Aggregatibacter (Actinobacillus) actinomycetemcomitans is a Gram-negative coccobacillus of the Pasteurellaceae family. It is implicated in periodontitis, a common low-grade bacterial infection, but it can also cause non-oral infections. The main aim of this project was to identify and characterize in A. actinomycetemcomitans novel cell surface components bearing virulence potential that could contribute to systemic immunoinflammatory burden. We first established and evaluated a method for preparing homogeneous cell suspensions of autoaggregating clinical isolates of A. actinomycetemcomitans. The chosen method is based on a gradual dispersion of bacterial colonies into solution, which generated homogeneous suspensions without losing cell viability or fimbriation. When sera from two patients with A. actinomycetemcomitans-associated infections were used to probe A. actinomycetemcomitans outer membrane protein (OMP) preparations in western blot, strong reactions were found at 17 kDa. Interestingly, antiserum against CsgA, a major subunit of Eschirichia coli curli, also reacted with A. actinomycetemcomitans OMP preparations at 17 kDa size, that is the size of E. coli CsgA, suggesting antigenic crossreactivity. The 17 kDa A. actinomycetemcomitans OMP was subsequently identified as peptidoglycan-associated lipoprotein (PAL; AaPAL) by using immunoproteomics methods. Studies on the pal gene and its gene product showed that they were conserved among the clinical A. actinomycetemcomitans isolates representing all currently known serotypes. AaPAL expression was shown under different nutritional and atmospheric conditions that resembled those in periodontal pockets. PAL deficiency in turn led to pleiotropic effects on the phenotype of A. actinomycetemcomitans, such as cell elongation and decreased growth rate. To purify AaPAL we employed affinity chromatography using anti-AaPAL peptide antibodies. The extensive characterization of the purified AaPAL by SDS-PAGE gel staining and mass spectrometry demonstrated that the final purification product did not contain other bacterial proteins than AaPAL. The protein had not lost its antigenicity during purification, since it was recognized by sera from patients with A. actinomycetemcomitans-associated oral and nonoral infections. AaPAL also appeared to be a strongly immunoreactive antigen in patients with periodontitis whose serum IgG antibodies recognized in western blot a 17 kDa OMP in the parental strain but not in the pal-deficient mutant. In addition to its immunogenicity, AaPAL also induced proinflammatory cytokine and chemokine response from human whole blood as determined by a cytokine antibody array. A cell culture insert model was designed to study how bacterial components could be introduced to the host in infections. The experiments demonstrated that live bacteria released extracellularly free-soluble AaPAL, but also other components, via an unknown outer membrane vesicle-independent mechanism. The immunogenicity and proinflammatory potential of the previously uncharacterized outer membrane lipoprotein of A. actinomycetemcomitans, AaPAL, suggests that it contributes to the pathogenicity of this bacterium. That live A. actinomycetemcomitans cells released free-soluble cell components may represent a new pathogenic mechanism.

Bordetella pertussis is a gram-negative, human pathogen and etiologic agent of whooping cough (pertussis), a highly contagious, acute respiratory illness. In this study, the analysis of whole immunproteome and subproteomes of this microorganism was performed. The soluble cytoplasmic proteomes of B. pertussis Tohama I strain and a local isolate Saadet were separated by 2DE. By Western blot analysis, we identified 25 immunogenic proteins of three categories. In the first group, there were well-known proteins of the pathogen The second group comprised proteins which were already shown antigenic in certain pathogenic bacteria, but not in B. pertussis before. The third group of proteins were those which have not been shown to be immunogenic in any pathogen till the present study such as putative chromosome partition protein, preprotein translocase SecA subunit, carbamoyl-phosphate synthase large chain, PRP synthase, putative substrate-CoA ligase, lysyl-tRNA synthetase, fumaryl acetoacetase, putative peptidyl-prolyl cis-trans isomerase, aspartate-semialdehyde dehydrogenase, putative DNA-binding