Biological Functions Of Glycolysis During Toxoplasma Gondii Growth And Development

Toxoplasma gondii is an important zoonotic pathogen infecting one-third of the world's population and numerous animals,causing significant healthcare burden and socioeconomic problems.T.gondii has complex life cycle and it can convert between tachyzoites and bradyzoites in intermediate hosts.Tachyzoites are responsible for the acute phase of infection and may lead to clinical symptoms,whereas bradyzoites are responsible for the lifelong chronic infection.The interconversion between tachyzoites and bradyzoites plays a critical role in pathogenesis,as well as transmission of T.gondii.Studying the growth and metabolism of tachyzoites and bradyzoites not only refines our understanding of T.gondii life cycle,but also provides significant insights to the design of anti-toxoplasmosis drug and vaccine.Glycolytic enzymes are differentially expressed in tachyzoites and bradyzoites.What are the benefits of having more than one isoform and what are the biological functions? The ubiquitous pathogen T.gondii can use glucose or glutamine as main carbon sources for rapid growth.Such metabolic flexibility is thought to make glycolysis dispensable by glutaminolysis.Then,what is the role of glycolysis in this parasite? To analyze the biological functions of glycolysis during T.gondii growth and development,we performed a systematic genetic,metabonomics and biochemical dissection of the LDH and PYK enzymes in T.gondii.(1)Critical roles and mechanism of lactate fermentation for parasite growth in vivoThe genome of T.gondii encodes two lactate dehydrogenases(LDH),which are differentially expressed in tachyzoites and bradyzoites.In this study,we knocked out the two LDH genes individually and in combination,and found that neither gene was required for tachyzoite growth in vitro under standard growth conditions.However during infection in mice,?ldh1 and ?ldh1?ldh2 mutants were unable to propagate and displayed significant virulence attenuation and cyst formation defects.LDH2 only played minor roles in these processes.To further elucidate the mechanisms underlying the critical requirement of LDH in vivo,we found that ?ldh1?ldh2 mutants replicated significantly more slowly than wild type parasites when cultured under conditions with physiological levels of oxygen(3%).In addition,?ldh1?ldh2 mutants were more susceptible to the oxidative phosphorylation inhibitor oligomycin A.Together these results suggest that lactate fermentation is critical for parasite growth under physiological conditions,likely because energy production from oxidative phosphorylation is insufficient when oxygen is limited and lactate fermentation becomes a key supplementation.(2)A lactate fermentation mutant of Toxoplasma stimulates protective immunity against acute and chronic toxoplasmosisWe recently discovered that the Toxoplasma mutant lacking both lactate dehydrogenases LDH1 and LDH2(?ldh)grew well in vitro but was unable to propagate in mice,making it a good live vaccine candidate.Here we tested the protection efficacy of ME49?ldh using a mouse model.Vaccinated mice were efficiently protected from the lethal challenge of a variety of wild type strains,including type 1 strain RH,type 2 strain ME49,type 3 strain VEG,and a field isolate of Chinese 1.The protection efficacies of a single vaccination were nearly 100% for most cases and it worked well against the challenges of both tachyzoites and tissue cysts.To examine the efficiency of even longer time protection,ME49?ldh vaccinated mice were challenged with WT parasites 125 days post-vaccination.The results showed that the immune protection against ME49 and VEG challenge was still 100%.Re-challenging parasites were unable to propagate in vaccinated mice,nor did they make tissue cysts.High levels of Toxoplasma specific IgG were produced 30 days after immunization and stayed high during the whole tests(at least 125 days).However passive immunization of na?ve mice with sera from vaccinated mice did reduce parasite propagation,but the overall protection against parasite infections was rather limited.On the other hand,?ldh immunization evoked elevated levels of Th1 cytokines like INF-? and IL-12,at early time points.In addition,splenocytes extracted from immunized mice were able to induce quick and robust INF-? and other proinflammatory cytokine production upon T.gondii antigen stimulation.INF-? is key to activate cellular immune clearance of T.gondii.Together these results suggest that cellular immune responses are the main contributors to the protective immunity elicited by ?ldh vaccination,and humoral immunity also contributes partially.(3)Critical role of pyruvate homeostasis to metabolic flexibility and parasite growth in Toxoplasma gondiiGlucose transporter(GT1)and hexokinase(HK)are dispensable for tachyzoite survival.The parasite no longer catabolizes host-derived glucose through glycolysis in the absence of GT1 or HK(glycolysis-deficient),and becomes strictly dependent on glutamine to sustain its bioenergetic requirements.Functional analysis of Toxoplasma lactate dehydrogenases suggests that lactate fermentation is critical for parasite growth under physiological conditions.The phenotypic discrepancy between the LDH-null mutants and the GT1/HK-knockout strains prompted us to revisit the significance of glycolysis in T.gondii.We focused on the biological functions of pyruvate kinase(PYK).Toxoplasma expresses two distinct PYKs,of which PYK1 localizes in the cytosol and PYK2 resides in the apicoplast.Inactivation of PYK1 that generated pyruvate resulted in global alteration of carbon metabolism,reduced cellular ATP and accumulated amylopectin,leading to severe growth defects that could be partially rescued by lactate or alanine.PYK2 is dispensable for the parasite reproduction.However,inactivation of PYK1 and PYK2 coincides with loss of apicoplast and completely blocked parasite growth.Here we showed that Toxoplasma did display unprecedented metabolic plasticity capable of utilizing lactate and alanine besides glucose and glutamine.However all these nutrients needed to be converted to pyruvate to support parasite growth.Pyruvate was at the center of Toxoplasma carbon metabolism and the pyruvate production step of glycolysis was absolutely crucial.Taken together,our work of LDH project found that lactate fermentation is critical for parasite growth under physiological conditions and that mutants lacking LDH are good candidates for live vaccines.By analyzing the biological functions of PYK,our results advocated that pyruvate homeostasis is key for the success of metabolic flexibility and parasite survival.Our results will shed new lights on the molecular mechanisms of toxoplasma parasitism,and also provide new ideas for the design of anti-toxoplasmic drugs and vaccines.