Heterologous expression and functional characterization of «Plasmodium falciparum» ABCG in mammalian cells

Malaria affects more than a quarter of this planet's population with 214 million infections and 438,000 deaths annually. In the absence of an effective malaria vaccine, the rise and spread of drug resistant strains coupled with the slow development of new antimalarials is a potential human tragedy. Research to date has led to the identification of several proteins that can mediate parasite resistance to most antimalarials. Two of the latter proteins (e.g., PfMDR1 and PfMRP1) are members of a large and evolutionary conserved family of ATP-dependent membrane transporters (e.g., ABC transporters). The P. falciparum genome encodes 16 different ABC transporters, including one member of the ABCG subfamily (e.g., PfABCG). By contrast, the human genome encodes 48 members of the ABC transporters, including five members of the ABCG subfamily (huABCG1, G2, G4, G5 and G8). With the exception of huABCG2, which transports many anti-cancer drugs and some normal cell metabolites, huABCG1, G4, G5 and G8 mediate the transport of cholesterol and other sterols. Earlier studies using PfABCG-knockout clones of P. falciparum have suggested that PfABCG may play a role in the parasite's sensitivity to ketotifen (an anti-histamine drug), and in the accumulation of neutral lipids in PfABCG-knockout clones. Moreover, we have shown that PfABCG shares 24.3 % and 26.5 % amino acid sequence identity with huABCG1 and huABCG2, respectively. Hence, it is presently not clear if PfABCG is functionally more like huABCG1, huABCG2, or both. In an effort to characterize the functions of PfABCG, it was of interest to compare its substrate specificity and subcellular localization to that of huABCG1 and G2 in the same expression system, using mammalian HEK-293 cells. Our results show the stable expression of PfABCG in HEK-293, as a fusion protein with GFP sequence linked to PfABCG N-terminal. In addition, relying on the fluorescence of GFP in PfABCG-HEK-293 transfectants, we have demonstrated the localization of GFP-PfABCG to the endosomal membranes, likely the endoplasmic reticulum. We also show that HEK-293 cells stably transfected with GFP-PfABCG are more sensitive to ketotifen in the presence of reseripine, a calcium channel blocker and an inhibitor of huABCB1 and huABCG2 expressed at low levels in HEK-293 cells. Efforts are on going to further characterize the functions of PfABCG and its substrate specificity and how these functions relate to those of huABCG1 and G2.Le paludisme affecte plus d'un quart de la population de cette planète avec 214 millions d'infections et 438 000 décès chaque année. De plus, en l'absence d'un vaccin efficace contre le paludisme, l'augmentation et la propagation de souches résistantes aux médicaments associées au développement lent de nouveaux antipaludiques est une tragédie humaine potentielle. La recherche à ce jour a permis d'identifier plusieurs protéines qui peuvent servir de médiateur à la résistance des parasites à la plupart des antipaludiques. Deux de ces protéines (par exemple, PfMDR1 et PfMRP1) sont des membres d'une importante famille évolutivement conservée de transporteurs membranaires dépendant de l'ATP (par exemple, des transporteurs ABC). Le génome de P. falciparum code pour 16 transporteurs ABC différent, y compris un seul membre de la sous-famille ABCG (par exemple, PfABCG). En revanche, le génome humain code pour 48 membres des transporteurs ABC, dont cinq membres de la sous-famille ABCG (huABCG1, G2, G4, G5 et G8). À l'exception de huABCG2 qui transporte de nombreux médicaments anti-cancéreux et certains métabolites cellulaires normaux, huABCG1, G4, G5 et G8 font la médiation du transport du cholestérol et d'autres stérols. Des études antérieures utilisant des clones de PfABCG-knock-out de P. falciparum ont suggéré que PfABCG peut jouer un rôle dans la sensibilité du parasite au ketotifène (un médicament anti-histaminique) et à l'accumulation de lipides neutres dans des clones PfABCG-knock-out. En outre, nous avons montré que PfABCG partage 24,3% et 26,5% de séquence d'acides aminés identique avec huABCG1 et huABCG2, respectivement. Par conséquent, il n'est présentement pas encore clair si PfABCG du point de vue fonctionnelle est comme huABCG1, huABCG2 ou les deux. Dans le but de caractériser les fonctions de PfABCG, il était intéressant de comparer sa spécificité de substrat et sa localisation à celle de huABCG1 et G2 dans le même système d'expression, en utilisant des cellules HEK-293 de mammifères. Nos résultats montrent une expression stable de PfABCG dans HEK-293, en tant que protéine de fusion avec une séquence de la GFP liée à une portion N-terminale de PfABCG. De plus, en prenant avantage de l'auto fluorescence de la GFP dans les transfectants PfABCG-HEK-293, nous avons démontré la localisation de GFP-PfABCG aux membranes endosomales, probablement le réticulum endoplasmique. Nous avons montré également que les cellules HEK-293 transfectées de manière stable avec GFP-PfABCG sont plus sensibles au kétotifène en présence de la réserpine, un bloqueur des canaux calciques et un inhibiteur de huABCB1 et huABCG2 qui sont exprimés à des niveaux faibles dans les cellules HEK-293. Des expériences sont en cours pour caractériser davantage les fonctions de PfABCG et sa spécificité de substrat ainsi que la façon dont ces fonctions se rapportent à celles de huABCG1 et G2

