An efficient strategy for gene targeting and phenotypic assessment in the Plasmodium yoelii rodent malaria model.

In this report, we describe a cloning procedure for gene replacement by double homologous recombination in Plasmodium yoelii, which requires only one digestion and ligation step. This significantly shortens the time required to complete the production of the targeting vector. Furthermore, for more efficient phenotypic evaluation of the gene knockout parasites, we have also introduced a fluorescent protein cassette into the targeting vector. This allows for a more rapid assessment of parasite growth in all of its developmental stages. In addition, the introduction of the fluorescent marker via the replacement strategy confers the stable integration of the marker.

A combined transcriptome and proteome survey of malaria parasite liver stages.

For 50 years since their discovery, the malaria parasite liver stages (LS) have been difficult to analyze, impeding their utilization as a critical target for antiinfection vaccines and drugs. We have undertaken a comprehensive transcriptome analysis in combination with a proteomic survey of LS. Green fluorescent protein-tagged Plasmodium yoelii (PyGFP) was used to efficiently isolate LS-infected hepatocytes from the rodent host. Genome-wide LS gene expression was profiled and compared with other parasite life cycle stages. The analysis revealed approximately 2,000 genes active during LS development, and proteomic analysis identified 816 proteins. A subset of proteins appeared to be expressed in LS only. The data revealed exported parasite proteins and LS metabolic pathways including expression of FASII pathway enzymes. The FASII inhibitor hexachlorophene and the antibiotics, tetracycline and rifampicin, that target the apicoplast inhibited LS development, identifying FASII and other pathways localized in the apicoplast as potential drug targets to prevent malaria infection.

Protracted sterile protection with Plasmodium yoelii pre-erythrocytic genetically attenuated parasite malaria vaccines is independent of significant liver-stage persistence and is mediated by CD8+ T cells.

Irradiation-attenuated sporozoite vaccinations confer sterile protection against malaria infection in animal models and humans. Persistent, nonreplicating parasite forms in the liver are presumably necessary for the maintenance of sterile immunity. A novel vaccine approach uses genetically attenuated parasites (GAPs) that undergo arrested development during liver infection. The fate of GAPs after immunization, their persistence in vaccinated animals, and the immune mechanisms that mediate protection are unknown. To examine the developmental defects of genetically attenuated liver stages in vivo, we created deletions of the UIS3 and UIS4 loci in the Plasmodium yoelii rodent malaria model (Pyuis3[-] and Pyuis4[-]). The low 50% infectious dose of P. yoelii in BALB/c mice provides the most sensitive infectivity model. We show that P. yoelii GAPs reach the liver, invade hepatocytes, and develop a parasitophorous vacuole but do not significantly persist 40 h after infection. A single dose of Pyuis4(-) sporozoites conferred complete protection, but full protection by Pyuis3(-) sporozoites required at least 2 immunizations. CD8(+) T cells were essential for protection, but CD4(+) T cells were not. Our results show that genetically distinct GAPs confer different degrees of protective efficacy and that live vaccine persistence in the liver is not necessary to sustain long-lasting protection. These findings have important implications for the development of a P. falciparum GAP malaria vaccine.

Plasmodium yoelii sporozoites with simultaneous deletion of P52 and P36 are completely attenuated and confer sterile immunity against infection.

Malaria infection starts when sporozoites are transmitted to the mammalian host during a mosquito bite. Sporozoites enter the blood circulation, reach the liver, and infect hepatocytes. The formation of a parasitophorous vacuole (PV) establishes their intracellular niche. Recently, two members of the 6-Cys domain protein family, P52 and P36, were each shown to play an important albeit nonessential role in Plasmodium berghei sporozoite infectivity for the rodent host. Here, we generated p52/p36-deficient Plasmodium yoelii parasites by the simultaneous deletion of both genes using a single genetic manipulation. p52/p36-deficient parasites exhibited normal progression through the life cycle during blood-stage infection, transmission to mosquitoes, mosquito-stage development, and sporozoite infection of the salivary glands. p52/p36-deficient sporozoites also showed normal motility and cell traversal activity. However, immunofluorescence analysis and electron microscopic observations revealed that p52/p36-deficient parasites did not form a PV within hepatocytes in vitro and in vivo. The p52/p36-deficient parasites localized as free entities in the host cell cytoplasm or the host cell nucleoplasm and did not develop as liver stages. Consequently, they did not cause blood-stage infections even at high sporozoite inoculation doses. Mice immunized with p52/p36-deficient sporozoites were completely protected against infectious sporozoite challenge. Our results demonstrate for the first time the generation of two-locus gene deletion-attenuated parasites that infect the liver but do not progress to blood-stage infection. The study will critically guide the design of Plasmodium falciparum live attenuated malaria vaccines.

Quantitative isolation and in vivo imaging of malaria parasite liver stages.

The liver stages of Plasmodium, the causative agent of malaria, are the least explored forms in the parasite's life cycle despite their recognition as key vaccine and drug targets. In vivo experimental access to liver stages of human malaria parasites is practically prohibited and therefore rodent model malaria parasites have been used for in vivo studies. However, even in rodent models progress in the analysis of liver stages has been limited, mainly due to their low abundance and associated difficulties in visualisation and isolation. Here, we present green fluorescent protein (GFP)-tagged Plasmodium yoelii rodent malaria parasite liver infections in BALB/c mice as an excellent quantitative model for the live visualisation and isolation of the so far elusive liver stages. We believe P. yoelii GFP-tagged liver stages allow, for the first time, the efficient quantitative isolation of intact early and late liver stage-infected hepatocyte units by fluorescence activated cell sorting. GFP-tagged liver stages are also well suited for intravital imaging, allowing us for the first time to visualise them in real time. We identify previously unrecognised features of liver stages including vigorous parasite movement and expulsion of 'extrusomes'. Intravital imaging thus reveals new, important information on the malaria parasite's transition from tissue to blood stage.

Host refractoriness of the tobacco hornworm, Manduca sexta, to the braconid endoparasitoid Cotesia flavipes.

Cotesia flavipes (Hymenoptera:Braconidae) is a gregarious endoparasitoid of several pyralid stemborer larvae of economic significance including the sugarcane borer, Diatraea saccharalis. In this study, the ability of this parasitoid to develop in a sphingid host, Manduca sexta, was tested. First, second, third, fourth, and even pharate fifth instar host tobacco hornworm larvae were readily parasitized by the female C. flavipes parasitoids but no wasp larvae hatched from the eggs in this refractory host. Instead, the parasitoid eggs were invariably encapsulated by the host's hemocytes and, ultimately, no parasitoids emerged from tobacco hornworm hosts. The first stages of encapsulation were evident at 2 h post-parasitization of the host M. sexta larvae, when the beginning stages of capsule formation were seen. The developmental fate of the host larvae with encapsulated parasitoids was variable. Most succumbed as abnormally small fifth instars or as post-wandering prepupal animals, while a few developed normally to the pupal stage. Dissection of all the larvae or pupae with encapsulated wasp eggs showed evidence of hemocytic encapsulation and melanization of the C. flavipes eggs. This report describes the association between C. flavipes and M. sexta, which appears to be an excellent model system for studying the physiological processes accompanying wasp egg encapsulation that result in death of the host as well as the parasitoid. Since the parasitoid egg never hatches, the system offers an excellent opportunity to identify and study the effects of parasitoid-injected polydnavirus and venom on host physiology.