protein and a putative outer membrane protein. In our surfaceome study, surface proteins of two strains were identified by 2DE followed by MALDI-TOF-MS/MS analysis and also geLC-MS/MS. With these techniques 45 proteins were identified by 2DE and 226 proteins by geLC-MS/MS. The immunogenicity of surface proteins on 2DE gels were analyzed by Western blotting and among 11 identified immunogenic proteins glutamine-binding periplasmic protein, leu/ile/val-binding protein, one putative exported protein, and iron-superoxide dismutase were found to be immunogenic for the first time in Bordetella. It was also found that 16 proteins were differentially expressed in B. pertussis Saadet and Tohama I. Five proteins were expressed only in Saadet (adhesin, chaperone protein DnaJ, fimbrial protein FimX, putative secreted protein Bsp22 and putative universal stress protein), and two (ABC transporter substrate-binding protein and a putative binding protein-dependent transport periplasmic protein) only in Tohama I. In the secretome study, we identified 40 proteins by 2DE and 357 proteins by geLC-MS/MS. It was found that 12 proteins were immunogenic by Western blot analysis and the immunogenicity of putative secreted protein (BP1047) was shown for the first time in this study. In our study, PT subunit 2 and putative outer protein D (BopD) were more abundant in Saadet while one protein, glutamate synthase subunit beta was expressed at a higher level in Tohama I. Four proteins were expressed only in Saadet (two capsular polysaccharide biosynthesis protein, protein FimX and putative outer membrane permeability protein). The present study comprehensively covered almost the entire proteome of a crucial pathogen, demonstrated many novel antigens and identified hundreds of membrane-bound proteins, cell surface-associated and extracellular proteins. Thus, it is anticipated to greatly aid in a better understanding of pathogen-host relations, rational design of novel drugs and developing new generation vaccines against B. pertussis.

Les pneumopathies d'hypersensibilité sont des maladies respiratoires liées à l'inhalation répétée de substances antigéniques. Le diagnostic nécessite la présence d'un ensemble d'arguments cliniques, radiologiques, fonctionnels et immunologiques car les symptômes sont peu spécifiques. La mise en évidence d'immunoglobulines G (IgG) spécifiques des agents étiologiques est un élément essentiel dans la prise en charge diagnostique et thérapeutique. L'objectif de ce travail de thèse était d'identifier des protéines bactériennes et fongiques reconnues par les IgG des patients atteints de la maladie du poumon de fermier (PDF) et du poumon de mécanicien (FDM), et de produire ces protéines de façon recombinante afin de développer des tests sérologiques standardisés de type ELISA. Deux micro-organismes impliqués dans le PDF (Saccharopolyspora rectivirgula et Eurotium amstelodami, un Aspergillus), et un micro-organisme impliqué dans le PDM (Mycobacterium immunogenum) ont été étudiés. L'approche immunoprotéomique développée a permis de sélectionner les protéines d'intérêt, puis de produire les antigènes recombinants correspondants par génie génétique. Des sérums de patients PDF, PDM et de témoins exposés ont été recueillis dans le cadre de protocoles cliniques multicentriques. Les performances (sensibilité, spécificité, aire sous la courbe) des tests ELISA-IgG utilisant les antigènes recombinants produits ont été évaluées par l'analyse en courbe ROV (Receiver operating characteristics). A partir des trois micro-organismes étudiés, 71 protéines immuno-réactives ont été identifiées et 28 protéines recombinantes ont été produites et testées en ELISA. Pour le diagnostic du PDM, deux antigènes recombinants, la dihydrolipoyl déshydrogénase and Facyl-CoA déshydrogénase, étaient particulièrement performants avec une sensibilité de 90% lorsqu'ils étaient utilisés en combinaison. Pour le diagnostic du PDF, deux antigènes recombinants à'Aspergillus, la Glu/Leu/Phe/Val déshydrogénase et la glucose-6-phosphate isomérase, ont permis d'obtenir une sensibilité de 89% et une spécificité de 84%. L'utilisation de trois antigènes recombinants de S. rectivirgula,SRl¥A (protéine de fonction inconnue), SRI? (catalase) et SR22 (kétol acide réducto-isomérase), ont permis d'obtenir une sensibilité et spécificité optimales de 83% et 77%. Les protéines identifiées étaient majoritairement