Abstract

The ability of Plasmodium falciparum to remodel the host’s cells drastically increases the virulence of this malaria parasite. One such source of remodelling is the formation of a parasitophorous vacuole (PV), which creates a controlled and hospitable environment within the host’s cells. Certain parasite proteins that traffic to the parasitophorous vacuole membrane (PVM) have been found to be essential for the parasite’s progression through its life-cycle. Some of these proteins share a conserved sequence in the C-terminus, which is thought to be a secretory signal motif. One such protein is ETRAMP 10.3, which is a protein up-regulated during the red blood cell (RBC) stage of the life-cycle and has been found to be essential for the intraerythrocytic asexual growth stage. Removal of this conserved sequence in another PVM protein, Pfs16, inhibits the proper trafficking of the protein to the PVM. It would be hypothesized that removal of the sequence of ETRAMP 10.3 would cause similar problems and possibly prevent the proliferation of the parasite in the blood. This proposal suggests the creation of two GFP (green fluorescent protein) tagged, transformed parasite lines and the use of epifluorescent microscopy to determine protein movement throughout parasite development with and without the conserved sequence.

Background

Each year millions of people are infected with the most severe malaria parasite, Plasmodium falciparum, and despite decades of vaccine research, an effective vaccine remains elusive. Falciparum’s virulence is in part due to the parasite’s ability to remodel its host’s cells. An example of the parasite’s ability to remodel is seen in the formation of a parasitophorous vacuole (PV), as the parasite invades both hepatocyte and erythrocyte cells (Cowman et al. 2006). The PV is surrounded by a membrane (PVM) which creates a controlled and hospitable environment for the parasite within the host cell (Cowman et al. 2006). Much of the PVM is created from the host membrane material; however, additional molecules are secreted from the parasite to the PVM (Cowman et al. 2006). Some Falciparum proteins, known as PVM proteins, are found to traffic to the PVM, including EXP-1 (PF11_0224), members of the ETRAMP gene family, and Pfs16. Studies have found some of these PVM proteins necessary for continuation of the parasite life-cycle. This includes the rodent malaria protein UIS4, which is essential to parasite proliferation in the liver stage of P. berghei and P. yoelii (Mueller et al. 2005). More recently, Mackeller et al. (2010) found evidence that ETRAMP 10.3 (PF10_0164), a protein up-regulated in the RBC stage during the transition from ring to trophozoite stage (Spielmann et al. 2002), is vital for intraerythrocytic asexual growth stage. 

In a recent a study of PVM protein trafficking, Eksi and Williams (2011) provided evidence for a secretory signal motif for PVM proteins located in the proteins’ C-terminus. The conservation of this domain was seen in members of the ETRAMP family, EXP-1, and Pfs16. They found that removal of this motif in Pfs16 inhibited the proper trafficking of Pfs16 to the PVM. ETRAMP 10.3 is known to possess this conserved sequence, but it is unknown whether the deletion of this sequence will also prevent movement of ETRAMP 10.3 to the PVM. Because it is essential for parasite proliferation in the RBC stage, the trafficking of ETRAMP 10.3 is of special interest (Mackeller et al. 2010). This study will use DNA constructs to determine the role of this motif in ETRAMP 10.3 trafficking. Previously, Mackeller et al. determined that ETRAMP 10.3 localizes to the PVM during the trophozoite intraerythrocytic stage and then progresses into the host erythrocyte, suggesting association with Maurer’s Clefts (2010). If this motif serves as a PVM secretion signal for ETRAMP 10.3, we would hypothesize the removal of this motif would prevent localization to the PVM during the trophozoite stage and subsequent export into the host erythrocyte.

Specific Aims

1.To use the Falciparum transformation plasmid, pDH.TgA.22, to construct two in vitro transformed parasite lines tagged with GFP, pDH.TgA.22.10.3.FL.GFP and pDH.TGA.22.10.3.NM.GFP, to express the full length ETRAMP 10.3 protein sequence and the ETRAMP 10.3 sequence excluding the C-terminal motif, respectively.

2.To use epifluorescent microscopy to track the movement of the two ETRAMP 10.3 constructs (FL and NM) throughout the three stages of intraerythrocytic parasite development: ring stage, trophozoite stage, and schizont stage.

Experimental Protocol

Reporter Constructs

Two GFP reporter constructs will be created to determine the role of a secretory motif in trafficking of ETRAMP 10.3. These constructs will then be utilized to create two transformed parasite lines, pDH.TgA.22.10.3.FL and pDH.TGA.22.10.3.NM.

Eski and Williamson (2011) showed that ETRAMP 10.3 and Pfs16 show some conservation of amino acid sequence in the C-terminus. In ETRAMP 10.3, this motif is between amino acid 64 and 105 and overlaps with the transmembrane region of the C-terminus (53-75aa). This is consistent with the overlap of motif and transmembrane region seen in Pfs16. Due to this overlap, we will include the entire transmembrane sequence with our conserved sequence (53-105aa) in our proposed signal sequence.

The GFP reporter constructs will be created following the methods outlined in Eksi and Williamson (2011). The first construct (FL), which will serve as a control, will contain the full length ETRAMP 10.3 amino acid sequence, and the second (NM) will include the entire amino acid sequence minus the signal sequence (Figure 1). First, the 5’- Flanking Region (FR; Eksi et al. 2008) containing the promoter and the exact sequence of the N-terminus will be determined using sequencing. Then, the Falciparum transformation plasmid pDH.TgA.22, which confers resistance to pyrimethamine, will be used to create these transformed parasite lines (Wu et al. 1996). Four sequence regions will be digested with specific restriction enzymes, amplified via PCR, and ligated into the pDH.TgA.22 plasmid at the corresponding restriction site. The regions are as follows: (1) the 5’- FR and the N-terminus, (2) the core amino acid region between the N- and C-termini, (3) the overlapped region containing the transmembrane and predicted secretory signal sequences, and (4) the remainder of the C-terminal region. Each region will be amplified using primers specific to that sequence.