Mutant/mutation
The mutant 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)
The CDC perfringolysin O (PFO) is a potent toxin that is expressed by the gram-positive bacterium, Clostridium perfringens (see Additional information).

Phenotype
Strongly reduced infectivity of sporozoites. Only a low number of mice developed blood stage parasitemia after infection with (high numbers of) sporozoites. In vivo, PbPFO(LS) parasites showed a strong attenuated phenotype. None of the BALB/c mice injected with 5,000 PbPFOLS sporozoites developed a parasitemia and only two of 10 of the susceptible C57BL/6 mice became positive when inected with 5,000 sporozoites of this parasite strain. High infective  doses of 25,000 PbPFO(LS) sporozoites confirmed that the attenuation was not complete: three of five mice developed a delayed blood parasitemia.

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.

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.

In vivo, PbPFO(LS) parasites showed a strong attenuated phenotype. None of the BALB/c mice injected with 5,000 PbPFOLS sporozoites developed a parasitemia and only two of 10 of the susceptible C57BL/6 mice became positive when inected with 5,000 sporozoites of this parasite strain. High infective  doses of 25,000 PbPFO(LS) sporozoites confirmed that the attenuation was not complete: three of five mice developed a delayed blood parasitemia.

Other mutants
RMgm-854: A mutant lacking expression of PDH E1α (pyruvate dehydrogenase E1 alpha subunit ) and expressing FPO