The sun is the primary source of all chemical energy on the planet. Starch granules have evolved as storage deposits for captured light energy. Many complex biological functions take place at the starch granule surface, including starch granule metabolism and defense. The starch granule-associated protein puroindoline is a known antimicrobial with unique functional and biological properties, attributed to the presence of a unique tryptophan-rich domain. To test puroindoline's tight association, puroindoline removed from the starch granule surface during water-washing was assessed. Washing more than eight times failed to further reduce puroindoline content of starch granules, suggesting a strong association of puroindoline with the starch granule surface. To identify the tryptophan-rich domain tightly associated with the starch granule surface, we used a combination of in situ tryptic digestion and mass spectrometry. We identified the tryptophan-rich domain of puroindoline directly bound to the starch granule surface of wheat. This is the first instance of the tryptophan-rich domain directly observed at the starch granule surface. In addition, using mass spectrometry, we determined that during development and maturation, wheat seeds appear to have resisted infection and lysed the pathogens where, upon desiccation, the molecular evidence remained fixed at the starch granule surface. Proteins with known antimicrobial activity were identified, as well as several proteins from the plant pathogens Agrobacterium tumefaciens, Pectobacterium carotovorum, Fusarium graminearum, Magnaporthe grisea, Xanthomonas axonopodis, and X. oryzae. Future characterization may reveal previously unknown host-pathogen interactions. Finally, we have demonstrated that puroindoline, when expressed in the seeds of transgenic corn, will localize and associate with the starch granule surface in a pattern similar to the puroindoline expression pattern observed in wheat. Surprisingly, puroindoline expression in transgenic corn is correlated with an increase in total seed oil content.

The sun is the primary source of all chemical energy on the planet. Starch granules have evolved as storage deposits for captured light energy. Many complex biological functions take place at the starch granule surface, including starch granule metabolism and defense. The starch granule-associated protein puroindoline is a known antimicrobial with unique functional and biological properties, attributed to the presence of a unique tryptophan-rich domain. To test puroindoline's tight association, puroindoline removed from the starch granule surface during water-washing was assessed. Washing more than eight times failed to further reduce puroindoline content of starch granules, suggesting a strong association of puroindoline with the starch granule surface. To identify the tryptophan-rich domain tightly associated with the starch granule surface, we used a combination of in situ tryptic digestion and mass spectrometry. We identified the tryptophan-rich domain of puroindoline directly bound to the starch granule surface of wheat. This is the first instance of the tryptophan-rich domain directly observed at the starch granule surface. In addition, using mass spectrometry, we determined that during development and maturation, wheat seeds appear to have resisted infection and lysed the pathogens where, upon desiccation, the molecular evidence remained fixed at the starch granule surface. Proteins with known antimicrobial activity were identified, as well as several proteins from the plant pathogens Agrobacterium tumefaciens, Pectobacterium carotovorum, Fusarium graminearum, Magnaporthe grisea, Xanthomonas axonopodis, and X. oryzae. Future characterization may reveal previously unknown host-pathogen interactions. Finally, we have demonstrated that puroindoline, when expressed in the seeds of transgenic corn, will localize and associate with the starch granule surface in a pattern similar to the puroindoline expression pattern observed in wheat. Surprisingly, puroindoline expression in transgenic corn is correlated with an increase in total seed oil content.

