Abstract
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.
Highlights
Toxoplasma gondii, an obligate intracellular protozoan pathogen, has the ability to infect a wide range of warm-blooded animals and humans (Pappas et al, 2009; Dubey, 2010; Andiappan et al, 2014; Chemoh et al, 2015; Liu et al, 2015)
We have investigated the role of 15 rhoptry organelle proteins (ROPs) genes in the type I T. gondii (RH strain), to determine whether they play critical roles in T. gondii-host interaction, on replication in human cells and virulence in the BALB/c mice
To assess the contribution of 15 ROPs to T. gondii infection, CRISPR-Cas9 system was used to disrupt these genes via the insertion of DHFR∗-Ts at the sgRNA-targeted coding region in the 15 endogenous ROP loci (Figure 1A)
Summary
Toxoplasma gondii, an obligate intracellular protozoan pathogen, has the ability to infect a wide range of warm-blooded animals and humans (Pappas et al, 2009; Dubey, 2010; Andiappan et al, 2014; Chemoh et al, 2015; Liu et al, 2015). Intracellular survival of this parasite is critically dependent on its ability to actively invade surrogate host cell, establish a replication-permissive vacuole and avoid host cell immune defenses (Melo et al, 2011; Hunter and Sibley, 2012) Another important feature of T. gondii is the dramatic difference between strains in terms of their virulence in animal models (Su et al, 2003, 2012; Lorenzi et al, 2016) and in human infections (Niedelman et al, 2012). Many ROPs contain a conserved serine/threonine protein kinase domain and may function as kinases or pseudokinases, which include only part of the catalytic triad, possibly modifying host cell pathways by phosphorylation of specific targets (Melo et al, 2011; Hunter and Sibley, 2012)
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