Analysis of the virulence determination mechanisms in a local Toxoplasma strain (T.gHB1) isolated from central China

Zhang, Weichao; Li, Longjiao; Xia, Ningbo; Zhou, Yanqin; Fang, Rui; He, Li; Hu, Min; Shen, Bang; Zhao, Junlong

doi:10.1007/s00436-016-5141-z

Cited by 10 publications

(9 citation statements)

References 42 publications

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“…Phylogenetic analyses of T. gondii isolates based on introns which are selectively neutral loci are thought to provide an unbiased estimation, while secretory antigens, known to be functionally related to virulence and infectivity in the host (GRA6, GRA7 and ROP18) (Shwab et al, 2016;Zhang et al, 2016), are also considered capable of revealing great genetic diversity because they are frequently under selective pressure (Miller et al, 2004;Pena et al, 2008;Sundar et al, 2008). However, on the basis of these genes, phylogenetic analysis found that the avirulent TgCtwh6and virulent Chinese I (e.g.…”

Section: Discussionmentioning

confidence: 99%

Genetic analyses of Chinese isolates of Toxoplasma gondii reveal a new genotype with high virulence to murine hosts

Gao

Xie

et al. 2017

Veterinary Parasitology

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A great deal of evidence demonstrates that a strongly clonal population structure of Toxoplasma gondii strains exists in humans and animals in North America and Europe, while the strains from South America are genetically separate and more diverse. Potential differences in virulence between different strains mean that an understanding of strain diversity is important to human and animal health. However, to date, only one predominant genotype, ToxoDB#9 (Chinese I), and a few other genotypes, including ToxoDB#205, have been identified in China. By using DNA sequence-based phylogenetic analyses, we have re-evaluated the population structure of T. gondii strains collected from China and compared them with other global strains. Based on phylogenetic analysis of restriction fragment length polymorphisms, multilocus sequence typing and intron sequences from T. gondii, we propose that the Chinese isolates described as Chinese I are divided into two groups called Chinese I and Chinese III. Our results demonstrate that significant differences were found in mouse mortality caused by some Chinese strains, and also the archetypal I, II, III strains in mice. Furthermore, a comparison of cyst loading in the brains of infected rats showed some Chinese strains to be capable of a high degree of cyst formation. Furthermore we show that genotyping using neutral genetic markers may not be a useful predictor of pathogenic phenotypes.

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Section: Discussionmentioning

confidence: 99%

Genetic analyses of Chinese isolates of Toxoplasma gondii reveal a new genotype with high virulence to murine hosts

Gao

Xie

et al. 2017

Veterinary Parasitology

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“…The infected cell cultures were monitored microscopically over 7 days after infection. Then, the cells were fixed with 4% paraformaldehyde and stained for 10 min with 2% crystal violet to visualize and count plaques formed by the growing parasites by using inverted microscope (Wang et al, 2016b; Zhang et al, 2016). …”

Section: Methodsmentioning

confidence: 99%

Functional Characterization of Rhoptry Kinome in the Virulent Toxoplasma gondii RH Strain

Wang

Elsheikha

et al. 2017

Front. Microbiol.

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Toxoplasma gondii is an obligatory intracellular apicomplexan protozoan which can infect any warm-blooded animal and causes severe diseases in immunocompromised individuals or infants infected in utero. The survival and success of this parasite require that it colonizes the host cell, avoids host immune defenses, replicates within an appropriate niche, and exits the infected host cell to spread to neighboring non-infected cells. All of these processes depend on the parasite ability to synthesis and export secreted proteins. Amongst the secreted proteins, rhoptry organelle proteins (ROPs) are essential for the parasite invasion and host cell manipulation. Even though the functions of most ROPs have been elucidated in the less virulent T. gondii (type II), the roles of ROPs in the highly virulent type I strain remain largely un-characterized. Herein, we investigated the contributions of 15 ROPs (ROP10, ROP11, ROP15, ROP20, ROP23, ROP31, ROP32, ROP33, ROP34, ROP35, ROP36, ROP40, ROP41, ROP46, and ROP47) to the infectivity of the high virulent type I T. gondii (RH strain). Using CRISPR-Cas9, these 15 ROPs genes were successfully disrupted and the effects of gene knockout on the parasite’s ability to infect cells in vitro and BALB/c mice in vivo were investigated. These results showed that deletions of these ROPs did not interfere with the parasite ability to grow in cultured human foreskin fibroblast cells and did not significantly alter parasite pathogenicity for BALB/c mice. Although these ROPs did not seem to be essential for the acute infectious stage of type I T. gondii in the mouse model, they might have different functions in other intermediate hosts or play different roles in other life cycle forms of this parasite due to the different expression patterns; this warrants further investigations.

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“…Also, this increased efficiently has made it possible to use non-laboratory adapted strains for genetic studies which were previously difficult to modify 13 . Even though in its infancy, CRISPR/Cas9 has already been used to make gene disruptions 2 13 17 18 48 49 , tag genes 17 , and make gene knockouts 16 19 50 51 52 53 54 in T. gondii , promising to be a useful tool for many other studies in the future.…”

Section: Discussionmentioning

confidence: 99%

QTL Mapping and CRISPR/Cas9 Editing to Identify a Drug Resistance Gene in <em>Toxoplasma gondii</em>

Shen

Powell

Behnke

2017

JoVE

Self Cite

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Scientific knowledge is intrinsically linked to available technologies and methods. This article will present two methods that allowed for the identification and verification of a drug resistance gene in the Apicomplexan parasite Toxoplasma gondii, the method of Quantitative Trait Locus (QTL) mapping using a Whole Genome Sequence (WGS) -based genetic map and the method of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 -based gene editing. The approach of QTL mapping allows one to test if there is a correlation between a genomic region(s) and a phenotype. Two datasets are required to run a QTL scan, a genetic map based on the progeny of a recombinant cross and a quantifiable phenotype assessed in each of the progeny of that cross. These datasets are then formatted to be compatible with R/qtl software that generates a QTL scan to identify significant loci correlated with the phenotype. Although this can greatly narrow the search window of possible candidates, QTLs span regions containing a number of genes from which the causal gene needs to be identified. Having WGS of the progeny was critical to identify the causal drug resistance mutation at the gene level. Once identified, the candidate mutation can be verified by genetic manipulation of drug sensitive parasites. The most facile and efficient method to genetically modify T. gondii is the CRISPR/Cas9 system. This system comprised of just 2 components both encoded on a single plasmid, a single guide RNA (gRNA) containing a 20 bp sequence complementary to the genomic target and the Cas9 endonuclease that generates a double-strand DNA break (DSB) at the target, repair of which allows for insertion or deletion of sequences around the break site. This article provides detailed protocols to use CRISPR/Cas9 based genome editing tools to verify the gene responsible for sinefungin resistance and to construct transgenic parasites.

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Analysis of the virulence determination mechanisms in a local Toxoplasma strain (T.gHB1) isolated from central China

Cited by 10 publications

References 42 publications

Genetic analyses of Chinese isolates of Toxoplasma gondii reveal a new genotype with high virulence to murine hosts

Genetic analyses of Chinese isolates of Toxoplasma gondii reveal a new genotype with high virulence to murine hosts

Functional Characterization of Rhoptry Kinome in the Virulent Toxoplasma gondii RH Strain

QTL Mapping and CRISPR/Cas9 Editing to Identify a Drug Resistance Gene in <em>Toxoplasma gondii</em>

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