During invasion, apicomplexan parasites form an intimate circumferential contact with the host cell, the tight junction (TJ), through which they actively glide. The TJ, which links the parasite motor to the host cell cytoskeleton, is thought to be composed of interacting apical membrane antigen 1 (AMA1) and rhoptry neck (RON) proteins. Here we find that, in Plasmodium berghei, while both AMA1 and RON4 are important for merozoite invasion of erythrocytes, only RON4 is required for sporozoite invasion of hepatocytes, indicating that RON4 acts independently of AMA1 in the sporozoite. Further, in the Toxoplasma gondii tachyzoite, AMA1 is dispensable for normal RON4 ring and functional TJ assembly but enhances tachyzoite apposition to the cell and internalization frequency. We propose that while the RON proteins act at the TJ, AMA1 mainly functions on the zoite surface to permit correct attachment to the cell, which may facilitate invasion depending on the zoite-cell combination.

Apicomplexa are obligate intracellular parasites that actively invade host cells using their membrane-associated, actin-myosin motor. The current view is that host cell invasion by Apicomplexa requires the formation of a parasite-host cell junction, which has been termed the moving junction, but does not require the active participation of host actin. Using Toxoplasma gondii tachyzoites and Plasmodium berghei sporozoites, we show that host actin participates in parasite entry. Parasites induce the formation of a ring-shaped F-actin structure in the host cell at the parasite-cell junction, which remains stable during parasite entry. The Arp2/3 complex, an actin-nucleating factor, is recruited at the ring structure and is important for parasite entry. We propose that Apicomplexa invasion of host cells requires not only the parasite motor but also de novo polymerization of host actin at the entry site for anchoring the junction on which the parasite pulls to penetrate the host cell.

We describe here an efficient method for conditional gene inactivation in malaria parasites that uses the Flp/FRT site-specific recombination system of yeast. The method, developed in Plasmodium berghei, consists of inserting FRT sites in the chromosomal locus of interest in a parasite clone expressing the Flp recombinase via a developmental stage-specific promoter. Using promoters active in mosquito midgut sporozoites or salivary gland sporozoites to drive expression of Flp or its thermolabile variant, FlpL, we show that excision of the DNA flanked by FRT sites occurs efficiently at the stage of interest and at undetectable levels in prior stages. We applied this technique to conditionally silence MSP1, a gene essential for merozoite invasion of erythrocytes. Silencing MSP1 in sporozoites impaired subsequent merozoite formation in the liver. Therefore, MSP1 plays a dual role in the parasite life cycle, acting both in liver and erythrocytic parasite stages.

The liver is the first organ infected by Plasmodium sporozoites during malaria infection. In the infected hepatocytes, sporozoites undergo a complex developmental program to eventually generate hepatic merozoites that are released into the bloodstream in membrane-bound vesicles termed merosomes. Parasites blocked at an early developmental stage inside hepatocytes elicit a protective host immune response, making them attractive targets in the effort to develop a pre-erythrocytic stage vaccine. Here, we generated parasites blocked at a late developmental stage inside hepatocytes by conditionally disrupting the Plasmodium berghei cGMP-dependent protein kinase in sporozoites. Mutant sporozoites are able to invade hepatocytes and undergo intracellular development. However, they remain blocked as late liver stages that do not release merosomes into the medium. These late arrested liver stages induce protection in immunized animals. This suggests that, similar to the well studied early liver stages, late stage liver stages too can confer protection from sporozoite challenge.Malaria is among the deadliest infectious diseases in the world. It is caused by protozoan parasites of the genus Plasmodium that undergo a complex life cycle in the mammalian host and the mosquito vector. A human malaria infection begins when a Plasmodium sporozoite delivered through the bite of an infected mosquito infects a hepatocyte in the host liver. Within an intrahepatic membrane-bound vacuole the sporozoite undergoes extensive physical transformation followed by nuclear divisions, cytoplasmic segmentation, and eventually the formation of thousands of merozoites (1). Merozoites exit the infected hepatocyte by budding off in membrane-bound vesicles termed merosomes (2). Merosomes extrude from the infected hepatocyte through the endothelial cell layer and are released into the neighboring sinusoids. Thus, hepatic merozoites are delivered directly into the blood stream where they initiate invasion of erythrocytes and the symptomatic phase of a malaria infection (2). Unlike other stages of the Plasmodium life cycle, the stages that develop inside the hepatocytes, called "liver stages" (LSs), 3 are relatively poorly understood. Although the execution of the LS developmental program must require a large repertoire of molecules, only a few have been functionally identified so far (3-10). LS are of significant clinical and biological interest. Inhibiting the growth of LS could prevent the pathology associated with the erythrocytic stages of a malaria infection. The morbidity associated with Plasmodium vivax, the major human species in South America and South Asia, partly results from its ability to form dormant liver stages, termed hypnozoites, against which there are few effective treatment options (11). Reactivated hypnozoites can cause disease relapse up to a year after initial infection. Finally, LS have long been recognized to be ideal targets for developing a pre-erythrocytic stage malaria vaccine. Animals immunized with irradiated or genetica...

