Conditional genome engineering in Toxoplasma gondii uncovers alternative invasion mechanisms
TL;DR: Using a new single-vector strategy that allows ligand-dependent, efficient removal of a gene of interest, three knockouts of apicomplexan genes considered essential for host-cell invasion are generated.
Abstract: We established a conditional site-specific recombination system based on dimerizable Cre recombinase-mediated recombination in the apicomplexan parasite Toxoplasma gondii. Using a new single-vector strategy that allows ligand-dependent, efficient removal of a gene of interest, we generated three knockouts of apicomplexan genes considered essential for host-cell invasion. Our findings uncovered the existence of an alternative invasion pathway in apicomplexan parasites.
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TL;DR: The RNA-guided Cas9 nuclease is used to efficiently generate knockouts without selection, and to introduce point mutations and epitope tags into the T. gondii genome, which will streamline the functional analysis of parasite genes and enable high-throughput engineering of their genomes.
Abstract: Toxoplasma gondii is a parasite of humans and animals, and a model for other apicomplexans including Plasmodium spp., the causative agents of malaria. Despite many advances, manipulating the T. gondii genome remains labor intensive, and is often restricted to lab-adapted strains or lines carrying mutations that enable selection. Here, we use the RNA-guided Cas9 nuclease to efficiently generate knockouts without selection, and to introduce point mutations and epitope tags into the T. gondii genome. These methods will streamline the functional analysis of parasite genes and enable high-throughput engineering of their genomes.
248 citations
Cites background from "Conditional genome engineering in T..."
...gondii rely on the generation of complex constructs for homologous recombination, which yield significant efficiency only in specific parasite strains and require antibiotic selection [1,2,18]....
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TL;DR: These effectors highlight novel mechanisms by which T. gondii has learned to harness host signaling to favor intracellular survival and will guide future studies designed to uncover the additional complexity of this intricate host-pathogen interaction.
Abstract: SUMMARY Early electron microscopy studies revealed the elaborate cellular features that define the unique adaptations of apicomplexan parasites. Among these were bulbous rhoptry (ROP) organelles and small, dense granules (GRAs), both of which are secreted during invasion of host cells. These early morphological studies were followed by the exploration of the cellular contents of these secretory organelles, revealing them to be comprised of highly divergent protein families with few conserved domains or predicted functions. In parallel, studies on host-pathogen interactions identified many host signaling pathways that were mysteriously altered by infection. It was only with the advent of forward and reverse genetic strategies that the connections between individual parasite effectors and the specific host pathways that they targeted finally became clear. The current repertoire of parasite effectors includes ROP kinases and pseudokinases that are secreted during invasion and that block host immune pathways. Similarly, many secretory GRA proteins alter host gene expression by activating host transcription factors, through modification of chromatin, or by inducing small noncoding RNAs. These effectors highlight novel mechanisms by which T. gondii has learned to harness host signaling to favor intracellular survival and will guide future studies designed to uncover the additional complexity of this intricate host-pathogen interaction.
240 citations
Cites background from "Conditional genome engineering in T..."
...However, it has also been argued that there may be alternative pathways for entry, as these mutants show some residual, albeit limited, ability to enter cells (107, 108)....
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TL;DR: The discovery of the principles that govern gliding motility, the characterization of the molecular machinery involved, and its impact on parasite invasion and egress from infected cells are discussed.
Abstract: Protozoan parasites have developed elaborate motility systems that facilitate infection and dissemination. For example, amoebae use actin-rich membrane extensions called pseudopodia, whereas Kinetoplastida are propelled by microtubule-containing flagella. By contrast, the motile and invasive stages of the Apicomplexa - a phylum that contains the important human pathogens Plasmodium falciparum (which causes malaria) and Toxoplasma gondii (which causes toxoplasmosis) - have a unique machinery called the glideosome, which is composed of an actomyosin system that underlies the plasma membrane. The glideosome promotes substrate-dependent gliding motility, which powers migration across biological barriers, as well as active host cell entry and egress from infected cells. In this Review, we discuss the discovery of the principles that govern gliding motility, the characterization of the molecular machinery involved, and its impact on parasite invasion and egress from infected cells.
