Orientador: Renata de Oliveira Mattos Graner / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-24T14:42:19Z (GMT). No. of bitstreams: 1 Alves_LiviaAraujo_M.pdf: 2021430 bytes, checksum: 07a84279508cd84bfef41550d34e6ca7 (MD5) Previous issue date: 2014 / Resumo: Streptococcus mutans (SM) é um patógeno oral da cárie dentária e endocardite infecciosa. Para se estabelecer no hospedeiro, SM precisa se adaptar às condições biofísicas e aos fatores de defesa do hospedeiro. Para isto, SM utiliza sistemas reguladores de transcrição de dois componentes (SDC). Dois SDCs, VicRK e CovR, são associados à virulência de SM. O objetivo deste trabalho foi investigar a participação dos reguladores de transcrição gênica VicRK e CovR na evasão de SM ao sistema complemento e fagocitose. Dessa forma, a marcação pelo complemento foi comparada entre os mutantes vicK- e covR- obtidos da cepa SM UA159 (UAvic e UAcov, respectivamente) e UA159, através da incubação com soro humano a 20% ou soro humano livre de C1q (30 min, 37 ?C, 10% CO2). A deposição de C3b sobre a superfície de SM foi analisada também nas cepas cultivadas em meio com 0,1% de sacarose. A quantidade de C3b nas superfícies bacterianas foi determinada através de reações com anticorpos IgG de cabra anti-C3 humano conjugado com FITC e análise de citometria. Bactérias incubadas com PBS foram usadas como controle negativo. O reconhecimento das cepas de SM por anticorpos séricos opsonizantes foi realizado com anticorpos anti-IgG humano conjugado com FITC e anti-IgM humano conjugado com APC. Para análise da fagocitose, as mesmas cepas coradas com FITC foram incubadas com neutrófilos (PMN) por 5 e 30 min. na presença ou ausência de soro humano a 20%; o número de PMN com bactérias fagocitadas foi determinado por citometria de fluxo. A deposição de C3b foi menor em UAcov (10,86%) e em UAvic (7,6%), comparado à UA159 (23,5%). Na presença de sacarose, a marcação por C3b caiu para 10,86% em UA159, e para os mutantes UAcov e UAvic foi reduzida a 6,6 e 3,96%, respectivamente (ANOVA p<0,001). Os mutantes UAcov e UAvic foram menos reativos contra anticorpos IgG e IgM. A fagocitose por PMN foi reduzida em UAcov e UAvic em cerca de 50 a 60% em relação à UA159 nos tempos de 5 e 30 min. (ANOVA p< 0,005). Assim, a inativação dos sistemas VicRK e CovR reduz significativamente a deposição de C3b do complemento e a frequência de fagocitose de SM, indicando que estes SDC regulam genes envolvidos no escape à opsonização pelo complemento / Abstract: Streptococcus mutans (SM) is an oral pathogen of dental caries and infective endocarditis. To get established in host sites, SM needs to adapt to biophysical conditions and host defense factors. To this purpose, SM applies transcriptional regulatory systems of two components (SDC). Two SDCs, VicRK and CovR, have been implicated in SM virulence. The aim of this study was to investigate the role of VicRK and CovR on SM evasion from complement system and phagocytosis by neutrophils (PMN). Thus, complement deposition was compared between vicK- and covR- mutants obtained strain UA159 (UAvic and UAcov, respectively) and the parent UA159 exposed to 20% human serum or human serum depleted of C1q (30 min, 37ºC, 10% CO2). Deposition of C3b on SM surfaces was also analyzed in strains cultivates in the presence of 0.1% sucrose. The amount of C3b on the bacterial surface was determined by reactivity with goat IgG antibody anti-C3 conjugated FITC and flow cytometry analysis. Bacteria incubated with PBS were used as negative control. The reactiveness of SM strains with human serum antibodies was quantified by flow citometry with anti- human IgG- FITC and anti- human IgM-APC antibodies. For analysis of phagocytosis, the strains stained with FITC were incubated with PMN during 5 and 30 