<i>Toxoplasma gondii</i>: Laboratory Maintenance and Growth

Khan, Asis; Grigg, Michael E.

doi:10.1002/cpmc.26

Cited by 44 publications

(27 citation statements)

References 60 publications

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“…All procedures were performed in accordance with the laws and conditions of the Guide for the Care and Use of Laboratory Animals and Institutional Animal Ethics Committee guidelines of Shanxi Medical University. T. gondii tachyzoites (RH strain) were kindly provided by the Peking University Health Science Center and were maintained via serial intraperitoneal passage in BALB/c mice [11].…”

Section: Methodsmentioning

confidence: 99%

Toxoplasma Gondii ROP17 Promotes Autophagy via the Bcl-2–Beclin 1 Pathway

Guo

Jia

Wang

et al. 2020

Preprint

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Abstract Background Toxoplasmosis caused by Toxoplasma gondii (T. gondii) has been and continues to be a major threat to public health worldwide. Proteins with serine/threonine (S/T) kinase activity, such as rhoptry organelle protein (ROP)16 and ROP18, are secreted by T. gondii rhoptries organelles that play important roles in modulating the survival and proliferation of Toxoplasma. ROP17 is another important effector kinase protein. Nevertheless, little is known about its function in regulating the interplay between T. gondii and host cells during infection. In this study, we investigated the function and the underlying mechanisms of ROP17 in vitro and in vivo. Methods The autophagy of cells in the small intestines of mice infected with T. gondii tachyzoite (RH strain) was detected by assessing the LC3B, Beclin 1 and p62 levels using immunohistochemical staining. ROP17 overexpression augmented starvation-induced autophagy in HEK 293T cells as measured by MDC staining, transmission electron microscopy (TEM), fluorescence microscopy and Western blot analysis. The interaction of ROP17 and Bcl-2 was confirmed using co-immunoprecipitation (co-IP) analysis. Moreover, the levels of phosphorylation of Bcl-2 and total Bcl-2 in the small intestines of mice infected with T. gondii tachyzoite were detected using immunohistochemical staining. In the end, Pearson coefficient was used to analyse correlations between the expression of ROP17 with the expression of autophagy markers LC3B, beclin 1 and p62, and with phosphorylation of Bcl-2 and Bcl-2. Results Infection with T. gondii resulted in autophagy of mouse small intestine cells accompanied by increased levels of LC3B and Beclin 1 and decreased levels of p62, which were measured by immunohistochemistry. Meantime, the elevated levels of Phosphorylated Bcl-2 and declined Bcl-2 in T. gondii-infected mouse small intestine tissue and in ROP17 overexpressing HEK 293T cells were detected by immunohistochemistry and Western blotting. ROP17 overexpression promoted serum starvation-induced cellular autophagy in the HEK293T cells, reflecting the augmentation of autophagosomes and GFP-LC3B puncta accompanied by increased levels of LC3B and Beclin 1 and decreased levels of p62. Moreover, we found that ROP17 interacted with Bcl-2 and phosphorylated and degraded it to liberate Beclin 1, thus promoting autophagy. Pearson coefficient analysis showed that there exsited strong positive correlations between the expression of ROP17 with the expression of LC3B, Beclin 1 and phosphorylation of Bcl-2, while strong negative correlations between the expression of ROP17 and the expression of p62 and Bcl-2. Conclusions Our findings indicate that ROP17 plays a pivotal role in maintaining Toxoplasma survival and proliferation in host cells by promoting cellular autophagy, which depends on the Beclin 1-Bcl-2 pathway.

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Section: Methodsmentioning

confidence: 99%

Toxoplasma Gondii ROP17 Promotes Autophagy via the Bcl-2–Beclin 1 Pathway

Guo

Jia

Wang

et al. 2020

Preprint

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“…Before being employed in the current study, the tachyzoites were peritoneally injected (5 × 10 4 parasites per mouse) into naive mice. The second generation of tachyzoites were then recovered from the mice (ensure they are unlysed), washed and resuspended to 5 × 10 6 parasites/ml in an invasion Endo buffer (100 ml physiological saline (NaCl) and 100 µL heparin (48,49). Transgenic parasites used were GFP-labeled and were therefore viewed under fluorescent microscope.…”

Section: Parasite Strainmentioning

confidence: 99%

CD209 C-Type Lectins Promote Host Invasion, Dissemination, and Infection of Toxoplasma gondii

Njiri

Zhang

et al. 2020

Front. Immunol.

