Alpha-tocopherol transfer protein disruption confers resistance to malarial infection in mice
© Herbas et al; licensee BioMed Central Ltd. 2010
Received: 4 February 2010
Accepted: 19 April 2010
Published: 19 April 2010
Various factors impact the severity of malaria, including the nutritional status of the host. Vitamin E, an intra and extracellular anti-oxidant, is one such nutrient whose absence was shown previously to negatively affect Plasmodium development. However, mechanisms of this Plasmodium inhibition, in addition to means by which to exploit this finding as a therapeutic strategy, remain unclear.
α-TTP knockout mice were infected with Plasmodium berghei NK65 or Plasmodium yoelii XL-17, parasitaemia, survival rate were monitored. In one part of the experiments mice were fed with a supplemented diet of vitamin E and then infected. In addition, parasite DNA damage was monitored by means of comet assay and 8-OHdG test. Moreover, infected mice were treated with chloroquine and parasitaemia and survival rate were monitored.
Inhibition of α-tocopherol transfer protein (α-TTP), a determinant of vitamin E concentration in circulation, confers resistance to malarial infection as a result of oxidative damage to the parasites. Furthermore, in combination with the anti-malarial drug chloroquine results were even more dramatic.
Considering that these knockout mice lack observable negative impacts typical of vitamin E deficiency, these results suggest that inhibition of α-TTP activity in the liver may be a useful strategy in the prevention and treatment of malaria infection. Moreover, a combined strategy of α-TTP inhibition and chloroquine treatment might be effective against drug resistant parasites.
Despite recent advances in understanding malaria and Plasmodium, the parasite responsible for the disease, 500 million cases of clinical malarial in over 100 countries still occur. This disease poses a public health problem for 3.3 billion people, a number representing a staggering 50% of the world's population. Furthermore, the global death figure for malaria reaches more than 1 million each year . A number of factors affect the severity of malaria, including the size of the sporozoite infective dose, host nutritional status, acquired immunity level, host genetic factors, parasite features and even certain associated socioeconomic factors [2–7]. Although micronutrient malnutrition is usually highly prevalent in areas in which malaria is endemic, the contribution of these micronutrient deficiencies to malarial symptoms is often overlooked.
Vitamin E is a powerful anti-oxidant that acts mainly in the lipid phase of cells and has a primary role in preventing the oxidation of polyunsaturated fatty acids . While vitamin E deficiency seems to have both protective and adverse effects in malarial infection, the involvement of vitamin E in the genesis of malarial illness is still controversial . The clinical observations that feeding famine victims with grain exacerbated the effects of cerebral malaria were attributed to the vitamin E content of the grain that subsequently influenced severity of malaria symptoms . In addition, according to the results of animal studies, dietary vitamin E deficiency is thought to protect against malarial infection, presumably because the absence of this anti-oxidant leads to an increase in oxygen radicals production derived from the immune response of the host against the infection, consequently making an inhospitable environment for the parasite [11, 12]. However, even if it were shown to be possible to utilize vitamin E deficiency for the prevention or treatment of malaria, it would be quite difficult to actually lower vitamin E in circulation via nutritional manipulation because the majority of daily foods in a normal diet contain significant amounts of vitamin E .
Vitamin E is transported in plasma lipoproteins and, unlike other fat-soluble vitamins, has no specific plasma carrier protein, however, alpha-tocopherol transfer protein (α-TTP), a liver cytosolic protein, acts as an important regulator of vitamin E concentration in circulation [13, 14]. It does this through binding specifically α-tocopherol amongst the other tocopherols, including β and γ-tocopherol, in the liver. Targeted disruption of the α-TTP gene revealed that α-tocopherol concentration in circulation was regulated by α-TTP [13, 15]; heterozygous mutant mice contained plasma concentrations of α-tocopherol half that found in wild type mice while homozygous mutants were shown to have undetectable levels of α-tocopherol in circulation . Actual mechanism is not known. However, it is postulated that chylomicrons remnants with extra amount of α-tocopherol leaked in to the circulation.
The ability to manipulate α-tocopherol levels, and consequently vitamin E levels, via α-TTP gene inhibition inspired us to revisit the impact of serum vitamin E levels on the severity of malarial infection using an established rodent malarial model. Thus, α-TTP inhibition was examined as a potential application for the prevention or treatment of malarial infection. The results indicated that α-TTP inhibition led to parasite DNA damage sufficient to inhibit proliferation. Moreover, a combined therapy with chloroquine (CQ) seems to be useful as a new strategy for the treatment of malaria.