min. in the presence or absence of 20% human serum; the number of internalized bacteria by PMN was determined by flow cytometry. The deposition of C3b on UAcov (10.86 %) and UAvic (7.6%) was lower, compared to UA159 (23.5%) (ANOVA p<0,001). In the presence of sucrose, C3b deposition decreased to 10.86 % in UA159, and to 6.6 and 3.96% in UAcov and UAvic , respectively (ANOVA p < 0.001). The UAcov and UAvic mutants were less reactive with IgG and IgM antibodies. The phagocytosis by PMN was 50 to 60% reduced in UAcov and UAvic compared to UA159, respectively at times 5 and 30 min. (ANOVA p < 0.005). Thus, inactivation of CovR and VicRK TCS systems significantly reduces deposition of complement C3b and phagocytosis by PMN in a serum-dependent way, indicating that these SDC regulate genes involved in the evasion of complement to opsonization / Mestrado / Microbiologia e Imunologia / Mestra em Biologia Buco-Dental

Orientador: Renata de Oliveira Mattos-Graner / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia / Made available in DSpace on 2018-08-18T21:04:54Z (GMT). No. of bitstreams: 1 Vizoto_NataliaLeal_M.pdf: 1501643 bytes, checksum: 855281de051e36237ad28b6d3f428f1b (MD5) Previous issue date: 2011 / Resumo: Streptococcus mutans é o principal patógeno da cárie dental em humanos, por ser capaz de se acumular no biofilme dentário principalmente na presença de sacarose e produzir e tolerar grandes concentrações de ácidos, os quais causam a desmineralização dos dentes. Além disto, S. mutans pode estar associado à etiologia da endocardite bacteriana, doença cuja evolução envolve a formação de biofilmes em válvulas cardíacas lesadas. S. mutans expressa diversas proteínas de superfície envolvidas na adesão e acúmulo bacteriano em biofilmes. Estas incluem as glucosiltransferases (codificadas por gtfB, gtfC e gtfD), as quais sintetizam glucanos extracelulares a partir da sacarose, e as proteínas ligantes de glucano (codificadas por gbpA, gbpB, gbpC e gbpD). Os genes gtfB/C/D, gbpB/C são controlados diretamente pela proteína reguladora CovR. Em diversas espécies patogênicas de Streptococcus, como Streptococcus pyogenes e Streptococcus agalactiae, CovR compõe o sistema regulador de transcrição de dois componentes CovRS (Cov, de control of virulence), o qual regula diversos genes de virulência. Em S. mutans, CovR foi identificado como um regulador órfão pois o gene que codifica o receptor de membrana cognato não foi identificado no mesmo locus gênico de covR. Em S. mutans, CovR regula diversos genes envolvidos na formação de biofilmes (gtfB/C, gbpC e gbpB), na biossíntese do envelope celular e na resposta a estresses. Os estímulos que ativam CovR em S. mutans ainda não são conhecidos. O presente projeto visa caracterizar a participação de CovR na resposta de S. mutans ao contato com a saliva e sangue humano. Para isto, uma cepa knock-out de covR- foi comparada com a respectiva cepa selvagem quanto ao crescimento e/ou sobrevida em saliva e sangue de voluntários saudáveis. O efeito da exposição à saliva na expressão dos genes regulados diretamente por CovR também foi avaliado / Abstract: Streptococcus mutans is the main pathogen of dental caries in humans, because it can accumulate in the biofilm in the presence of sucrose and produce and tolerate high concentrations of acids, which cause demineralization of teeth. Moreover, S. mutans is involved in the etiology of bacterial endocarditis, a disease whose evolution involves the formation of biofilms on damaged heart valves. S. mutans expresses several surface proteins involved in bacterial adhesion and accumulation in biofilms. These include the glucosiltransferases (gtfB, gtfC and gtfD), which synthesize extracellular glucans from sucrose, and glucan binding proteins (encoded by gbpA/B/C/D). The