We describe here a highly efficient procedure for conditional mutagenesis in Plasmodium. The procedure uses the site-specific recombination FLP-FRT system of yeast and targets the pre-erythrocytic stages of the rodent Plasmodium parasite P. berghei, including the sporozoite stage and the subsequent liver stage. The technique consists of replacing the gene under study by an FRTed copy (i.e., flanked by FRT sites) in the erythrocytic stages of a parasite clone that expresses the flip (FLP) recombinase stage-specifically--called the 'deleter' clone. We present the available deleter clones, which express FLP at different times of the parasite life cycle, as well as the schemes and tools for constructing new deleter parasites. We also outline and discuss the various strategies for exchanging a wild-type gene with an FRTed copy and for generating conditional gene knockout or knockdown parasite clones. Finally, we detail the protocol for obtaining sporozoites that lack a protein of interest and for monitoring sporozoite-specific DNA excision and depletion of the target protein. The protocol should allow the functional analysis of any essential protein in the sporozoite, liver stage or hepatic merozoite stages of rodent Plasmodium parasites.

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Testis-specific protein, Y-encoded (TSPY) is a gene found on the Y chromosome that, like many genes on the Y chromosome of mammalian species, has multiple copies. Humans have between 20–60 copies, whereas cattle can have up to 200 copies. Genomic copy number of TSPY is of interest because it has been linked to fertility. In previous studies, enormous bull-to-bull variation in TSPY copy number has been found (Hamilton et al. 2009 Sex. Dev. 3, 205–213). The aims of this study were to a) examine the copy number of TSPY in brother embryos to see if variation exists among embryos of a single generation and b) to determine if TSPY mRNA (mRNA) is expressed in the early bovine embryo since there is evidence from transgenic mice studies that human TSPY may play a role in development. 80 Holstein blastocysts were produced using standard in vitro fertilization techniques with the semen of a single bull. Individual blastocysts (n = 50) were lysed and quantitative polymerase chain reaction (qPCR) was used to sex the embryos and to measure TSPY copy number in the embryos that were found to be male. The male-specific TSPY and SRY (sex determining region Y) genes were measured in order to sex the embryos along with ZAR1 (zygote arrest protein 1), as an autosomal control. TSPY copy number was measured with a modified 2ΔΔCT method (Hamilton et al. 2009 Sex. Dev. 3, 205–213) and SRY as the single copy reference gene. Messenger RNA was extracted from two pools of blastocysts (n = 20, n = 10) and reverse transcribed into cDNA. The presence of TSPY mRNA in these pools was determined with reverse transcription PCR (RT–PCR) along with the reference gene GAPDH, following standard protocols. Briefly, two sets of TSPY specific primers and one set of GAPDH specific primers were used to amplify the target mRNAs in both pools of embryos using AmpliTaq Gold DNA polymerase (Applied Biosystems) and an annealing temperature of 60°C. PCR products were visualised on a 2% gel using gel electrophoresis and then sequenced to verify specificity of the primers. The results of the embryo sexing experiment showed a 50:50 sex ratio (25 males, 25 females) in the group of individual embryos. TSPY copy number was found to vary significantly (using a one-way ANOVA, P < 0.0001) among brother embryos produced from the semen of the same Hostein bull (n = 25) and ranged from 21.9 ± 7.1 to 159.0 ± 14.0 copies, with an average of 65.9 ± 6.3 copies. Furthermore, the results of the expression experiment showed that TSPY mRNA transcripts were present in both pools of blastocysts. These results provide the first evidence that TSPY is expressed in bovine blastocysts and that copy number can vary within a single generation. This data suggests that the bovine Y chromosome is a dynamic chromosome that is not clonally inherited but can vary its genetic composition within a single generation. The fact that TSPY transcripts were found in the early embryo suggests that it might have another function other than simply a role in spermatogenesis. Supported by National Sciences Engineering and Research Council grant; Canadian Research Chair program; and L’Alliance Boviteq Inc.