Animals and malaria infection
Since C57BL/6J mice are frequently used as an experimental model in biomedical sciences, α-TTP knockout mice were generated with a C57BL/6J background . These knockout mice were bred in our own colony. BALB/c mice were known to be murine malaria resistant strain. Adult α-TTP knockout mice with a C57BL/6J genetic background, C57BL/6J and BALB/c mice were infected with 4 × 105 Plasmodium berghei NK65 or 4 × 104 Plasmodium yoelii 17XL infected red blood cells (IRBCs) by intraperitoneal injection, and their survival was monitored. Six animals were used in each experimental group. Mice were fed with a commercial diet (CE-2, containing 45 mg/kg of D-α-tocopherol, CLEA Japan, Tokyo, Japan) or α-tocopherol supplementation (CE-2 with supplementary D-α-tocopherol, 600 mg/kg, CLEA Japan, Tokyo, Japan). Furthermore, to determine the effect of vitamin E deficiency on the virulence of P. berghei NK65, α-TTP knockout or C57BL/6J mice were inoculated with P. berghei NK65 (4 × 105 IRBCs) recovered from α-TTP knockout or wild type mice at day 9 post-infection, and their survival was monitored. In a part of the experiments, mice infected with P. berghei NK65 were administered 0-7.5 mg/kg of CQ (C6628, Sigma-Aldrich, St. Louis, USA) on day 0, 1 and 2 after infection, and their survival was monitored. All experiments described in the present study were conducted in accordance with the Guiding Principles for the Care and Use of Research Animals of the Obihiro University of Agriculture and Veterinary Medicine, Japan.
Haematological parameters, such as the number of red blood cells (RBCs), white blood cells (WBCs), haemoglobin concentration, and haematocrit were determined with an autohaematology analyzer (Celltac α, MEK-6358, Nihon Kohden, Tokyo, Japan) during the course of infection. Six mice were used for each experimental group. For the reticulocyte count, 2 μl of whole blood taken from the tail vein were mixed with 2 μl of Brilliant Crystal Blue (Wako, Tokyo, Japan). Then thin smears of sample were prepared, dried at room temperature and stained with Giemsa (Merck, Darmstadt, Germany). The numbers of infected and uninfected reticulocytes, as well as mature RBCs, were determined under light microscopy.
The comet assay
DNA damage of the parasites infecting the mice was assessed by single-cell gel electrophoresis (comet assay) . Evaluation of the shape of the DNA "comet" tail and migration pattern gives an assessment of DNA damage. Whole blood was obtained by cardiac puncture, washed with cold phosphate buffered saline (PBS), and then centrifuged twice at 5,000 rpm for 5 min at 4°C. After removing the buffy coat, RBCs were washed twice in PBS. Cell mixture was suspended in Comet LMAgarose (1% low-temperature-melting agarose, Trevigen, Gaithersburg, MD, USA) at a ratio of 1:10 (v/v), 25 μl of the cell suspension was immediately placed on a CometSlideTM (Trevigen), slides were placed flat in a refrigerator at 4°C for 10 min, and then submerged in 23 ml of lysis solution (Trevigen) at 4°C for 60 min. Subsequently, slides were then maintained in an alkaline solution (>pH13) for 60 min at room temperature in the dark and washed two times in 1× TBE buffer (Tris-borate EDTA) for two times (5 min. each). Finally, slides were subjected to electrophoresis in 1× TBE buffer at 25 V for 10 min, stained with SYBR Green (Trevigen), and analysed under microscopy (IX-70, Olympus Co., Tokyo, Japan). Denatured cleaved DNA fragments migrated out of the cell under the stimulus of an electric potential, whereas the undamaged supercoiled DNA remained within the confines of the cell membrane when a current was applied. Parasites from α-TTP knockout with 18-29% of parasitaemia were recovered on day 21 after the infection. Parasites from C57BL/6J with 40-49% of parasitaemia were collected on day 12 after the infection. Four mice were used each experimental group.
