Trophozoites

BackgroundThe activity of several well-known anti-malarials, including chloroquine (CQ), is attributed to their ability to inhibit the formation of haemozoin (Hz) in the malaria parasite. The formation of inert Hz, or malaria pigment, from toxic haem acquired from the host red blood cell of the parasite during haemoglobin digestion represents a pathway essential for parasite survival. Inhibition of this critical pathway therefore remains a desirable target for novel anti-malarials. A recent publication described the results of a haem fractionation assay used to directly determine haemoglobin, free haem and Hz in Plasmodium falciparum inoculated with CQ. CQ was shown to cause a dose-dependent increase in cellular-free haem that was correlated with decreased parasite survival. The method provided valuable information but was limited due to its low throughput and high demand on parasite starting material. Here, this haem fractionation assay has been successfully adapted to a higher throughput method in 24-well plates, significantly reducing lead times and starting material volumes.MethodsAll major haem species in P. falciparum trophozoites, isolated through a series of cellular fractionation steps were determined spectrophotometrically in aqueous pyridine (5 % v/v, pH 7.5) as a low spin complex with haematin. Cell counts were determined using a haemocytometer and a rapid novel fluorescent flow cytometry method.ResultsA higher throughput haem fractionation assay in 24-well plates, containing at most ten million trophozoites was validated against the original published method using CQ and its robustness was confirmed. It provided a minimum six-fold improvement in productivity and 24-fold reduction in starting material volume. The assay was successfully applied to amodiaquine (AQ), which was shown to inhibit Hz formation, while the antifolate pyrimethamine (PYR) and the mitochondrial electron transporter inhibitor atovaquone (Atov) demonstrated no increase in toxic cellular free haem.ConclusionsThis higher throughput cellular haem fractionation assay can easily be applied to novel anti-malarials with a significantly decreased lead time, providing a valuable tool with which to probe the mechanisms of action of both new and established anti-malarials.

Drug discovery is a key part of malaria control and eradication strategies, and could benefit from sensitive and affordable assays to quantify parasite growth and to help identify the targets of potential anti-malarial compounds. Bioluminescence, achieved through expression of exogenous luciferases, is a powerful tool that has been applied in studies of several aspects of parasite biology and high throughput growth assays. We have expressed the new reporter NanoLuc (Nluc) luciferase in Plasmodium falciparum and showed it is at least 100 times brighter than the commonly used firefly luciferase. Nluc brightness was explored as a means to achieve a growth assay with higher sensitivity and lower cost. In addition we attempted to develop other screening assays that may help interrogate libraries of inhibitory compounds for their mechanism of action. To this end parasites were engineered to express Nluc in the cytoplasm, the parasitophorous vacuole that surrounds the intraerythrocytic parasite or exported to the red blood cell cytosol. As proof-of-concept, these parasites were used to develop functional screening assays for quantifying the effects of Brefeldin A, an inhibitor of protein secretion, and Furosemide, an inhibitor of new permeation pathways used by parasites to acquire plasma nutrients.

Giardia trophozoites attach to the intestinal microvilli (or inert surfaces) using an undefined "suction-based" mechanism, and remain attached during cell division to avoid peristalsis. Flagellar motility is a key factor in Giardia's pathogenesis and colonization of the host small intestine. Specifically, the beating of the ventral flagella, one of four pairs of motile flagella, has been proposed to generate a hydrodynamic force that results in suction-based attachment via the adjacent ventral disc. We aimed to test this prevailing "hydrodynamic model" of attachment mediated by flagellar motility. We defined four distinct stages of attachment by assessing surface contacts of the trophozoite with the substrate during attachment using TIRF microscopy (TIRFM). The lateral crest of the ventral disc forms a continuous perimeter seal with the substrate, a cytological indication that trophozoites are fully attached. Using trophozoites with two types of molecularly engineered defects in flagellar beating, we determined that neither ventral flagellar beating, nor any flagellar beating, is necessary for the maintenance of attachment. Following a morpholino-based knockdown of PF16, a central pair protein, both the beating and morphology of flagella were defective, but trophozoites could still initiate proper surface contacts as seen using TIRFM and could maintain attachment in several biophysical assays. Trophozoites with impaired motility were able to attach as well as motile cells. We also generated a strain with defects in the ventral flagellar waveform by overexpressing a dominant negative form of alpha2-annexin::GFP (D122A, D275A). This dominant negative alpha2-annexin strain could initiate attachment and had only a slight decrease in the ability to withstand normal and shear forces. The time needed for attachment did increase in trophozoites with overall defective flagellar beating, however. Thus while not directly required for attachment, flagellar motility is important for positioning and orienting trophozoites prior to attachment. Drugs affecting flagellar motility may result in lower levels of attachment by indirectly limiting the number of parasites that can position the ventral disc properly against a surface and against peristaltic flow.

Entamoeba histolytica, a protozoan parasite, causes amebiasis, which is a global public health problem. Clinical manifestation and pathogenesis of amebiasis are closely associated with the proliferation and tissue invasion abilities of E. histolytica trophozoites. At the same time, E. histolytica trophozoites are studied in a broad range of biology research fields. As such, the development of in vitro culture of E. histolytica trophozoites greatly facilitated amebic research. Now, a standard method for cryopreservation of E. histolytica trophozoites is required because available methods either do not give a high enough revival rate, which can significantly delay projects, or they are not widely adopted. Here, we attempted to optimize the conditions for E. histolytica trophozoite cryopreservation, including cell density, cooling rate, cell freezing reagent, and freezing profile. We found an optimized condition that reproducibly yielded >30% revival, regardless of storage period in liquid nitrogen (up to 365 days). This optimized condition is that E. histolytica trophozoites are suspended in 0.5 mL CELLBANKER 2 in a 1 mL cryotube at 2 × 106 cells/mL and frozen from 4 to -40°C at a rate of -0.2°C/min using VIA Freeze Uno. This cryopreservation method can minimize the risk of losing E. histolytica trophozoite lines required to continue projects, facilitating amebic research.ImportanceAmebiasis, which is caused by Entamoeba histolytica infection, is the third deadliest parasitic disease globally. Proliferation of E. histolytica trophozoites and their invasion into the host tissues cause amebiasis symptoms and pathogenesis. E. histolytica trophozoites are also important in multiple biology research topics. Therefore, E. histolytica trophozoites are a common subject in academic as well as clinical fields. A standard method for in vitro culture of E. histolytica trophozoites is well established. By contrast, a widely adopted practical method for cryopreservation of E. histolytica trophozoites is not yet available. This hampers the advancement of amebic research, as the required E. histolytica trophozoite lines sometimes cannot be revived from cryopreservation. In this study, we varied parameters critical to the revival rate, namely, cell density, cooling rate, freezing reagent, and freezing profile, and present an optimized cryopreservation method for E. histolytica trophozoites, which gives reproducibly high revival rates.