PP2Acα-B'/PR61 Holoenzyme of Toxoplasma gondii Is Required for the Amylopectin Metabolism and Proliferation of Tachyzoites.

Here, we report that the inhibition of the PP2A subfamily by okadaic acid results in an accumulation of polysaccharides in the acute infection stage (tachyzoites) of Toxoplasma gondii, which is a protozoan of global zoonotic importance and a model for the apicomplexan parasites. The loss of the catalytic subunit α of PP2A (ΔPP2Acα) in RHΔku80 leads to the polysaccharide accumulation phenotype in the base of tachyzoites as well as residual bodies and significantly compromises the intracellular growth in vitro and the virulence in vivo. A metabolomic analysis revealed that the accumulated polysaccharides in ΔPP2Acα are derived from interrupted glucose metabolism, which affects the production of ATP and energy homeostasis in the T. gondii knockout. The assembly of the PP2Acα holoenzyme complex involved in the amylopectin metabolism in tachyzoites is possibly not regulated by LCMT1 or PME1, and this finding contributes to the identification of the regulatory B subunit (B'/PR61). The loss of B'/PR61 results in the accumulation of polysaccharide granules in the tachyzoites as well as reduced plaque formation ability, exactly the same as ΔPP2Acα. Taken together, we have identified a PP2Acα-B'/PR61 holoenzyme complex that plays a crucial role in the carbohydrate metabolism and viability in T. gondii, and its deficiency in function remarkably suppresses the growth and virulence of this important zoonotic parasite both in vitro and in vivo. Hence, rendering the PP2Acα-B'/PR61 holoenzyme functionless should be a promising strategy for the intervention of Toxoplasma acute infection and toxoplasmosis. IMPORTANCE Toxoplasma gondii switches back and forth between acute and chronic infections, mainly in response to host immunologic status, which is characterized by flexible but specific energy metabolism. Polysaccharide granules are accumulated in the acute infection stage of T. gondii that have been exposed to a chemical inhibitor of the PP2A subfamily. The genetic depletion of the catalytic subunit α of PP2A leads to this phenotype and significantly affects the cell metabolism, energy production, and viability. Further, a regulatory B subunit PR61 is necessary for the PP2A holoenzyme to function in glucose metabolism and in the intracellular growth of T. gondii tachyzoites. A deficiency of this PP2A holoenzyme complex (PP2Acα-B'/PR61) in T. gondii knockouts results in the abnormal accumulation of polysaccharides and the disruption of energy metabolism, suppressing their growth and virulence. These findings provide novel insights into cell metabolism and identify a potential target for an intervention against a T. gondii acute infection.

Redundant targeting signals of the apicoplast-resident protein TgMnmA in Toxoplasma gondii involve trans-organellar function.

The apicoplast, which is the result of secondary endosymbiosis, is a distinctive subcellular organelle and a crucial therapeutic target for apicomplexan parasites. The majority of apicoplast-resident proteins are encoded by the nuclear genome and target the apicoplast via bipartite targeting signals consisting of a signal peptide and a transit peptide. The properties and functions of these peptides are poorly understood, which hinders the identification of apicoplast proteins and the study for plastid evolution. Here, the targeting signals of the recently discovered apicoplast tRNA thiouridylase TgMnmA of Toxoplasma gondii were analyzed. Our data using a reporter (the enhanced green fluorescent protein) fused with individual fragments containing various numbers of its N-terminal amino acids unequivocally revealed that the first 28 amino acids of TgMnmA functioned as a signal peptide for cellular secretion. The N-terminal 150 amino acids were sufficient to direct the fusion protein to the apicoplast, whereas its deletion caused the fusion protein to be localized to the mitochondrion. Our data further demonstrated that the apicoplast, rhoptry, and mitochondrion shared similar targeting signals, indicating that the apicoplast localization peptide was trans-organellar in function. In addition, the apicoplast localization peptide was important for the healthy proliferation of tachyzoites. In conclusion, the targeting signals of the nucleus-encoded apicoplast-targeted protein TgMnmA have been mapped out and the importance of this localization peptide has been elucidated in the current study.

Host autophagy limits Toxoplasma gondii proliferation in the absence of IFN-γ by affecting the hijack of Rab11A-positive vesicles.