Detection of biomarker for oxidative stress with anti-8-hydroxy-2'-deoxyguanosine (8-OHdG) antibody
Collected blood from infected animals (n = 4) was mixed with an equivalent volume of PBS and then was centrifuged at 5,000 rpm for 5 min at 4°C, then the supernatant was removed (this step was repeated 3 times). The percentages of parasitaemia in α-TTP knockout and C57BL/6J mice were 27% and 35%, respectively. The pellet was resuspended with 0.1 ml of PBS and 0.1 ml of 3% foetal calf serum. The suspension was fixed with methanol for 10 min. Anti-8OHdG (2 ng/ml), a biomarker for the oxidative damage of DNA , monoclonal antibody (N45.1; MOG-20P; Japan Institute for the Control of Aging, Nikken SEIL Co. Ltd., Shizuoka, Japan) was labelled with Biotin-XX Mouse IgG1 according to the manufacturer's instructions (Z25052: ZenonTM Biotin-XX mouse IgG1, Molecular Probes, Eugene, USA), then this complex was incubated with the samples for 45 min at 37°C. The sample was washed twice with PBS for 5 min, and was incubated with streptavidine-Alexa Fluor 488 conjugate (Molecular probes) for 45 min at 37°C. The incubated sample was washed twice with PBS for 5 min. Parasite DNA was stained with propidium iodide (P1304 MP; Molecular Probes) containing RNase A (10109142001; Roche Applied Science, Mannheim, Germany) for 10 min at 37°C. After washing with PBS, the sample was treated with 1% n-propyl gallate (102747; MP Biomedicals, Irvine, CA USA), an anti-oxidant, and observed with a confocal laser microscope (DMRB/E, TCS NT; Leica Microsystems, Wetzlar, Hessen, Germany).
Quantification of anti-oxidative stress enzymes of P. berghei NK65
Primer and Probe sequences used in real--time quantitative PCR
Statistical analysis was performed using one-way variance analysis (S-plus6 software for windows) (Insightful corporation, Seattle Washington USA). Data are expressed as means of the standard error of the mean (SEM). P values less than 0.05 were considered to be significant. Survival rate % analysis was performed using the Kaplan-Meier method.
Resistance of α-TTP knockout mice to malaria infection
The spleen plays an important role during malarial infection removing uninfected and infected red blood cells from circulation. Histological analysis of spleens revealed the presence of haemozoin, a toxic haem product derived from the haemoglobin digestion thought to be an indication of macrophage phagocytic activity and parasite maturation , in the red pulp was visible by day 6 that increased in intensity by day 9 in wild type animals (Figure 1e). In addition, during the acute phase of the infection an increase in mature RBCs could be found indicating erythropoietic activity might be diminished in the wild type mice, a condition that could lead to anaemia. In contrast to wild type animals, α-TTP knockout mice displayed very little pigmentation or increase in mature RBCs even by day 9 indicative of delayed parasite maturation in these mice. In addition, mature RBCs and erythroblasts were intermixed in the red pulp of the spleen in α-TTP knockout mice indicating that red blood cell production is not affected in the infected mice early after infection. Moreover, the mRNA expression of erythropoietin receptor (EPOR) in bone marrow was significantly decreased in both genotypes on day three post infection, however the expression of EPOR was significantly increased in liver and spleen suggesting such organs compensate bone marrow function in order to avoid anaemia during the acute phase of the infection (manuscript under submission). These results indicate that α-TTP knockout animals are displaying no signs of infection even at a time when most wild type animals are dying.
DNA damage of Plasmodium in α-TTP knockout mice
To elucidate whether the virulence of parasites in α-TTP knockout mice might be affected during infection due to parasite DNA damage, mice were inoculated with P. berghei NK65 that had been recovered from previously infected α-TTP knockout or wild type mice 9 days post infection. Tellingly, both the α-TTP knockout and wild type mice infected with parasites recovered from the α-TTP knockout mice survived significantly longer than the mice infected with parasites recovered from wild type mice (Figure 3c). Surprisingly, despite increased longevity, parasitaemia kinetics in wild type mice and knockout mice were similar irrespective of the source of the parasites (Figure 3d). These results suggest that the virulence of the parasites existing in α-TTP knockout mice had decreased during their time within that host.