genes gtfB/C/D, and gbpB/C are directly controlled by the regulatory protein CovR. In several pathogenic species of Streptococcus, such as S. pyogenes and S. agalactiae, CovR is the transcriptional regulator that compose the two-component system CovRS (Cov for control of virulence), which regulates several virulence genes. In S. mutans, CovR was identified as an orphan regulator, because the gene encoding its cognate membrane receptor was not identified at the same locus of covR. In S. mutans, CovR regulates several genes involved in biofilm formation (gtfB/C, and gbpB/C), in biosynthesis of the cell envelope and in stress responses. The stimuli which activate CovR in S. mutans are not yet known. This project aims to characterize the participation of CovR in S. mutans response to contact with saliva and blood. To this purpose, a knock-out mutant of covR was compared with parent strain regarding growth and/or survival in human saliva and blood. The effect of exposure to saliva in the expression of genes directly by covR was also analyzed / Mestrado / Histologia e Embriologia / Mestre em Biologia Buco-Dental

Orientador: Wander José da Silva / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-20T13:31:39Z (GMT). No. of bitstreams: 1 Goncalves_LeticiaMachado_M.pdf: 2717766 bytes, checksum: fc9c2fda8a63938d55e0dc22f2155635 (MD5) Previous issue date: 2012 / Resumo: Os materiais reembasadores para próteses dentais removíveis, após a exposição à cavidade bucal, apresentam alterações estruturais que facilitam a colonização por espécies de Candida. Neste contexto, o ácido undecilênico (AUD) tem sido incorporado na formulação deste material na tentativa de reduzir o desenvolvimento de biofilmes fúngicos. No entanto, concentrações de AUD liberadas pelo material reembasador e os efeitos destas sobre o desenvolvimento dos biofilmes de Candida ainda não foram elucidados. Por isso, a proposta deste estudo foi investigar a cinética de liberação do AUD a partir do material reembasador e avaliar o efeito deste sobre o desenvolvimento de biofilmes de C. albicans ou C. glabrata. Inicialmente, simulou-se in vitro a liberação do AUD na cavidade bucal através da imersão de corpos de prova de material reembasador (10 mm x 2 mm) em saliva artificial, sendo o produto da liberação quantificado através de cromatografia gasosa. Em seguida, a suscetibilidade de um isolado de referência e dois isolados clínicos de C. albicans (ATCC 90028, P01 e P34) e C. glabrata (ATCC 2001, P11 e P31) ao AUD foi investigada através da concentração inibitória mínima (CIM), concentração fungicida mínima (CFM) e tempo de morte celular (time-kill). Para avaliar o efeito do AUD sobre os biofilmes de Candida, películas de saliva foram formadas na superfície de corpos de prova de material reembasador contendo AUD (grupo experimental) ou não (controle) e, em seguida, biofilmes dos isolados citados foram formados sobre estas superfícies. Nos períodos de adesão, 24, 48 e 72 h de desenvolvimento dos biofilmes, foram realizadas análises de contagem celular através de diluição decimal seriada; de atividade metabólica através da redução mitocondrial do XTT; de estrutura dos biofilmes através da microscopia confocal a laser; e secreção enzimática de proteinases e fosfolipases através de métodos colorimétricos. Os dados foram submetidos à análise de variância seguida de teste de Tukey com nível de significância de 5%. As concentrações de AUD liberadas pelo material reembasador estavam dentro dos intervalos de CIM e CFM encontrados para todos os isolados avaliados. Pelo teste de time-kill foi possível observar que na CIM, o AUD teve ação fungistática por até 8 horas de exposição, para todos os isolados investigados. A presença de AUD não alterou a contagem celular, atividade