Vector-borne diseases such as malaria, leishmaniasis, and African trypanosomiasis are a major scourge to humans and animals in some of the most impoverished nations across the globe. Enabling the transmission of these disease-causing pathogens is a highly sophisticated molecular arsenal of surface proteins. My research focuses on biophysical characterization of these proteins with the ultimate goal of deciphering the molecular crosstalk between pathogen and vector. In support of this goal, I have selected the tsetse fly-transmitted parasites of the genus Trypanosoma, the etiological agent of African sleeping sickness, as a model system. Towards elucidating the molecular mechanism of transmission, I have attempted to characterize structurally three novel proteins; TbFam50.360, TbPSSA2, and TcCISSA and get insight into their functions. Before this study, GARP (Glutamic Acid Rich Protein from T. congolense), and VSG (Variant Surface Glycoprotein from T. brucei) were the only proteins to be structurally characterized in the vector stages of the parasite. Our structural analysis revealed that while the N- terminal region of TbFam50.360 adopted a three-helical structure similar to previously characterized trypanosome surface proteins, ectodomains of both TbPSSA2 and TcCISSA adopted a previously uncharacterized bilobed architecture. The structural analysis further identified putative ligand binding regions in TbFam50.360 and TcCISSA. However, in the absence of binding partners, the exact function of these proteins could not be established. Our lab in conjunction with our collaborators is investigating the binding partners of these proteins within the tsetse. The structures of TbFam50.360, TbPSSA2, and TcCISSA can be added to the repertoire of structurally characterized surface proteins expressed by trypanosomes. The information gained from these first structures of trypanosome surface proteins offer insight into their role in the trypanosome life cycle, and may, in the future, contribute to the control of African trypanosomiasis. / Graduate

Breast cancer (BC) is the second leading cause of death in Canadian women. Detection of the disease at an early stage greatly increases the average 5-year survival rate, however non-invasive early detection methods are not available to-date. Cells release various types of extracellular vesicles (EVs) to mediate intercellular communication by transferring signals in the form of bioactive molecules such as proteins, metabolites, and nucleic acids. Understanding the composition of these biomolecules may shed light on the physiological state of the cell of origin. Therefore, EVs are a promising source of biomarkers for non-invasive detection of BC. However, the surface proteome of EVs is not yet understood well enough to propose BC biomarkers that could be detected directly from biofluids. In this study, small EVs (sEVs) and medium EVs (mEVs) were isolated by differential ultracentrifugation from breast cancer MDA-MB-231 and MCF7, and non-cancerous breast epithelial MCF10A cell lines and analyzed by nano-liquid chromatography coupled to tandem mass spectrometry. EV proteins were analyzed by two approaches: (1) global proteomic analysis and (2) enrichment of EV surface proteins by labelling surface-accessible proteins with a Sulfo-NHS-SS-Biotin reagent. Potential BC biomarkers were obtained from the first approach (1) by identifying the presence of cell line specific sEV proteins, filtering for membrane/surface proteins using UniProt annotations, and predicting the co-localization of proteins on sEVs with known EV marker proteins (CD63, CD9, CD81) by correlation analysis. This resulted in 11 potential BC sEV biomarkers (C8A, AXL, ST14, FAM20B, PROM2, CLDN3, ITGA7, MEGF10, SHISA2, GJC1, IFNGR1); the presence of ST14, CLDN3 and ITGA7 was validated by Western blot analysis. The surface labelling approach (2) enriched proteins previously not identified using the first approach (1). Potential general BC biomarkers were selected from surface proteins commonly identified from MDA-MB-231 and MCF7, but not identified in MCF10A EVs. Annotation with known BC disease associations from DisGeNET yielded 9 and 2 potential surface proteins on sEVs and mEVs, respectively. This study demonstrates the emerging role of EVs as a rich source of known and novel biomarkers which may be used for non-invasive detection of BC.