Expression of anti-oxidative enzymes in infected parasites
Parasite invasion into RBCs
Effect of chloroquine administration coupled with α-TTP disruption
Previous reports have indicated that reduction of host Vitamin E concentration through dietary restriction negatively impacted the development of Plasmodium and malaria development [11, 12, 20]. From 1954 to 1971, the notion that malnutrition was protective against malaria infection was promoted. This idea was strongly supported by epidemiological studies made from 1975 to 1980 by Murray et al . In addition, animal studies appeared to support this idea. For example, Levander et al  demonstrated that vitamin E and selenium status have a profound impact on the ability of the host to resist acute infectious diseases. The relation between nutritionally-induced oxidative stress and malaria was firstly studied using the Chinese traditional anti-malarial drug, Qinghaosu. This compound is thought to destroy the parasite by generating oxy-free radicals. Therefore, the idea of dietary deprivation of vitamin E, a free radical scavenger, might be expected to enhance the anti-malarial activity of the drug . In this study, rather than reduce vitamin E via diet, the α-TTP gene responsible for regulation of host vitamin E concentration was inhibited. Modification of host nutritional status through inhibition of α-TTP and subsequent lowering of vitamin E concentration conferred resistance to malarial infection (Figure 1).
Parasite proliferation was dramatically inhibited in the knockout mice infected with P. yoelii compared to the knockout mice infected with P. berghei NK65. The increased sensitivity to oxidative damage of P. yoelii compared to P. berghei has been demonstrated . In these studies, mice infected with P. yoelii and treated with intravenous injection of H2O2 almost cleared parasites compared to those infected with P. berghei in which parasitaemia still increased. This phenomenon was due to an earlier and higher production of O2 in the splenic macrophages as compared with the mice infected with P. berghei. Moreover, mice infected with P. yoelii sporozoites showed a decrement in the cellular inflammatory response against infection . Therefore, it is speculated that vitamin E deficiency enhances the susceptibility of P. yoelii to oxidative damage due to the exacerbative production of ROS from the immune response of the host and likely due to a decreased immune response. Moreover, α-TTP knockout mice exposed to 10-20 P. berghei ANKA infected mosquitoes displayed similar survival rates to the α-TTP knockout mice infected by IRBCs via intraperitoneal injection (Additional file 1). Thus, it seems that α-TTP gene disruption does not influence development of the sporozoite stage of parasites in the liver (pre-erythrocytic stage). It is well known that vitamin E, through its free radical scavenging properties, prevents damage of molecules such as DNA, lipids and proteins in the face of production and biotransformation of ROS in biological systems . Consistent with the notion that malaria resistance in α-TTP knockout mice resulted from lost vitamin E scavenging activity, reduced Plasmodium proliferation was reversible upon vitamin E supplementation (Figure 2). The absence of vitamin E in the host cells would lead to an oxidative stressful environment that would require parasites to efficiently use their anti-oxidant systems for survival such as the thioredoxin system [25–27].
Furthermore, anti-oxidant activity in these parasites was raised and DNA damage was evident. After initial high levels of expression the transcription of γ-GCS, Grx and TrxR genes dropped in the knockout mice, while parasitaemia increased presumably as the parasites adapted to the oxidative environment . This is in contrast to parasites hosted within wild type mice where the expression of γ-GCS and Grx increased over time, as haemoglobin consumption would be expected to occur and consequently create an oxidative environment  (Figures 3a-b). Taken together, it appears that inhibition of α-TTP gene activity leads to an inhospitable environment for parasites residing within the host. Since the nature of dietary fat alters the lipid composition of RBC membranes and malarial parasites can not biosynthesize their own fatty acids, likely, parasites and RBC membranes of mice fed with vitamin E deficient diets enriched with fish oil containing diets are highly susceptible to peroxidation due to a normal production of ROS . In this study, knockout mice fed with normal diet of vitamin E did not show early after infection stressing that one of the reasons of inhibition of parasite proliferation in the knockout mice is clearly related to oxidative damage of the parasite. This suggests that knockout mice might possess additional mechanisms to keep normal levels of vitamin E in the RBC membranes . It is impressive that α-TTP knockout mice survived with rising levels of parasitaemia at the later stage of the infection. Possible explanation might be that hosts surviving initial infection selected parasites less able to cause death to hosts. However, it is important to point out that whilst parasitaemia increases, anaemia status is more severe, suggesting that anaemia is a defense mechanism of the host for the prevention of parasite proliferation. Also, it can be speculated that parasites at this point are less virulent that the ones during the acute phase of the infection. Evidence for this notion comes from Comet assay revealing DNA damage to parasites hosted within the α-TTP knockout mice at the earliest points after infection. Furthermore, parasites recovered from these mice were less virulent than those recovered from the wild type mice; indeed, wild type mice infected with knockout recovered parasites survived significantly longer than those infected with wild type recovered parasites (Figures 3c-d). While these finding potentially explain the survival of knockout mice despite high levels of parasitaemia, they do not explain the observation of wild type mice surviving initial parasitaemia. This phenomenon, however, is certainly worth future study and may be used to gain insight into similar dynamics in human populations with malaria.