<p><italic>Cryptococcus neoformans</italic> is an opportunistic fungal pathogen that initiates infection following inhalation. As a result, the pulmonary immune response provides a first line of defense against <italic>C. neoformans</italic>. Surfactant protein D (SP-D) is an important regulator of pulmonary immune responses and is typically host protective against bacterial and viral respiratory infections. However, SP-D is not protective against <italic>C. neoformans</italic>. This is evidenced by previous work from our laboratory demonstrating that SP-D-deficient mice infected with a highly virulent <italic>C. neoformans</italic> strain (H99 Stud) have a lower fungal burden and live longer compared to wild-type (WT) control animals. We hypothesized that SP-D alters susceptibility to <italic>C. neoformans</italic> by dysregulating the innate pulmonary immune response following infection. For this reason, inflammatory cells and cytokines were compared in the bronchoalveolar lavage fluid from WT and SP-D<super>-/-</super> mice after <italic>C. neoformans</italic> infection. Post-infection, mice lacking SP-D had reduced eosinophil infiltration and IL-5 in lung lavage fluid. To further explore the interplay of SP-D, eosinophils, and IL-5, mice expressing altered levels of eosinophils and/or IL-5 were used to assess the role these innate immune mediators play during the host response to <italic>C. neoformans</italic>. IL-5 overexpressing mice had increased pulmonary eosinophilia and were more susceptible to <italic>C. neoformans</italic> infection as compared to WT mice. Furthermore, the response to <italic>C. neoformans</italic> infection in SP-D<super>-/-</super> mice could be restored to that of WT mice by increasing IL-5 and eosinophils, via crossing the IL-5 transgene onto the SP-D<super>-/-</super> background. Together, these studies support the conclusion that SP-D increases susceptibility to <italic>C. neoformans</italic> infection by promoting <italic>C. neoformans</italic>-driven pulmonary IL-5 and eosinophil infiltration.</p> / Dissertation

The sun is the primary source of all chemical energy on the planet. Starch granules have evolved as storage deposits for captured light energy. Many complex biological functions take place at the starch granule surface, including starch granule metabolism and defense. The starch granule-associated protein puroindoline is a known antimicrobial with unique functional and biological properties, attributed to the presence of a unique tryptophan-rich domain. To test puroindoline's tight association, puroindoline removed from the starch granule surface during water-washing was assessed. Washing more than eight times failed to further reduce puroindoline content of starch granules, suggesting a strong association of puroindoline with the starch granule surface. To identify the tryptophan-rich domain tightly associated with the starch granule surface, we used a combination of in situ tryptic digestion and mass spectrometry. We identified the tryptophan-rich domain of puroindoline directly bound to the starch granule surface of wheat. This is the first instance of the tryptophan-rich domain directly observed at the starch granule surface. In addition, using mass spectrometry, we determined that during development and maturation, wheat seeds appear to have resisted infection and lysed the pathogens where, upon desiccation, the molecular evidence remained fixed at the starch granule surface. Proteins with known antimicrobial activity were identified, as well as several proteins from the plant pathogens Agrobacterium tumefaciens, Pectobacterium carotovorum, Fusarium graminearum, Magnaporthe grisea, Xanthomonas axonopodis, and X. oryzae. Future characterization may reveal previously unknown host-pathogen interactions. Finally, we have demonstrated that puroindoline, when expressed in the seeds of transgenic corn, will localize and associate with the starch granule surface in a pattern similar to the puroindoline expression pattern observed in wheat. Surprisingly, puroindoline expression in transgenic corn is correlated with an increase in total seed oil content.

Salmonella enterica serovar Typhimurium is a facultative intracellular pathogen that has evolved to take advantage of the eukaryotic host cells it inhabits during infection. It uses bacterial effectors translocated into the host cell cytosol to manipulate host cell machinery and establish a replicative niche. In this thesis I study the function of two of these effectors, SopD and SopB, which have been shown to act cooperatively to induce phenotypes associated with gastroenteritis (fluid secretion and neutrophil influx into the intestinal lumen). In addition to promoting gastroenteritis, SopD has also been implicated in systemic and persistent infection of mice. Although recently implicated in invasion, the precise function of SopD has remained elusive. Here I show that SopD affects membrane dynamics during S. Typhimurium invasion of epithelial cells. SopD promotes membrane sealing and macropinosome formation, events that may have important consequences for efficiency of bacterial cell entry in vivo. Furthermore, we demonstrate that SopD is recruited to the invasion site membranes through the phosphatase activity of SopB, suggesting a mechanism for their cooperative action during induction of gastroenteritis. Unlike SopD, SopB has been a focus of intense research efforts and its role in invasion as a phosphoinositide phosphatase is well documented. However, we have observed that SopB also inhibits fusion of lysosomes with Salmonella-containing vacuoles (SCVs) following invasion. This ability depends on SopB-mediated reduction of negative membrane charge of the SCV during invasion by hydrolysis of the phosphoinositide PI(4,5)P2. Membrane charge alterations driven by SopB result in removal of Rab GTPases from the SCV that depend on electrostatic interactions for their targeting. Two of these Rabs, Rab23 and Rab35 were previously shown to promote phagosome-lysosome fusion. Therefore their removal from the SCV may promote SCV trafficking away from the degradative endocytic pathway of host cells. This represents a new mechanism by which an invasion associated effector controls SCV maturation. Together, this work advances our knowledge of the interaction between S. Typhimurium and its host. This research also suggests a new mechanism by which pathogens other than S. Typhimurium could promote their intracellular survival.

