In vivo uptake of a haem analogue Zn protoporphyrin IX by the human malaria parasite P. falciparum‐infected red blood cells

Abstract

The cellular traffic of haem during the development of the human malaria parasite Plasmodium falciparum, through the stages R (ring), T (trophozoite) and S (schizonts), was investigated within RBC (red blood cells). When Plasmodium cultures were incubated with a fluorescent haem analogue, ZnPPIX (Zn protoporphyrin IX) the probe was seen at the cytoplasm (R stage), and the vesicle-like structure distribution pattern was more evident at T and S stages. The temporal sequence of ZnPPIX uptake by P. falciparum-infected erythrocytes shows that at R and S stages, a time-increase acquisition of the porphyrin reaches the maximum fluorescence distribution after 60 min; in contrast, at the T stage, the maximum occurs after 120 min of ZnPPIX uptake. The difference in time-increase acquisition of the porphyrin is in agreement with a maximum activity of haem uptake at the T stage. To gain insights into haem metabolism, recombinant PfHO (P. falciparum haem oxygenase) was expressed, and the conversion of haem into BV (biliverdin) was detected. These findings point out that, in addition to haemozoin formation, the malaria parasite P. falciparum has evolved two distinct mechanisms for dealing with haem toxicity, namely, the uptake of haem into a cellular compartment where haemozoin is formed and HO activity. However, the low Plasmodium HO activity detected reveals that the enzyme appears to be a very inefficient way to scavenge the haem compared with the Plasmodium ability to uptake the haem analogue ZnPPIX and delivering it to the food vacuole.

Direct Tests of Enzymatic Heme Degradation by the Malaria Parasite Plasmodium falciparum

“…Plotting the observed absorbance increase at 415 nm as a function of PfHO concentration and fitting with a quadratic binding equation gives an apparent dissociation constant ( K d ) of 9 μ m (Fig. 6 D ), indicating modest affinity and consistent with prior reports (22, 23). A nearly identical heme affinity was determined by measuring quenching of PfHO tryptophan fluorescence upon heme binding (Fig.…”

“…On the basis of in vitro assays performed with the recombinant HO-like domain of PfHO and recombinant P. falciparum ferredoxin, it was proposed that PfHO catalyzes the degradation of heme to bilirubin (22). A subsequent study of recombinant full-length PfHO (amino acids 1–305) by a different group concluded that PfHO catalyzes degradation of heme to biliverdin (23). To evaluate whether PfHO is indeed a heme-degrading enzyme, we first analyzed its sequence features and then tested the ability of recombinant PfHO to bind and degrade heme.…”

“…Nevertheless, recent in vitro studies of a recombinant HO-like protein (PF3D7_1011900, formerly PF10_0116) from P. falciparum (termed PfHO) showing sequence similarity to known HO enzymes have suggested that parasites may enzymatically degrade some heme to BV or BR, using an apicoplast-targeted ferredoxin as a reductant co-factor (22, 23). Because of conflicting suggestions and assumptions in the literature and because no direct tests of enzymatic heme degradation by parasites have been reported, confusion and uncertainty persist regarding the presence of a heme oxygenase pathway within blood stage P. falciparum parasites (2, 19, 24, 25).…”

Iron and heme metabolism in Plasmodium falciparum and the mechanism of action of artemisinins

“…Earlier studies indicated that iron might be released during degradation of hematin by hydrogen peroxide in the digestive vacuole or by reaction with reduced glutathione in the parasite cytoplasm (see review by Scholl et al [8]). A putative heme oxygenase was recently identified in the parasite [20,21]; however, no heme oxygenase activity has been detected in infected RBCs and the designation of these enzymes as heme oxygenases has been questioned [22].…”

Papel do IP3 na transdução de sinal e função da heme oxigenase em Plasmodium falciparum.

“…(Sherman et al, 1965). A via alternativa de detoxificação da hemina formando BV e BR catalisada pela PfHO foi proposta pelo Okada (2009), entretanto a PfHO apresenta uma baixa ativida em converter hemina em BR (Sartorello et al, 2010) sugerindo uma função alternativa da detoxificação da hemina.…”