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Projects / Programmes source: ARIS

Mechanism of action and interplay between the three main listerial virulence factors

Research activity

Code Science Field Subfield
1.05.00  Natural sciences and mathematics  Biochemistry and molecular biology   

Code Science Field
P340  Natural sciences and mathematics  Lipids, steroids, membranes 

Code Science Field
1.06  Natural Sciences  Biological sciences 
Keywords
virulence factors, proteins, lipid membranes, three-dimensional structure, mechanism of action
Evaluation (rules)
source: COBISS
Researchers (16)
no. Code Name and surname Research area Role Period No. of publicationsNo. of publications
1.  37453  PhD Saša Aden  Biochemistry and molecular biology  Researcher  2020 - 2021  37 
2.  24290  PhD Matej Butala  Biochemistry and molecular biology  Researcher  2018 - 2021  237 
3.  32099  PhD Maja Grundner  Biochemistry and molecular biology  Researcher  2018 - 2021  36 
4.  53283  Maja Hostnik  Biochemistry and molecular biology  Researcher  2019 - 2021  19 
5.  38255  PhD Andreja Kežar  Biochemistry and molecular biology  Researcher  2020 - 2021  46 
6.  35382  PhD Matic Kisovec  Biochemistry and molecular biology  Researcher  2018 - 2021  86 
7.  53439  PhD Magdalena Kulma  Biochemistry and molecular biology  Researcher  2021  32 
8.  36425  PhD Tea Lenarčič  Biochemistry and molecular biology  Researcher  2018 - 2019  43 
9.  33923  PhD Polona Mrak  Natural sciences and mathematics  Researcher  2018 - 2021  103 
10.  35371  PhD Maruša Novak  Biotechnology  Researcher  2018  34 
11.  50709  Nejc Petrišič  Biochemistry and molecular biology  Junior researcher  2018 - 2021  17 
12.  12048  PhD Marjetka Podobnik  Biochemistry and molecular biology  Head  2018 - 2021  329 
13.  15328  PhD Kristina Sepčić  Biochemistry and molecular biology  Researcher  2018 - 2021  741 
14.  38479  PhD Aleksandra Šakanović  Neurobiology  Researcher  2019 - 2021  25 
15.  38473  Tomaž Švigelj    Technical associate  2018 - 2021  11 
16.  16381  PhD Nada Žnidaršič  Biology  Researcher  2018 - 2021  243 
Organisations (2)
no. Code Research organisation City Registration number No. of publicationsNo. of publications
1.  0104  National Institute of Chemistry  Ljubljana  5051592000  21,271 
2.  0481  University of Ljubljana, Biotechnical Faculty  Ljubljana  1626914  67,269 
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.
Significance for science
While pores of LLO homologes have been observed before by cryo-EM, at relatively low to medium resolution (Gilbert et al., 2014; van Pee et al., 2017), LLO pores have been imaged so far only by negative stain TEM and AFM (Podobnik et al., 2015, Mulvihill et al., 2015; Ruan et al., 2016). One of the main reasons is probably the high plasticity and thus heterogeneity of LLO oligomeric formations in comparison to other CDCs. We expect that screening of various lipid and detergent combinations at particular time points could lead to successful capture and separation of various stable and structured aggregates of LLO. We plan to develop novel methodological approaches and protocols to obtain (near) atomic resolution insights into these structures and thus interactions, which will represent an immense success and thus a huge and original contribution to studies involving the key virulence factor LLO. Namely, not only ring shaped pores, but also arcs have been shown to play important roles in functioning of pore forming proteins, and especially in the case of LLO (Gilbert et al., 2014; Rojko and Anderluh, 2015; Podobnik et al., 2015, Mulvihill et al., 2015; Ruan et al., 2016). This initial step will be an important basis to study the interplay or synergy of LLO and the two listerial phospholipases, as well as their interactions with lipid membranes at the mechanistic level, using various imaging biophysical and structural biology methods, which has not been done to date. We aim to perform a detailed screening of these intermolecular interactions (direct or indirect, protein-protein & protein-lipid) at a molecular level as well as study potential effects of phospholipases on structures of LLO oligomers, again aiming at highest possible resolutions, which will represent a major and by all means original contribution to this research field. It is thus important to study the mechanism of action of individual virulent factors like LLO and phospholipases, to thoroughly understand the background of Listeria pathogenesis and thus learn about potential ways to prevent it. This research will in long term contribute to the improvement of preventive measures and interventions, leading to lower incidence of listeriosis and better therapy outcomes in humans and animal stock. This project will include also PhD, MSc and BSc students, thus applying the ideas, experiences and knowledge on Listeria and various research methods to young generations of scientists. We aim to publish the outcomes of our research in scientific journals of high impact and will also present them at scientific conferences, in forms of lectures and posters. We will also attempt to present our data to general public via publishing in social media, newspapers and other media (lectures for general public, radio, etc).
Significance for the country
While pores of LLO homologes have been observed before by cryo-EM, at relatively low to medium resolution (Gilbert et al., 2014; van Pee et al., 2017), LLO pores have been imaged so far only by negative stain TEM and AFM (Podobnik et al., 2015, Mulvihill et al., 2015; Ruan et al., 2016). One of the main reasons is probably the high plasticity and thus heterogeneity of LLO oligomeric formations in comparison to other CDCs. We expect that screening of various lipid and detergent combinations at particular time points could lead to successful capture and separation of various stable and structured aggregates of LLO. We plan to develop novel methodological approaches and protocols to obtain (near) atomic resolution insights into these structures and thus interactions, which will represent an immense success and thus a huge and original contribution to studies involving the key virulence factor LLO. Namely, not only ring shaped pores, but also arcs have been shown to play important roles in functioning of pore forming proteins, and especially in the case of LLO (Gilbert et al., 2014; Rojko and Anderluh, 2015; Podobnik et al., 2015, Mulvihill et al., 2015; Ruan et al., 2016). This initial step will be an important basis to study the interplay or synergy of LLO and the two listerial phospholipases, as well as their interactions with lipid membranes at the mechanistic level, using various imaging biophysical and structural biology methods, which has not been done to date. We aim to perform a detailed screening of these intermolecular interactions (direct or indirect, protein-protein & protein-lipid) at a molecular level as well as study potential effects of phospholipases on structures of LLO oligomers, again aiming at highest possible resolutions, which will represent a major and by all means original contribution to this research field. It is thus important to study the mechanism of action of individual virulent factors like LLO and phospholipases, to thoroughly understand the background of Listeria pathogenesis and thus learn about potential ways to prevent it. This research will in long term contribute to the improvement of preventive measures and interventions, leading to lower incidence of listeriosis and better therapy outcomes in humans and animal stock. This project will include also PhD, MSc and BSc students, thus applying the ideas, experiences and knowledge on Listeria and various research methods to young generations of scientists. We aim to publish the outcomes of our research in scientific journals of high impact and will also present them at scientific conferences, in forms of lectures and posters. We will also attempt to present our data to general public via publishing in social media, newspapers and other media (lectures for general public, radio, etc).
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