Introduction: Autophagy has been recognized as a bona fide immunological process. Evidence has shown that this process in IFN-γ stimulated cells controls Toxoplasma gondii proliferation or eliminates its infection. However, little is known about the effect of T. gondii infection on the host cell autophagy in the absence of IFN-γ. Methods: Multiple autophagy detection methods and CRISPR/CAS9 technology were used to study T. gondii-induced autophagy in HeLa and several other mammalian cell lines. Results: Here, we report increased LC3 II, autophagosome-like membrane structures, enhanced autophagic flux, and decreased lysosomes in a range of mammalian cell lines without IFN-γ treatment after T. gondii infection. Specifically, disruption of host atg5 (a necessary gene for autophagy) in HeLa cells promoted the intracellular replication of T. gondii, with the transcript level of rab11a increased, compared with that in wild-type cells. Further, after T. gondii infection, the abundance of Rab11A remained stable in wild-type HeLa cells but decreased in atg5 -/- mutant. Disruption of rab11a in the HeLa cells compromised the proliferation of T. gondii, and increased the transcription of gra2 in the parasite. Compared to the T. gondii wild-type RH∆ku80 strain, the ∆gra2 mutant induces enhanced host autophagy in HeLa cells, and results in slower replication of the parasite. Discussion: Collectively, these results indicate that host cell autophagy can limit T. gondii proliferation in an IFN-γ independent manner, possibly by affecting the hijack of host Rab11A-positive vesicles by the parasite which involved TgGRA2. The findings provide novel insights into T. gondii infection in host cells and toxoplasmosis research.

The first apicoplast tRNA thiouridylase plays a vital role in the growth of Toxoplasma gondii.

Toxoplasmosis caused by the protozoan Toxoplasma gondii is one of the most common parasitic diseases in humans and almost all warm-blooded animals. Lys, Glu, and Gln-specific tRNAs contain a super-modified 2-thiourea (s2U) derivatives at the position 34, which is essential for all living organisms by maintaining the structural stability and aminoacylation of tRNA, and the precision and efficiency of codon recognition during protein translation. However, the enzyme(s) involved in this modification in T. gondii remains elusive. In this report, three putative tRNA-specific 2-thiolation enzymes were identified, of which two were involved in the s2U34 modification of tRNALys, tRNAGlu, and tRNAGln. One was named TgMnmA, an apicoplast-located tRNA-specific 2-thiolation enzyme in T. gondii. Knockout of TgMnmA showed that this enzyme is important for the lytic cycle of tachyzoites. Loss of TgMnmA also led to abnormities in apicoplast biogenesis and severely disturbed apicoplast genomic transcription. Notably, mice survived from the infection with 10 TgMnmA-KO RH tachyzoites. These findings provide new insights into s2U34 tRNA modification in Apicomplexa, and suggest TgMnmA, the first apicoplast tRNA thiouridylase identified in all apicomplexans, as a potential drug target.

Development of an Immunochromatographic Test Based on Rhoptry Protein 14 for Serological Detection of Toxoplasma gondii Infection in Swine.

Toxoplasma gondii, a worldwide distributed apicomplexan protozoan, can infect almost all warm-blooded animals and may cause toxoplasmosis. In order to provide a point-of-care detection method for T. gondii infection, an immunochromatographic test (ICT) was established. The proposed test uses recombinant T. gondii rhoptry protein 14 (ROP14) conjugated with 20 nm gold particles, recombinant protein A as the detection line and monoclonal antibody TgROP14-5D5 as the control line. The specificity, sensitivity, positive predictive value, negative predictive value and stability of this new ICT were evaluated. rTgROP14 was specifically recognized by positive serum of T. gondii but not negative serum. mAb TgROP14-5D5 showed higher specific recognition of T. gondii antigens and was therefore selected for subsequent colloidal gold strip construction. The new ICT based on TgROP14 exhibited good diagnostic performance with high specificity (86.9%) and sensitivity (90.9%) using IHA as a "reference standard". Among 436 field porcine sera, ICT and IHA detected 134 (30.7%) and 99 (22.7%) positive samples, respectively. The relative agreement was 87.8%. These data indicate that this new ICT based on TgROP14 is a suitable candidate for routine testing of T. gondii in the field.

Synthesis and application of poly(fluorene-alt-naphthalene diimide) as an n-type polymer for all-polymer solar cells.

A new alternating copolymer of fluorene and naphthalene diimide, PF-NDI, was synthesized and characterized. The highest power conversion efficiency of all-polymer solar cells based on P3HT:PF-NDI reached 1.63% with a relatively high fill factor of 0.66 by using 1,8-diiodooctane as a solvent additive to optimize the mixing morphology.

Formation of uniform ferrocenyl-terminated monolayer covalently bonded to Si using reaction of hydrogen-terminated Si(111) surface with vinylferrocene/n-decane solution by visible-light excitation.

Electrochemically active self-assembled monolayers (SAM) have been successfully fabricated with atomic-scale uniformity on a silicon (Si)(111) surface by immobilizing vinylferrocene (VFC) molecules through Si-C covalent bonds. The reaction of VFC with the hydrogen-terminated Si (H-Si)(111) surface was photochemically promoted by irradiation of visible light on a H-Si(111) substrate immersed in n-decane solution of VFC. We found that aggregation and polymerization of VFC was avoided when n-decane was used as a solvent. Voltammetric quantification revealed that the surface density of ferrocenyl groups was 1.4×10(-10) mol cm(-2), i.e., 11% in substitution rate of Si-H bond. VFC-SAMs were then formed by the optimized preparation method on n-type and p-type Si wafers. VFC-SAM on n-type Si showed positive photo-responsivity, while VFC-SAM on p-type Si showed negative photo-responsivity.