Systems-Based Analysis of the Sarcocystis neurona Genome Identifies Pathways That Contribute to a Heteroxenous Life Cycle

Blazejewski, Tomasz; Nursimulu, Nirvana; Pszenny, Viviana; Dangoudoubiyam, Sriveny; Namasivayam, Sivaranjani; Chiasson, Melissa A.; Chessman, Kyle; Tonkin, Michelle; Swapna, Lakshmipuram S.; Hung, Stacy S.; Bridgers, Joshua; Ricklefs, Stacy M.; Boulanger, Martin J.; Dubey, Jitender P.; Porcella, Stephen F.; Kissinger, Jessica C.; Howe, Daniel K.; Grigg, Michael E.; Parkinson, John

The S. neurona annotation effort predicted a gene for alphaglucosidase (EC 3.2.1.20) (see Text S1 and Fig. S4 in the supplemental material). Since conversion of sucrose to fructose and glucose by alpha-glucosidase would add functionality to the metabolic reconstruction, we tested in silico for its potential impact on growth. S. neurona was predicted to grow faster in the presence of sucrose and the absence of glucose than in the presence of glucose and the absence of sucrose (doubling time of 11.4 h versus 13.8 h). This is due, in part, to an increase in the concentration of fructose-6-phosphate caused by the action of hexokinase (EC 2.7. 1.1, Fig. 6C ). Consequently, under conditions of sucrose uptake, knockout of enzymes involved in glycolysis has a greater impact on the growth rate than conditions of glucose uptake (see Table S2 in the supplemental material). When we examined the impact of combining access to different carbohydrates, our simulations suggested that S. neurona has the capacity to significantly enhance its growth by utilizing fructose, with an even greater effect when sucrose is used as an additional energy source (Fig. 6D) . For example, while fructose supplementation alters parasite growth to 120%, supplementation with sucrose extends parasite growth to 180% of its original rate. Interestingly, glucose-6phosphate isomerase, the enzyme responsible for the conversion of glucose-6-phosphate to fructose-6-phosphate, operates in the reverse direction under glucose or sucrose uptake conditions. When only sucrose is available, more glucose-6-phosphate is produced from the conversion of fructose-6-phosphate, which is pre-dicted to feed into other pathways (e.g., the pentose phosphate pathway), resulting in the elevated production of NADPH and an increased growth rate. Importantly, glycolysis is utilized more when sucrose is available, so there is less reliance on the TCA cycle. Furthermore, the breakdown of sucrose makes fructose available for the synthesis of other key metabolites (e.g., branched-chain amino acids), decreasing the parasite's dependency on the TCA cycle for their production. Hence, the deletion of individual TCA cycle reactions has a greater impact on the growth rate in the presence of glucose than in the presence of sucrose (Fig. 6C) .