The PV is formed when the parasite invades the host cell. During this process most contain proteins of the host cell membrane are removed to form a ��vesicle�� which is not detectable for the immune system [14]. The parasite secretes many proteins in the newly formed PV; a few of them are also transported to the PV membrane and into the host cell cytosol [15]. One group of these important proteins is that of the Rhoptry proteins (ROP). The gene loci determining virulence of T. gondii highlighted the ROP2 family, a family of several proteins containing a protein-kinase-like domain [16�C18]. Expression of a virulent ROP18 allele in avirulent strains resulted in faster growing parasites and enhanced mortality by 4 to 5 logs in mouse in vivo infection experiments [17].
First experiments demonstrated the importance of the pseudokinase ROP5 for the correct ROP18 localisation to the PV [19, 20]. The family member ROP16 on the other hand interacts with host cell signal transduction pathways as it activates regulatory cytokine pathways like IL-4 via STAT6 phosphorylation [21]. In the current study, we compare the capacity of astrocytes to combat virulent and avirulent strains of T. gondii in terms of parasite replication and kinetics of accumulation of the two important IRGs Irga6 and Irgb6. We further characterized the localization of both IRGs at one individual vacuole and analysed the host cell manipulation of virulent and avirulent strains in coinfection experiments. 2. Methods2.1. In Vitro Passage of T. gondiiVirulent T.
gondii strains RH-YFP [22] and BK [23] were maintained in L929 fibroblasts (ATCC, Manassas, USA) and harvested after three days. Harvested parasites in the supernatants were purified from host cell debris by differential centrifugation (5min at 50��g, 15min at 500��g), counted, and used for reinfection. For cultivation of RH-YFP, chloramphenicol (Sigma, St. Louis, USA) was added to maintain selection pressure. Avirulent ME49 [24, 25], NTE [26], and 76K [27] Toxoplasma were maintained in HS27 fibroblast (ATCC) and cultivated as described previously [13]. 2.2. Preparation and Cultivation of AstrocytesAstrocytes were isolated from the brains of neonatal C57BL/6 mice. After decapitation, the brain was prepared, the cortices were isolated, and the meninges were removed.
A homogenized cell suspension was prepared as described earlier [13], and 1 �� 106cells/well were seeded in 6-well tissue culture plates in DMEM (10%FCS, 2mM glutamine, 50��M 2-mercaptoethanol; Invitrogen, Karlsruhe, Germany). Batimastat 2.3. Depletion of CD11b+ MicrogliaAfter the neonatal cell culture has formed a confluent monolayer (day 10�C12), cells were harvested using accutase (Invitrogen). CD11b-positive microglia were depleted using anti-CD11b microbeads based on the manufacturer’s protocol (Miltenyi Biotec, Bergisch Gladbach, Germany).
No related posts.