RTX (Repeat in ToXin) superfamily consists of many proteins divided into several groups according to their different functions and characteristics: toxins, metalloproteases, lipases, proteins of the S-layer, bacteriocins and proteins with unknown function. However, all of them can be characterized by the following features: i) they contain tandemly repeated (6-50) nonapeptide glycine-rich calcium-binding consensus sequences GGXGXDX[L/I/V/W/Y/F]X (where X is any amino acid residue) in the C-terminal part of the protein. The presence of these repeats is a sine qua non condition for RTX protein family membership; ii) secretion from the cell occurs without a periplasmic intermediate by a mechanism which involves recognition of a signal sequence at the C-terminus of the protein by membrane-associated proteins that export the toxin across a channel spanning the entire bacterial envelope directly to the outside of the cell (Type I Secretion System); iii) the genes for protein synthesis, activation and secretion are mostly grouped together on the chromosome and form rtx operons. RTX toxins are the largest protein group of the RTX family. To this group belong mostly the proteins with molecular weight ranging from 100 to 200 kDa, with posttranslational fatty acid acylation mediated by a specific activating...

Zugl.: Hannover, Tierärztliche Hochsch., Diss., 2009

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2016-12-23T12:26:14Z No. of bitstreams: 1 texto completo.pdf: 716349 bytes, checksum: 491af184849bdcb2477fa46e3081a75f (MD5) / Made available in DSpace on 2016-12-23T12:26:14Z (GMT). No. of bitstreams: 1 texto completo.pdf: 716349 bytes, checksum: 491af184849bdcb2477fa46e3081a75f (MD5) Previous issue date: 2016-07-15 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Os insetos respondem à infecção através da montagem de reações imunes do tipo celular e humoral. Os reguladores primários dessas respostas são células chamadas hemócitos, os quais medeiam importantes respostas celulares, incluindo a fagocitose, encapsulamento, nodulação, e também segregam fatores humorais, tais como opsoninas, fatores de melanização e peptídeos antimicrobianos. Os hemócitos circulam ao longo da hemocele (cavidade corporal do inseto) pelo fluxo rápido da hemolinfa (sangue), além disso, partes desses hemócitos também existem como células sésseis que estão associados aos tecidos. As larvas de Galleria mellonella são uma alternativa viável para os modelos tradicionais dos mamíferos para estudar a eficácia de drogas antimicrobianas e a patogênese de microrganismos in vivo. No entanto, apesar da sua importância como um modelo de infecção, aspectos biológicos sobre as células do sistema imunológico, tais como a densidade e dinâmica dos hemócitos das larvas são mal compreendidos. No presente trabalho, investigamos a resposta imune celular dos hemócitos circulantes das larvas de G. mellonella contra diferentes cepas da bactéria Gram-negativa Actinobacillus pleuropneumoniae: baixa virulência (780), alta virulência (1022), e cepas de referência do sorotipo 8 (R8). Os hemócitos foram classificados com base no seu tamanho, morfologia, coloração e seus papeis na resposta imune, incluindo cinco tipos: prohemócitos, plasmatócitos, granulócitos, esferulócitos, e oenocitóides. Contagem total de hemócitos, contagem diferencial de hemócitos, atividade dos fagolisossomos, resposta autofágica, viabilidade celular, e a ativação da caspase-3 (como indicador de apoptose) foram determinados em hemócitos circulantes provenientes de larvas desafiadas e controle. Demostramos pela primeira vez no modelo de G. mellonella que os plasmatócitos e granulócitos ativam suas respostas autofágicas através da formação dos autofagossomos após o contato com A. pleuropneumoniae. Além disso, nossos dados demonstram que a imunidade celular do presente modelo de infecção muda dependendo do grau de virulência das cepas bacterianas. / Insects respond to infection by mounting cellular and humoral immune reactions. The primary regulators of these immune responses are cells called hemocytes, which mediate important cellular immune responses including phagocytosis, encapsulation, nodulation and also secrete immune factors such as opsonins, melanization factors and antimicrobial peptides. Hemocytes circulate through the hemocoel (body cavity) by the swift flow of hemolymph (blood), and part of these hemocytes population are sessile and are attached to tissues. Larvae of Galleria mellonella is a widely used factitious host as a viable alternative to traditional mammalian models to study the efficacy of antimicrobial drugs and the microbial pathogenesis in vivo. However, despite their importance as an infection model, biological aspects about the immune cells, such as density and hemocyte dynamic of larvae are poorly understood. In the present study, we investigated the cellular immune response of hemocytes from G. mellonella larvae against three strains of the gram-negative bacterium Actinobacillus pleuropneumoniae: low virulent (780), high virulent (1022), and the serotype 8 reference strain (R8). Five types of larval hemocytes, prohemocytes, plasmatocytes, granulocytes, oenocytoids, and spherulocytes, were distinguished according to size, morphology, detection by molecular probes, dye-staining properties, and their role in the immune response. Total hemocyte count, differential hemocyte count, lysosome activity, autophagic response, cell viability, and caspase-3 activation were determined in circulating hemocytes of naïve and infected larvae. Granulocytes and plasmatocytes were the major hemocyte types involved in the cellular defense against A. pleuropneumoniae; these hemocytes activated phagolysosome activities associated with an autophagic response against the bacteria. Moreover, our results showed that apoptosis in circulating hemocytes after exposure to virulent bacterial strains was related to an excessive autophagic cell death response induced by stress and subsequent caspase-3 activation.

