Toxoplasma gondii is a widespread intracellular pathogen infecting humans and a variety of animals. Previous studies have shown that Toxoplasma uses glucose and glutamine as the main carbon sources to support asexual reproduction, but neither nutrient is essential. Such metabolic flexibility may allow it to survive within diverse host cell types. Here, by focusing on the glycolytic enzyme pyruvate kinase (PYK) that converts phosphoenolpyruvate (PEP) into pyruvate, we found that Toxoplasma can also utilize lactate and alanine. We show that catabolism of all indicated carbon sources converges at pyruvate, and maintaining a constant pyruvate supply is critical to parasite growth. Toxoplasma expresses two PYKs: PYK1 in the cytosol and PYK2 in the apicoplast (a chloroplast relict). Genetic deletion of PYK2 did not noticeably affect parasite growth and virulence, which contrasts with the current model of carbon metabolism in the apicoplast. On the other hand, PYK1 was refractory to disruption. Conditional depletion of PYK1 resulted in global alteration of carbon metabolism, amylopectin accumulation, and reduced cellular ATP, leading to severe growth impairment. Notably, the attenuated growth of the PYK1-depleted mutant was partially rescued by lactate or alanine supplementation, and rescue by lactate required lactate dehydrogenase activity to convert it to pyruvate. Moreover, depletion of PYK1 in conjunction with PYK2 ablation led to accentuated loss of apicoplasts and complete growth arrest. Together, our results underline a critical role of pyruvate homeostasis in determining the metabolic flexibility and apicoplast maintenance, and they significantly extend our current understanding of carbon metabolism in T. gondii. IMPORTANCE Toxoplasma gondii infects almost all warm-blooded animals, and metabolic flexibility is deemed critical for its successful parasitism in diverse hosts. Glucose and glutamine are the major carbon sources to support parasite growth. In this study, we found that Toxoplasma is also competent in utilizing lactate and alanine and, thus, exhibits exceptional metabolic versatility. Notably, all these nutrients need to be converted to pyruvate to fuel the lytic cycle, and achieving a continued pyruvate supply is vital to parasite survival and metabolic flexibility. Although pyruvate can be generated by two distinct pyruvate kinases, located in cytosol and apicoplast, respectively, the cytosolic enzyme is the main source of subcellular pyruvate, and cooperative usage of pyruvate among multiple organelles is critical for parasite growth and virulence. These findings expand our current understanding of carbon metabolism in Toxoplasma gondii and related parasites while providing a basis for designing novel antiparasitic interventions.

Pichinde virus (PICV) is a bisegmented enveloped RNA virus that targets macrophages and dendritic cells (DCs) early in infection and induces strong innate and adaptive immunity in mice. We have developed a reverse genetics system to produce live recombinant PICV (strain P18) with a trisegmented RNA genome (rP18tri), which encodes all four PICV gene products and as many as two foreign genes. We have engineered the vector to express the green fluorescent protein (GFP) reporter gene (abbreviated as G in virus designations) and either the hemagglutination (HA [H]) or the nucleoprotein (NP [P]) gene of the influenza A/PR8 virus. The trisegmented viruses rP18tri-G/H and rP18tri-G/P showed slightly reduced growth in vitro and expressed HA and NP, respectively. Mice immunized with rP18tri-G/H were completely protected against lethal influenza virus challenge even 120 days after immunization. These rP18tri-based vectors could efficiently induce both neutralizing antibodies and antigen-specific T cell responses via different immunization routes. Interestingly, the immune responses were significantly increased upon a booster dose and remained at high levels even after three booster doses. In summary, we have developed a novel PICV-based live vaccine vector that can express foreign antigens to induce strong humoral and cell-mediated immunity and is ideal for a primeand-boost vaccination strategy. IMPORTANCE We have developed a novel Pichinde virus (PICV)-based live viral vector, rP18tri, that packages three RNA segments and encodes as many as two foreign genes. Using the influenza virus HA and NP genes as model antigens, we show that this rP18tri vector can induce strong humoral and cellular immunity via different immunization routes and can lead to protection in mice. Interestingly, a booster dose further enhances the immune responses, a feature that distinguishes this from other known live viral vectors. In summary, our study demonstrates a unique feature of this live rP18tri vector to be used as a novel vaccine platform for a prime-and-boost vaccination strategy.A renaviruses are enveloped RNA viruses with a bisegmented genome and mostly use rodents as natural hosts. There are at least 27 members that are geographically, serologically, and phylogenetically divided into Old World and New World arenaviruses (1). The prototypic lymphocytic choriomeningitis virus (LCMV) infection of mice has long been used as a valuable model with which to study viral persistence and virus-induced immunity and immunopathology (2, 3). The arenavirus is composed of a total of four genes on two genomic RNA segments in opposite orientations (1). The Z protein, produced from the large (L) genomic segment, is a small RING domain-containing matrix protein that mediates virus budding, regulates viral RNA synthesis, and mediates host immune suppression (4, 5). The large L protein (ϳ200 kDa), also encoded on the L segment, is the RNA-dependent RNA polymerase (RdRp), which is required for viral RNA synthesis (6). The glycoprotein (GPC), encoded...

Glycolysis was thought to be the major pathway of energy supply in both fast-replicating tachyzoites and slowly growing bradyzoites of Toxoplasma gondii. However, its biological significance has not been clearly verified. The genome of T. gondii encodes two lactate dehydrogenases (LDHs), 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, Δldh1 and Δldh1 Δldh2 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 Δldh1 Δldh2 mutants replicated significantly more slowly than wild-type parasites when cultured under conditions with physiological levels of oxygen (3%). In addition, Δldh1 Δldh2 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.

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