Tetracycline analogue-regulated transgene expression in
            <i>Plasmodium falciparum</i>
            blood stages using
            <i>Toxoplasma gondii</i>
            transactivators

Meissner, Markus; Krejany, Efrosinia O.; Gilson, Paul R.; Koning-Ward, Tania F. de; Soldati, Dominique; Crabb, Brendan S.

doi:10.1073/pnas.0500112102

Cited by 101 publications

(92 citation statements)

References 23 publications

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“…Regulated transgene expression has also been described in P. falciparum, in which a tetracycline analog is used to repress transactivated gene expression 27 . Combining our integrase system with these other techniques offers the prospect of developing efficient methods for complementation and change or loss-of-function studies.…”

Section: Discussionmentioning

confidence: 99%

Efficient site-specific integration in Plasmodium falciparum chromosomes mediated by mycobacteriophage Bxb1 integrase

et al. 2006

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Here we report an efficient, site-specific system of genetic integration into Plasmodium falciparum malaria parasite chromosomes. This is mediated by mycobacteriophage Bxb1 integrase, which catalyzes recombination between an incoming attP and a chromosomal attB site. We developed P. falciparum lines with the attB site integrated into the glutaredoxin-like cg6 gene. Transfection of these attB + lines with a dual-plasmid system, expressing a transgene on an attPcontaining plasmid together with a drug resistance gene and the integrase on a separate plasmid, produced recombinant parasites within 2 to 4 weeks that were genetically uniform for single-copy plasmid integration. Integrase-mediated recombination resulted in proper targeting of parasite proteins to intra-erythrocytic compartments, including the apicoplast, a plastid-like organelle. Recombinant attB × attP parasites were genetically stable in the absence of drug and were phenotypically homogeneous. This system can be exploited for rapid genetic integration and complementation analyses at any stage of the P. falciparum life cycle, and it illustrates the utility of Bxb1-based integrative recombination for genetic studies of intracellular eukaryotic organisms.In response to the worsening burden of Plasmodium falciparum malaria, intense research efforts have led to the development of various tools for the genetic manipulation of this organism. The application of techniques such as gene disruption and allelic replacement has afforded important insights into parasite mechanisms of drug resistance, pathogenesis, host cell invasion and transmission 1 . Yet genomic integration studies are plagued by low transfection and recombination efficiencies. In addition, transgene expression assays have been COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests. restricted to episomally replicating plasmids that parse unequally during mitotic segregation and hence cannot be selected in sexual stages and propagated through the life cycle 1 . NIH Public AccessIntegrative recombination offers a powerful approach to target foreign DNA into specific genomic locations. Bacteriophage serine integrase-based systems are particularly suitable because of their intrinsically high efficiency of recombination between host attB and phage attP sites, with integration and excision having distinct reaction requirements that preclude spontaneous reversion events 2 . These recombinases, unlike their tyrosine counterparts such as lambda integrase (used to develop the Invitrogen GATEWAY site-specific recombination system), are sufficient to catalyze recombination and do not require bacterial host factors. Among the serine integrases, the most extensively used has been Streptomyces sp. phage phiC31, which efficiently integrates plasmid vectors into prokaryotic or eukaryotic hosts containing att or pseudo-att sites 2,3 .The serine integrase of the mycobacteriophage Bxb1, which is a temperate phage whose genome recombines into an attB site located wi...

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Section: Discussionmentioning

confidence: 99%

Efficient site-specific integration in Plasmodium falciparum chromosomes mediated by mycobacteriophage Bxb1 integrase

et al. 2006

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“…The development of a system for conditional gene expression in T. gondii (Meissner et al, 2002), also applied recently to P. falciparum (Meissner et al, 2005), has provided a means to study the function of essential genes in apicomplexan parasites. In the second successful application of this system we have shown directly that TgAMA1 functions in host cell invasion.…”

Section: Discussionmentioning

confidence: 99%

Conditional Expression ofToxoplasma gondiiApical Membrane Antigen-1 (TgAMA1) Demonstrates That TgAMA1 Plays a Critical Role in Host Cell Invasion