des protéines enzymatiques, dont certaines ont été mis en évidence comme facteurs de virulence dans d'autres pathologies. Des études complémentaires, sur des modèles animaux et sur des modèles cellulaires, devront être mennées pour explorer l'implication de ces protéines dans l'induction de la maladie. Ce travail a permis d'améliorer les connaissances sur les protéines bactériennes et fongiques reconnues par les anticorps des patients atteints de PDF et de PDM, de développer des antigènes recombinants, et de mettre au point des tests sérologiques standardisés et performants. Ces tests pourront faire l'objet d'une valorisation vers l'industrie. En effet, les sérologies pour le diagnostic des PHS sont des demandes courantes dans les laboratoires d'analyse médicale. / The hypersensitivity pneumonitis is a pulmonary disease caused by repetitive inhalation of antigens. The diagnosis requires clinical, radiological, functional and immunological features because of unspecific symptoms. The identification of specifie antibodies to causative antigens is an essential way for the diagnosis and therapeutic management.The aims of this thesis work were to identify bacterial and fungal immunogenic proteins specifie to patient with farmer's lung (FL) and machine operator's lung (MOL) diseases, to synthesize specifie recombinant antigens for the development of a standardized ELISA. Two microorganisms involved in FL (Saccharopolyspora rectivirgula and Eurotium amstelodami (Aspergillus sp)), and one involved in MOL had been srudied. The immunoproteomics approach has permit to select interesting proteins and to synthesize recombinant antigens by genomics techniques. The sera from FL patients (n=52), MOL patients (n=16) and sera from exposed control subjects were recruited. ELISA-IgG using recombinant antigens efficacies were evaluated by Receiver operating characteristics analysis (sensitivity, specifïcity, area under the curve).From the three srudied microorganisms, 71 immunogenic proteins were identified and 28 recombinant antigens were produced and tested by ELISA. For the MOL diagnosis, the recombinants dihydrolipoyl dehydrogenase and acyl-CoA dehydrogenase were useful with a sensitivity of 90% when used in combination. For the FL diagnosis, two recombinant proteins from Aspergillus, Glu/Leu/Phe/Val dehydrogenase and glucose-6-phosphate isomerase had a sensitivity of 89% and a specifïcity of 84%. A combination of the three recombinant antigens from S. rectivirgula, SRI FA (unknown function), SRI 7 (catalase) and SR22 (ketol acid reductoisomerase) allowed to obtain a sensitivity of 83% and a specificity of 77%. The immunogenic proteins were mainly enzymes, and some of these have been implicated in important functions for survival or the virulence of others pathogens. Further studies are required to determine the role of these proteins in immunological or virulence processes by animal and cellular model application. This present work has improved our knowledge about bacterial and fungal proteins recognized by FL and MOL patient's antibodies, and to develop useful standardized serological tests with new recombinant antigens. These tests could be exported to the health management in industry. Indeed, hypersensitivity pneumonitis serodiagnosis tests are common requests in medical analysis laboratories

Orientador: Sandra de Moraes Gimenes Bosco / Resumo: A pitiose, cujo agente etiológico é o oomiceto Pythium insidiosum, é uma doença emergente que ocorre principalmente em países tropicais e subtropicais, afetando diversas espécies animais, sendo mais frequente em cavalos no Brasil e humanos na Tailândia. A doença é difícil de diagnosticar, porque as hifas do patógeno são frequentemente confundidas com mucormicoses ou entomoftoromicoses em cortes histológicos. Além disso, não há antígeno eficiente e específico que possa ser utilizado para o diagnóstico rápido e o tratamento eficiente da pitiose em diferentes espécies. Os antígenos são moléculas que o sistema imunológico reconhece, tornando essas moléculas importantes alvos para o diagnóstico de micro-organismos. Para a identificação de antígenos, a imunoproteômica é considerada uma ferramenta poderosa. Nesse sentido, investigamos quais antígenos de P. insidiosum são reconhecidos pelo soro de equinos no Brasil e por soro de pacientes tailandeses com pitiose, a fim de encontrar proteínas semelhantes que possam ser utilizadas para futuros testes de diagnóstico ou terapia para pitiose. Para identificar as proteínas imunorreativas de P. insidiosum, foram utilizadas abordagens imunoproteômicas, técnica de Western blot, seguida pela identificação de antígenos por espectrometria de massas. Consequentemente, foi possível identificar 23 antígenos reconhecidos entre os soros de equinos e humanos. Sete antígenos foram correspondentes aos soros em ambas espécies: heat shock cognate 70 KDa p... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Pythiosis, whose etiological agent is the oomycete Pythium insidiosum, is an lifethreatening disease that occurs mainly in tropical and subtropical countries, affecting several animal species, being more frequent in horses in Brazil and humans in Thailand. The disease is difficult to diagnose, because the pathogen's hyphae are often confused with Mucorales or Entomophthorales fungi in histological sections. In addition, there is no efficient and specific antigen that can be used for the rapid diagnosis and efficient treatment of pythiosis in different species. Antigens are molecules that the immune system recognizes, making these molecules important targets for the diagnosis of microorganisms. For the identification of antigens, immunoproteomics is considered a powerful tool. In this sense, we investigated which P. insidiosum antigens are recognized by horses in Brazil and Thai humans’ serum with pythiosis, in order to find similar proteins that could be used for future diagnostic tests or therapy approaches for pythiosis. To identify the immunoreactive proteins of P. insidiosum, immunoproteomic approaches were used, using the Western blot technique followed by the identification of antigens by mass spectrometry. Consequently, it was possible to identify 23 antigens recognized between the serum of horses and humans. Seven antigens were similar to the two types of serum, being heat shock cognate 70 KDa protein, heat shock 70 KDa protein, glucan 1,3-beta-glucosidase, fructosebi... (Complete abstract click electronic access below) / Doutor

The genus Bordetella contains several pathogenic species generally associated with upper respiratory tract infections in warm-blooded animals. Bordetella pertussis is the etiologic agent of whooping cough. Whooping cough is presently one of the ten most common causes of death from infectious diseases and reported by the World Health Organisation (WHO) to cause 50 million cases and 350000 deaths worldwide per year, mainly among unvaccinated individuals in poor countries. The term proteome, in analogy to the term genome, was coined to describe the complete set of proteins that an organism has produced under a defined set of conditions. Proteomics has been used to identify novel bacterial vaccine candidates against several human pathogens. Fueled by growing DNA sequence information, the analysis of the proteome becomes a valuable and useful tool for antigen discovery. Much of information about immunogenic component can be derived from proteomics coupled to Western blotting, namely immunoproteomics. v In the present study, we report first immunoproteomics analysis to identify candidate antigens of B. pertussis for vaccine development. Different sera from mice, which were immunized or challenged with B. pertussis, were analyzed for reactivity by Western blot against whole cell extracts of B. pertussis Tohama and Saadet strains separated by 2-DE. We identified 15 immunogenic proteins of Bordetella pertussis as a total (60 kDa chaperonin, heat shock protein, serum resistance protein, putative substrate-CoA ligase, ATP-dependent protease, preprotein translocase secA subunit, S-adenosylmethionine synthetase, elongation factor Tu, RNA polymerase alpha subunit, ketol-acid reductoisomerase, pertactin, lysyl-tRNA synthetase, serum resistance protein, carbamoyl-phosphate synthase large chain, 30S ribosomal protein S1 subunit), 6 of which being identified as immunogenic in a pathogenic microbe (ATP-dependent protease, carbamoylphosphate synthase large chain, lysyl-tRNA synthetase, putative chromosome partition protein, preprotein translocase secA subunit, 30S ribosomal protein S1 subunit) and 5 identified as immunogenic for Bordetella pertussis (RNA polymerase alpha subunit, S-adenosylmethionine synthatase, putative substrate-CoA ligase, elongation factor Tu, ketol-acid reductoisomerase) for the first time.

