| Name: | Description: | Size: | Format: | |
|---|---|---|---|---|
| 1.99 MB | Adobe PDF |
Advisor(s)
Abstract(s)
A bactéria estreptococos do grupo B (EGB), comensal dos tratos intestinal e geniturinário de adultos saudáveis, é a principal causa de infeções bacterianas em recém-nascidos, incluindo a meningite. A morbidade associada é elevada e até 50 % das crianças que sobrevivem à infeção apresentam sequelas neurológicas permanentes. Para o desenvolvimento de novas estratégias terapêuticas e neuro protetoras é fundamental uma maior compreensão sobre a fisiopatologia da doença. No entanto, os métodos clássicos de quantificação da infeção não permitem estudos longitudinais, pois implicam a morte do animal. Assim, o objetivo deste estudo foi o desenvolvimento de um novo sistema bioluminescente com base na luxABCDE que permita monitorizar a disseminação da EGB, através do uso de imagens in vivo em murganhos. Para isso, a estirpe hipervirulenta da EGB (BM110) foi transformada com o plasmídeo pSL101P32, que contém o operão luxABCDE (EGB BM110+pSL101P32). Estudos in vitro mostraram que o sinal de bioluminescência e densidade ótica foram proporcionais ao número de bactérias. Além disso, a presença do plasmídeo não afetou o metabolismo da EGB, uma vez que a sua taxa de crescimento não foi alterada e o plasmídeo permaneceu estável na ausência de pressão seletiva durante 8 dias. A virulência da EGB BM110+pSL101P32 no contexto da colonização do hospedeiro foi avaliada através da capacidade de adesão e invasão às linhas celulares raw 264.7 e Caco-2. Nenhuma diferença significativa foi detetada em relação à estirpe selvagem. De forma a avaliar a virulência in vivo, murganhos recém-nascidos foram inoculados intraperitonealmente com uma dose letal da estirpe selvagem ou bioluminescente. Não foram observadas diferenças na taxa de sobrevivência nem na carga bacteriana presente no cérebro, fígado, pulmões e intestino entre os grupos. No entanto, a bioluminescência só foi detetada em crias infetadas com um inóculo inferior e apenas na região abdominal (local da infeção), o que sugere que este sistema tem uma limitada capacidade de deteção. Assim, foi gerada outra estirpe luminescente, usando um novo plasmídeo que contém a luciferase do pirilampo “red-shifted” [FFluc(PTA)], uma vez que este apresenta uma maior capacidade de penetração dos tecidos. Resultados preliminares revelaram que a emissão de luz pode ser expressa em função do crescimento celular e a bioluminescência obtida foi maior do que com a luxABCDE. Estes resultados sugerem que o FFluc(PTA) é um sistema promissor para monitorizar a disseminação da EGB em recém-nascidos através de técnicas de imagem in vivo, permitindo o estudo de meningite neonatal comprovada.
Group B Streptococcus (GBS), a commensal of the intestinal and genitourinary tracts of healthy adults, is the main bacterial cause of severe neonatal invasive disease, including meningitis. Morbidity is high and up to 50 % of surviving infants experience long-term neurologic sequalae. To define new therapeutic and neuroprotective strategies, we must gain deeper insights into the pathophysiology of disease. The classical methods for quantifying infection do not allow longitudinal studies as it entails the animal death. Thus, the aim of this work was the development of a luxABCDE-based bioluminescent reporter system to monitor GBS dissemination, using whole-mouse in vivo imaging. For that purpose, a hypervirulent GBS strain (BM110) was transformed with the plasmid pSL101P32, containing the luxABCDE operon (GBS BM110+pSL101P32). After confirming that GBS was efficiently transformed, the new strain was validated in vitro. The obtained results revealed that the bioluminescence signal and optical density were proportional to the number of bacteria. Moreover, the presence of the plasmid did not affect GBS fitness [measured by GBS growth rate and compared with wild-type (WT) strain] and the plasmid remained stable in the absence of selective pressure, for at least 8 days. The virulence of GBS BM110+pSL101P32 in the context of host colonization was evaluated in vitro by analyzing the adhesion and invasion ability to the raw 264.7 and Caco-2 cell lines, relative to the WT strain. No significant differences were detected between both strains. The infectivity was also evaluated in vivo by inoculating pups intra-peritoneally with a lethal dose of the WT strain or its bioluminescent derivate. No differences were observed between groups either in the survival rate or in bacterial load present in brain, liver, lungs and gut. However, the bioluminescence signal was only detected in living pups infected with a lower inoculum and only in the abdominal area (infection zone), suggesting that this lux-based reporter system has a limited ability of being detected. Thus, we decided to use another plasmid, FFluc(PTA), containing the red-shifted firefly luciferase that has a higher ability to penetrate deeper tissues. Our preliminary results revealed that the light emission can be expressed as a function of cell growth. Importantly, the radiance signal was higher than the one obtained with the luxABCDE operon. These results suggest that FFluc(PTA) is a promising reporter system to monitor GBS dissemination in neonates using in vivo imaging technics, allowing the study of pups with proved meningitis.
Group B Streptococcus (GBS), a commensal of the intestinal and genitourinary tracts of healthy adults, is the main bacterial cause of severe neonatal invasive disease, including meningitis. Morbidity is high and up to 50 % of surviving infants experience long-term neurologic sequalae. To define new therapeutic and neuroprotective strategies, we must gain deeper insights into the pathophysiology of disease. The classical methods for quantifying infection do not allow longitudinal studies as it entails the animal death. Thus, the aim of this work was the development of a luxABCDE-based bioluminescent reporter system to monitor GBS dissemination, using whole-mouse in vivo imaging. For that purpose, a hypervirulent GBS strain (BM110) was transformed with the plasmid pSL101P32, containing the luxABCDE operon (GBS BM110+pSL101P32). After confirming that GBS was efficiently transformed, the new strain was validated in vitro. The obtained results revealed that the bioluminescence signal and optical density were proportional to the number of bacteria. Moreover, the presence of the plasmid did not affect GBS fitness [measured by GBS growth rate and compared with wild-type (WT) strain] and the plasmid remained stable in the absence of selective pressure, for at least 8 days. The virulence of GBS BM110+pSL101P32 in the context of host colonization was evaluated in vitro by analyzing the adhesion and invasion ability to the raw 264.7 and Caco-2 cell lines, relative to the WT strain. No significant differences were detected between both strains. The infectivity was also evaluated in vivo by inoculating pups intra-peritoneally with a lethal dose of the WT strain or its bioluminescent derivate. No differences were observed between groups either in the survival rate or in bacterial load present in brain, liver, lungs and gut. However, the bioluminescence signal was only detected in living pups infected with a lower inoculum and only in the abdominal area (infection zone), suggesting that this lux-based reporter system has a limited ability of being detected. Thus, we decided to use another plasmid, FFluc(PTA), containing the red-shifted firefly luciferase that has a higher ability to penetrate deeper tissues. Our preliminary results revealed that the light emission can be expressed as a function of cell growth. Importantly, the radiance signal was higher than the one obtained with the luxABCDE operon. These results suggest that FFluc(PTA) is a promising reporter system to monitor GBS dissemination in neonates using in vivo imaging technics, allowing the study of pups with proved meningitis.
Description
Keywords
EGB Meningite neonatal Operão luxABCDE Bioluminescência IVIS GBS Neonatal meningitis LuxABCDE operon Bioluminescence