Unlike the synchronous Plasmodium falciparum and P. chabaudi infection, the P. berghei and P. yoelii asynchronous infections are not affected by melatonin

Abstract

We have previously reported that Plasmodium chabaudi and P. falciparum sense the hormone melatonin and this could be responsible for the synchrony of malaria infection. In P. chabaudi and P. falciparum, melatonin induces calcium release from internal stores, and this response is abolished by U73122, a phospholipase C inhibitor, and luzindole, a melatonin-receptor competitive antagonist. Here we show that, in vitro, melatonin is not able to modulate cell cycle, nor to elicit an elevation in intracellular calcium concentration of the intraerythrocytic forms of P. berghei or P. yoelii, two rodent parasites that show an asynchrononous development in vivo. Interestingly, melatonin and its receptor do not seem to play a role during hepatic infection by P. berghei sporozoites either. These data strengthen the hypothesis that host-derived melatonin does not synchronize malaria infection caused by P. berghei and P. yoelii. Moreover, these data explain why infections by these parasites are asynchronous, contrary to what is observed in P. falciparum and P. chabaudi infections.

Keywords: calcium; cell cycle; malaria; melatonin; rhythm; sporozoite.

Figure 1

Calcium mobilization in Fluo-3 labeled P. berghei-isolated parasites. A) Addition of thapsigargin (THG, 5 μM) in 1 mM Calcium medium. B) Addition of 25 μM monensin (MON) to medium containing 1 mM Calcium. C) Addition of 5 μM THG to calcium-free medium. D) Addition of 25 μM MON to calcium-free medium. E) Addition of 20 μM MON to P. yoelii parasites, in 1 mM calcium medium.

Figure 2

Effects of melatonin addition in isolated, Fluo-3 labeled, P. berghei and P. yoelii parasites. A) Addition of 20 μM melatonin (MEL) to P. berghei in medium containing 1mM calcium. B) Addition of 20 μM MEL to P. berghei in calcium-free medium. C)Addition of 40 μM melatonin (MEL 40) followed by 25 μM monensin to P. yoelii in medium containing 1mM calcium. D) Addition of 50 μM melatonin (MEL 50) followed by THG (5 μM) to P. yoelii in medium containing 1 mM calcium. These experiments show that melatonin was not able to elicit a calcium response on these parasites.

Figure 3

In vitro culture of P. berghei A) and P. yoelii B) incubated with different melatonin concentrations. The figure shows the distribution of P. berghei and P. yoelii life forms after 18 or 13 hours incubation, respectively, with different melatonin concentrations (1 nM, 10 nM, 100 nM e 1 μM). There are no statistical differences in the distribution. The results are presented as the mean of three independent experiments.

Figure 4

In vitro culture of P. berghei A) and P. yoelii B) incubated with different melatonin concentrations. The figure shows P. berghei- and P. yoelii-infected red blood cells (iRBC) after 18 or 13 hours incubation, respectively, melatonin concentrations (1 nM, 10 nM, 100 nM and 1 μM). There are no statistical differences in the number of iRBC. The results are presented as the mean of three independent experiments.

Figure 5

Distribution of P. berghei in infected Wistar rats, five days after inoculation of 10⁷ infected erythrocytes. Notes: To assess life forms distributions, no less than 1000 cells were counted in Giemsa-stained smears. No statistical differences were observed between the percentage of rings and trophozoites. Schizonts were not present in peripheral bloodstream due to microvasculature sequestration. Abbreviations: R, rings; T, trophozoites; S, schizonts.

Figure 6

A) Effect of inhibition of melatonin receptor in C3H mice infected by P. berghei sporozoites. Liver infection load was measured by qRT-PCR analysis of P. berghei 18 S rRNA in liver extracts taken 40 h after sporozoite i.v. injection, and plotted as a percentage of the mean of negative control samples. The plot represents three independent experiments (n = 18). No statistical significances were observed. B) Effect of 400 nM melatonin on infection of mouse primary hepatocytes by P. berghei sporozoites. Infection rates were calculated for each sample well as the number of EEFs, plotted as a percentage relative to the mean of negative control samples. Results are expressed as the mean ± SD of triplicate in three independent experiments. Abbreviations: EEFs, exoerythrocytic forms; qRT-PCR, quantitative reverse transcriptase polymerase chain reaction; SD, standard deviation.