Genome-Wide CRISPR/Cas9 Screen Identifies New Genes Critical for Defense Against Oxidant Stress in Toxoplasma gondii.

Toxoplasma gondii is one of the most widespread apicomplexans and can cause serious infections in humans and animals. Its antioxidant system plays an important role in defending against oxidant stress imposed by the host. Some genes encoding the antioxidant enzymes of T. gondii have been identified; however, critical genes that function in response to reactive oxygen species (ROS) stress are still poorly understood. Here, we performed genome-wide CRISPR/Cas9 loss-of-function screening in the T. gondii RH strain to identify potential genes contributing to the ROS stress response. Under hydrogen peroxide treatment, 30 single guide RNAs targeting high-confidence genes were identified, including some known important antioxidant genes such as catalase and peroxiredoxin PRX3. In addition, several previously uncharacterized genes were identified, among which five hypothetical protein-coding genes, namely, HP1-HP5, were selected for further functional characterization. Targeted deletion of HP1 in T. gondii RH led to significant sensitivity to H2O2, suggesting that HP1 is critical for oxidative stress management. Furthermore, loss of HP1 led to decreased antioxidant capacity, invasion efficiency, and proliferation in vitro. In vivo results also revealed that the survival time of mice infected with the HP1-KO strain was significantly prolonged relative to that of mice infected with the wild-type strain. Altogether, these findings demonstrate that the CRISPR/Cas9 system can be used to identify potential genes critical for oxidative stress management. Furthermore, HP1 may confer protection against oxidative damage and contributes to T. gondii virulence in mice.

Antiaging Effects of Urolithin A on Replicative Senescent Human Skin Fibroblasts.

Although the health benefits attributed to urolithin A, such as anticancer, anti-inflammatory, and antioxidant effects, are based on numerous, diverse studies carried out in vitro, the biological effects of urolith A are still not entirely understood. In this study, we explored the biological effects of urolithin A using senescent human skin fibroblasts (HSFs) to determine whether urolithin A has any antiaging potential. Our results showed that urolithin A significantly increased type I collagen expression and reduced matrix metalloproteinase 1 (MMP-1) expression. Urolithin A also reduced intracellular reactive oxygen species, which may be partially due to activation of the Nrf2-mediated antioxidative response. These results indicate that urolithin A is a promising antiaging agent. Meanwhile, we noticed that the 50 µM urolithin A could cause changes in cell morphology and inhibition in cell proliferation, which were due to cell cycle arrest in G2/M phase. However, SA-ß-gal (senescence-associated ß-galactosidase) staining and γH2AX immunofluorescence staining showed cellular senescence status of HSFs did not change. Results of DAPI (4'6-diamidino-2-phenylindole) staining (no significant change) increased BCL2 gene expression and mitochondrial membrane potential (no significant change) after urolithin A treatment showed that the cells did not undergo apoptosis. These results provided further insights into the molecular mechanism of urolithin A. In conclusion, urolithin A showed a strong potential of antiaging.

Identification and characterization of an immunogenic antigen, enolase 2, among excretory/secretory antigens (ESA) of Toxoplasma gondii.

An immunogenic protein, enolase 2, was identified among the secreted excretory/secretory antigens (ESAs) from Toxoplasma gondii strain RH using immunoproteomics based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Enolase 2 was cloned, sequenced, and heterologously expressed. BLAST analysis revealed 75-96% similarity with enolases from other parasites. Immunoblotting demonstrated good immunoreactivity of recombinant T. gondii enolase (Tg-enolase 2) to T. gondii-infected animal serum. Purified Tg-enolase 2 was found to catalyze dehydration of 2-phospho-d-glycerate to phosphoenolpyruvate. In vitro studies revealed maximal activity at pH 7.5 and 37 °C, and activity was inhibited by K(+), Ni(2+), Al(3+), Na(+), Cu(2+) and Cr(3+). A monoclonal antibody against Tg-enolase 2 was prepared, 1D6, with the isotype IgG2a/κ. Western blotting revealed that 1D6 reacts with Tg-enolase 2 and native enolase 2, present among T. gondii ESAs. The indirect immunofluorescence assays showed that enolase 2 could be specifically detected on the growing T. gondii tachyzoites. Immunoelectron microscopy revealed the surface and intracellular locations of enolase 2 on T. gondii cells. In conclusion, our results clearly show that the enzymatic activity of T. gondii enolase 2 is ion dependent and that it could be influenced by environmental factors. We also provide evidence that enolase 2 is an important immunogenic protein of ESAs from T. gondii and that it is a surface-exposed protein with strong antigenicity and immunogenicity. Our findings indicate that enolase 2 could play important roles in metabolism, immunogenicity and pathogenicity and that it may serve as a novel drug target and candidate vaccine against T. gondii infection.