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.
|Date of Award||24 Feb 2021|
- Universidade Católica Portuguesa
|Supervisor||Elva Bonifácio Andrade (Supervisor) & Paula Ferreira da Silva (Co-Supervisor)|
- Neonatal meningitis
- LuxABCDE operon
- Mestrado em Microbiologia Aplicada