved in metabolic pathways, transport, among other predicted functions. The complete cDNAs encoding aromatic L-amino acid decarboxylase (Pbddc), lumazine synthase (Pbls) and a homologue of the high affinity copper transporter (Pbctr3) were obtained. Recombinant proteins PbDDC and PbLS were obtained; a peptide was synthesized for PbCTR3. The proteins and the synthetic peptide were recognized by sera of patients with confirmed PCM and not by sera of healthy patients. Using the vivo-induced antigen technology (IVIAT) we identified immunogenic proteins expressed at high levels during infection. Quantitative real - time RT-PCR demonstrated high transcript levels of Pbddc, Pbls and Pbctr3 in yeast cells infecting macrophages. Transcripts in yeast cells derived from spleen and liver of infected mice were also measured by qRT-PCR. Our results suggest a putative role for the immunogenic proteins in the infectious process of P. brasiliensis. / Paracoccidioides brasiliensis é o agente etiológico da paracoccidioidomicose (PCM), uma micose sistêmica, prevalente nos países da América Latina. Uma biblioteca de cDNA de expressão de Paracoccidioides brasiliensis foi construída e rastreada com soros de pacientes acometidos por paracoccidioidomicose (PCM). Foram identificados 35 clones de cDNAs que codificam proteínas relacionadas com metabolismo celular, transporte, energia, transcrição, endereçamento de proteínas, transdução de sinal e componentes celulares. Os cDNAs codificantes da L- aminoacido aromatico - descarboxilase (Pbddc), da lumazina sintase (Pbls) e do transportador de cobre de alta afinidade foram obtidos. As proteínas recombinantes PbDDC e PbLS e o peptídio sintético PbCTR3 foram reconhecidos por soros de pacientes com PCM e não reagiram com soros controle. A técnica de IVIAT (tecnologia de antígenos induzidos in vivo) propiciou a identificação de proteínas imunogênicas mais expressas durante o processo infecçioso. RT-PCR em tempo real quantitativa (qRT-PCR) demostrou altos níveis de transcritos de Pbddc, Pbls e Pbctr3 em células leveduriformes infectando macrófagos. O transcritos de células leveduriformes de P. brasiliensis recuperadas de fígado e baço de camundongos foram medidos por qRT-PCR. Estes resultados sugerem o provável papel das proteínas imunogênicas no processo infeccioso de P. brasiliensis.