Thesis (Ph.D. in Microbiology & Immunology) -- University of Colorado Denver, 2007. / Typescript. Includes bibliographical references (leaves 200-212).

Submitted by Rodnei Dennis Rossoni (rodnei.rossoni@fosjc.unesp.br) on 2017-12-11T11:19:52Z No. of bitstreams: 1 Tese_Pronta.pdf: 4361864 bytes, checksum: 4e8e22ae0f0744e73ce578cec2fbcce4 (MD5) / Submitted by Rodnei Dennis Rossoni (rodnei.rossoni@fosjc.unesp.br) on 2017-12-11T18:47:11Z No. of bitstreams: 1 Tese_Pronta.pdf: 4361864 bytes, checksum: 4e8e22ae0f0744e73ce578cec2fbcce4 (MD5) / Submitted by Rodnei Dennis Rossoni (rodnei.rossoni@fosjc.unesp.br) on 2017-12-14T11:25:03Z No. of bitstreams: 1 Tese_Pronta.pdf: 4361864 bytes, checksum: 4e8e22ae0f0744e73ce578cec2fbcce4 (MD5) / Submitted by Rodnei Dennis Rossoni (rodnei.rossoni@fosjc.unesp.br) on 2017-12-14T13:50:32Z No. of bitstreams: 1 Tese_Pronta.pdf: 4361864 bytes, checksum: 4e8e22ae0f0744e73ce578cec2fbcce4 (MD5) / Approved for entry into archive by Silvana Alvarez null (silvana@ict.unesp.br) on 2017-12-18T15:12:51Z (GMT) No. of bitstreams: 1 rossoni_rd_dr_sjc.pdf: 4361864 bytes, checksum: 4e8e22ae0f0744e73ce578cec2fbcce4 (MD5) / Made available in DSpace on 2017-12-18T15:12:51Z (GMT). No. of bitstreams: 1 rossoni_rd_dr_sjc.pdf: 4361864 bytes, checksum: 4e8e22ae0f0744e73ce578cec2fbcce4 (MD5) Previous issue date: 2017-12-04 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O estudo da atividade inibitória de Lactobacillus pode contribuir na descoberta de novas estratégias terapêuticas nas infecções por Candida. Nesse contexto, o objetivo desse estudo foi isolar e identificar Lactobacillus da cavidade bucal de indivíduos livres de cárie e avaliar seu potencial de inibição de C. albicans por meio de estudos in vitro e in vivo. Primeiramente, foram avaliados os efeitos de 30 isolados clínicos de Lactobacillus sobre o número de células viáveis (UFC) em biofilme de C. albicans e sobre a formação de hifas. Os isolados que obtiveram os maiores efeitos inibitórios sobre C. albicans foram selecionados para os testes de determinação da biomassa total dos biofilmes pela absorbância do cristal violeta, análise da arquitetura dos biofilmes por microscopia eletrônica de varredura (MEV) e quantificação da expressão de genes de C. albicans (ALS3, HWP1, EFG1 e CPH1) por qPCR. Esses isolados também foram submetidos a estudos in vivo usando os modelos de Galleria mellonella e Caenorhabditis elegans. Para o estudo em G. mellonella, a infecção experimental foi avaliada pela curva de sobrevivência, quantificação da carga fúngica na hemolinfa, densidade hemocitária, quantificação da expressão gênica de peptídeos antifúngicos (Gallerymicina e Galiomicina) e monitoramento da infecção de C. albicans por análise de bioluminescência. No modelo de C. elegans, a infecção foi avaliada por meio dos ensaios de curva de sobrevivência e estudo da filamentação de C. albicans. Os resultados dos ensaios in vitro demonstraram que L. paracasei 28.4, L. rhamnosus 5.2 e L. fermentum 20.4 foram as cepas com maior atividade antimicrobiana sobre os biofilmes de C. albicans. Nessas cepas, todos os genes analisados foram regulados negativamente na associação com Lactobacillus quando comparados com o grupo controle. No estudo in vivo, a injeção de L. paracasei 28.4 em G. mellonella infectadas com C. albicans aumentou a sobrevida das larvas, o número de hemócitos e a expressão de peptídeos antifúngicos, reduzindo assim a UFC de C. albicans. Em C. elegans, L. paracasei 28.4 também foi capaz de aumentar a sobrevida dos vermes infectados com C. albicans e reduzir a filamentação. Conclui-se que L. fermentum 20.4, L. paracasei 28.4 e L. rhamnosus 5.2 