Antibodies directed against AMPAR and GABA_BR subunits have been implicated in forms of limbic encephalitis (LE), a disease characterised by memory loss and seizures. Patients with LE show clinical improvement with immunomodulatory treatment, suggesting that the associated antibodies are pathogenic. To explore further the AMPAR and GABA_BR antibodies, an in house cell based assay (CBA) was established for screening and potential pathogenicity was explored using a series of in vitro experiments. Human embryonic kidney (HEK) cells transfected with AMPAR and GABA_BR subunits and primary neuronal cultures were used to detect antibodies in patient sera and CSF. In total, 15/1361 (1.1%) AMPAR antibody positive samples and 24/1438 (1.7%) GABABR antibody positive samples were identified. The predominant antibody subclass for AMPAR and GABA_BR antibodies was shown to be IgG1. Interestingly, on transfected cells, only AMPAR antibodies showed complement deposition, and therefore had the potential to activate the classical pathway of the complement cascade. Application of IgG purified from AMPAR antibody positive patients, but not GABA_BR antibody positive patients caused a down regulation of the receptor from the cell surface of transfected HEK cells and primary hippocampal cultures. Electrophysiological analysis showed changes in Up state duration and spike rate in the entorhinal cortex following application of purified GABA_BR antibody IgG on brain slices. These findings suggest that GABABR antibodies are having a direct short term effect on GABA_BRs, and by extension cortical networks. Finally, we attempted to study whether or not viral infection could be a trigger for antibody production to known and novel antigen targets in a cohort of Japanese encephalitis viral (JEV) samples. A JEV cohort of 44 children was screened for antibodies to neuronal surface proteins by CBA. Twenty-seven percent of patients had antibodies to known neuronal surface antigens, with NMDAR and CASPR2 as the most common antigen targets. Interestingly, screening the cohort on primary neuronal cultures revealed that 65.9% of children with JEV have neuronal surface antibodies. The identification of the novel antigen target was attempted using immunoprecipitation and mass spectrometry techniques. In total, 4 neuronal surface proteins were identified that could be potential targets in JEV patients. In summary, antibodies directed against previously described antigenic targets were explored further for pathogenic potential, and a cohort of patients with a viral infection with potential novel antigen targets was investigated.

Toxoplasma gondii is a eukaryotic, intracellular parasite capable of infecting any vertebrate animal and establishing a life-long latent infection. Despite the prevalence of T. gondii infections, the molecular mechanisms by which these parasites gain access to the host cell remain largely unknown. Recent knockout studies have implicated a select group of T. gondii surface proteins, termed SRSs (Surface Antigen Glycoprotein Related Sequences), in directing parasite attachment and persistence. Follow-up structural studies with the prototypical SRS antigen, SAG1, revealed a novel fold, termed the SRS fold, and a dimeric structure with a topologically defined basic groove predicted to play a role in ligand binding. While these initial results were very exciting, follow-up work has failed to identify a host cell ligand for SAG1, and no other members out of more than 160 members of the SRS superfamily have been structurally characterized. As a result, conservation of the SRS fold and, more specifically, structural determinants of molecular recognition remain elusive. While sequence alignments of the SRS superfamily suggested conservation of the SRS fold, several insertions and deletions presented the possibility of localized structural elements that may be essential in molecular recognition. To characterize how these insertions/deletions are represented at the structural level, the X-ray crystal structures of two members of the SRS superfamily, BSR4 and SRS2, were solved. Structural analysis revealed an unexpected degree of diversity in the SRS fold. Divergent connectivity of the beta-strands in studied proteins indicates that the SRS superfamily may be more structurally diverse than previously thought, while structural variations in the beta-strands and the loops of D1 domain suggests a possible mechanism to recognize diverse host cell ligands, such as heparan sulfate proteoglycans (HSPGs). To probe HSPGs binding and determine the role of homodimerization, the dimer constructs of SRS2 and BSR4 were engineered, produced and tested in a carbohydrate binding macro-array. Selective binding of the SRS2 dimer to heparin was detected during screening and validated using heparin-agarose pull-down and native gel shift assays. Possible molecular mechanism for SRS-HPGS interaction and the implications in T. gondii virulence are also discussed.

The Lyme disease spirochete, B. burgdorferi is atypical in that a large portion of its genome is in the form of plasmids. Many of the plasmid-carried genes form extensive paralogous gene families and encode outer-surface proteins. In this report we have assessed the humoral immune response to proteins belonging to the paralogous protein family, family 163. We have cloned and expressed BBP39, BBO40, BBQ47 and BBN39 and used these recombinant proteins to monitor the temporal nature of the antibody response to these antigens during experimental infection of mice. The immunodominant regions of each protein have also been assessed through immunoblot analyses of a series of truncations of each protein. These analyses have led to the delineation of the targets of the antibody response during infection and of the specificity of the antibody response to family 163 proteins. In addition, we quantified the expression of each gene using real time RT-PCR.