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Toxoplasma gondii, the causative agent of toxoplasmosis and a major opportunistic parasite associated with AIDS, is able to invade host cells of animals and humans. Studies suggested that the ability of host invasion by the tachyzoite, the infectious form of T. gondii, is essential for the pathogenicity to promote its dissemination to other parts of animal hosts. However, the detailed molecular mechanisms for host invasion and dissemination of the parasites are not clear. On the other hand, viruses and bacteria are able to interact with and hijack DC-SIGN (CD209) C-type lectin on antigen presenting cells (APCs), such as dendritic cells and macrophages as the Trojan horses to promote host dissemination. In this study, we showed that invasion of T. gondii into host cells was enhanced by this parasite-CD209 interaction that were inhibited by ligand mimickingoligosaccharides and the anti-CD209 antibody. Furthermore, covering the exposures of DC-SIGN by these oligosaccharides reduced parasite burden, host spreading and mortality associated with T. gondii infection. These results suggested that interaction of T. gondii to APCs expressing DC-SIGN might promote host dissemination and infection. Can the blockage of this interaction with Mannan and/or anti-CD209 antibody be developed as a prevention or treatment method for T. gondii infection?

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“…We implement our network on TensorFlow framework [Keeton and Roscoe, 2016] with Tesla K40C and 128G memory in Ubuntu 16.04 system, and use the Adam solver [Kingma and Ba, 2014] for FCGAN network with a learning rate of 2e-4 and closely follow the settings of Cycle GAN and Vgg Network to train generators, discriminators and feature extractors. After jointly training for 4000 epoches, we apply early stop when the fuzzy loss no longer decreases for about 5 epochs (usually takes 4000 jointly training epochs to reach a desired point).…”

Section: Training Detailsmentioning

confidence: 99%

“…T oxoplasma gondii (T.gondii) is an ubiquitous, single cell protozoan parasite that can infect all warm-blooded animals as well as one-third of human population worldwide [Khan and Grigg, 2017]. Most infections in humans are life-long and several studies have suggested that such infections may contribute to severe neurological and psychiatric symptoms.…”

Section: Introductionmentioning

confidence: 99%

A Novel Transfer Learning Approach for Toxoplasma Gondii Microscopic Image Recognition by Fuzzy Cycle Generative Adversarial Network

Lara

et al. 2019

Preprint

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T oxoplasma gondii, one of the world's most common parasites, can infect all types of warm-blooded animals, including one-third of the world's human population. Most of current routine methods of diagnosis are costly, time-consuming, and laborintensive. The parasite can be directly observed under the microscope in tissue or spinal fluid samples, and this form of testing is cheap, but used less frequently because of the difficulty in operation, and needs well trained professionals. Nevertheless, observation of parasites in patient or animal specimens under the microscope is still performed by a large number of laboratories. The identification of Toxoplasma parasite with costeffectiveness solution is still a health challenge in developing countries. Here, we propose an Artificial Intelligence (AI)-powered microscopic image analysis method for T oxoplasma gondii. The method uses Fuzzy Cycle Generative Adversarial Network (FCGAN) with transfer learning, by applying knowledge gained by the parasitologists that T oxoplasma gondii is in banana or crescent shaped form. Our approach aims to build connection between micro and macro associated objects by embedding fuzzy C-means cluster algorithm into Cycle GAN. We show the high accuracy and effectiveness of our approach in the newly collected 28,130 unlabeled microscopic images from Toxoplasma infection samples.

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Toxoplasma gondii: Laboratory Maintenance and Growth

Cited by 44 publications

References 60 publications

Toxoplasma Gondii ROP17 Promotes Autophagy via the Bcl-2–Beclin 1 Pathway

Toxoplasma Gondii ROP17 Promotes Autophagy via the Bcl-2–Beclin 1 Pathway

CD209 C-Type Lectins Promote Host Invasion, Dissemination, and Infection of Toxoplasma gondii

A Novel Transfer Learning Approach for Toxoplasma Gondii Microscopic Image Recognition by Fuzzy Cycle Generative Adversarial Network

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