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.