Mestrado em Genética e Biologia Molecular (ICB)
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Navegando Mestrado em Genética e Biologia Molecular (ICB) por Por Orientador "Rocha, Juliana Alves Parente"
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Item Identificação de proteínas de superfície de Staphylococcus saprophyticus e análise de fatores de virulência(Universidade Federal de Goiás, 2014-06-09) Carvalho, Alex Jesus de; Rocha, Juliana Alves Parente; http://lattes.cnpq.br/7089231795367245; Rocha, Juliana Alves Parente; Coelho, Alexandre Siqueira Guedes; Baeza, Lilian CristianeThe 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.Item Perfil comparativo do exoproteoma de 3 isolados clínicos de Staphylococcus saprophyticus(Universidade Federal de Goiás, 2016-09-30) Oliveira, Andrea Santana de; Rocha, Juliana Alves Parente; http://lattes.cnpq.br/7089231795367245; Rocha, Juliana Alves Parente; Marval, Marcia Giambiagi de; Paccez, Juliano DomiraciStaphylococcus saprophyticus is a Gram-positive bacterium responsible for genitourinary infections, mainly affecting young sexually active women. Along with Escherichia coli is responsible for 90% of infections in fertile women, however, can cause infections in men and women of all ages. The repertoire of proteins secreted by pathogenic microorganisms is used to ensure success in the establishment of infection and persistence in the host. In this sense, this study aimed to comparative characterization of extracellular proteome of 3 clinical strain of S. saprophyticus, in order to detect possible differences in the secretion of proteins related to virulence and adaptation of the microorganism. The strains used in the study are called ATCC 15305, 7108 and 9325. Ultra-Performance Liquid Chromatography coupled to Mass Spectrometry in tandem (UPLC-MSE) have identified a total of 159 proteins. Among them, 44 were found in exoproteome of 3 strain, while 20 only in strains ATCC 15305 and 7108, 11 in ATCC 15305 and 9325, and 12 in 7108 and 9325. Fifteen peptides were expressed exclusively by S. saprophyticus 9325, 21 by ATCC 15305 strain and 36 by 7108. The three strain secreted molecules with biological function related to the glycolytic pathway, such as the triosephosphate isomerase (TPI), enolase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and fructose-bisphosphate aldolase, molecules clearly involved in extracellular processes from other pathogenic organisms, proteins known as "moonlighting". Proteins involved in defense against stress, such as catalase, alkyl hydroperoxide reductase subunit C (AhpC), superoxide dismutase (SOD), were also detected in the analysis, among others related to metabolic processes such as lactic fermentation, cell wall synthesis, protein processing, and iron metabolism. Staphylococcal secretory antigen A (SsaA), a immunogenic molecule previously detected in ATCC 15305 strain, was not detected in strain 7108, as confirmed by Western-blotting assay. PCR reactions with specific primers and genomic DNA of the 3 strains and subsequent sequencing ssaA gene, showed that the strain has the gene under conditions identical to those found in S. saprophyticus ATCC 15305 and 9325, but is not able to secrete the antigen into the extracellular milieu, under environmental conditions in study. In exoproteome S. saprophyticus 7108 was found to take a greater amount of unique proteins, including four of the class of peptidases, enzymes often identified as potential virulence factors of bacterial. Thus, the differences found in repertoire of extracellular proteins of three clinical strain of S. saprophyticus, demonstrate a metabolic flexibility used by this uropathogen to promote pathogenesis in human genitourinary tract.Item Clonagem e expressão heteróloga do antígeno SsaA de Staphylococcus saprophyticus e avaliação da secreção durante interação com macrófagos(Universidade Federal de Goiás, 2016-06-28) Rosa, Isabella I. R.; Rocha, Juliana Alves Parente; http://lattes.cnpq.br/7089231795367245; Rocha, Juliana Alves Parente; http://lattes.cnpq.br/7089231795367245; Paccez, Juliano Domiraci; Amaral, Andre CorreaStaphylococcus saprophyticus is a pathogenic bacterium of the urinary tract and the main etiological agent of urinary tract infections by Gram-positive bacteria. Although S. saprophyticus potentially can cause serious infections such as pyelonephritis, septicemia, endocarditis and nephrolithiasis, and also multidrug resistance has been reported, not much is known about the mechanisms used by this bacterium during infection. Secreted proteins might be essential on those mechanisms if their role is accomplished during phagocytosis by their assistance of an active infection in phagocytic cells, protecting against oxidative stress and increasing the persistence of bacterial cells within phagocytes, and / or causing lysis of the host cell. Recently our group identified the immunogenic protein SsaA in the secretome of S. saprophyticus. This protein had been previously identified in S. aureus proteome, and it appears to be controlled by regulatory systems for known virulence factors. It also presents similarities with lytic proteins and proteins that assist the persistence within phagocytic cells. However, no approach had analyzed the contribution of SsaA during