Aeromonas hydrophila is an opportunistic pathogen that causes a wide range of symptoms and diseases in fish. Development of a commercial vaccine has been problematic due to the heterogenicity between isolates of A. hydrophila. A new approach using immunoproteomics was used in this study to try to develop a vaccine that would protect against a wide range of A. hydrophila strains. The virulence of 14 isolates of A. hydrophila from different geographical regions was determined in common carp (Cyprinus carpio) indicating that 6 isolates were virulent, while 8 isolates were avirulent. Expression of cellular and extracellular products (ECP) of six of these isolates (4 virulent and 2 avirulent isolates) were examined following culture of the bacterium in vitro, in tryptic soy broth, and in vivo, in dialysis tubing placed within the peritoneal cavity of carp. Two types of molecular weight cut off tubes (25 and 100 kDa) were used for the implants. Whole cell (WC), outer membrane protein (OMP) and ECPs of the bacteria grown in vitro and in vivo were analysed by 1 dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (1D SDS-PAGE). Additionally, 2D SDS-PAGE was used to analyse WC preparations of A. hydrophila grown in vitro and in vivo. The production of unique proteins and up and down-regulation of protein expression were observed in all the preparations of bacteria grown in vitro and in vivo. Unique bands were seen in the 1D SDS-PAGE at 58 and 55 kDa for WC and OMP preparations, respectively, for all the isolates cultured in vivo. Bands of increased intensity were observed at 70, 55, 50 and 25 kDa with WC preparations for the virulent isolates cultured in vivo. Analysis of WC preparations by 2D SDS-PAGE indicated differences in the expression of spots between bacteria cultured in vitro and in vivo. A number of unique spots, mostly between 30 and 80 kDa with pI values ranging from 5.0-6.0 were observed in the bacteria grown in vivo. The protein profiles of different preparations (WC, OMP, ECP) of bacteria cultured in vitro and in vivo were screened by 1D Western blot using antibodies from carp artificially infected with different isolates of A. hydrophila to identify potential vaccine candidates. The WC preparations of A. hydrophila (T4 isolate) grown in vitro were also analysed by 2D Western blot. A 50 kDa protein of A. hydrophila was found to be the most immunogenic molecule in both WC and OMP of bacteria grown both in vitro and in vivo. The protection efficacy of this protein was determined in goldfish by vaccinating fish with electro-eluted 50 kDa protein then challenging the fish with A. hydrophila. Fish were also passively immunised with fish sera raised to the 50 kDa protein and then challenged. The relative percentage survival (RPS) was 67 % in the vaccination trial, while the results were inconclusive for the passive immunisation trial. The 50 kDa protein was confirmed to be the S-layer protein of A. hydrophila following identification using matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS). Recombinant S-layer protein was then produced and the cross-protection efficacy of this protein against six virulent isolates of A. hydrophila was confirmed in a large scale vaccination trial using carp. The RPS value for the 6 isolates of A. hydrophila ranged from between 56 and 87 %. The results of this project suggest that the immunogenic S-layer protein of A. hydrophila could be used as a common antigen to protect fish against infection by different isolates of this pathogenic bacterium.

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No. of bitstreams: 2 Dissertação - Lucas Silva de Oliveira - 2014.pdf: 3199133 bytes, checksum: cb42a7815c2701477249ff4d0cb2a05b (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2014-04-23 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Staphylococcus saprophyticus is characterized as uropathogenic bacteria that causes urinary tract infections especially in young women. Some virulence factors were elucidated, however, little is known about how this bacteria install itself at host human. The urease was the first virulence factor described in S. saprophyticus, being responsible by increasing of the bladder pH in infected patients. This is the first study about S. saprophyticus in proteomics and immunoproteomics perspective of the secreted proteins (exoproteome) of this bacterium. A total of 44 new secreted proteins were detected by mass spectrometry. Among the proteins found, five of them have a crucial role in the glycolytic pathway: Triosephosphate isomerase (TPI), enolase (ENO), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Glucose-6-phosphate isomerase (GPI), however, its role as moonlighting proteins have been observed in various microorganisms. Through of the immunoproteomics, it was possible to detect 18 protein species that reacted onto Western-blotting, and 5 of them could be identified by mass spectrometry. The most abundant protein specie identified as immunogenic in S. saprophyticus, it was transglycosilase IsaA (Immunodominant staphylococcal antigen A), being it well described in Staphylococcus aureus as virulence factor. Another abundant protein