|dc.description.abstracteng||Toxoplasma gondii is an intracellular protozoan parasite that infects warm-blooded animals including an estimated thirty percent of humans world-wide. In immunocompetent host, primary infection is usually asymptomatic but fast-replicating tachyzoites partially undergo developmental switching into slow-replicating, dormant bradyzoites preferentially within muscular and neural tissues. Stage differentiation from tachyzoites to bradyzoites enables the parasite to establish chronic infection and facilitates transmission of the parasite to new hosts via predation or ingestion of raw or undercooked meat from infected livestock. Bradyzoite differentiation and tissue cyst development is spontaneously triggered in terminally differentiated skeletal muscle cells (SkMCs), i.e in myotubes but not in proliferating myoblasts or fibroblasts. The factors that trigger bradyzoite differentiation in myotubes are only partially known. Herein, host cell transcriptomes, metabolomes and cell cycle regulation were determined to identify host cell factors that might regulate bradyzoite differentiation in myotubes.
RNA sequencing-based transcriptome analysis of non-infected and T. gondii-infected myotubes and myoblasts revealed that these cell types differed significantly in the expression of ~6500 genes (DEGs) irrespective of T. gondii infection. Gene ontology analysis revealed that these DEGs predominantly regulated cellular component organization or biogenesis, cell cycle processes including mitotic cell cycle, muscle structure development and cellular metabolic processes. Surprisingly, infection with T. gondii had only a minor impact on gene expression in myoblasts and myotubes. Further analyses of the transcriptomes from infected and non-infected myoblasts and myotubes showed differential expression of various enzymes of the central carbohydrate metabolism. For example, genes encoding most glycolytic enzymes, some tricarboxylic acid (TCA) cycle enzymes, particularly the pyruvate carboxylase of TCA cycle anaplerosis, and multiple genes encoding glycogen metabolic enzymes were upregulated in myotubes as compared to myoblasts. In contrast, genes encoding enzymes of the pentose phosphate pathway (PPP) including glucose-6-phosphate dehydrogenase (G6PDH)2 or G6PDH(X-linked) and 6-phosphogluconate dehydrogenase (6PGDH) were upregulated in myoblasts compared to myotubes. In addition, myotubes showed upregulation of various cell cycle inhibitors including p21Waf1/Cip1 as compared to myoblasts whereas many cyclin-dependent kinases and their cyclins were upregulated in myoblasts compared to myotubes irrespective of whether the cells were non-infected or infected with T. gondii. Differential expression of these molecules in myoblasts and myotubes irrespective of infection was further confirmed by RT-qPCR.
GC-MS analyses of non-infected and T. gondii-infected myotubes and myoblasts after labeling with 13C-glucose indeed indicated increased PPP activities in myoblasts, accelerated TCA cycle anaplerosis via the pyruvate carboxylase in myotubes, but no differences in glycolysis between the cell types. Remarkably, pharmacological inhibition of the PPP using the G6PDH inhibitor dehydroepiandrosterone and the 6PGDH inhibitor 6-aminonicotinamide upregulated T. gondii bradyzoite antigen (BAG) 1 mRNA expression and tissue cyst formation while it reduced parasite replication in both cell types but to a higher extent in myoblasts. This indicated that lower PPP activities as observed in myotubes compared to myoblasts favors T. gondii stage conversion. In contrast, addition of the TCA cycle intermediate analogue dimethyl-α-ketoglutarate accelerated BAG1 mRNA expression without increasing tissue cyst formation by T. gondii in myoblasts but not in myotubes. Modulation of anaerobic glycolysis using sodium-L-lactate showed a trend of increased T. gondii bradyzoite differentiation in myoblasts only. Lower PPP activity as observed in myotubes led to reduced levels of NADPH and higher NADP+/NADPH ratios in myotubes than in myoblasts. Despite lower NADPH levels in myotubes, reactive oxygen species (ROS) levels were however nearly doubled in myoblasts than in myotubes. Modulation of endogenous ROS using the antioxidant N-acetyl cysteine inhibited T. gondii bradyzoite differentiation in both myotubes and myoblasts. Interestingly, inducing ROS in myotubes and myoblasts using the oxidants luperox or H2O2 accelerated T. gondii bradyzoite differentiation in both cell types. Thus, physiological concentration of endogenous ROS as observed in myotubes but not myoblasts might favor T. gondii stage conversion in myotubes.
Modulation of the host cell cycle of T. gondii-infected myotubes and myoblasts using pharmacological inhibitors aphidicolin and mimosine accelerated BAG1 mRNA expression in T. gondii within SkMCs but only partially induced tissue cyst formation. Furthermore, complete halting of parasite replication by higher concentrations of these inhibitors suggested a possibly direct effect on the parasites. To specifically modulate the host cell cycle and since myotubes showed upregulation of the cell cycle inhibitor p21Waf1/Cip1 as compared to myoblasts, p21 was inhibited in SkMCs using RNA interference. Knock-down of p21 in myoblasts sustained cell cycle progression and inhibited differentiation of myoblasts into myotubes. However, host cell p21 knock-down surprisingly even increased BAG1 mRNA expression by T. gondii but also strongly accelerated parasite replication in SkMCs. Thus, differentiation of SkMCs to myotubes promotes T. gondii bradyzoite formation independently of p21Waf1/Cip1.
Together these data unravel large differences in the transcriptomes, the central carbohydrate metabolism and the cell cycle regulation between myoblasts and myotubes before and after infection with T. gondii. Out of these differences, the lower PPP activities, the higher TCA cycle activities and the physiological concentrations of endogenous ROS as observed in mature myotubes can regulate T. gondii bradyzoite formation in SkMCs.||de