In this study, the mRNA expression of IL-10, INF-γ and TNF-α in liver, kidney, and spleen throughout infection was similar in the knockout mice as compared to the wild type mice suggesting that the inflammatory-immune response of the knockout mice is not altered by α-TTP disruption (manuscript in preparation). It has been suggested that the acquired immune response of the host likely play an important role in the parasite elimination . When α-TTP knockout mice were infected with P. yoelii and re-infected with P. berghei ANKA on day 14 post-infection, 80% of the knockout mice were able to survive showing a very low parasitaemia. In contrast, 20% of the knockout mice infected with P. berghei ANKA or P. yoelii and P. berghei ANKA simultaneously were able to survive, indicating that knockout mice might be able to generate an acquired immunity or produce malarial antibodies against parasites. In this study parasite infecting the knockout mice were able to proliferate from day 15 after infection suggesting that either parasites might develop a resistance characteristic against malarial antibody or host protective immune response is impaired in these mice due to a mechanism of adaptation between parasite and mice. The mRNA expression of IL-10, INF-γ, and TNF in liver, kidney, and spleen throughout infection was similar in the knockout mice as compared to the wild type mice.
With the finding that inhibition of α-TTP leads to malaria resistance, potential exploitation as a novel therapeutic strategy becomes possible. Interestingly, inhibition of α-TTP did not trigger development of anaemia as indicated by the finding that no significant differences in RBC/reticulocyte ratio or total numbers of RBCs could be seen between the α-TTP knockout and wild type mice in uninfected mice (Figure 5a). Moreover, RBCs from the α-TTP knockout were normal in appearance and quality, the maximum and minimum resistance to osmotic stress at different concentrations of NaCl were the same (Additional file 2). Lastly, the concentration of reduced glutathione, an indicator of oxidative status, in the liver unchanged 19.3 ± 1.28 and 17.0 ± 1.20 for the C57BL/6J and α-TTP knockout mice, respectively (Additional file 3). Either α-TTP knockout mice absorb vitamin E from their diet to maintain transiently acceptable levels of vitamin E within the RBCs, as has been demonstrated in patients with familial isolated vitamin E deficiency , or other anti-oxidants such as ascorbate and β-carotene compensate for missing vitamin E in RBC membranes of α-TTP knockout mice . Based on these observations it appears that α-TTP gene inhibition is a potentially valid therapeutic strategy.
Another prerequisite of α-TTP gene disruption as a potential therapeutic strategy lies in the ability to chemically inhibit the protein. Fortunately, in humans, a binding pocket in α-TTP, specific for α-tocopherol, has been revealed by crystallography and is considered to be responsible for the homeostasis of vitamin E . Disruption of α-TTP inhibits α-tocopherol transfer from the liver and thus, would serve as an ideal target in drug design.
One of the most exciting findings from this study lies in the use of combinations of CQ , a classic anti-malarial drug, and gene disruption; levels of CQ found to leave parasites remaining in wild type mice removed all traces of the parasite from α-TTP knockout mice. By using such a combination, levels of CQ known to be toxic to patients could be avoided. Furthermore, drug resistance of Plasmodium, a serious problem in many parts of the world, could be avoided in a manner similar to avoidance of HIV drug resistant strains through combinations of drugs . In terms of genetics, this duel approach is similar to synthetic lethality whereby simultaneous disruption of two genes results in a synergistic effect that is greater than either single gene disruption [32–34]. The finding that the presence of CQ is able to impair the intracellular alpha tocopherol transport suggests that CQ treatment and disruption of α-TTP likely are impinging on the same cellular process and therefore prone to such a synthetic therapeutic approach . This synthetic lethal approach has been used recently for drug discovery  and has begun to be exploited in cancer research [37, 38]. Lastly, it is possible that this type of strategy might be extended to other parasites as α-TTP gene disruption was found to be protective against a variety of parasites .
Inhibition of α-TTP activity in host leads to a resistance characteristic against malaria infection due to parasite's DNA oxidative damage. Therefore, α-TTP inhibition in host might be a new strategy for its prevention and control. Moreover, a combined strategy of α-TTP inhibition and chloroquine treatment might be effective against drug resistant parasites.
The authors thank Dr. Bryce Nelson for his valuable advice. This work was funded by a grant from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
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