tem potencial para serem usados como probióticos na cavidade bucal devido sua ação anti-biofilme e sua regulação negativa dos genes de virulência de C. albicans. L. paracasei 28.4 foi capaz de prolongar a sobrevida nos dois modelos experimentais infectados com C. albicans por apresentarem ação antifúngica e imunomodulatória. / The study of the antifungal activity of Lactobacillus may contribute to the discovery of new therapeutic strategies for Candida infections. In this context, the objective of this study was to isolate and identify Lactobacillus from the oral cavity of caries-free subjects and to evaluate its effects through in vitro and in vivo studies. First, the effects of 30 clinical isolates of Lactobacillus on the number of viable cells (CFU) in biofilms of C. albicans and on hyphae formation were evaluated. The isolates that obtained the highest inhibitory effects on C. albicans were selected for biofilm biomass determination by violet crystal absorbance, analysis of biofilm architecture by scanning electron microscopy (SEM) and quantification of the expression of C. albicans (ALS3, HWP1, EFG1 and CPH1) by real time PCR. These isolates were also submitted to in vivo studies using the Galleria mellonella and Caenorhabditis elegans models. For the study in the model of Galleria mellonella, the experimental infection was evaluated by the survival curve, quantification of the fungal load in the hemolymph, hemocitary density, the gene expression of antifungal peptides (Gallerymicin and Galiomicin) and monitoring of C. albicans infection by bioluminescence analysis. In the Caenorhabditis elegans model, the infection was evaluated by the survival curve assays and the study of C. albicans filamentation. The results of in vitro tests demonstrated that L. paracasei 28.4, L. rhamnosus 5.2 and L. fermentum 20.4 were the strains with the highest antimicrobial activity on the biofilms of C. albicans. In these strains, all analyzed genes were negatively regulated in association with Lactobacillus when compared to the control group. In the in vivo study, the injection of L. paracasei 28.4 into the G. mellonella increased survival of the larvae, the number of hemocytes and the expression of antifungal peptides, thus reducing the CFU of C. albicans. In C. elegans, L. paracasei 28.4 was also able to increase the survival of worms infected with C. albicans and reduce the filamentation. We conclude that L. fermentum 20.4, L. paracasei 28.4 and L. rhamnosus 5.2 have potential to be used as probiotics in the oral cavity due to their anti-biofilm action and their negative regulation of virulence genes of C. albicans. L. paracasei 28.4 was able to prolong survival of both experimental models infected with C. albicans for having antifungal and immunomodulatory action. / 13/25181-8 / 14/12458-4

Apicomplexan parasites are leading causes of human and livestock diseases such as toxoplasmosis and malaria caused by Toxoplasma gondii and Plasmodium falciparum respectively. These organisms are varied in their morphologies and astoundingly complex on their life cycles that include infections of more than one host organism, differentiation through several morphologically distinct forms, and both sexual and asexual replication. What we and others have initially proposed was that the control of gene expression and cellular differentiation are particularly interesting in these organisms, as the apparent lack of large families of recognizable transcription factors typically found in other eukaryotic organisms suggests that they may be unusually reliant on epigenetic mechanisms. The initial hypothesis had to be re-assessed in light