Submitted by Simone Souza (simonecgsouza@hotmail.com) on 2017-10-18T12:24:30Z No. of bitstreams: 1 DISS_2014_Laila Natasha Santos Brandão.pdf: 1739750 bytes, checksum: 16eb165dd6c3a1476424ddf9bd18fe73 (MD5) / Approved for entry into archive by Jordan (jordanbiblio@gmail.com) on 2017-11-07T15:29:45Z (GMT) No. of bitstreams: 1 DISS_2014_Laila Natasha Santos Brandão.pdf: 1739750 bytes, checksum: 16eb165dd6c3a1476424ddf9bd18fe73 (MD5) / Made available in DSpace on 2017-11-07T15:29:45Z (GMT). No. of bitstreams: 1 DISS_2014_Laila Natasha Santos Brandão.pdf: 1739750 bytes, checksum: 16eb165dd6c3a1476424ddf9bd18fe73 (MD5) Previous issue date: 2014-03-05 / CAPES / A possibilidade de detecção de agentes patogênicos representou um grande passo para a ciência. O desenvolvimento e melhorias nas técnicas de diagnóstico como a técnica de Reação em Cadeia da Polimerase (PCR), ao longo dos anos ainda requer infraestrutura laboratorial, apesar de sua sensibilidade e custo decrescente, investimentos em equipamentos e cuidados com fontes de contaminação externa. Técnicas que possam ser executas com facilidade e pouca mão de obra ou exigência de pessoas capacitadas, têm grande possibilidades de aplicabilidade. Neste estudo desenvolveu-se uma técnica de rápida execução e baixo custo, para detecção de um dos principais agentes causadores de pneumonias em granjas de suínos o Actinobaccilus pleuropneumoniae, com sensibilidade 93,8% e especificidade de 84,6% em amostras de pulmões com e sem lesão. O teste Kappa entre a PCR e nanopartícula de ouro foi 0,684 representando boa concordância. A técnica pode ser utilizada como alternativa aos testes convencionais, já que é de fácil e rápida execução e não exige infraestrututra e mão de obra especializada. / The possibility of detection of pathogens was a major step for science as a whole, developing and improving these techniques over the years to increase visibly. The development of the technique of Polymerase Chain Reaction ( PCR ) , although its sensitivity and decreasing cost over the years, it's still a handy little technique that requires laboratory infrastructure, high investments in equipment and care with sources of external contamination. Techniques that can be performed through the ease and little manpower or requirement of skilled people, always have great scope of applicability. This study develops a technique for quick and low- cost detection of a major causative agent of pneumonia in swine herds the Actinobaccilus pleuropneumoniae , with 93.8 % sensitivity and 84.6% specificity in samples of lungs with and without injury, the Kappa test between PCR and gold nanoparticle was 0,684 representing good agreement . The technique can be used as an alternative to conventional tests, since it is easy and quick to implement and does not require infrastructure and skilled labor.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal

Actinobacillus pleuropneumoniae (App) est l’agent étiologique de la pleuropneumonie porcine, une infection pulmonaire contagieuse chez les porcs. Parmi les nombreux mécanismes de virulence retrouvés chez les bactéries, la formation de biofilms joue souvent un rôle important dans la pathogenèse. Il a été récemment démontré qu’App avait la capacité de former des biofilms in vitro. Dans notre laboratoire, la formation de biofilms par App a été évaluée en microplaques dans différents milieux de culture. Nous avons démontré que la souche de référence de sérotype 1 est capable de former des biofilms. Le but de ce travail est d’identifier des gènes impliqués dans la biosynthèse et dans la régulation de l’expression des biofilms chez App. L’objectif de cette étude était de générer une banque de mutants d’App 4074NalR à l’aide du transposon mini-Tn10. Cette banque de 1200 mutants a été criblée à l’aide du modèle in vitro de formation de biofilms en microplaques et en tubes : 24 mutants démontrant une formation de biofilms modifiée par rapport à la souche mère App 4074NalR ont été sélectionnés et identifiés, nous permettant ainsi de localiser le site d’insertion du transposon. Une analyse a permis d’identifier de nouveaux gènes impliqués dans la biosynthèse et dans la régulation de l’expression des biofilms chez App. Notre criblage a permis d’identifier 16 gènes connus impliqués dans la formation de biofilms chez App (hns) ou chez d’autres pathogènes (potD2, ptsI, tig and rpmF) mais également de nouveaux gènes impliqués dans la formation de biofilm (APL_0049, APL_0637 and APL_1572). Une caractérisation plus poussée de ces gènes nous permettra d’améliorer la compréhension des mécanismes impliqués dans la formation de biofilm chez App. / A. pleuropneumoniae (App) is the causative agent of porcine pleuropneumonia, a contagious pulmonary infection in swine. Among the numerous virulence mechanisms found in bacteria, the formation of biofilms often plays an important role in pathogenesis. It has been recently demonstrated that App has the ability to form biofilms in vitro. In our laboratory, the formation of biofilms by App has been evaluated in microplates under different growth conditions. We showed that the reference strain of serotype 1 is capable of forming biofilms when cultured in a specific growth medium. The objective of this work is to identifiy genes implicated in the biosynthesis and regulation of biofilm formation in App. The objective of this study was to generate a mutant library of App using the mini-Tn10 transposon. A total of 1200 mutants has been screened with the help of in vitro models for biofilm formation which use microtiter plates or test tubes; 24 mutants exhibited modified biofilm formation when compared to the parental strain 4074NalR. The selection and identification of these mutants allowed the identification of the insertion site of the transposon. Analysis revealed novel genes implicated in biosynthesis and regulation of the biofilm formation in App. Our screen allowed the identification of genes already associated in biofilm formation of App (hns) or other pathogens (potD2, ptsI, tig and rpmF). Genes (APL_0049, APL_0637 and APL_1573) that have not yet been associated with biofilm formation were also identified. Further characterization of the genes mentioned above would permit a greater understanding of the mechanisms implicated in biofilm formation of App.

Immunological stressors, in the form of clinical and sub-clinical disease are currently controlled using both prophylactic antibiotics in-feed, and therapeutic antibiotic treatment. Respiratory disease, primarily Actinobacillus pleuropneumoniae (App) infection, is recognised as a major factor causing reduced productivity in pigs. This thesis reports investigations into the use of novel immunomodulators in particular Interleukin 4 (IL-4) and Interleukin 1 receptor antagonist (IL-1ra) as alternatives to antibiotics to treat App infection. Immunological and molecular biological assays were used to investigate and accumulate data. An in vitro study undertaken to find potential anti-inflammatory substances, revealed that Interleukin 8 (IL-8) mRNA production stimulated by PMA or LPS in whole pigs' blood was suppressed by IL-4. IL-1ra also suppressed stimulated IL-8 mRNA production by heat killed App bacteria (KB) in vitro. An acute LPS challenge in pigs in vivo however, showed no variation in illness or weight loss between pigs treated prophylactically with anti-inflammatory substance (IL-4 and IL-1ra) and saline treated pigs. The use of plasmids as a delivery system for anti-inflammatory substance did not show promise since it did not enhance growth or prolong the expression of the substances in the pigs. However, in the chronic App challenge model IL-4 and IL-1ra administered prophylactically in vivo showed an ability to improve growth. The therapeutic administration of IL-4 and IL-1ra to App challenged pigs showed no difference in pigs' growth, regardless of the treatment or control administered. To conclude, IL-4 and IL-1ra showed promise when administered prophylactically and improved growth and abrogated disease under conditions of App challenge. However when IL-4 and IL-1ra where administered therapeutically they did not perform as well. Moreover these compounds have potential as a commercial application to reduce the growth reduction caused by disease such as App.