found as immunogenic was Ssa (Staphylococcal secretory antigen), which, it was identified under 3 isoforms. The enolase was the last protein identified at immunoproteome of S. saprophyticus. It was possible to conclude from these results that S. saprophyticus has a wide of extracellular proteins capable to promote bacteria adaption, and some of them are involved in oxidative/nitrosative stress (SOD and AhpC), besides possess proteins that have the capacity to incite the humoral immune response in BalbC mice. All this lead us to hypothesize that S. saprophyticus have defense and virulence mechanisms in order to protect itself against extracellular stresses and appropriate mechanisms to promote urinary tract infections. / Staphylococcus saprophyticus caracteriza-se por ser uma bactéria uropatogênica que causa infecção no trato genito-urinário especialmente de mulheres jovens. Alguns fatores de virulência já foram elucidados, no entanto, pouco se conhece do modo pelo qual esta bactéria acomete o hospedeiro humano. A uréase foi o primeiro fator de virulência descrito em S. saprophyticus, sendo responsável pela alcalinização do pH da bexiga de pacientes contaminados. Este estudo é o pioneiro tratando-se de S. saprophyticus na abordagem da proteômica e imunoproteômica do perfil de proteínas secretadas (exoproteoma) por esta bactéria. Um total de 44 proteínas secretadas inéditas foram detectadas por espectrometria de massas. Dentre as proteínas encontradas, cinco delas possuem papel fundamental na via glicolítica: triose-fosfato isomerase (TPI), enolase (ENO), gliceraldeído-3-fosfato-desidrogenase (GAPDH) e glicose-6-fosfato isomerase (GPI), no entanto, seu papel como proteína “moonlighting” já foi observado em diversos microrganismos. Através da imunoproteômica, foi possível detectar 18 espécies proteicas que reagiram no Wester-blotting, e 5 delas puderam ser identificadas por espectrometria de massas. A espécie proteica mais abundante identificada como imunogênica em S. saprophyticus, foi a transglicosilase IsaA (Immunodominant staphylococcal antigen A), sendo bem descrita em S. aureus como um fator de virulência. Outra proteína abundante identificada como imunogênica foi a Ssa (Staphylococcal secretory antigen), no qual, foi identificada em 3 isoformas. A enolase também foi identificada no imunoproteoma de S. saprophyticus. Foi possível concluir através destes resultados que S. saprophyticus possui uma gama de proteínas extracelulares capazes de promover a adaptação desta bactéria, algumas delas envolvidas na proteção contra o estresse oxidativo/nitrosativo (SOD e AhpC), além de possuir proteínas que tem a característica de incitar a resposta imune humoral de camundongos BalbC. Isso tudo nos leva a hipotetizar que S. saprophyticus possui mecanismos de defesa e virulência a fim de se proteger contra estresses exteriores e mecanismos para promover a patogênese no trato genito-urinário.

Cryptococcus neoformans, an opportunistic fungal pathogen ubiquitously present in the environment, causes cryptococcal meningitis (CM) mainly in immunocompromised patients, such as AIDS patients. We aimed to identify disease-associated cryptococcal protein antigens targeted by the human humoral immune response. Therefore, we used sera from Colombian CM patients, with or without HIV infection, and from healthy individuals living in the same region. Serological analysis revealed increased titers of anti-cryptococcal IgG in HIV-negative CM patients, but not HIV-positive CM patients, compared to healthy controls. In contrast, titers of anti-cryptococcal IgM were not affected by CM. Furthermore, we detected pre-existing IgG and IgM antibodies even in sera from healthy individuals. The observed induction of anti-cryptococcal IgG but not IgM during CM was supported by analysis of sera from C. neoformans-infected mice. Stronger increase in IgG was found in wild type mice with high lung fungal burden compared to IL-4Ra-deficient mice showing low lung fungal burden. To identify the proteins targeted by human anti-cryptococcal IgG antibodies, we applied a quantitative 2D immunoproteome approach identifying cryptococcal protein spots preferentially recognized by sera from CM patients or healthy individuals followed by mass spectrometry analysis. Twenty-three cryptococcal proteins were recombinantly expressed and confirmed to be immunoreactive with human sera. Fourteen of them were newly described as immunoreactive proteins. Twelve proteins were classified as disease-associated antigens, based on significantly stronger immunoreactivity with sera from CM patients compared to healthy individuals. The proteins identified in our screen significantly expand the pool of cryptococcal proteins with potential for (i) development of novel anticryptococcal agents based on implications in cryptococcal virulence or survival, or (ii) development of an anti-cryptococcal vaccine, as several candidates lack homology to human proteins and are localized extracellularly. Furthermore, this study defines preexisting anti-cryptococcal immunoreactivity in healthy individuals at a molecular level, identifying target antigens recognized by sera from healthy control persons.

