Mechanism of action and interplay between the thee main listerial virulence factors
ARRS project code: J1-9174
Period: 1.7.2018 - 30.6.2021
Head: Assoc. Prof. Dr. Marjetka Podobnik
ABSTRACT
Listeria monocytogenes is a facultative intracellular bacterium, and it is a leading cause of food‐borne illness that causes serious disease called listeriosis in immunocompromised individuals. Although rare in humans, the mortality rate can be as high as 20 ‐30 %. Importantly, listeriosis is far more common in domestic animals (mammals and poultry) and especially ruminants. Listeriosis in people and animals can sometimes be cured with antibiotics when diagnosed early, but especially in animals, it is generally fatal.
The virulence of L. monocytogenes is supported by a highly complex and coordinated intracellular life cycle that comprises several crucial steps: host cell adhesion and invasion, intracellular multiplication and motility, and intercellular spread. The completion of each stage is dependent on the orchestrated activity of specialized bacterial factors, in turn tightly controlled by a specific set of regulators.
Once the bacterium is internalized inside the target cell into phagosomal vacuole, it uses listeriolysin O (LLO) and two phospholipases, PI‐PLC and PC‐PLC, for vacuolar rupture, escape, and intercellular spread, which are crucial steps in L. monocytogenes pathogenesis. The disintegration of the vacuolar membrane is achieved by the pore forming activity of LLO, which has been found as a key event during bacterial infection, with the two phospholipases acting as a support to LLO for successful bacterial virulence. LLO is secreted as a monomeric soluble protein, which binds to membranes with high concentration of cholesterol. Upon binding to membranes it starts assembling into oligomeric structures that get inserted into the membrane lipid bilayer resulting in formation of functional transmembrane pores. In contrast to homologous toxins from other pathogenic bacteria, LLO does not form typically ring‐shaped pores with inner diameters of 40 nm, but instead has been found to form transmembrane perforations of irregular shapes and sizes, finally reaching dimensions that may act as gates for bacterial escape from vacuoles.
Previous studies on LLO in vitro by our group and others have shown that the mechanism of pore formation is affected by environmental cues like temperature, pH and membrane lipid content. While these studies brought important contribution to understanding of LLO mechanism of action, high resolution details on the unique plasticity of LLO pores and its dependence on environmental conditions are still largely missing. Moreover, the effect of bacterial phospholipases on membranes, the details of their structure and mechanism of action at the molecular level, as well as the interplay with LLO are still unknown.
Therefore, this study aims at resolving these yet unanswered questions with a use of wide range of modern methodological approaches from molecular biology, protein and lipid biochemistry and biophysics, to structural biology, including x‐ray crystallography and cryo‐electron microscopy. We believe that the proposed study will largely reveal important properties of LLO pores, such as plasticity, which is potentially responsible for exact tuning of events during infection by L. monocytogenes. Importantly, we aim to obtain data (close) to atomic resolutions, which will give crucial and detailed insights into the pore formation mechanism of LLO, alone and synergistically with the two phospholipases, which is fundamental for understanding of in vivo events during infection. We believe that the results of this research will contribute to understanding of Listeria pathogenesis and building of therapeutic strategy to prevent dissemination of L. monocytogenes and systemic infection in humans as well as livestock.
