Suppression of experimental cerebral malaria by disruption of malate:quinone oxidoreductase

Abstract

Background: Aspartate, which is converted from oxaloacetate (OAA) by aspartate aminotransferase, is considered an important precursor for purine salvage and pyrimidine de novo biosynthesis, and is thus indispensable for the growth of Plasmodium parasites at the asexual blood stages. OAA can be produced in malaria parasites via two routes: (i) from phosphoenolpyruvate (PEP) by phosphoenolpyruvate carboxylase (PEPC) in the cytosol, or (ii) from fumarate by consecutive reactions catalyzed by fumarate hydratase (FH) and malate:quinone oxidoreductase (MQO) in the mitochondria of malaria parasites. Although PEPC-deficient Plasmodium falciparum and Plasmodium berghei (rodent malaria) parasites show a growth defect, the mutant P. berghei can still cause experimental cerebral malaria (ECM) with similar dynamics to wild-type parasites. In contrast, the importance of FH and MQO for parasite viability, growth and virulence is not fully understood because no FH- and MQO-deficient P. falciparum has been established. In this study, the role of FH and MQO in the pathogenicity of asexual-blood-stage Plasmodium parasites causing cerebral malaria was examined.

Results: First, FH- and MQO-deficient parasites were generated by inserting a luciferase-expressing cassette into the fh and mqo loci in the genome of P. berghei ANKA strain. Second, the viability of FH-deficient and MQO-deficient parasites that express luciferase was determined by measuring luciferase activity, and the effect of FH or MQO deficiency on the development of ECM was examined. While the viability of FH-deficient P. berghei was comparable to that of control parasites, MQO-deficient parasites exhibited considerably reduced viability. FH activity derived from erythrocytes was also detected. This result and the absence of phenotype in FH-deficient P. berghei parasites suggest that fumarate can be metabolized to malate by host or parasite FH in P. berghei-infected erythrocytes. Furthermore, although the growth of FH- and MQO-deficient parasites was impaired, the development of ECM was suppressed only in mice infected with MQO-deficient parasites.

Conclusions: These findings suggest that MQO-mediated mitochondrial functions are required for development of ECM of asexual-blood-stage Plasmodium parasites.

Keywords: Fumarate hydratase (FH); Luciferase–luciferin system; Malate:quinone oxidoreductase (MQO); Plasmodium berghei.

Fig. 1

Establishment of fumarate hydratase (FH)- and malate:quinone oxidoreductase (MQO)-deficient P. berghei. a Expression of luciferase (Luc2) and fh in control and Δfh parasites. b Expression of Luc2 and mqo in control and Δmqo parasites. The genes were subjected to semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR) using specific primers (see Additional files 2, 3). hsp70 was used as a positive control. Samples treated with DNase-treated RNA template [hsp70 (−)] were used as a negative control that is the control of eventual DNA contamination of the RNA preparations. c FH activity in infected (left) and uninfected (right) erythrocytes. d MQO activity in infected erythrocytes. Results are expressed as mean ± SD of triplicate determinations

Fig. 2

Parasite growth is delayed by deficiency of FH and MQO. a C57BL/6 mice were infected with 5 × 10⁶ to 5 × 10⁷ schizonts of control, Δfh and Δmqo parasites. At 6, 12, 18 and 24 h after inoculation, the proportion of asexual-blood-stage forms in 100 infected erythrocytes was determined. Results are expressed as mean ± SD of three mice. Asterisks indicate statistically significant differences (*, vs. control parasites). b Absolute luciferase activity values in trophozoites of wild-type (WT), control, Δfh and Δmqo parasites. Trophozoites (5 × 10⁶) were obtained from C57BL/6 mice at 8 and 16 h post-inoculation with purified schizonts. Results are expressed as mean ± SD absolute luciferase activity values of three wells at 20 min after addition of d-luciferin. Experiments were performed in duplicate, and representative data are shown. Asterisks indicate statistically significant differences (**, vs. control and Δfh parasites)

Fig. 3

Mitochondrial membrane potential (MMP) in trophozoites of control, Δfh and Δmqo parasites. a Bright-field and fluorescence microscopy images of control, Δfh and Δmqo parasites (left panel). MMP was assessed using the MMP-sensitive fluorochrome MitoTracker^® Red CMXRos (MitoTracker, red). The inner membrane and cell nuclei were stained with BODIPY FL C16 (BODIPY, green) and Hoechst 33342 (Hoechst, blue), respectively. Bar indicates 5 µm. b Mean fluorescence intensity (MFI) of MitoTracker (right panel). Results are expressed as mean ± SD of 100 control, Δfh and Δmqo trophozoites. Experiments were performed in duplicate, and representative data are shown

Fig. 4

Parasite viability is restored by addition of fumarate or malate. Fold change of luciferase activity in control, Δfh and Δmqo parasites after cultivation. Infected erythrocytes (1 × 10⁷) were cultured for 3 h in RPMI 1640 medium supplemented with 5 mM fumarate (Fum), 5 mM malate (Mal) or control PBS (Ctrl). Results are expressed as mean ± SD fold changes of three wells in relative luciferase units (RLU) compared with the control at 5 min after addition of d-luciferin. Experiments were performed in duplicate, and representative data are shown. Asterisks indicate statistically significant differences (*, vs. control)

Fig. 5

Experimental cerebral malaria (ECM) is suppressed by deficiency of parasite MQO but not FH. Female C57BL/6 mice were infected with 1 × 10⁴ infected erythrocytes of control, Δfh and Δmqo parasites. a Course of parasitaemia. Asterisks indicate a statistically significant difference (*, control vs. Δfh-infected mice; **, control vs. Δmqo-infected mice). b Survival rate. Note that neurologic signs of ECM were observed in mice infected with control and Δfh parasites on day 7 post-inoculation. c Brains of mice injected with Evans blue (top panels). Brains were obtained from uninfected and control- and Δfh-infected mice on day 7 post-infection, and from Δmqo-infected mice on days 7 and 14 post-infection. Scale bar 10 mm. Quantitative analysis of Evans blue extravasation in the brain (bottom). Results are expressed as mean ± SD of three mice. Experiments were performed in duplicate, and representative data are shown. Asterisks indicate statistically significant differences (**, control vs. Δmqo-infected mice)