A Sphingolipid Inhibitor Induces a Cytokinesis Arrest and Blocks Stage Differentiation in Giardia lamblia

Abstract

Sphingolipid biosynthesis pathways have recently emerged as a promising target for therapeutic intervention against pathogens, including parasites. A key step in the synthesis of complex sphingolipids is the glucosylation of ceramide, mediated by glucosylceramide (GlcCer) synthase, whose activity can be inhibited by PPMP (1-phenyl-2-palmitoylamino-3-morpholino-1-propanol). In this study, we investigated whether PPMP inhibits the proliferation and differentiation of the pathogenic parasite Giardia lamblia, the major cause of parasite-induced diarrhea worldwide. PPMP was found to block in vitro parasite replication in a dose-dependent manner, with a 50% inhibitory concentration of 3.5 μM. The inhibition of parasite replication was irreversible at 10 μM PPMP, a concentration that did not affect mammalian cell metabolism. Importantly, PPMP inhibited the completion of cell division at a specific stage in late cytokinesis. Microscopic analysis of cells incubated with PPMP revealed the aberrant accumulation of cellular membranes belonging to the endoplasmic reticulum network in the caudal area of the parasites. Finally, PPMP induced a 90% reduction in G. lamblia differentiation into cysts, the parasite stage responsible for the transmission of the disease. These results show that PPMP is a powerful inhibitor of G. lamblia in vitro and that as-yet-uncharacterized sphingolipid biosynthetic pathways are potential targets for the development of anti-G. lamblia agents.

Giardia lamblia is one of the most common parasites found in the intestinal tracts of vertebrates, including humans (21), and is the causative agent of giardiasis, an acute or chronic infection of the intestine. The binucleated parasite is found worldwide, but the incidence of giardiasis in humans is highest in developing countries, where the disease is a significant cause of morbidity, especially in children (11). In addition, giardiasis is regarded as one of the most common causes of traveler's diarrhea (7, 25, 38, 40).

The simple life cycle of G. lamblia consists of replicating trophozoites, responsible for pathogenesis, and nonreplicating, environmentally resistant cysts, responsible for disease transmission (35). Currently available anti-G. lamblia drugs target primarily the trophozoite stage of the parasite. Examples are metronidazole, the current drug of choice, and nitrofuran compounds such as furazolidone and albendazole. All of these drugs cause serious in vivo side effects, often in the gastrointestinal tract, which frequently necessitate treatment interruption (9). In vitro genotoxic and cytotoxic effects of nitroimidazoles have been shown as well (20, 29). Moreover, the development of resistance has been demonstrated both in vitro and in vivo (for comprehensive reviews, see references 3 and 36). Thus, there is a need for safe and improved drugs to treat giardiasis.

Sphingolipids are essential membrane components of virtually all eukaryotic cells. An important step in the sphingolipid biosynthesis pathways is the glucosylation of ceramide mediated by glucosylceramide (GlcCer) synthase, whose activity can be modulated by pharmacological inhibitors, including PPMP (dl-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-pro- panol) (1, 37). In the present study, we examined the sphingolipid biosynthetic pathways as a potential target for anti-G. lamblia drugs. Previous studies of G. lamblia lipid metabolism revealed that the parasite has only a limited capability for de novo lipid synthesis but that it is capable of taking up lipids from the environment (6), including gangliosides (26) and ceramide (13). However, data about G. lamblia lipid requirements and lipid biosynthesis pathways are still scarce.

Here, we investigated whether G. lamblia is inhibited by the GlcCer synthase inhibitor PPMP. To evaluate the effects of PPMP on both trophozoites and encysting G. lamblia cells, we analyzed essential processes of parasite replication, the integrity of intracellular structures, adhesion, and cyst formation.

MATERIALS AND METHODS

Biochemical reagents.

Unless otherwise stated, all chemicals were purchased from Sigma and cell culture reagents were from GIBCO BRL. PPMP stock solutions were prepared at 10 mM in methanol. PPMP was freshly diluted to the concentrations required for the individual experiments.

Parasite and tissue culture.

Trophozoites of the G. lamblia strain WBC6 (ATCC catalog number 50803) were grown axenically in 11-ml culture tubes (Nunc, Roskilde, Denmark) containing Diamond's TYI-S-33 medium (6a) supplemented with 10% adult bovine serum and bovine bile. Parasites were harvested by chilling the culture tubes on ice for 30 min to detach adherent cells, and cells were collected by centrifugation at 1,000 × g for 10 min. Cells were then resuspended in phosphate-buffered saline (PBS) and counted using the improved Neubauer chamber. New subcultures were obtained by inoculating 5 × 10⁴ trophozoites from confluent cultures into new tubes.

Two-step encystation was induced as described previously (10, 12), by cultivating the cells for ∼44 h in medium without bile (preencysting medium) and subsequently in medium with a higher pH and porcine bile (encysting medium).

Mammalian cells used in this study were Caco-2 (human colon adenocarcinoma; ATCC HTB 37), intestine 407 (human embryonic jejunoileum; ATCC CCL-6), and MDBK (Madin-Darby bovine kidney; ATCC CCL-22) cells. Cells were routinely cultured in Dulbecco's modified Eagle medium supplemented with 10% fetal calf serum, 2 mM glutamine, 50 U of penicillin/ml, and 50 μg of streptomycin/ml at 37°C with 5% CO₂ in tissue culture flasks. Cultures were trypsinized at least once a week.