Mannose-binding lectin (MBL) and ficolins are complement-activating proteins, and both play an important role in innate immunity by recognizing specific carbohydrate moieties on the surface of wide range of microorganisms. Previous studies have shown that porcine ficolin-α and MBL-A bind to surface polysaccharides of bacteria cultured in suspension, but their interactions with bacteria in biofilm culture have not been studied. The objectives of this thesis were to determine whether porcine plasma ficolin and MBL bind to Actinobacillus pleuropneumoniae in biofilm cultures. APP serotype 5a (APP5a) was used because it produced pronounced biofilm in plastic culture dishes, in comparison with APP5b that was previously reported to bind ficolin in suspension cultures. N-acetylglucosamine (GlcNAc) in the biofilm produced by APP5a was stained with wheat germ agglutinin conjugated with Alexa Fluor-555 and identified by confocal laser scanning microscopy (CLSM). Dispersin B prevented APP5a biofilm formation indicating the requirement of poly N-acetylglucosamine (PNAG) for bacterial cohesion. Bound purified ficolin or ficolin in plasma both were eluted with GlcNAc from APP5a biofilm cultures. To address preferential binding of ficolin-α to biofilm matrix, ficolin-α was eluted with GlcNAc from extracellular polymeric substances (EPS) in supernatant after pelleting the bacteria. Biotinylated-ficolin that retained GlcNAc-binding activity for APP5b planktonic cultures was shown to bind strongly to APP5a biofilm, as detected by fluorescent NeutrAvidin staining and CLSM, but not in the presence of GlcNAc. Further, MBL-A in ficolin-depleted porcine plasma also bound to APP5a biofilm and was eluted with a sugar solution containing GlcNAc, galactose, mannose and glucose. These studies demonstrate that both porcine ficolin-α and MBL-A bind to biofilm cultures of APP5a in a carbohydrate-dependent manner, and suggest that the production of PNAG in biofilm is a binding target for ficolin. / Natural Sciences and Engineering Research Council of Canada (NSERC)

Actinobacillus pleuropneumoniae (App) est l’agent étiologique de la pleuropneumonie porcine, une infection pulmonaire contagieuse chez les porcs. Parmi les nombreux mécanismes de virulence retrouvés chez les bactéries, la formation de biofilms joue souvent un rôle important dans la pathogenèse. Il a été récemment démontré qu’App avait la capacité de former des biofilms in vitro. Dans notre laboratoire, la formation de biofilms par App a été évaluée en microplaques dans différents milieux de culture. Nous avons démontré que la souche de référence de sérotype 1 est capable de former des biofilms. Le but de ce travail est d’identifier des gènes impliqués dans la biosynthèse et dans la régulation de l’expression des biofilms chez App. L’objectif de cette étude était de générer une banque de mutants d’App 4074NalR à l’aide du transposon mini-Tn10. Cette banque de 1200 mutants a été criblée à l’aide du modèle in vitro de formation de biofilms en microplaques et en tubes : 24 mutants démontrant une formation de biofilms modifiée par rapport à la souche mère App 4074NalR ont été sélectionnés et identifiés, nous permettant ainsi de localiser le site d’insertion du transposon. Une analyse a permis d’identifier de nouveaux gènes impliqués dans la biosynthèse et dans la régulation de l’expression des biofilms chez App. Notre criblage a permis d’identifier 16 gènes connus impliqués dans la formation de biofilms chez App (hns) ou chez d’autres pathogènes (potD2, ptsI, tig and rpmF) mais également de nouveaux gènes impliqués dans la formation de biofilm (APL_0049, APL_0637 and APL_1572). Une caractérisation plus poussée de ces gènes nous permettra d’améliorer la compréhension des mécanismes impliqués dans la formation de biofilm chez App. / A. pleuropneumoniae (App) is the causative agent of porcine pleuropneumonia, a contagious pulmonary infection in swine. Among the numerous virulence mechanisms found in bacteria, the formation of biofilms often plays an important role in pathogenesis. It has been recently demonstrated that App has the ability to form biofilms in vitro. In our laboratory, the formation of biofilms by App has been evaluated in microplates under different growth conditions. We showed that the reference strain of serotype 1 is capable of forming biofilms when cultured in a specific growth medium. The objective of this work is to identifiy genes implicated in the biosynthesis and regulation of biofilm formation in App. The objective of this study was to generate a mutant library of App using the mini-Tn10 transposon. A total of 1200 mutants has been screened with the help of in vitro models for biofilm formation which use microtiter plates or test tubes; 24 mutants exhibited modified biofilm formation when compared to the parental strain 4074NalR. The selection and identification of these mutants allowed the identification of the insertion site of the transposon. Analysis revealed novel genes implicated in biosynthesis and regulation of the biofilm formation in App. Our screen allowed the identification of genes already associated in biofilm formation of App (hns) or other pathogens (potD2, ptsI, tig and rpmF). Genes (APL_0049, APL_0637 and APL_1573) that have not yet been associated with biofilm formation were also identified. Further characterization of the genes mentioned above would permit a greater understanding of the mechanisms implicated in biofilm formation of App.