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Year: 2010

The role of P-glycoprotein in host-pathogen interactions

Bottova, Iveta

Abstract: P-glycoprotein (P-gp) is an exceptional membrane transporter because of its ability to carry a large variety of compounds across the plasma membrane. Thanks to this substrate promiscuity, P-gp over-expression is responsible for multidrug resistance (MDR) in a variety of cells from microorganisms to humans. The cellular MDR is responsible for failure of treatment in over 90% of patients with metastatic cancer, as well as in yeast and bacteria infections and in life-threatening human parasites such as Plas- modium causing . P-gp is not only expressed in multidrug resistant cells but also in non-resistant cells at basal levels, but its physiological functions in the absence of drug pressure are not well under- stood. So far P-gp was proposed to be involved in several important cellular processes ranging from immunological functions to membrane lipid remodelling. Given the importance of P-gp in human and microbial MDR, there was an important need to identify the substrates and transport activities of this protein and to understand its physiological functions as a basis for the rational design of new drugs and MDR inhibitors. P-gp is localized mainly in the plasma membrane. Because the plasma membrane is the first barrier that intracellular pathogens need to cross to infect the host cell, we wanted toinves- tigate whether P-gp plays a role in this first line host-pathogen interaction. As a model pathogen we used the human parasite , which causes , a potentially life-threatening disease in individuals with immunodeficiency. As both host cell and parasite have a P-gp localized inthe plasma membrane, we hypothesised that both P-gps could be involved in events during parasite infection and replication. Our project had two goals: i) to characterize the physiological functions of host cell P-gp and its contribution to host-parasite interactions and ii) to characterize the T. gondii P-gp and its function in host-parasite interactions. To investigate the physiological functions of host cell P-gp and its contribution to host-parasite interactions, we compared three mouse embryonic fibroblast cell lines with different P-gp expression: wild type cells (WT) and P-gp knockout (P-gp KO) cells, as wellasP-gp complemented P-gp KO (P-gp KO/P-gp) cells. We used these cell lines to analyse the role of host P-gp in parasite invasion, replication and cholesterol transport, processes that are crucial for successful T. gondii propagation and survival. We showed that host P- gp is essential for normal T. gondii replication and specifically required for cholesterol transport from host endo- to the parasite. Importantly, using cholesterol auxotrophic T. gondii replication as a bio- indicator of cholesterol homeostasis in the host cell, we found that P-gp is involved in host cholesterol trafficking from endo-lysosomes to the plasma membrane. To characterize the T. gondii P-gp and its function in host-parasite interactions, we phar- macologically inhibited parasite P-gp (TgP-gp) with the P-gp specific inhibitor GF120918 (GF) and we used P- gp KO host cells to avoid the contribution of host P-gp to the observed phenotype. We analysed the role of parasite P-gp in invasion, replication and lipid metabolism. We showed that T. gondii P-gp is crucial for parasite Ca2+-depended attachment and invasion. Moreover, we propose that TgP- gp is involved in Ca2+ signalling which is important for the regulation of these processes and essential for T. gondii survival. Importantly, TgP-gp functionality is required for successful T. gondii replication and involved in the parasite lipid synthesis and transport. In summary, in this study we showed for the first time that both host cell and parasite P-gp have an important role in host-parasite interactions. I was also involved in a joint project focusing on the epigenetic regulation of stage conversion of Giardia lamblia. G. lamblia is an intestinal pathogen which infects millions of people all over the world causing the disease giardiasis. G. lamblia resides in the upper small intestine of humans and other vertebrates which become infected by ingestion of cysts from contaminated water or food. Giardia is characterized by two different developmental stages: the motile, proliferating trophozoites responsible for the clinical manifestation of the disease such as diarrhoea and malabsorption, and the cyst forms, the infective stage of the parasite. Infectious cysts are environmentally resistant and protected by a rigid extracellular ma- trix, the cyst wall. Stage conversion of G. lamblia is essential for transmission and survival of this human pathogen, but the understanding of the molecular mechanism which regulates this fundamental process still remains poorly understood. In this project we aimed to analyse the effect of histone deacetylase in- hibitor FR235222 on gene regulation of stage conversion in G. lamblia. We showed for the first time that inhibition of a histone deacetylase activity in Giardia with FR235222 induced transcriptional changes in proliferating and differentiating stages and potently blocked formation of cysts. We propose thatG. lamblia stage conversion is under the control of epigenetic regulation and dependent on the acetylation state of histones. In addition, histone deacetylase activity could be a promising target for developing new anti-Giardia drugs since the inhibition of histone deacetylase activity is blocking G. lamblia cyst formation and thus has potential to reduce disease transmission. Zusammenfassung Das P-Glykoprotein (P-gp) ist ein außergewöhnlicher Membrantransporter, vor allem wegen seiner Eigenschaft, eine Vielzahl verschiedener Stoffe über die Plasmamembran befördern zu können. Aufgrund der geringen Substrat- präferenz führt das Überexprimieren von P-gp zu einer gesteigerten Antibiotikaresistenz in einer Vielzahl von Zellen sowohl in Mikroorganismen als auch beim Menschen. Diese zelluläre Antibiotikaresistenz, auch „multidrug resistance“ (MDR) genannt, ist verantwortlich für einen Misserfolg bei mehr als 90% der klinischen Behandlungen von Patienten mit metastatischem Krebs aber auch bei der Behandlung von pathogenen Pilzen oder Bakterien und lebensbedrohlichen menschlichen Parasiten wie dem Malariaer- reger . Da P-gp nicht nur in Antibiotika-resistenten, sondern auch in nicht-resistenten Zellen exprimiert wird, ist es von großem Interesse die allgemeine physiologische Funktion dieses Proteins zu verstehen. Nach dem heutigen Wissensstand wird vermutet, dass P-gp in mehreren wichtigen zellulären Prozessen involviert ist und sowohl immunologische Funktionen übernimmt als auch bei der Modellierung von Membranlipiden mitwirkt. Aufgrund der Bedeutung von P- gp bei der mikrobiellen und humanen MDR ist es von großer Wichtigkeit, die Substratspezifität, Transportaktivität und physiologische Funk- tion des Proteins zu verstehen – letztlich, um bei der Entwicklung von neuen Antibiotka- oder MDR- Inhibitoren zu helfen. P-gp ist vor allem in der Plasmamembran lokalisiert. Da die Plasmamembran für pathogene Organismen die erste Barriere darstellt, die für die Infektion der Wirtszelle überwunden werden muss, untersuchten wir zunächst, ob P- gp eine Funktion bei der Interaktion zwischen Wirt und Pathogen übernimmt. Dabei diente der den Menschen infizierende Parasit Toxoplasma gondii, der in immunsupprimierten Individuen eine lebensbedrohliche Toxoplasmose hervorrufen kann, als Modellor- ganismus. Unser Projekt besaß zwei Zielsetzungen: i) die Erforschung der physiologischen Funktion von Wirtszellen P-gp und dessen Beitrag zur Wirt-Parasit-Interaktion und ii) die Charakteri