Mital

Meissner

Soldati

et al. 2005

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Toxoplasma gondii is an obligate intracellular parasite and an important human pathogen. Relatively little is known about the proteins that orchestrate host cell invasion by T. gondii or related apicomplexan parasites (including Plasmodium spp., which cause malaria), due to the difficulty of studying essential genes in these organisms. We have used a recently developed regulatable promoter to create a conditional knockout of T. gondii apical membrane antigen-1 (TgAMA1). TgAMA1 is a transmembrane protein that localizes to the parasite's micronemes, secretory organelles that discharge during invasion. AMA1 proteins are conserved among apicomplexan parasites and are of intense interest as malaria vaccine candidates. We show here that T. gondii tachyzoites depleted of TgAMA1 are severely compromised in their ability to invade host cells, providing direct genetic evidence that AMA1 functions during invasion. The TgAMA1 deficiency has no effect on microneme secretion or initial attachment of the parasite to the host cell, but it does inhibit secretion of the rhoptries, organelles whose discharge is coupled to active host cell penetration. The data suggest a model in which attachment of the parasite to the host cell occurs in two distinct stages, the second of which requires TgAMA1 and is involved in regulating rhoptry secretion. INTRODUCTIONToxoplasma gondii is one of the one most common protozoan parasites of humans, infecting approximately one-third of the world's population. The disease caused by an acute T. gondii infection, toxoplasmosis, can be life threatening in the congenitally infected fetus and immunocompromised hosts. Like all members of the Phylum Apicomplexa, including Plasmodium spp. (the causative agents of malaria) and Cryptosporidium parvum (a significant worldwide cause of waterborne illness), T. gondii is an obligate intracellular parasite. Host cell invasion is a critical step in the pathogenesis of the diseases caused by apicomplexan parasites, including toxoplasmosis. T. gondii represents an excellent model system for studying the relatively conserved process of apicomplexan invasion, because of the ease with which this parasite can be cultured and genetically manipulated (Roos et al., 1994;Black and Boothroyd, 2000;Kim and Weiss, 2004).Host cell invasion by apicomplexan parasites is a complex, multistep process (reviewed in Black and Boothroyd, 2000;Chitnis and Blackman, 2000). In T. gondii, the asexual stage tachyzoites move over solid surfaces, including cells, by an unusual form of substrate-dependent gliding motility (Sibley et al., 1998;Hakansson et al., 1999). After the apical end of a parasite comes in contact with the host cell membrane, apical secretory organelles (micronemes and rhoptries) sequentially discharge their contents (Dubremetz et al., 1993;Carruthers and Sibley, 1997) and a zone of tight interaction forms between the two cells (Nichols and O'Connor, 1981;Dubremetz et al., 1985;Grimwood and Smith, 1995). An invagination in the host cell plasma membrane develops at the point ...

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“…The two artificial transactivators functioning in T. gondii and P. falciparum correspond to a short stretch of rather hydrophobic amino acids. Neither transactivators function in HeLa cells, underlining the differences between the transcription machinery in apicomplexans and higher eukaryotes [65]. This new system led to the generation of the first conditional knockout for an essential gene with no apparent reversion effect and operates on the parasites in the animal model [64].…”

Section: Experimental Approaches and Tools Developed To Regulate Genementioning

confidence: 99%

“…Further characterisation of these transactivators revealed that they do not function in HeLa cells but can activate transcription in P. falciparum [65]. This restricted range of activity suggests that considerable differences exist between the core transcription machinery of these protozoa and other eukaryotes.…”

Section: The Basic Machinery Of Transcription In Apicomplexamentioning

confidence: 99%

The transcription machinery and the molecular toolbox to control gene expression in Toxoplasma gondii and other protozoan parasites

Meissner¹,

Soldati

2005

Microbes and Infection

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The phylum of Apicomplexa groups a large variety of obligate intracellular protozoan parasites that exhibit complicated life cycles, involving transmission and differentiation within and between different hosts. Little is known about the level of regulation and the nature of the factors controlling gene expression throughout their life stages. Unravelling the mechanisms that govern gene regulation is critical for the development of adequate tools to manipulate these parasites and modulate gene expression, in order to study their function in molecular terms in vivo. A comparative analysis of the transcriptional machinery of several apicomplexan genomes and other protozoan parasites has revealed the existence of a primitive eukaryotic transcription apparatus consisting only of a subset of the general transcription factors found in higher eukaryotes. These findings have some direct implications on development of tools.

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Tetracycline analogue-regulated transgene expression in Plasmodium falciparum blood stages using Toxoplasma gondii transactivators

Cited by 101 publications

References 23 publications

Efficient site-specific integration in Plasmodium falciparum chromosomes mediated by mycobacteriophage Bxb1 integrase

Efficient site-specific integration in Plasmodium falciparum chromosomes mediated by mycobacteriophage Bxb1 integrase

Conditional Expression ofToxoplasma gondiiApical Membrane Antigen-1 (TgAMA1) Demonstrates That TgAMA1 Plays a Critical Role in Host Cell Invasion

The transcription machinery and the molecular toolbox to control gene expression in Toxoplasma gondii and other protozoan parasites

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