of the discovery in Apicomplexa of an expanded family of plant-like transcription factors (TFs) harbouring APETALA2 (AP2)-like domains. Yet, a growing body of evidence tends to favor epigenetic as one of the main contributor to parasite developmental programs and adjustments to fluctuant environment. One way to examine dynamic changes in post-translations modifications (PTMs) patterns is to alter the histone code writing. We therefore took advantage of HDAC inhibitors and showed that specific inhibition of TgHDAC3 by the cyclopeptide FR235222 disrupts the genome wide steady-state level of histone H4 acetylation inducing derepression of stage-specific genes. Yet, many questions about TgHDAC3 modus operandi remain unanswered. During my thesis, I uncovered the TgHDAC3-regulated proteome-wide acetylome typified by the presence of non-histone proteins including AP2 TFs and novel PTMs, e.g. the acetylation at Lys31 within the globular domain of histone H4. H4K31ac promotes a relaxed chromatin state at the promoter of active genes through nucleosome disassembly in both parasites. We identified TgGCN5B and TgHDAC3 as two antagonist enzymes regulating H4K31 acetylation in T. gondii. In contrast, H4K31monomethylation is enriched throughout the gene body of T. gondii active genes and contributes to transcription, whereas it is enriched at transcriptionally inactive pericentromeric heterochromatin regions in P. falciparum, a region that is lacking H3K9me3 and heterochromatin protein 1 in this parasite. We also showed that treating T. gondii cystogenic strains with a low dose of FR235222 induces the levels of proteins known to be expressed exclusively in cat (sporozoite and merozoite) or in murine chronic stage (bradyzoite). Lastly, we determined the specific interactome of TgHDAC3 and found as partners a MORC protein (CR230), several AP2 TFs, and ELM2 domain-containing scaffolding proteins. Collectively, these data established TgHDAC3 family as a central regulator of gene expression and stage conversion in T. gondii and, likely, other Apicomplexa. / Apicomplexan genome architecture is typified by a binary chromatin structure, with a major fraction of the bulk genome packaged as transcriptionally permissive euchromatin while few loci are embedded in silenced heterochromatin. There is evidence that histone modifications occurring at the lateral surface of the nucleosome play a substantial role in shaping chromatin structure, yet our understanding of the exact mechanism of action is poor. Here, we address how versatile modifications at Lys31 within the globular domain of histone H4 contribute to genome organization and expression in Apicomplexa. H4K31 acetylation was found at the promoter of active genes. The residue lies where the DNA wraps around the histone and its acetylation may enhance nucleosome disassembly, thereby favoring a more relaxed, open chromatin state. This residue tends also to be monomethylated and depending of the parasite examined different patterns were found. H4K31me1 was enriched in the core body of Toxoplasma active genes, yet its occupancy was inversely correlated with transcripts levels likely because the mark by reducing histone turnover impedes RNA polymerase progression across transcribed units. In contrast to the methylation of H3, it is the first time that a methylated residue of H4 has been clearly associated with transcriptional regulation. In Plasmodium, H4K31me1 was exclusively enriched at transcriptionally inactive genomic regions and peculiarly at pericentromeric heterochromatin, likely to replace the missing H3K9me3 that commonly decorated pericentric nucleosomes in other species.