Clostridium chauvoei is a gram-positive, spore-forming anaerobic bacterium. It is the pathogenic agent of blackleg, a disease causing serious toxemia and high mortality in cattle, sheep and many other domestic and wild animals. It is considered the most important Clostridium producing economic losses in livestock. Typically, animals infected with blackleg die rapidly without any signs of illness. Animals quickly die within 12 to 48 hours after contracting the disease. Therefore, the control of this disease is done by commercial vaccines consisting of whole formolized cultures. Immunity against C. chauvoei is associated with whole cell, including its somatic and flagellar antigens while in other clostridial diseases, protective immunity is obtained by the use of vaccines containing toxoids. Moreover, it is essential to obtain new information about the somatic antigens of C. chauvoei. Proteomics is the study of the proteome, the protein complement of the genome. The proteome has been defined as the entire complement of proteins expressed by a cell, organism, or tissue type, and accordingly, proteomics is the study of this complement expressed at a given time or under certain environmental conditions. 2-DE with Immobilized pH Gradients (IPGs) combined with protein identification by Mass Spectrometry (MS) is currently the workhorse for proteomics. Much of information about immunogenic component can be derived from proteomics coupled to Western blotting, namely immunoproteomics. Our study constitutes the first immunoproteomic analysis of C. chauvoei to identify candidate immunogenic antigens for development of new vaccines. Analyses were performed by Western blot and dot blot techniques against the whole cell extract proteins of C. chauvoei separated by 2-DE. Firstly, the growth conditions of two different strains, C. chauvoei ATCC 11957 and C. chauvoei 20 were optimized. After mice immunization studies with experimental vaccines prepared, sera were obtained for evaluation of the immunoglobulin G antibody level by ELISA. After high level of antibody response determination, 1-DE, 2-DE and immunoblot studies were performed for the characterization of immunogenic proteins. In the study, a total of 460 protein spots could be detected on the 2-DE gels by the help of Delta2D image analysis software and 30 of them were reacted with polyclonal antibodies against inactivated whole cells of C. chauvoei. Among these 30 spots, and 8 of them could be characterized by MALDI-TOF MS analyses. Of these 8 spots revealed four different gene products (distinct ORFs). Ornithine decarboxylase, methionine adenosyltransferase, glucose-6-phosphate isomerase, and flagellin protein FliB (C) are the characterized proteins. Glucose-6-phosphate isomerase has been identified as an immunogenic protein for a pathogenic microbe and in C. chauvoei for the first time. Methionine adenosyltransferase and ornithine decarboxylation were identified as immunogenic for C. chauvoei for the first time. The last defined protein is the flagellin protein FliB(C) which is known to be major immunogenic protein of C. chauvoei.

Das magenbesiedelnde Bakterium Helicobacter pylori gehört zu den am weitesten verbreiteten Infektionserregern. Obwohl die Infektion meist lebenslang symptomlos verläuft, kann H. pylori bei einigen Menschen schwere Erkrankungen bis hin zum Magenkarzinom verursachen. Ziele dieser Arbeit waren Magenkarzinom-assoziierte Antigene für einen diagnostischen Test zu finden und Methoden zur Untersuchung von Spotkompositionen mittels MALDI-TOF/TOF Massenspektrometrie zu entwickeln. Im ersten Teil der Promotion wurden die Antigenerkennungsmuster von 30 Magenadenokarzinom- mit 30 Ulkus duodeni-Patienten mithilfe hochauflösender zweidimensionaler Immunoblots von H. pylori Lysat verglichen. Diese fokussierte Gegenüberstellung eignet sich gut für diese Fragestellung, da beide Erkrankungen von diesem Bakterium verursacht werden, aber nur sehr selten gemeinsam auftreten. Durch univariate statistische Analysen wurden 14 Magenkarzinom korrelierte Spots gefunden (p / The stomach-colonizing bacterium Helicobacter pylori is one of the most widespread infectious agents. Although infection mostly persists unnoticed, it may cause serious diseases like gastric carcinoma. Aims of this project were to find gastric carcinoma-associated antigens for a diagnostic test and to develop methods to analyze spot compositions using MALDI-TOF/TOF mass spectrometry. In the first part of this project antigen recognition patterns of 30 gastric carcinoma- and 30 duodenal ulcer- patients were compared using high-resolution two-dimensional immunoblots of H. pylori lysate. This focused comparison lent itself to this question because both diseases are caused by the bacterium but rarely occur conjointly. Utilizing univariate statistical tests 14 gastric carcinoma-associated spots were found (p