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Uncovering the UIS4 protein interacting network and its role during the malaria liver stage infection
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Malaria, which is caused by Plasmodium parasites, is one of the most prevalent infectious diseases worldwide, with an annual death rate around 435,000, accounting for more than half of all deaths by a vector-borne disease, despite innumerous efforts to eliminate it. The infection initiates when the liver form of the parasite, called sporozoites, are injected into the host skin through a bite by the female Anopheles mosquito. Hepatocyte infection by Plasmodium sporozoites is the first natural step towards the establishment of malaria disease. Inside hepatocytes, the parasite lives in a specialized compartment enveloped by a membrane called parasitophorous vacuole membrane (PVM). The PVM represents the interface between the parasite and the host cell, and most likely plays different roles that may range from protection and signalling to waste elimination. Nevertheless, little is known about the molecular players and interactions occurring at this interface.
A subset of proteins encoded by the “Upregulated in Infective Sporozoites” (UIS) genes have been identified, and it has been shown that some of them are exported to the PVM. One such protein is UIS4, which has been shown to be essential for parasite survival. For instances, sporozoites of the rodent malaria parasites species, Plasmodium berghei (P. berghei) and Plasmodium yoelii (P. yoelii) lacking UIS4 can invade liver cells and begin to grow. However, they do not efficiently develop and replicate.
Despite its important role for malaria parasites during the liver phase of the disease, the biological function of UIS4 remains to be elucidated.
The aims of this work were twofold:
Aim 1. To study the biological function of the UIS4 in the liver stage on the PVM, by revealing its interaction partners.
Aim 2. To search for new host or parasite proteins localized on the PVM during the liver stage of infection.
Our working hypothesis to fulfil the first aim is that UIS4 protects the parasite and that this role is achieved through a network of interacting proteins. Although UIS4-KO parasites have been previously generated in the NK65 parasite line, they did not infect the mosquito efficiently. Two new UIS4-KO parasites in WT and GFP-expressing P. berghei ANKA lines were created.
To generate these knock-out parasite lines, the P. berghei ANKA UIS4 open reading frame (ORF) was replaced by the hDHFR gene (human Dihydrofolate Reductase) using a double cross-over recombination strategy. The modified parasite line has resistance to pyrimethamine.
The UIS4-KO parasite lines were genotyped to ensure that the transfection constructs integrated into the correct locus. The absence of UIS4 expression in mutant parasites was confirmed by RT-PCR (for RNA expression), immunofluorescence and Western Blot (WB), for protein expression. Assessment of parasite load agreed with previously published studies, both in vivo and in vitro.
To identify the UIS4 interacting partners, immunoprecipitation using an antibody against UIS4 followed by mass spectrometry and WB of infected cells with UIS4-KO and wild-type parasites was performed. Independent experiments identified actin as a major UIS4 interacting partner. Moreover, a similar immunoprecipitation experiment was carried out on mammalian cells transfected with a plasmid expressing the soluble domain of UIS4. Again, actin was immunoprecipitated with UIS4. Notably, in vitro assays of actin polymerization revealed that UIS4 promotes actin polymerization.
In order to find new PVM proteins, an approach based on proximity-dependent biotin labelling, named BioID, was designed. To that end, a parasite line expressing a UIS4-BirA* fusion protein, PbUIS4-BirA*-HA, was generated by transfecting P. berghei ANKA parasites, via a single cross-over recombination, with a plasmid containing approximately 800bp of the UIS4 gene sequence in frame with the BirA*-coding sequence, followed by pyrimethamine treatment and clonal selection. The transgenic parasites were characterized to ensure the correct size of the fusion protein by WB and localization to the PVM by microscopy. Most importantly, the data show that PbUIS4-BirA*-HA parasites behave as wild-type parasites throughout the entire parasite life cycle, and that the biotin supplementation leads to specific biotinylation of proteins on the PVM.
Such immunoprecipitation approach followed by mass spectrometry analysis revealed several novel parasite and host proteins as potential PVM-resident candidates.
Altogether, this study revealed: a) a previously unknown interaction between a critical parasite protein essential for the success of the liver stage of infection and host actin, and b) it also identified new parasite and host proteins that may play a critical role in the interface between Plasmodium parasites and its first obligatory host cell on the PVM.
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Esporozoítos Proteínas Membrana do vacúolo parasitário Plasmodium Etiologia Proteína UIS4 Teses de doutoramento - 2020