metabólica, estrutura dos biofilmes e secreção enzimática nos estágios iniciais de colonização (adesão e 24 h) para ambas as espécies investigadas (p > 0.05). A exposição ao AUD resultou em menor contagem celular (p < 0.05) e atividade metabólica (p = 0.001) para os biofilmes maduros (48 e 72 h) de C. albicans, embora alterações estruturais e de secreção enzimática não foram identificadas (p > 0.05). Em contraste, biofilmes maduros de C. glabrata apresentaram maior quantidade de células (p = 0.004), atividade metabólica (p < 0.001) e secreção de proteinases no grupo experimental (p < 0.001). Considerando as limitações deste estudo, pôde-se concluir que a liberação de AUD pelo material reembasador não evitou a colonização de biofilmes de Candida / Abstract: After exposure to oral cavity, denture liners exhibit structural alterations which facilitate colonization by Candida species. In this context, undecylenic acid (UDA) has been incorporated into denture liner formulation in an attempt to reduce the development of fungal biofilms. However, released concentrations of UDA from denture liner and its effects on Candida biofilms have not yet been elucidated. Therefore, the purpose of this study was to investigate the UDA-released concentrations from denture liner and evaluate its effects on C. albicans or C. glabrata biofilms development. Initially, it was simulated, in vitro, the UDA-releasing at oral cavity by immersing specimens of denture liner (10 mm x 2 mm) in artificial saliva, and the released product was quantified by gas chromatography. Then, susceptibility tests of a reference strain and two clinical isolates of C. albicans (ATCC 90028, P01 and P34) and C. glabrata (ATCC 2001, P11 and P31) for UDA were performed by minimal inhibitory concentration (MIC), minimal fungicidal concentration (MFC) and time-kill assays. For evaluate the effects of UDA-released on Candida biofilms, specimens of denture liner containing UDA (experimental group) or not (control group) were saliva-coated and then, biofilms of mentioned strains were developed on such surfaces. At adhesion phase, 24, 48 and 72 h, developed biofilms had their cells counts analyzed by serial dilution; metabolic activity by XTT reduction assay; biofilm structure by confocal laser microscopy; and enzymatic activity of proteinases and phospholipases by colorimetric methods. Data were subjected by analysis of variance followed by Tukey test with a significance level of 5%. UDA-released concentrations from denture liner were within the ranges of MIC and MFC for all strains evaluated. Time-kill results demonstrated that UDA at MIC had a fungistatic behavior for at least 8 hours of exposition for all strains investigated. The presence of UDA did not alter the cell counting, metabolic activity, biofilm structure and enzymatic secretion in the early stages of colonization (adhesion and 24 h) for both species investigated (p > 0.05). At UDA exposure, mature biofilms (48 and 72 h) of C. albicans presented lower cell counts (p < 0.05) and metabolic activity (p = 0.001), although structural changes and enzymatic secretion were not identified (p > 0.05). In contrast, C. glabrata mature biofilms showed higher cell counts (p = 0.004), metabolic status (p < 0.001) and also high secretion of proteinases in the experimental group (p < 0.001). Within the limitations of this study, it could be concluded that UDA-released from DL did not avoid Candida biofilms colonization / Mestrado / Protese Dental / Mestre em Clínica Odontológica