Cell surface proteins play an important role in understanding cell-cell communication, cell signaling pathways, cell division and molecular pathogenesis in various diseases. Commonly used biotinylation regents for cell surface mapping have shown some potential drawbacks such as crossing the cell membrane, difficult recovery of biotinylated proteins from streptavidin/avidin beads, interference from endogenous biotin and nonspecific nature of streptavidin. With aim to solve these problems, we introduced sulfo-N-hydroxysuccinimidyl (NHS) ester functionalized magnetic nanoparticles containing cleavable groups to label solvent exposed primary amine groups of proteins. Silica coated iron oxide magnetic nanoparticles (Fe3O4@SiO2 MNPs) were linked to NHS ester groups via a cleavable disulfide bond. Additionally, the superparamagnetic properties of Fe3O4@SiO2 MNPs facilitate efficient separation of the labeled peptides and removal of the detergent without any extra step of purification. In the last step, the disulfide bond between the labeled peptides and MNPs was cleaved to release the labeled peptides. The disulfide linked NHS ester modified Fe3O4@SiO2 MNPs were tested using a small peptide, and a model protein (bovine serum albumin) followed by liquid chromatography-tandem mass spectrometry analysis (LC-MS/MS) of labeled peptides. In the next step, disulfide linked, NHS ester modified Fe3O4@SiO2 MNPs (150 nm) successfully labeled the solvent exposed cell surface peptides of Saccharomyces cerevisae. Electron microscopic analysis confirmed the cell surface binding of NHS ester modified Fe3O4@SiO2 MNPs. Mass spectrometric analysis revealed the presence of 30 unique proteins containing 56 peptides. Another MNPs based labeling reagent was developed to target solvent exposed carboxyl acid residues of peptides and proteins. The surface of Fe3O4@SiO2 MNPs was modified with free amine groups via a disulfide bond. Solvent exposed carboxyl groups of ACTH 4-11 and BSA were labeled by using1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) chemistry. Upon cleaving the disulfide bond, labeled peptides were analyzed by LC-MS/MS. The MNPs containing labeling reagents offers specific labeling under physiological conditions and rapid magnetic separation of labeled peptides prior to mass spectrometric analysis. The ability of large Fe3O4@SiO2 MNPs to specifically attach to cell surface makes them a potential candidate to study the surface of variety of different cell types and complex proteins surrounded by lipid bilayer.

Malaria affects over half of the world's population and causes half a million deaths annually, especially in Sub-Saharan Africa. Four species of the apicomplexan Plasmodium parasite (P. falciparum, P. ovale, P. malariae and P. vivax) are responsible for malaria in Africa. Both parasite and host factors contribute to heterogeneity in the risk of developing malaria, clinical manifestation of the disease as well as the number of treatments required to clear parasites. The epidemiology of the different species, and the role of exposure to mixed-species Plasmodium co-infections in generating heterogeneity remains poorly studied. Being an obligate intracellular parasite the blood-stage life cycle of the Plasmodium parasite takes place in the erythrocytes of the human host. The surfaces of these erythrocytes are the medically important ABO blood group antigens that have been reported to influence the susceptibility or otherwise of an individual developing severe malaria. In this thesis I have considered the contributions of the species of Plasmodium parasites and the ABO blood group of the host in driving heterogeneity in human malaria. The aims of this thesis were to determine: (i) the seroepidemiology of the different Plasmodium species in two mesoendemic African populations (Zimbabwe and Sudan); (ii) to determine if heterogeneity in clinical presentations of malaria (history of fever, body temperature and parasitaemia) and response to drug treatment is related to exposure to single vs. mixed-Plasmodium species infection; (iii) the spatial and temporal dynamics of malaria prevalence and Plasmodium species distribution in a mesoendemic village in eastern Sudan; (iv) gene expression changes in 3D7 P. falciparum parasites as they infect erythrocytes of different ABO blood group donors. For aims (i to iii) I developed an enzyme-linked immunosorbent assay using antigens derived from Plasmodium merozoite surface protein 1, also known as MSP-119, to detect IgG antibodies to all four malaria parasite species in Zimbabwean and Sudanese