Biophysical Characterization of Phospholipase C (PlcB) in Pseudomonas aeruginosa رسالة ماجستير

Abstract

Pseudomonas aeruginosa (P. aeruginosa), a bacteria considered by the World Health Organization (WHO) as a high-priority pathogen. It mainly affects people with a weak immune system, especially Cystic Fibrosis (CF) patients, caused by a mutation in the Cystic Fibrosis Transmembrane conductance Regulator gene (CFTR). This leads to the formation of mucous in various organs, plugging the airways and the secretion of enzymes (pancreas). Pseudomonas aeruginosa occupies the mucus in the lungs of CF patients, causing lung infections, which can lead to lung damage and even death. Also, P. aeruginosa is known for its ability to resist many Antibiotics, making treatment hard. Its ability to express virulent factors gives P. aeruginosa an advantage in being virulent. Phospholipases are one of those factors, supporting bacteria’s severity on human beings. They are divided into groups, one of them is Phospholipase C (PlcB). The aim of my project is to study the biophysical characterizations of PlcB. This is achieved by cloning PlcB in a plasmid, then finding the right temperature and expression strain that expresses PlcB in its soluble state. Afterward, purification using Ni-NTA, and checking functionality using an activity assay. Then, starting with biophysical characterizations, opening up many doors allowing understanding its structure. In this study, expression of PlcB in different expression strains and temperatures ended up in inclusion bodies formation. Different expression conditions were tried to overcome this problem, like the co-expression of PlcB with its chaperone, lowering the temperature or the concentration of used inducer. In conclusion, I could isolate and purify PlcB using the denaturation-refolding process that was done using urea. SDS PAGE showed that no impurities were isolated during purification. Activity was tested by a kit, confirming protein’s activity. This will allow proceeding to the step of structural analysis. Further experiments must be done to solve the structure of PlcB, by applying experiments that belong to biophysical characterization like Nuclear Magnetic Resonance (NMR), Circular dichroism (CD) and X-ray crystallography. This research was done in Hamburg-Germany, from October 2021 to June 2022.