La pathogénicité des bactéries a toujours été attribuée à des facteurs de virulence et les bactéries pathogènes sont considérées comme étant mieux armées, comparé à des bactéries ne provoquant pas de maladies. Selon les premières études génomiques, le fait de supprimer un certain nombre de gènes des bactéries pathogènes, limiterait leur capacité à infecter leurs hôtes. Au contraire, des études de génomique comparatives récentes, démontrent que la spécialisation des bactéries dans les cellules eucaryotes est associée à une perte de gènes massive, en particulier pour les endosymbiontes allopatriques qui sont isolés depuis longtemps dans une niche intracellulaire. En effet, les bactéries sympatriques, extracellulaires, ont souvent des génomes plus grands et présentent une résistance et une plasticité plus importante. Ces bactéries constituent, de fait, plutôt des complexes d’espèces que de vraies espèces. Certaines bactéries spécialistes, comme les bactéries pathogènes, arrivent à s’échapper de ces complexes et à coloniser une niche, bénéficiant alors d’un nom d’espèce. Leur spécialisation leur permet de devenir allopatriques et leurs pertes de gènes favorisent une évolution réductive. Ces observations nous ont conduits à réaliser une étude afin de quantifier le taux de perte de gènes lors de l’évolution de ces bactéries extracellulaires vers celle de bactéries spécialistes intracellulaires. Notre objectif était de vérifier que ce qui caractérise l’évolution des bactéries intracellulaires est bien la réduction génomique, en prenant en compte tous les événements possibles de gains de gènes. Par ailleurs, dans une étude neutre comparant les 12 espèces pandémiques les plus dangereuses pour l’homme avec les espèces non-épidémiques les plus proches, nous avons voulu identifier des spécificités génomiques associées à la capacité virulente de bactéries pathogènes et démontrer que, à part les toxines et les modules toxine-antitoxine, ce qui caractérise ces espèces ce ne sont pas les facteurs de virulence, mais la perte des gènes de régulation. Au final, les bactéries pathogènes ont un répertoire virulent dans lequel les gènes absents sont aussi importants que les gènes présents. / The virulence of pathogenic bacteria has been attributed to virulence factors and pathogenic bacteria are considered to have more genes compared to bacteria that do not cause disease. According to the first genomic studies, removing a certain number of genes from pathogenic bacteria impairs their capacity to infect hosts. However, more recent studies have demonstrated that the specialization of bacteria in eukaryotic cells is associated with massive gene loss, especially for allopatric endosymbionts that have been isolated for a long time in an intracellular niche. Indeed, bacteria living in sympatry often have bigger genomes and exhibit greater resistance and plasticity and constitute species complexes rather than true species. Specialists, including specific pathogenic bacteria, escape these bacterial complexes and colonize a niche; thereby gaining a species name. Their specialization allows them to adopt allopatric lifestyle and experience reductive genome evolution. These observations led us to design a study to quantify the rate of gene losses during the evolution of free-living bacteria to intracellular specialists. Our objective was to verify that what characterizes the evolution of intracellular bacteria is genomic reduction, taking under consideration all possible gene gain events. Furthermore, in another neutral study comparing the 12 most dangerous pandemic bacteria to Humans to their closest non-epidemic species, we wished to identify any genomic specificities associated to the virulent capacity of pathogenic bacteria and demonstrate that, besides toxins and surprisingly, toxin-antitoxin modules, pathogenic bacteria are not characterized by more virulence factors, but rather by a loss of regulatory genes. Finally, virulent bacteria exhibit a genomic repertoire in which absent genes are as important as present ones.

