Relative expression and proteomics data derived from the malaria parasite’s red blood cell cycle provide valuable data which could be used to identify potential new diagnostic target proteins (Foth et al., 2011; LeRoch et al., 2003; www.PlasmoDB.org). Ideally an immuno- diagnostic target should be present throughout the Plasmodium red cell cycle as this is when disease symptoms appear and patients seek medical help (Antia et al., 2008; Golgi, 1886;
Miller et al., 1994). The target proteins should also be present at relatively high concentrations, therefore using these data sets parasite proteins, of which there are 5554 (Foth et al., 2011), can be ranked according to their relative abundance which dramatically narrows the search. The targets should also be unique, or have a unique structural component to the parasite homologue, which could be exploited for diagnosis. Using the bioinformatics approach outlined here, two possible diagnostic target proteins were identified and are briefly introduced. The first potential target was glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is involved in glycolysis and the second was phosphoethanolamine-N- methyltransferase (PMT), which is involved in phospholipid metabolism.
During a malaria infection a single merozoite divides into 14-36 new parasites within 24-72 hours in a human Plasmodium infection (Antia et al., 2008; Baldacci and Menard, 2004;
Collins and Jeffery, 2007). This process has a high energy demand resulting in up to 100-fold greater glycolytic rates in infected compared to uninfected red blood cells (Daubenberger et al., 2000; Mehta et al., 2006). Since two current RDT target proteins, aldolase and LDH are both involved in glycolysis, targeting proteins which functions within the same pathway and are suggested to be present at higher concentrations than LDH and aldolase (Foth et al., 2011;
LeRoch et al., 2003) would be attractive. Another important metabolic pathway is phospholipid metabolism. The parasite needs to produce sufficient phospholipid membranes to envelope each newly developing daughter merozoite. The phospholipid content of P. falciparum therefore increases between five to six fold after infecting a red blood cell for example (Dechamps et al., 2010). Current RDTs targeting a metabolic enzyme also have the
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Glucose 2 ATP
2 ADP
4 reactions not shown
4 ADP 4 ATP
4 reactions not shown OH
H
C – C – CH2OPO32- + Pi H
O 2
Glyceraldehyde-3- phosphate
2 C – C – CH2OPO32-
+ H+
-2
3OPO
O OH
H
1,3-Bisphosphoglycerate
2 C – C – CH3 + H+ O -
O O
Pyruvate 2 C – C – CH3
O -
O OH
H
L-lactate
GAPDH
LDH 2 NAD+
2 NADH
advantage, of monitoring treatment outcome. This was shown with RDTs using LDH as the target antigen, which was no longer detected in patient blood two to seven days after the clearance of an infection (Iqbal et al., 2004, Murray et al., 2008).
Anaerobic glycolysis yields two ATP molecules from the breakdown of glucose to pyruvate and L-lactate. In the absence of oxygen, the pathway has ten reactions of which the fifth and last reactions are coupled by a dinucleotide cofactor NAD+(H) and involve two dehydrogenases: GAPDH and LDH respectively (Voet and Voet, 2004).
Figure 3.1 NAD+(H) linked glycolytic reactions involving GAPDH and LDH
The respective reduction and oxidation of NAD+(H) by GAPDH and LDH highlighted in bold (Voet and Voet, 2004).
From a glycolysis perspective, an excess of either GAPDH or LDH would be redundant due to limiting NAD+(H) levels. In vitro, however, P. falciparum GAPDH mRNA levels exceed LDH levels by three to four fold (LeRoch et al. 2003) and semi-quantitative proteome data also suggest greater GAPDH to LDH levels (Foth et al., 2011; Lasonder et al., 2002;
Nirmalan et al., 2004; Smit et al., 2010). This suggests a greater demand for GAPDH in relation to LDH and that Plasmodium GAPDH may have additional functions to glycolysis.
These non-glycolytic functions, often termed “moonlighting” functions have been identified in seven of the ten glycolytic enzymes (Alam et al., 2014; Gómez-Arreaza et al., 2014). These moonlighting functions of GAPDH as well as the possible post-translational modifications
67 involved will be discussed later as possible explanations for comparatively higher levels of GAPDH to LDH.
The second new target, PMT, is involved in phospholipid synthesis. Interestingly, no homologs of higher or lower eukaryote phosphatidylethanolamine methyltransferases were identified in the P. falciparum genome. Instead, Pessi et al. (2004 and 2005) identified a plant-like phosphoethanolamine methyltransferase gene homolog (PfPMT) in P. falciparum and hypothesised that the protein was involved in an alternate serine decarboxylase- phosphoethanolamine methyltransferase pathway. Several plant-like P. falciparum proteins are targeted to the apicoplast (Foth et al., 2003), however PfPMT lacks any signal or transit peptides (Pessi et al., 2004) and localises as a soluble protein within the Golgi (Witola et al., 2006). In culture PfPMT expression increased by three-fold as the parasite progressed from its initial ring to trophozoite stages (Pessi et al., 2004; LeRoch et al., 2003) and was expressed during the gametocyte and sporozoite stages of the life cycle (www.PlasmoDB.org). Analysis of the P. falciparum proteome also confirmed the presence of PfPMT throughout the red blood cell cycle ranking it within the top 20 most abundant soluble proteins (Foth et al., 2011;
Nirmalan et al., 2004). PMT homologs were identified in Burkholderia pseudomallei, B.
oklahomensis, Xenopus laevis, X. tropicalis, Caenorhabditis briggsae, Danio rerio, Branchiostoma floridae, Caenorhabditis elegans and Anopheles gambiae, but critically no human homologs exist (Pessi et al., 2004; Bobenchik et al., 2011, 2013). In theory antibodies raised against such a target are unlikely to cross-react with human proteins, therefore reducing misdiagnosis.
Differences in the PMT and GAPDH primary amino acid sequences could potentially be exploited to develop immune-reagents for species identification, as demonstrated by Hurdayal et al. (2010), targeting unique LDH peptide epitopes. This strategy was adopted in this work and the aim was to identify and exploit unique and conserved amino acid sequences on both GAPDH and PMT. The LDH sequences identified by Hurdayal et al. (2010) were also included in this study. This is therefore outlined in chapter 3 in which the bioinformatics approach taken to identify the two novel protein targets and their respective surface epitopes is described.
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