Submitted by Erika Demachki (erikademachki@gmail.com) on 2017-03-07T13:37:49Z No. of bitstreams: 4 Dissertação - Aparecido Ferreira de Souza - 2017 - Parte 01.pdf: 17508857 bytes, checksum: 1d04cb16c6e33e54fe0304cecb5e491b (MD5) Dissertação - Aparecido Ferreira de Souza - 2017 - Parte 02.pdf: 14162361 bytes, checksum: b19921eb48893ef7a1aa4ddc2a2e1553 (MD5) Dissertação - Aparecido Ferreira de Souza - 2017 - Parte 03.pdf: 17178000 bytes, checksum: aaf5cfd215078190a7900eafd1e47790 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-03-07T15:14:15Z (GMT) No. of bitstreams: 4 Dissertação - Aparecido Ferreira de Souza - 2017 - Parte 01.pdf: 17508857 bytes, checksum: 1d04cb16c6e33e54fe0304cecb5e491b (MD5) Dissertação - Aparecido Ferreira de Souza - 2017 - Parte 02.pdf: 14162361 bytes, checksum: b19921eb48893ef7a1aa4ddc2a2e1553 (MD5) Dissertação - Aparecido Ferreira de Souza - 2017 - Parte 03.pdf: 17178000 bytes, checksum: aaf5cfd215078190a7900eafd1e47790 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-03-07T15:14:15Z (GMT). No. of bitstreams: 4 Dissertação - Aparecido Ferreira de Souza - 2017 - Parte 01.pdf: 17508857 bytes, checksum: 1d04cb16c6e33e54fe0304cecb5e491b (MD5) Dissertação - Aparecido Ferreira de Souza - 2017 - Parte 02.pdf: 14162361 bytes, checksum: b19921eb48893ef7a1aa4ddc2a2e1553 (MD5) Dissertação - Aparecido Ferreira de Souza - 2017 - Parte 03.pdf: 17178000 bytes, checksum: aaf5cfd215078190a7900eafd1e47790 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-02-21 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / Iron (Fe) is an indispensable metal for most biological systems and is a target of competition in host-pathogen interactions. In humans, most Fe is complexed to the cofactor heme, present in hemoglobin, a molecule that is exploited by pathogens as an iron source. Paracoccidioides spp., a complex of thermodymorphic pathogenic fungi, are the etiological agents of paracoccidioidomycosis (PCM), a systemic mycosis endemic in Latin America. Paracoccidioides spp. are able to use hemoglobin in a receptor(PbRbt5)-mediated process. However experimental evidence points to the existence of a complex system with the presence of other proteins. In the present work, we demonstrated the similarity between PAAG_02225 (PbPga7), from Paracoccidioides lutzii, and the sequence of the heme/hemoglobin receptor Pga7 from Candida albicans, and expressed PbPga7 in Escherichia coli. Due to the presence of rare codons in P. lutzii sequence, compared to the codons preferably used by Escherichia coli, chemical synthesis of the gene was employed. The expression of PbPga7 protein opens perspectives for PbPga7 characterization. In addition, nanoUPLC-MSE was employed to analyze P. lutzii cell wall proteome. We observed that the treatment of fungus with hemoglobin promotes induction of potential adhesins and defense-related enzymes against reactive oxygen species. These data indicate that these proteins may be important for the pathogen to access hemoglobin by adhering and lysing erythrocytes, besides counteracting the toxicity generated by heme/hemoglobin released from erythrocytes, allowing the uptake and use of these molecules. The results obtained in the present work reinforce the complexity of the interaction event between pathogen and host and, in addition, contribute to broaden the understanding of the biology of Paracoccidioides spp. / O Ferro (Fe) é um metal indispensável para a maioria dos sistemas biológicos e é alvo de competição em interações patógeno-hospedeiro. Em humanos, a maioria do Fe encontra-se complexada ao cofator heme, presente na hemoglobina, uma molécula que é explorada por patógenos como uma fonte de Fe. Paracoccidioides spp., um complexo de fungos patogênicos termodimórifocos, são agentes etiológicos da paracoccidioidomicose (PCM), uma micose sistêmica endêmica na América Latina. Paracoccidioides spp. é hábil em utilizar hemoglobina por um processo mediado por receptor (PbRbt5). Contudo, evidências experimentais apontam para a existência de um sistema complexo com a presença de outras proteínas. No presente trabalho, foi demonstrada a similaridade de PAAG_02225 (PbPga7), de Paracoccidioides lutzii, com a sequência do receptor de heme/hemoglobina Pga7 de Candida albicans. A expressão heteróloga de PbPga7 foi realizada em Escherichia coli. Devido a presença de códons raros na sequência de P. lutzii, comparados aos códons utilizados preferencialmente por E. coli, a síntese química do gene foi empregada. A expressão da proteína PbPga7 abre perspectivas para estudos de caracterização da mesma. Adicionalmente, nanoUPLC-MSE foi utilizada para analisar o proteoma de parede celular de P. lutzii. Observou-se que o tratamento do fungo com hemoglobina promove a regulação positiva de potenciais adesinas e enzimas relacionadas com a defesa contra espécies reativas de oxigênio. Estes dados indicam que estas proteínas podem ser importantes para que o patógeno possa acessar a hemoglobina, aderindo e lisando eritrócitos, além de contrapor a toxicidade gerada pelo heme/hemoglobina liberados, permitindo a captação e utilização dessas moléculas. Os resultados obtidos no presente trabalho reiteram a complexidade do evento de interação entre patógeno e hospedeiro e, adicionalmente, contribuem para a ampliação do entendimento da biologia de Paracoccidioides spp.

