Mutant/mutation
The mutant lacks expression of PDH E1α (pyruvate dehydrogenase E1 alpha subunit ) and expresses (the toxin) CDC (cholesterol-dependent cytolysins) perfringolysin O (PFO) under the control of the liver stage-specific promoter of PBANKA_100300. FPO contains a C-terminal V5-tag. In addition, the mutant expresses GFP under the control of the constitutive eef1 promoter.

Protein (function)
Acetyl-CoA is synthesized from pyruvate by the enzyme complex pyruvate dehydrogenase (PDH). PDH is a member of the α-ketoacid dehydrogenase multienzyme complexes and consists of three subunits: pyruvate dehydrogenase (E1), dihydrolipoyl acetyltransferase (E2) and lipoamide dehydrogenase (dihydrolipoyl dehydrogenase; E3). Acetyl-CoA is an essential precursor of fatty acid synthesis in plastids and fuels the tricarboxylic acid (TCA) cycle in mitochondria, which generates energy in the form of ATP.
Analyses of P. yoelii and P. berghei mutants (RMgm-376, RMgm-677) lacking expression of PDH E1α (see below) indicate a non-essential role of PDH E1α for blood stages and mosquito stages but an important role during late liver stage development.

The CDC perfringolysin O (PFO) is a potent toxin that is expressed by the gram-positive bacterium, Clostridium perfringens (see Additional information). See also RMgm-853 for a mutant expressing FPO in maturing liver stages.

Phenotype
Strongly reduced infectivity of sporozoites. Only a very low number of mice developed blood stage parasitemia after infection with high numbers of sporozoites.
Injection of 5,000 PbΔPDH-E1-PFO(LS) sporozoites per mouse did not result in the development of a blood infection. Even when we used higher numbers (25,000 and 100,000) of sporozoites per mouse, only one of five mice in each group developed parasitemia and this with a long delay in patency.

Maturing liver stages abort development as a result of a combination of the lack of expression of PDH-E1α (see RMgm-677) and expression of FPO (see RMgm-853).

Evidence is presented for FPO expression in (the cytosol of) liver stages (evidence is presented for partial co-localisation with the PVM). Evidence is presented for premature rupture/damage of the PVM in maturing liver stages that express FPO (see RMgm-853).

Additional information
Pore-forming toxins are widespread in prokaryotes. Approximately one-third of all characterised bacterial toxins are PFPs. They are expressed as stable water-soluble monomers, which bind to their target membrane. The membrane binding is followed by a lateral diffusion and self-assembly with other PFP monomers, which leads to oligomerization to a so-called pre-pore complex. After further conformational changes, some parts of the pre-pore reach through the lipid bilayer to finally form a membrane-spanning pore. This, in turn, results in perforation of the target membrane. A huge group of PFPs consists of cholesterol-dependent  cytolysins (CDCs). As their name indicates, these proteins need cholesterol to become active. Cholesterol is a component of many membranes and is believed to function as a receptor for several CDCs and to trigger the conformational shift from the pre-pore to the pore form.
The CDC perfringolysin O (PFO) is a potent toxin that is expressed by the gram-positive bacterium, Clostridium perfringens. By oligomerization of up to 50 monomers, PFO forms pores with a final pore diameter between 30 and 45 nm. Although  PFO-membrane binding is maximal at pH 5.5–6, it also occurs at neutral pH. PFO is able to perforate different kinds of membranes such as phagosomal, vacuole and plasma membranes and it was expected to perforate the cholesterol-rich PV membrane (PVM) (Bano et al., 2007; Silvie et al., 2008) in P. berghei-infected hepatocytes.

At later stages of liver stage development (48 h p.i.), the PVM and the plasma membrane of mutant parasites were damaged because the GFP was no longer restricted to the parasite cytosol, but was distributed  throughout the host cell. In HepG2 cells infected with control parasites, GFP remained in the parasite at this stage of development.  Furthermore, in the mutant parasites, Exp1 distribution was clearly affected, again indicating  damage of the PVM. These results clearly show that the
expression of PFO leads to the expected premature rupture of the PVM, as well as the parasite plasma membrane. The parasites were able to express the putative cysteine protease, SERA2, a marker of late schizonts, confirming that the parasites can reach this stage before PVM rupture.
The premature PVM rupture had no direct negative effect on the host cell since the cell shape and motility, as well as contacts to neighbouring cells, were not affected.

Injection of 5,000 PbΔPDH-E1-PFO(LS) sporozoites per mouse did not result in the development of a blood infection. Even when we used higher numbers (25,000 and 100,000) of sporozoites per mouse, only one of five mice in each group developed parasitemia and this with a long delay in patency

Other mutants
RMgm-677: A P. berghei mutant lacking expression of PDH E1α (pyruvate dehydrogenase E1 alpha subunit ) .
See also RMgm-853 for a mutant expressing FPO in maturing liver stages.