populations. In the Zimbabwean study, plasma samples from 100 individuals each (aged 5-18 years) from three villages (Burma Valley, Mutoko and Chiredzi) were screened for exposure to Plasmodium parasites. In Daraweesh, Sudan, plasma samples from 333 individuals (aged 1-74 years) who had experienced a first malaria episode between 1990 and 2000 were recruited into the study. For study aim (iv) I cultured a single clone of 3D7 P. falciparum parasite using erythrocytes of individuals of different ABO blood group types, harvested parasite RNA and sequenced it to determine gene expression changes in the different hosts. I showed that human IgG antibodies to MSP-119 antigens of the four Plasmodium species are species-specific and do not cross-react. In both study populations almost all antibody responses involved P. falciparum, and single-species responses were almost exclusively directed against P. falciparum antigens. Mixed-species responses accounted for more than a third of responses, and were associated with chloroquine treatment failure, with significantly high proportion of individuals with mixed-species infections requiring repeated treatment with chloroquine/sulfadoxine-pyrimethamine for parasite clearance. This finding highlights the need for a sensitive method for detecting mixed-species malaria infections to enable the assessment of the true prevalence and magnitude of the disease burden caused by the non-falciparum species in endemic populations. Drug treatment failures associated with mixed species infections have significant impact on malaria morbidity and mortality. Treatment failure or partial parasite clearance has the potential to allow dormant liver stages of P. vivax and P. ovale to become a source of parasite reservoir for onward transmission. Furthermore, untreated low-grade chronic infections caused by P. malariae have been reported to cause systemic diseases many years after the primary infection. Spatial analysis of malaria epidemiology showed that malaria parasite transmission in Daraweesh was focal, and that infections are not randomly distributed in the village. Two space-time clusters of significantly increased malaria risk were identified (1993- 1999, and 1998-1999) with marked variations between households, but little or no variation in the species of Plasmodium over time. Similarly, multiple significant clusters were identified for the parasite species; three for P. falciparum, two for P. vivax and P. malariae, and one for P. ovale. These clusters had overlapping time frames, with some of the species significantly infecting the same households. This suggests that even in a small geographic area malaria transmission shows heterogeneity, and that such data can provide useful information to guide malaria control efforts. Finally, I demonstrated that 3D7 P. falciparum parasite growth was similar in the erythrocytes of different blood group donors, and provide preliminary data to show that the non-coding RNA gene, PF3D7_1370800, is differentially expressed in blood group A donors relative to blood groups B and O donors. Further research is needed to better understand the role of this gene in malaria pathology. All together, these findings will aid malaria researchers and other stakeholders in making informed choices about tools for diagnosing Plasmodium species, and control programmes targeting eradication of malaria caused by all Plasmodium species, as is the case of incorporating these findings into current malaria research in Sudan.

Les bactéries lactiques, en particulier Lactococcus lactis sont utilisées en industrie agroalimentaire. Ces bactéries sont connues pour avoir une activité réductrice, désignant leur aptitude à abaisser le potentiel redox (Eh) d’un milieu. Le génome de L. lactis MG1363 code plusieurs protéines possédant un motif CXXC potentiellement liées à une activité redox. Pour comprendre le rôle des protéines de surface riches en cystéines, deux approches ont été utilisées. Par l’approche bioinformatique, notre intérêt s'est porté sur deux protéines de surface de fonctions inconnues et à motif CX2CX10CX2C : Llmg_0524 et Llmg_0526. Leurs gènes forment un opéron induit temporairement en début de croissance. Dans les deux protéines, le motif chélate un ion de zinc par les résidus cystéines, formant un complexe très stable. Nos données suggèrent que cet opéron contribue à l'intégrité de la paroi cellulaire et que le zinc participe à la stabilité des protéines. L'identification des protéines à thiols exofaciaux par une approche biochimique indique la présente d’AhpF à la surface de L. lactis. La