Des souches de Vibrio appartenant au clade Splendidus sont retrouvées de manière récurrente lors des mortalités estivales d’huîtres juvéniles. La souche V. tasmaniensis LGP32 est un pathogène intracellulaire facultatif des hémocytes d’huître, dont elle altère les fonctions de défense. Nous nous intéressons ici aux interactions entre les phagocytes et V. tasmaniensis LGP32, à diverses échelles (moléculaire, cellulaire et environnementale). Dans une première partie, nous avons découvert un mécanisme antimicrobien, encore inconnu chez C. gigas : la formation de pièges d'ADN extracellulaire (ETs). Ces ETs sont associés à des histones antimicrobiennes et sont capables de piéger des bactéries. Comme chez les vertébrés, la formation de ces ETs est dépendante de la production d'espèces réactives de l'oxygène. De plus, la présence de ces ETs a été confirmée in vivo et a été associée à une accumulation d'histones antimicrobiennes dans les tissus, suite à une blessure ou à une infection. Dans une seconde partie,nous avons étudié les interactions entre V. tasmaniensis LGP32 et des protistes hétérotrophes présents dans l’environnement des huîtres, tels que les amibes et les ciliés, qui se nourrissent de micro-organismes par phagocytose. Un résultat important de cette thèse a été de montrer que V. tasmaniensis LGP32 résiste également à la phagocytose des protistes hétérotrophes environnementaux, de manière intracellulaire. C'est à notre connaissance la première fois que les mécanismes d’interaction entre des bactéries pathogènes d'origine marine et des amibes marines sont décrits. Les amibes que nous avons isolées étant présentes dans l'environnement direct des huîtres, nous pouvons donc supposer que la pression de sélection exercée par les phagocytes environnementaux pourrait favoriser l’émergence de traits de virulence et/ou de résistance à la phagocytose parmi les bactéries marines, comme c’est le cas pour V. tasmaniensis LGP32. / Vibrio strains belonging to the Splendidus clade have been repeatedly found in juvenile diseased oysters affected by summer mortalities. V. tasmaniensis LGP32 is an intracellular pathogen of oyster hemocytes which has been reported to alter the oxidative burst and inhibit phagosome maturation.We here focus on the interactions between phagocytes and V. tasmaniensis LGP32, at molecular, cellular and environmental scales. In the first part of this work, we uncover anunknown antimicrobial mechanism of C. gigas hemocytes: the formation of DNA extracellular traps (ETs). These ETs are associated with antimicrobial histones and are able to entrap bacteria. As in vertebrates, ETs formation depends on reactive oxygen species production. In addition, the presence of ETs was confirmed in vivo and has been associated with antimicrobial histones accumulation in tissues, in response to injury or infection. In the second part of this work, we studied the interactions between V. tasmaniensis LGP32 and heterotrophic protists found in the oyster’s environment, such as amoebae and ciliates, which feed on microorganisms by phagocytosis. An important result of this workwas that V. tasmaniensis LGP32 resists to phagocytosis by environmental heterotrophic protists, as well as to oyster hemocytes. To our knowledge, this is the first mechanical description of an interaction between marine amoebae and marine pathogenic bacteria. As the amoebae were isolated from the direct environment of oysters, we can presume that the selective pressure exerted by environmental phagocytes could select for virulence and/or phagocytosis resistance traits in marine bacteria as in the case of V. tasmaniensis LGP32.