Orientador: Tomomassa Yano / Dissertação (mestrado) - Universidade Estadual de Campinas,. Instituto de Biologia / Made available in DSpace on 2018-08-14T11:00:37Z (GMT). No. of bitstreams: 1 Thome_JacquelineDarcSilva_M.pdf: 1223668 bytes, checksum: 17699f260f82fb8a09136004fcc1e28a (MD5) Previous issue date: 2006 / Mestrado / Microbiologia / Mestre em Genética e Biologia Molecular

Orientador: Elisa Maria Aparecida Giro / Resumo: O peróxido de hidrogênio (H2O2) é considerado uma das principais fontes endógenas de estresse oxidativo para bactérias orais. Contudo, o seu papel no processo de desenvolvimento da cárie dentária ainda não está completamente elucidado. O objetivo deste estudo in vitro foi gerar uma cepa de Streptococcus mutans tolerante ao H2O2, e avaliar a habilidade do H2O2 em afetar a formação e a virulência de biofilmes de S. mutans. Para gerar a cepa tolerante ao H2O2, a cepa de S. mutans UA159, foi reativada em placas de agar BHI e incubadas (48 horas, a 37°C e 5% de CO2). Duas a cinco colônias foram transferidas para tubos falcon contendo 2 mL de caldo BHI suplementado com 1% de glicose e incubadas por 18 horas, sob as mesmas condições. As culturas foram diluídas 1:20 em meio de cultura fresco, incubadas até atingirem a fase exponencial de crescimento, centrifugadas, lavadas, ressuspensas em glicina, e submetidas a tratamento com 80 mM de H2O2. Esses procedimentos foram repetidos por oito vezes, e as colônias sobreviventes foram consideradas tolerantes. Em seguida, para o crescimento do biofilme, foi usado um modelo de aderência ativa. Lamínulas de vidro estéreis, jateadas com óxido de alumínio, foram imersas em saliva filtrada para a formação da película adquirida. Posteriormente, foram imersas verticalmente em 2,8 mL de caldo BHI com 1% de sacarose (n=12 por grupo) inoculado com 106 UFC/mL da cepa controle (grupo GC) ou da cepa tolerante ao H2O2. Para a cepa tolerante ao H2O2, em um ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Hydrogen peroxide (H2O2) is one of the main endogenous sources of oxidative stress for oral bacteria. However, its role in the dental caries development has not been fully elucidated. The objective of this in vitro study was to generate a strain of S. mutans tolerant to H2O2, and to evaluate the ability of H2O2 to affect the biofilm formation and virulence of S. mutans. To generate the H2O2 tolerant strain, S. mutans UA159 was reactivated on BHI agar plates, and incubated (48 hours at 37 oC and 5% CO2). Two to five colonies of the microorganism were transferred to falcon tubes containing 2 mL of BHI broth supplemented with 0.8% glucose and incubated under the same conditions for 18 hours. The culture was diluted 1:20 in fresh culture medium, incubated again until reaching the mind-log growth phase, centrifuged, washed, suspended in glycine, and treated with 80 mM H2O2. These procedures were repeated for eight times, and then the surviving colonies were considered tolerant. Next, the biofilm were grown on glass slides using an active adherence model. First, sterile glass slides, sandblasted with aluminum oxide, were immersed in filtered saliva to form the acquired pellicle. After that, the slides were immersed vertically in 2.8 mL of BHI broth with 1% sucrose (n = 12 per group) and inoculated with 106 CFU/mL of the control strain (GC Group) or H2O2 tolerant strain. For the H2O2 tolerant strain, in a group (GTcPH group) 80 mM H2O2 were added in the culture medium and in the oth... (Complete abstract click electronic access below) / Mestre

Background P. gingivalis, a non-motile, rod-shaped, anaerobic, Gram-negative bacterium is one of the principal sources of periodontal disease. It possesses a number of potential virulence factors thought to be important in the disease process including 5 hemagglutinins (Hag). One of these is HagB. It is a well characterized nonfimbrial adhesin expressed on the surface of P. gingivalis. HagB is very pro-inflammatory and induces robust chemokine and cytokine responses in vitro and in vivo. Since the chemokine and cytokine responses seen from single cells grown in tissue culture often are not representative of the chemokine and cytokine profiles seen in clinical samples or biopsy specimens, we devised a co-culture model of keratinocytes, dendritic cells, and T-cells to test the hypothesis that chemokine and cytokine responses from