délétion du gène ahpF entraîne une forte sensibilité du mutant au cumène hydroperoxyde, mais aucune au peroxyde d'hydrogène. Le cumène hydroperoxyde provoque une modification de la proportion en acide gras chez le mutant ahpF, le mécanisme de cyclopropanation contribue à sa survie en réponse à un stress oxydatif. La compréhension des fonctions impliquées dans l'activité réductrice des lactocoques permettra une meilleure maîtrise du Eh dans la fabrication des produits fermentés et un meilleur contrôle des flores pathogènes et d’altérations. Le projet Food-Redox a été financé par l'ANR. / Lactic acid bacteria, particularly Lactococcus lactis are used in dairy industry. These bacteria are known to have a reducing activity, indicating their ability to lower the redox potential (Eh) of a medium. L. lactis MG1363 genome encodes several proteins with a CXXC motif, potentially linked with a redox activity. To understand the role of proteins rich in cysteine located at the surface of L. lactis, two approaches were used, one bioinformatics and biochemical another. For bioinformatic approach, interest was focused on two proteins of unknown function and CX2CX10CX2C motif: Llmg_0524 and Llmg_0526. Their corresponding genes form an operon temporarily induces in early growth phase. In these two proteins, the pattern chelate a zinc ion via its cysteine residues. The zinc-cysteine complexe is very stable, it suggests a probable role in protein stability. Data suggest that this operon contributes to the cell wall integrity. The identification of exofacial thiol proteins by a biochemical approach indicates that AhpF is present at the surface of L. lactis. The ahpF gene deletion causes a strong sensitivity to the cumene hydroperoxide, but no sensibility for hydrogen peroxide. In the mutant ahpF incubation with cumene hydroperoxide modified fatty acid proportion, cyclopropanation mechanism thus contributes to the survival in response to oxidative stress. Understanding the lactococci functions involved in the reduction activity allows a better control of redox potentiel in the fermented food production and thus a better control of foodbornes microorganisms in these products. Food-Redox project is financially supported by the French National Research Agency.

Neospora caninum é um parasita intracelular obrigatório do filo Apicomplexa, intimamente relacionado a Toxoplasma gondii e responsável por abortamento e perda da fertilidade em bovinos, o que acarreta prejuízos significativos na pecuária mundial. Como parte de seu ciclo intracelular, a primeira interação do parasita com a célula alvo é realizada por proteínas de superfície conhecidas como superfamília SRS (Surface Antigen Glycoprotein - Related Sequences). Proteínas SRS ou SAG tem sido alvo de intensas pesquisas devido ao seu padrão imunodominante, exibindo grande potencial como ferramenta de diagnóstico e/ou candidatos vacinais. Atualmente existem cinco genes pertencentes à extensa família de proteínas SRSs descritos na literatura científica para N. caninum, dos quais dois foram caracterizados de taquizoítas por serem altamente reconhecidos por soros de animais infectados: NcSRS29B (SAG1) e NcSRS29C (SRS2). Diante disso, este trabalho foi realizado com o objetivo de caracterizar as proteínas de superfície SRS, NcSRS57 e NcSRS67. Além disso, foi obtido um panorama geral de proteínas ancoradas por GPI de N. caninum na linhagem Nc-1. Dentre os homólogos apicomplexas, NcSRS67 apresentou maior identidade e similaridade com Hammondia hammondi (HHA_450490), enquanto NcSRS57 revelou maior identidade e similaridade com Toxoplasma gondii (TgSRS57). NcSRS67 e NcSRS57 apresentaram a terceira maior semelhança entre os homólogos envolvidos no alinhamento estrutural. Estas duas proteínas SRS possuem doze resíduos de cisteína conservados que por predição formam seis pontes dissulfeto distribuídas em dois domínios SRS (D1 e D2), formando sanduiches de folhas ? e ? hélices associadas entre si. A sequência codificadora de NcSRS67 (sem o peptídeo sinal e sem a âncora GPI) foi clonada e expressa constitutivamente no plasmídeo pCR-Blunt II-TOPO-His6x. A forma nativa de NcSRS67 apresentou massa molecular de 35 kDa (predito 30.6 kDa sem peptídeo sinal e sem âncora GPI). A sequência rNcSRS57 (sem o peptídeo sinal e sem a âncora GPI) foi clonada em pET32, entretanto apenas um fragmento de 92 pb foi traduzido em relação a sequência clonada de 1074pb, devido a presença de stop códon oculto. Este evento gerou rNcSRS57 com massa molecular abaixo do esperado (19,5 kDa). NcSRS57 nativa apresentou massa de 43 kDa (predito sem