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Characterising Plasmodium falciparum FIKK protein kinases

thesis
posted on 26.02.2017, 23:32 by Siddiqui, Ghizal
Human malaria remains a major global health problem. In 2013, over 600,000 deaths were reported to be due to malaria infection, with greater than 90% of these cases caused by Plasmodium falciparum. Although the number of deaths per year has decreased over the last decade, the number of parasites resistant to current anti-malarial drugs including artemisinin, the current front line treatment against malaria is rising. This underpins the requirement for the discovery of new anti-malarial drugs. Protein kinases are considered attractive drug targets and have been used successfully as various anti-cancer treatments. The focus of this work has been on characterising a unique family of protein kinases specific to P. falciparum, known as FIKK kinases. The P. falciparum kinome consists of 86 protein kinases and the majority of these clusters within the major families of “eukaryotic-like kinases”, with the exception of the FIKK family. A kinome wide reverse genetic strategy identified 35 of the “eukaryotic-like kinases” as being essential for parasite survival during the red blood cell stage, however this study did not include the analysis of FIKK kinases. Here using a gene knockout approach, this study has identified five FIKKs that are essential for parasite survival in the red blood cell stage. Furthermore, it was shown that three of the essential FIKKs were exported into the infected red blood cell cytoplasm, with some associated with structures known as Maurer’s clefts. Surprisingly, two were not exported past the parasitophorous vacuole membrane, despite having a signal sequence and a functional PEXEL motif that signals parasite proteins for export. Together, this suggests that each FIKK has a unique function in infected red blood cells. Seven of the exported FIKKs were not essential for parasite survival in the red blood cell, but are still of great interest since most exported parasite proteins that contribute to the virulence of the parasite are not essential in vitro. In this thesis three non-essential FIKKs (FIKK4.2, 9.4 and 5) have been characterised in detail, and their specific role in RBC remodeling and other related virulence factors were determined. FIKK4.2 was found to be an exported kinase that requires its PEXEL motif and a small region upstream of its PEXEL (53 amino acids) for correct targeting to the infected red blood cell cytoplasm. The N-terminal region of FIKK4.2 was not sufficient for protein interactions and the entire protein appears to be required for identification of its interacting partner/s. Preliminary studies on FIKK4.2 showed that FIKK4.2 has a role in modifying the infected red blood cell, specifically red blood cells infected with FIKK4.2-knockout parasites were less rigid and less adhesive compared to wild type. This phenotype was observed as a result of severely altered knob structures on FIKK4.2-deleted infected red blood cells. Global phosphoproteomic approach was used in an attempt to identify FIKK4.2 substrates, however, despite high proteomic coverage and numerous differentially phosphorylated sites in the knockout line, phosphorylated FIKK4.2 substrate/s were not identified. FIKK9.4 was found to be a novel cytoplasmic associated Maurer’s cleft protein kinase that required both its PEXEL motif and regions within its N-terminal domain for correct targeting to the Maurer’s clefts. FIKK9.4 was shown to interact with and phosphorylate Pf332, a large exported parasite protein that has a role in Maurer’s cleft integrity and infected red blood cell rigidity. In the absence of FIKK9.4, the delivery of Pf332 to the infected red blood cell cytoskeleton was affected. Furthermore, FIKK9.4 was found to interact with the host cytosolic chaperone complex, CCT, strongly suggesting that the function of FIKK9.4 involves chaperone-mediated transport of Pf332 to its target destination at the infected red blood cell cytoskeleton. FIKK5 was found to be localised in the rhoptry bulb of the parasite and not exported, despite encoding a functional PEXEL motif. FIKK5 did not appear to play an essential role in invasion of the parasite into red blood cells, as deletion of FIKK5 did not affect parasite survival in vitro, however, RAP1 expression was up regulated. RAP1 is another rhoptry bulb protein with an unknown function. This thesis expands our understanding of protein kinases in P. falciparum and demonstrates that particular members in this unique, novel FIKK family of kinases have evolved to encompass different functions that could be utilised as novel therapeutic targets against P. falciparum malaria infection.

History

Principal supervisor

Brian M. Cooke

Year of Award

2016

Department, School or Centre

Microbiology

Campus location

Australia

Course

Doctor of Philosophy

Degree Type

DOCTORATE

Faculty

Faculty of Medicine Nursing and Health Sciences