co-cultured cells would be more representative of responses seen in clinical samples from individuals with periodontal disease than single cell models. Methods and materials HagB was prepared by cloning hagb of P. gingivalis (1.4 kb) into the vector pQE31 (QIAGEN Inc., Valencia, CA); expressed in E. coli M15(pREP4)pQE31-TX1; and isolated from E. coli lysates by affinity chromatography using a Ni-charged resin (Profinity IMAC Resin, BioRad, Hercules, CA) and examined by SDS-PAGE. Co-culture models were treated with 10 µg/ml HagB (Test) or 10 µg/ml HagB diluent (Control). At 64 hours the supernatants were collected. Chemokine and cytokine biomarkers GM-CSF, CCL3 (MIP-1α), CCL4 (MIP-1β), CCL5 (RANTES), IL-1α, IL-6, IL-8, TNFα, IL-12(p40), and VEGF responses were determined using Milliplex immunoassays. HagB responses were corrected by subtracting the constitutive responses detected in supernatants incubated with HagB diluent. Statistical differences among groups were determined on Log10 transformed biomarker concentration using JMP 10 (version 10.0, SAS, CAR; NC). Results Buffers (e.g. HagB diluent) did not induce a chemokine or cytokine response, however there was a gradual increase in chemokine and cytokine responses from cells at 64 hours. These were subtracted from HagB induced responses. Responses generally fell in 2 groups; in one group containing VEGF, IL-12(p40), IL-6, RANTES and GM-CSF, there were no significant differences among groups (p>0.05). In another group containing IL-1α, IL-8, MIP-1α, MIP-1β and TNF-α, there were significant differences among groups (p< 0.05). Interestingly these resulting responses fell in 2 categories- GM-CSF, IL-12, IL-1α, IL-6 were less than 25pg/ml and IL-8, MIP-1α, MIP-1β, RANTES, TNFα and VEGF were more than 25pg/ml. Some responses were driven by a particular cell type e.g. GM-CSF produced by dendritic cells, RANTES produced by T- cells and VEGF produced by T cells. There were similar responses in HagB-induced IL-8, MIP-1β, MIP-1α and TNFα responses by dendritic cells + keratinocytes and dendritic cells + keratinocytes + T cells. Conclusions Co-culture models can more realistically produce chemokine and cytokine responses to agonists than individual cultures of cells, which is important for predicting and assessing novel therapeutic treatments of periodontal disease.

Orientador: Ary Fernandes Júnior / Resumo: O aumento no número de bactérias multirresistentes aos fármacos antibacterianos é preocupação de saúde pública e tem motivado pesquisas na buscsa por antimicrobianos alternativos para minimizar este problema, e na obtenção de substâncias com capacidade de matar bactérias e/ou interferir com a sua patogenicidade. O Staphylococcus aureus é uma bactéria altamente virulenta, capaz de causar inúmeras doenças, incluindo intoxicações alimentares. Esta bactéria se tornou resistente aos diversos antimicrobianos ao longo dos anos com destaque para o S. aureus meticilina-resistente (MRSA). O peptídeo antimicrobiano (AMP) nisina, bacteriocina produzida por Lactococcus lactis é um que vem sendo estudado na forma de nanoemulsões e nanopartículas. O objetivo desse estudo foi sintetizar, caracterizar e testar nanoemulsões e nanopartículas de ouro (AuNPs) de nisina, para a verificação da ação antibacteriana, através da Concentração Inibitória Mínima (CIM), atividade antibiofilme, antienterotoxina, atividade hemolítica, ação sobre a membrana bacteriana determinada pelo extravasamento de proteínas, sobre linhagens padrões ATCC de S. aureus, e teste de viabilidade celular em linhagem HCT-116 por citometria de fluxo. Os tratamentos utilizados foram nisina, cinco nanoemulsões com nisina (Nano-Nis), AuNPs com nisina (AuNPs-Nis), AuNPs com Nano-Nis (AuNPs + Nano-Nis) e AuNPs-Nis com nanoemulsão (AuNPs-Nis + Nano). De acordo com os resultados obtidos para CIM, observou-se que para a cepa ATCC 33591 d... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The growth in the number of multiresistant bacteria resistant to traditional antimicrobial drugs is a public health concern which has been motivated researchs worldwide, seeking new antimicrobial drugs to minimize this problem besides getting new substances able to erradicate bacteria and/or interfer with their pathogenicity. Staphylococcus aureus is a highly virulent bacteria, able to cause countless diseases including food poisoning. This bacteria got resistant to many antimicrobials within the years, highlighting the S. aureus metchilin-resistant (MRSA). The antimicrobial peptide (AMP) nisin, bacteriocin synthesized by Lactococcus lactis, is one that has been studied in nanoemulsions and nanoparticles. The objective of this study was to synthesize, characterize and evaluate nanoemulsions and gold nanoparticles (AuNPs) with nisin, to verify their antibacterial action, using the Minimum Inhibitory Concentration (MIC), antibiofilm activity, antienterotoxin activity, hemolytic activity, action on bacterial membrane determined by protein leakage, on MRSA ATCC strains, and cell viability in HCT-116 strain by flow citometry. The treatments used were nisin, five nanoemulsions with nisin (Nano-Nis), AuNPs with nisin (AuNPs-Nis), AuNPs with Nano-Nis (AuNPs + Nano-Nis) and AuNPs-Nis with nanoemulsion (AuNPs-Nis + Nano). According to the results obtained for MIC, it was observed that for the MRSA strain ATCC 33591, the number 1 formulation of nanoemulsions was the more efficient betwe... (Complete abstract click electronic access below) / Mestre

The Bordetellae are aerobic Gram-negative coccobacilli colonizing the upper respiratory tract of mammals and thereby causing diseases with similar symptoms but different host specificity. The bacteria produce a variety of adhesins and toxins that facilitate their ability to promote infection and evade the innate immune system. Among them, the filamentous hemagglutinin (FHA) and the adenylate cyclase toxin (CyaA) are the major virulence factors providing the adherence to the host epithelial cells and the protection against bactericidal activity of phagocytic cells, respectively. Moreover, CyaA along with the Escherichia coli α-hemolysin (HlyA) and the Kingella kingae cytotoxin (RtxA) represent a prominent group of Repeats in ToXin (RTX) cytotoxins/hemolysins that undergo post-translational acylation on conserved lysine residues. Here, different mass spectrometry approaches were employed to analyze the structural features of FHA and to characterize the acylation status of the RTX toxins and their various hybrid molecules. First, the differential 16O/18O labeling revealed that the mature FHA proteins of B. pertussis (Bp-FHA) and the B. bronchiseptica (Bb-FHA) are processed at different sites, after Ala2348 and Lys2479 of the FhaB precursor, respectively. Second, the bottom-up proteomics of the...

Indiana University-Purdue University Indianapolis (IUPUI) / Lyme disease is a multi-system inflammatory disorder that is currently the fastest growing arthropod-borne disease in the United States. The Lyme disease pathogen, Borrelia burgdorferi, exists within an enzootic cycle consisting of Ixodes tick vectors and a variety of vertebrate hosts. Borrelia lies within a distinct clade of microorganisms known as spirochetes which exhibit a unique spiral morphology. The underlying genetic mechanisms controlling for borrelial morphologies are still being discovered. One flagellar protein, FlaB, has been indicated to affect both spiral shape and motility of the organisms and significantly impacts the organism’s ability to establish infection. Due to the potential connection between morphological characteristics and pathogenesis, we sought to screen and identify morphological mutants in an attempt to identify genes associated with morphological phenotypes of Borrelia burgdorferi. Among Borrelia’s unique features is the presence of abundant lipoproteins making up its cellular membrane as opposed to the typical lipopolysaccharides. These proteins confer a wide variety of functions to the microorganism, among which include the abilities to circulate between widely differing hosts and to establish infection. Two important outer surface proteins, OspC and OspA, are found to be inversely expressed throughout the borrelial life cycle. OspC, in particular, becomes highly expressed during tick-feeding and transmission to the mammalian host. It has been found to be essential for establishment of infection. A global regulatory pathway has been shown to control for OspC, however there are missing links in this pathway between the external stimuli (such as temperature, pH, and cell density) and the regulatory pathway. We have performed a screening process to identify OspC expression mutants in order to identify novel genes associated with this pathway.