peptídeo sinal e sem âncora GPI 31.14 kDa). Os efeitos inibitórios dos anticorpos policlonais anti-rNcSRS67, anti-rNcSRS57 e a associação destes sobre a adesão/invasão de taquizoítas foram investigados in vitro, resultando em uma inibição de 20% para o anticorpo anti-rNcSRS67, 16% para o anticorpo anti-rNcSRS57 e 11% para a associação destes dois anticorpos. NcSRS67 foi localizada sobre parte da superfície de taquizoítas, ao contrário de NcSRS57, que abrangeu toda a área da superfície destes parasitas. Apesar das inúmeras tentativas, as formas nativas de NcSRS67 e NcSRS57 obtidas por eletroforese 2D não foram identificadas por MS/MS. O tratamento de taquizoítas de N. caninum com a enzima fosfolipase C fosfatidilinositol (PI-PLC) específica, seguido de análises por MS/MS também gerou a identificação de proteínas de N. caninum, ii dentre elas as proteínas mais abundantes já identificadas no secretoma de N. caninum, NcSRS29B (SAG1) e NcSRS29C (SRS2). Dessa forma, os resultados obtidos neste estudo agregam conhecimentos sobre o parasita N. caninum e revelam-se úteis na busca e seleção de novos alvos a serem investigados contra a neosporose. / Neospora caninum is an obligate intracellular parasite of the Apicomplexa phylum, closely related to Toxoplasma gondii and responsible for abortion and loss of fertility in cattle, resulting in significant losses in the worldwide livestock. As part of its intracellular cycle, the first interaction of the parasite with the target cell is performed by surface proteins known as SRS superfamily (Surface Antigen Glycoprotein - Related Sequences). SRS or SAG proteins have been subject of intensive research due to their immunodominant pattern, exhibiting great potential as a diagnostic tool and/or vaccine candidates. Currently there are five genes belonging to the SRS family of proteins described in the scientific literature for N. caninum. Two of these genes were isolated from tachyzoites due to their high sera reactivity of infected animals: NcSRS29B (SAG1) and NcSRS29C (SRS2). Therefore, this work was carried out with the aim of characterizing SRS surface proteins, NcSRS57 and NcSRS67. In addition, we have performed an overview of N. caninum GPI anchored proteins in the Nc-1 lineage. Our results showed that; among the apicomplexan homologues, NcSRS67 presented higher identity and similarity with Hammondia hammondi (HHA_450490), while NcSRS57 revealed greater identity and similarity with Toxoplasma gondii (TgSRS57). NcSRS67 and NcSRS57 presented the third major similarity between the homologues involved in the structural alignment. These two SRS proteins have twelve conserved cysteine residues predicted to form six disulfide bonds distributed in two SRS domains (D1 and D2), forming ?-sheet sandwiches and ?-helices associated with each other. The coding sequence of NcSRS67 (without the signal peptide and without the GPI anchor) was cloned and constitutively expressed in the plasmid pCR-Blunt II-TOPO-His6x. The native form of NcSRS67 has a molecular mass of 35 kDa (predicted 30.6 kDa without signal peptide and without the GPI anchor). The rNcSRS57 sequence (without the signal peptide and without the GPI anchor) was cloned into pET32, however only a 92 bp fragment was translated in contrast to the cloned sequence of 1074 bp, due to the presence of a hidden stop codon. This event generated rNcSRS57 with molecular mass lower than expected (19.5 kDa). Native NcSRS57 has 43 kDa mass (predicted without signal peptide and without GPI anchor 31.14 kDa). The inhibitory effects of the anti-rNcSRS67 polyclonal antibodies, anti-rNcSRS57 and the association of both on the adhesion/invasion of tachyzoites were investigated in vitro, resulting in a 20% inhibition for the anti-rNcSRS67 antibody, 16% rNcSRS57 and 11% for the association. NcSRS67 was localized on part of the surface of tachyzoites, unlike NcSRS57, which covered the entire surface area of these parasites. Despite of the iv numerous attempts, native forms of NcSRS67 and NcSRS57 obtained by 2D electrophoresis were not identified by MS/MS. The treatment of N. caninum tachyzoites with the specific phospholipase C phosphatidylinositol (PI-PLC) enzyme, followed by MS/MS analysis also generated the identification of N. caninum proteins, among them the most abundant proteins already identified in the secretome of N. caninum, NcSRS29B (SAG1) and NcSRS29C (SRS2). Thus, the results obtained in this study increase the knowledge of the parasite N. caninum and demonstrate to be useful in the search and selection of new targets to be investigated against neosporosis.

