Copper ions are essential trace nutrients for all higher plants and animals at extremely low concentrations. They play an extensive role in living organisms, from microbes to plants and animals, by regulating the activities of several critical copper-binding proteins such as cytochrome c oxidase, Cu/Zn superoxide dismutase, dopamine β-hydroxylase, prion protein, tyrosinase, X-linked inhibitor of apoptosis protein, lysyl oxidase, metallothionein, ceruloplasmin, and other proteins
[12, 13]. Particularly in relation to microbes, copper ions are critical participants in the mitochondrial respiratory reaction and in energy generation, regulation of iron acquisition, oxygen transport, the cellular stress response, antioxidant defense, and several other important processes. The yeast Saccharomyces cerevisiae provides an accessible model for eukaryotic copper transport. Uptake of the Cu2+ ion by yeast cells is accompanied by reduction of Cu2+ to Cu1+ by a metalloreductase in the plasma membrane. Subsequent transport of the Cu1+ ion across the plasma membrane is carried out by a copper transporter (Ctr). Within the cell, Cu1+ ions are bound to the copper chaperones Atx1, Cox17, and CCS for specific delivery to the Golgi complex, mitochondria, and Cu/Zn superoxide dismutase, respectively
. Although there is no comprehensive understanding of copper metabolism and function in P. falciparum, the proteins involved in copper pathways and trafficking have been identified in Plasmodium spp. These include a putative copper channel, a copper transporter, a putative COX17, and a copper-transporting ATPase
[6, 15, 16].
TTM has been known inhibit copper-binding proteins that regulate copper physiology through formation of a sulfur-bridged copper–molybdenum cluster, rather than by direct chelation of copper ions
. In the current study, TTM caused profound cessation of the growth of P. falciparum; this arrest resulted from inhibition of schizogony of the parasite. In contrast, treatment of uninfected RBCs with higher concentrations of TTM caused only slight growth arrest. Thus, the target molecule(s) of TTM may be present predominantly in the parasite, although the molecule(s) involved in the growth arrest of the parasite remain to be determined. Also, the possibility that the excess TTM affects, directly or indirectly, various proteins that do not bind to copper, and thus causes developmental arrest of the parasite, remains to be elucidated.
Chelation with Neocuproine, which selectively removes Cu1+
, inhibited the successive ring–trophozoite–schizont progression of P. falciparum effectively at extremely low concentration; blockage of trophozoite progression from the ring stage was shown at higher concentrations. In contrast, the growth of P. falciparum pretreated with Neocuproine was arrested only to a very small extent, even when treated with much higher concentrations. This is quite different from the profound developmental arrest of P. falciparum maintained in the presence of Neocuproine throughout the culture period. We surmise that either the binding of Neocuproine may be reversible or copper ions may be replenished by host cells. RBCs contain copper at levels as high as a mean value of 18 μM, although most of the copper present in RBCs is bound to the enzyme superoxide dismutase
Developmental arrest of P. falciparum, similar to that in CDRPMI and GFSRPMI in the presence of Neocuproine and TTM, was detected in the parasite cultured in CDM-C16alone. We have demonstrated previously, using genome-wide transcriptome profiling and various CDMs, profound down-regulation of the putative copper channel in parasites cultured in CDM-C16alone. This was associated with the blockage of trophozoite progression from the ring stage of the parasite. In the current study, the expression of genes encoding copper-binding proteins of P. falciparum was investigated, in detail, with cultures in CDM-C16alone, CDRPMI, and GFSRPMI. Transcript levels of not only a putative copper channel, which has previously been detected by genome-wide transcriptome profiling
, but also a copper transporter were profoundly decreased during the arrested development of the parasite at the ring stage in CDM-C16alone. The severe down-regulation of copper-binding proteins of the parasite cultured in CDM-C16alone is considered to affect copper pathways and trafficking; this maybe involved in the perturbation of copper homeostasis and developmental arrest of the parasite, similar to the growth arrest seen with TTM and Neocuproine. However, the additional involvement of other proteins, such as merozoite surface protein 2, a putative DEAD/DEAH box RNA helicase, a serine repeat antigen 3, and a palmitoyl acyltransferase, which have been demonstrated to be associated with developmental arrest of the early stage of the parasite cultured in CDM-C16alone
, is not excluded.
In addition to their basal functions, such as acting as important intermediates in lipid biosynthesis, there is evidence that various NEFAs are involved in numerous biological processes, including activation of protein kinases and cell proliferation, differentiation, and death
[19–21]. NEFAs also affect numerous cellular systems and functions, including regulation of gene expression, ion-channel functions, and membrane fusion
[22–24]. Saturated NEFAs such as C16:0 have been reported to cause a significant increase in mitochondrial reactive oxygen species, mitochondrial DNA damage, mitochondrial dysfunction, induction of Jun-N-terminal kinase, apoptosis, and inhibition of insulin signaling in skeletal muscle cells. In this study, we detected, for the first time, a profound down-regulation of the transcripts of copper-binding proteins when the parasites were cultured in CDM-C16alone, which contains C16:0. In addition, developmental arrest of the parasite at the ring/trophozoite stage occurred in tandem with the profound decrease in transcript levels. C18:1 (oleic acid) has been reported to prevent the mitochondrial dysfunction and apoptosis induced by C16:0 (palmitic acid)
. Similarly, P. falciparum cultured in CDRPMI containing both C18:1 and C16:0 showed similar growth to the parasite in GFSRPMI, which implies that C18:1 protected the parasite from the developmental arrest induced by C16:0 and the decrease in transcript levels. The mechanisms responsible for the profound down-regulation of copper-binding proteins in the parasite associated with C16:0 remain to be elucidated.