Listeria monocytogenes (Lm) is the intracellular bacterial agent of listeriosis, a potentially fatal foodborne infection. The three main virulence factors of Lm are listeriolysin O (LLO) and two phospholipases: broad-range phospholipase C (LmPC-PLC) and phosphatidylinositol-specific phospholipase C (LmPI-PLC). The main research focus of this PhD thesis was the functional and structural analysis of LmPC-PLC and its interactions with LLO. We have determined the crystal structure of LmPC-PLC and through additional biochemical and biophysical experiments we have shown the factors that regulate the function of LmPC-PLC. The crystal structure reveals that LmPC-PLC has evolved several structural features to regulate its activity, including the position of the N-terminal tryptophan side group (W1), a structurally plastic active site, Zn2+-dependent activity, and a tendency to form inactive oligomers. Next, we found the activation of LmPC-PLC by cholesterol, which suggests a directed action on the host membranes. We described the specific inhibition of LmPC-PLC by the addition of its propetide in trans, which opens the way for the development of specific inhibitors for the treatment of infections with antibiotic-resistant strains of Lm. We also studied the synergy between LmPC-PLC and LLO and found a synergistic effect of LmPC-PLC on the binding of LLO to membranes and its premobilization activity. We also showed that preincubation of lipid membranes with LmPC-PLC affects the morphology of oligomers formed by LLO. This research provides important insight into the structure and function of LmPC-PLC, which has potential as a therapeutic target in the treatment of listeriosis, and uncovers new mechanisms of synergy between LmPC-PLC and LLO, which significantly contributes to the understanding of the pathophysiology of Lm.
|