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No. of bitstreams: 2 Dissertação - Alex Jesus de Carvalho - 2014.pdf: 1685689 bytes, checksum: 5b4f38809e4a3fe6252bba20b25d949b (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2014-06-09 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The Gram-positive bacterium Staphylococcus saprophyticus, one of the coagulasenegative staphylococci, is the second most common causative agent of urinary tract infection, affecting mainly sexually active women. Staphylococcus saprophyticus can cause acute diseases as pyelonephritis, sepsis, nephrolithiasis, endocarditis, urethritis, epididymitis and prostatitis. This work aims to identify Staphylococcus saprophyticus surface proteins by using a proteolytic shaving approach, a methodology that was established to identify surface-exposed protein domains by tripsinization of intact cells. The peptides obtained were treated by trypsin, reduced, alkylated and identified by nano-chromatography using a nanoACQUITY UPLCTM system (Waters) coupled to a SYNAPT Q-TOF mass spectrometer (Waters). The homology analysis was performed using the software ProteinLynx 2.3 (Waters). Through the shaving, it was possible to identify 219 proteins, many of them, described as virulence factors. Of total, 01 is cell wall protein, 09 are extracelular proteins, 19 are membrane proteins and 190 are citoplasmatic proteins. Besides of the lysis process, the presence of cytoplasmic proteins on cell surface can be due to the activity of export pathways not yet identified and many of these proteins can be proteins with moonlighting function, in other words, proteins that plays more of one function, it can, in this case, plays functions on S. saprophyticus cell surface related to bacterial virulence. The main proteins with moonlighting function include metabolic enzymes of the glycolytic pathway; enzymes of other metabolic pathways, such as, glyoxalate cycle; chaperones and proteins related with the proteic folding. The prediction of cellular localization was performed through LocateP database. The results of this research help to elucidate the strategies and machineries used by proteins during the adhesion, infection and proliferation, leading us to understand the interaction between the pathogenic bacteria S. saprophyticus and the human host. The knowledge about the proteins present on the cell surface is of extreme importance, because many of these proteins represent targets to new drugs, therapeutic antibodies or vaccines, since the pathogen cell surface is the first to contact with the host cells during the infection process. / A bactéria Gram-positiva Staphylococcus saprophyticus, uma das bactérias estafilococos coagulase negativa, é o segundo agente mais comum causador de infecções do trato urinário, afetando principalmente mulheres sexualmente ativas. S. saprophyticus pode causar doenças agudas como pielonefrite, sepse, nefrolitíase, endocardite, uretrite, epididimite e prostatite. Este trabalho teve como objetivo identificar proteínas de superfície de S. saprophyticus pela abordagem de shaving proteolítico, uma metodologia que foi estabelecida para identificar proteínas que possuem domínios proteicos na superfície celular utilizando a tripsinização de células intactas. Posteriormente, os peptídeos obtidos foram tripsinizados, reduzidos, alquilados e identificados através de nano-cromatografia utilizando um sistema nanoACQUITY UPLCTM (Waters) acoplado a um espectrômetro de massas SYNAPT Q-TOF (Waters). Com isso foi possível identificar 219 proteínas, muitas delas descritas como fatores de virulência. Do total, 01 proteína é de parede celular, 09 extracelulares, 19 de membrana e 190 citoplasmáticas. Além do processo de lise, a presença de proteínas citoplasmáticas na superfície celular pode ser devida à atividade de vias de exportação ainda não identificadas e muitas dessas proteínas podem ser proteínas com função moonlighting, ou seja, proteínas que desempenham mais de uma função, podendo, neste caso, desempenhar funções na superfície de S. saprophyticus relacionadas à virulência bacteriana. As principais proteínas com função moonlighting incluem enzimas metabólicas da via glicolítica; enzimas de outras vias metabólicas, tais como, ciclo do glioxalato; chaperonas e proteínas relacionadas com o dobramento proteico. A predição de localização celular foi realizada com o banco de dados LocateP. Os resultados desta pesquisa contribuíram na elucidação das estratégias e maquinarias utilizadas pelas proteínas durante a adesão, infecção e proliferação, levando-nos a compreender a interação entre a bactéria patogênica S. saprophyticus e o hospedeiro humano. O conhecimento acerca das proteínas presentes na superfície celular é de extrema importância, visto que muitas dessas proteínas representam alvos para novas drogas, anticorpos terapêuticos ou vacinas, uma vez que a superfície celular do patógeno é a primeira a entrar em contato com as células do hospedeiro durante o processo de infecção.