226 citations
TL;DR: SLI allowed us to functionally analyze targets at the gene and protein levels, thus permitting mislocalization of native proteins, and demonstrated the power and robustness of this approach by validating it for more than 12 targets, including eight essential ones.
Abstract: Selection-linked integration in the Plasmodium genome allows for rapid selection of genes inserted at a genomic locus or induction of the inactivation of gene products.
222 citations
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TL;DR: It is shown that replication of the apicomplexan plastid (apicoplast) genome in Toxoplasma gondii tachyzoites can be specifically inhibited using ciprofloxacin, and that this inhibition blocks parasite replication.
Abstract: Parasites of the phylum Apicomplexa include many important human and veterinary pathogens such as Plasmodium (malaria), Toxoplasma (a leading opportunistic infection associated with AIDS and congenital neurological birth defects), and Eimeria (an economically significant disease of poultry and cattle). Recent studies have identified an unusual organelle in these parasites: a plastid that appears to have been acquired by secondary endosymbiosis of a green alga. Here we show that replication of the apicomplexan plastid (apicoplast) genome in Toxoplasma gondii tachyzoites can be specifically inhibited using ciprofloxacin, and that this inhibition blocks parasite replication. Moreover, parasite death occurs with peculiar kinetics that are identical to those observed after exposure to clindamycin and macrolide antibiotics, which have been proposed to target protein synthesis in the apicoplast. Conversely, clindamycin (and functionally related compounds) immediately inhibits plastid replication upon drug application-the earliest effect so far described for these antibiotics. Our results directly link apicoplast function with parasite survival, validating this intriguing organelle as an effective target for parasiticidal drug design.
590 citations
TL;DR: A system for tagging genes endogenously with yellow fluorescent protein (YFP) in a Δku80 strain that is more effective and efficient in integrating the YFP-tagged constructs into the correct locus than wild-type strain RH.
Abstract: As with other organisms with a completed genome sequence, opportunities for performing large-scale studies, such as expression and localization, on Toxoplasma gondii are now much more feasible. We present a system for tagging genes endogenously with yellow fluorescent protein (YFP) in a Δku80 strain. Ku80 is involved in DNA strand repair and nonhomologous DNA end joining; previous studies in other organisms have shown that in its absence, random integration is eliminated, allowing the insertion of constructs with homologous sequences into the proper loci. We generated a vector consisting of YFP and a dihydrofolate reductase-thymidylate synthase selectable marker. The YFP is preceded by a ligation-independent cloning (LIC) cassette, which allows the insertion of PCR products containing complementary LIC sequences. We demonstrated that the Δku80 strain is more effective and efficient in integrating the YFP-tagged constructs into the correct locus than wild-type strain RH. We then selected several hypothetical proteins that were identified by a proteomic screen of excreted-secreted antigens and that displayed microarray expression profiles similar to known micronemal proteins, with the thought that these could potentially be new proteins with roles in cell invasion. We localized these hypothetical proteins by YFP fluorescence and showed expression by immunoblotting. Our findings demonstrate that the combination of the Δku80 strain and the pYFP.LIC constructs reduces both the time and cost required to determine localization of a new gene of interest. This should allow the opportunity for performing larger-scale studies of novel T. gondii genes.
494 citations
TL;DR: A genetic screen to generate a tetracycline-inducible transactivator system in T. gondii caused severe impairment in host cell invasion and parasite spreading in cultured cells, and unambiguously established the pathogenic function of this motor in an animal model.
Abstract: Obligate intracellular apicomplexan parasites rely on gliding motion powered by their actomyosin system to disperse throughout tissues and to penetrate host cells. Toxoplasma gondii myosin A has been implicated in this process, but direct proof has been lacking. We designed a genetic screen to generate a tetracycline-inducible transactivator system in T. gondii. The MyoA gene was disrupted in the presence of a second regulatable copy of MyoA. Conditional removal of this myosin caused severe impairment in host cell invasion and parasite spreading in cultured cells, and unambiguously established the pathogenic function of this motor in an animal model.
446 citations
TL;DR: The feasibility of HXGPRT as both a positive and negative selectable marker for stable transformation of T. gondii was demonstrated under selection with mycophenolic acid andKinetic analysis of purified recombinant enzyme revealed no significant differences between the two isoforms.
434 citations