NK Cells in Protection from HIV Infection

Some people, known as HIV-exposed seronegative (HESN) individuals, remain uninfected despite high levels of exposure to HIV. Understanding the mechanisms underlying their apparent resistance to HIV infection may inform strategies designed to protect against HIV infection. Natural Killer (NK) cells are innate immune cells whose activation state depends on the integration of activating and inhibitory signals arising from cell surface receptors interacting with their ligands on neighboring cells. Inhibitory NK cell receptors use a subset of major histocompatibility (MHC) class I antigens as ligands. This interaction educates NK cells, priming them to respond to cells with reduced MHC class I antigen expression levels as occurs on HIV-infected cells. NK cells can interact with both autologous HIV-infected cells and allogeneic cells bearing MHC antigens seen as non self by educated NK cells. NK cells are rapidly activated upon interacting with HIV-infected or allogenic cells to elicit anti-viral activity that blocks HIV spread to new target cells, suppresses HIV replication, and kills HIV-infected cells before HIV reservoirs can be seeded and infection can be established. In this manuscript, we will review the epidemiological and functional evidence for a role for NK cells in protection from HIV infection

The Frequency and Function of NKG2C⁺CD57⁺ Adaptive NK Cells in Cytomagalovirus Co-Infected People Living with HIV Decline with Duration of Antiretroviral Therapy

Human cytomegalovirus (CMV) infection drives the expansion and differentiation of natural killer (NK) cells with adaptive-like features. We investigated whether age and time on antiretroviral therapy (ART) influenced adaptive NK cell frequency and functionality. Flow cytometry was used to evaluate the frequency of adaptive and conventional NK cells in 229 CMV+ individuals of whom 170 were people living with HIV (PLWH). The frequency of these NK cell populations producing CD107a, CCL4, IFN-γ or TNF-α was determined following a 6-h antibody dependent (AD) stimulation. Though ART duration and age were correlated, longer time on ART was associated with a reduced frequency of adaptive NK cells. In general, the frequency and functionality of NK cells following AD stimulation did not differ significantly between treated CMV+PLWH and CMV+HIV- persons, suggesting that HIV infection, per se, did not compromise AD NK cell function. AD activation of adaptive NK cells from CMV+PLWH induced lower frequencies of IFN-γ or TNF-α secreting cells in older persons, when compared with younger persons

The Frequency and Function of NKG2C+CD57+ Adaptive NK Cells in Cytomagalovirus Co-Infected People Living with HIV Decline with Duration of Antiretroviral Therapy

Human cytomegalovirus (CMV) infection drives the expansion and differentiation of natural killer (NK) cells with adaptive-like features. We investigated whether age and time on antiretroviral therapy (ART) influenced adaptive NK cell frequency and functionality. Flow cytometry was used to evaluate the frequency of adaptive and conventional NK cells in 229 CMV+ individuals of whom 170 were people living with HIV (PLWH). The frequency of these NK cell populations producing CD107a, CCL4, IFN-γ or TNF-α was determined following a 6-h antibody dependent (AD) stimulation. Though ART duration and age were correlated, longer time on ART was associated with a reduced frequency of adaptive NK cells. In general, the frequency and functionality of NK cells following AD stimulation did not differ significantly between treated CMV+PLWH and CMV+HIV- persons, suggesting that HIV infection, per se, did not compromise AD NK cell function. AD activation of adaptive NK cells from CMV+PLWH induced lower frequencies of IFN-γ or TNF-α secreting cells in older persons, when compared with younger persons