infection, therefore, through the construction a cloning vector containing the S. saprophyticus gene ssaA, heterologous expression of the recombinant protein and the production of specific polyclonal antibodies, it was able to verify the interaction of SsaA and proteins from macrophages infected by bacterial cells. Through immunofluorescence microscopy, it was verified that the dispersion of SsaA is not limited to phagocytic cells but it was throughout their cytoplasm after internalization of the bacterium. These findings together with other evidence in the literature suggest that SsaA is used during infection by S. saprophyticus, more specifically during phagocytosis. Further approaches are required to confirm if SsaA has a lytic activity and also characterize this protein as a virulence factor, contributing to elucidate strategies used by S. saprophyticus during infection in the human host.Item Caracterização do antígeno proteico SsaA de Staphylococcus saprophyticus utilizando estratégias in silico e modelo ex vivo de infecção(Universidade Federal de Goiás, 2020-03-02) Silva, Guilherme Algusto Alves; Rocha, Juliana Alves Parente; http://lattes.cnpq.br/7089231795367245; Rocha, Juliana Alves Parente; Tomazett, Mariana Vieira; Amaral, André CorrêaStaphylococcus saprophyticus is a Gram-positive bacterium and stands out as the second pathogen responsible for diagnosed cases of urinary tract infection (UTI), affecting mainly young women. Some factors may explain the ability of S. saprophyticus to colonize periurethral, urinary and genital regions, such as the ability to bind to the epithelial tissue of the genitourinary tract and the high activity of the urease enzyme. However, few mechanisms that this bacterium uses to efficiently infect and colonize the host are fully elucidated. In species of the Staphylococcus genus, most of the known virulence factors are proteins or the pathogen's surface. Our research group identified proteins secreted from S. saprophyticus isolates that demonstrated a strong ability to stimulate the immune response in mice and one of the main immunogenic proteins identified was the Staphylococcal Antigen Secreted A (SsaA), not yet characterized in this species. In other species of the Staphylococcus genus, the SsaA protein seems to be related to virulence factors regulated by the same systems, but its specific role during infection has not yet been fully elucidated. In this sense, we propose the characterization of the SsaA protein in S. saprophyticus. Bioinformatics analyzes using the database revealed that the SsaA protein has a CHAP domain with an expected amidase function. The 3D structure of the SsaA protein was predicted through the modeling of proteins using an online server following the validation protocols, the ABCpred server was used to predict epitopes in the SsaA protein. Analysis of the phagocytosis assay revealed that blocking the SsaA protein by sera containing anti-SsaA antibody reduces the number of cells recovered thus indicating that SsaA may be important during the infectious process. Our work will contribute to elucidate the biological function of the SsaA protein, an immunogenic protein that can be useful as a diagnostic target and / or vaccine in this model.Item Regulação do metabolismo de ferro em função do pH e caracterização da produção de sideróforos em Staphylococcus saprophyticus(Universidade Federal de Goiás, 2018-03-08) Souza, Bianca Silva Vieira de; Bailão, Mirelle Garcia Silva; http://lattes.cnpq.br/8844829751376156; Rocha, Juliana Alves Parente; http://lattes.cnpq.br/7089231795367245; Rocha, Juliana Alves Parente; Paccez, Juliano Domiraci; Cardoso, Juliana LamaroStaphylococcus saprophyticus is a coagulase negative bacteria that is part of the human microbiota and may also be present in surfaces, food and the environment. It can act as a pathogen causing urinary tract infections (UTIs) in humans. The ability to capture micro and macro nutrients is related to the ability to survive and establish infection in pathogenic microorganisms. One of the micronutrients is iron, which can be acquired by microorganisms through secret siderophores or iron-reducing system on the cell surface. When S. saprophyticus infection is initiated the bacteria causes changes in the medium, resulting in a change in the pH of the urine. Iron can be found in soluble form (Fe 2+) in higher concentration at acid pH, and insoluble (Fe 3+) in higher concentration at basic pH. In this study, the proteomic profile of S. saprophyticus when grown at acidic and basic pH was evaluated. The results demonstrate that S. saprophyticus respond to the instances of modulating the pH of proteins related to iron metabolism. A siderophores production capacity by S. saprophyticus was also investigated after culturing in minimal SSD medium without iron. The results demonstrate that this bacteria produces siderophores of the carboxylate type when cultivated in the absence of iron. Phagocytosis assays in macrophages demonstrate that S. saprophyticus is more susceptible to death after infection when they are deprived of iron, demonstrating that this element is important to ensure infection. The present study increased the knowledge of the proteomic and metabolic flexibility of S. saprophyticus in response to extracellular iron levels.