Drug incubation, adhesion, and reversibility assays.

The incubation of trophozoites with PPMP was performed with freshly inoculated subcultures. Parasites were allowed to adhere for 8 h and then incubated for an additional 16 h with PPMP at the concentrations indicated in the figures corresponding to the individual experiments. The incubation of encysting cells with the drug was performed in two steps: 7 h of incubation with PPMP in preencysting medium and an additional 16 h of incubation in encysting medium. Cells were then harvested and counted as described above.

The adhesion assay was performed by enumerating nonadherent and adherent parasites from the same culture tube. Nonadherent trophozoites floating in the culture medium were transferred into a new tube, the tube was chilled on ice to prevent adhesion to the tube wall, and the trophozoites were collected by centrifugation. The remaining adherent trophozoites in the original culture tube were harvested as previously described.

The reversibility assay was performed with exponentially growing and stationary-phase trophozoites. In the first case, freshly inoculated subcultures were incubated with the inhibitor for 16 h as described above, harvested, and washed to remove the drug. Collected parasites were then counted and reinoculated in the absence of the inhibitor for an additional 24 h, followed by counting. In the second case, confluent cultures were incubated with the inhibitor for 16 h, harvested, and washed in PBS and 5 × 10⁴ trophozoites were used for new subcultures in the absence of the inhibitor. After an additional 24 h of incubation, cells were collected and counted as described above.

Nile red staining.

Nile red stock solution was prepared at a concentration of 0.5 mg/ml in acetone. Trophozoites were fixed onto glass slides with 3% formaldehyde solution in PBS for 45 min, blocked with a solution of 2% bovine serum albumin and 100 mM glycine in PBS for 1 h, and stained with Nile red at a concentration of 0.5 μg/ml in PBS for 20 min. Cells were extensively washed in PBS and mounted in VECTASHIELD antifade agent (Vector Laboratories, Inc., Burlingame, CA) containing 4′,6-diamidino-2-phenylindole (DAPI) for nuclear staining. Lipid fluorescence was analyzed by employing a filter set for rhodamine (530- to 560-nm-wavelength band-pass excitation).

Immunofluorescence analysis.

Trophozoites and encysting cells were harvested as described above, washed twice in ice-cold PBS, and fixed as before onto glass slides. Fixed cells were permeabilized with 0.2% Triton X-100 in PBS for 20 min, blocked, and incubated with primary antibodies for 1 h. The primary antibodies used in this study were anti-protein disulfide isomerase 2 (anti-PDI2) mouse antiserum at a 1:1,000 dilution, anti-IscS rabbit antiserum (a kind gift of J. Tovar) at a 1:500 dilution, anti-Flex mouse antiserum (corresponding to Giardia Genome Database open reading frame 8855) at a 1:1,000 dilution, and Cy3-conjugated anti-cyst wall protein 1 (anti-CWP1) mouse monoclonal antibody (Waterborne, New Orleans, LA) at a 1:60 dilution. Fluorophore-conjugated secondary antibodies were purchased from Invitrogen (Basel, Switzerland) and used at a 1:200 dilution. Immunofluorescence analysis was performed on an SP2 acousto-optical beam splitter confocal laser-scanning microscope (Leica Microsystems, Wetzlar, Germany) using the appropriate settings. Image stacks of optical sections were further processed using the Huygens deconvolution software package, version 2.7 (Scientific Volume Imaging, Hilversum, The Netherlands). Three-dimensional reconstruction and surface rendering were done with the Imaris software suite (Bitplane, Zurich, Switzerland) using the surpass functions.

Flow cytometric analysis.

Fresh G. lamblia subcultures were treated with PPMP as described above, harvested, and washed in PBS. Parasites (2 × 10⁶) were then stained for 30 min at 4°C with propidium iodide (10 μg/ml) in PBS containing 1% Triton X-100. After washing in PBS, cells were incubated for 30 min at 37°C with DNase-free RNase (10 μg/ml; Roche, Mannheim, Germany) and analyzed for DNA content on a FACSCalibur flow cytometer (Becton Dickinson, Basel, Switzerland).

Mammalian cell metabolic assay.

The metabolic activity of mammalian cells was tested by using the Alamar blue assay. Briefly, Caco-2, CCL-6, and CCL-22 cells were grown to confluence in 96-well plates, incubated for 24 h with PPMP at the concentrations indicated in the figures, and processed according to the instructions of the assay manufacturer (Biosource, Camarillo, CA).

RESULTS

PPMP inhibits G. lamblia trophozoite replication.

In order to characterize the effect of the sphingolipid inhibitor PPMP on G. lamblia, we treated parasite subcultures with different concentrations of the drug, as described in Materials and Methods. Control cells grown for 24 h in the absence of the drug completed an average of three replication cycles (with an average replication time of 8 h). In contrast, the addition of PPMP to the culture medium severely inhibited parasite replication in a dose-dependent manner, with 3.5 μM being the 50% inhibitory concentration (IC₅₀) and with 10 μM PPMP arresting parasite replication (Fig. 1A). Incubation with 20 μM PPMP caused parasite lysis and was therefore not included in the subsequent analyses.

FIG. 1.

PPMP inhibits the replication of G. lamblia trophozoites. (A) Freshly inoculated cultures containing equal numbers of trophozoites (day 1) were allowed to adhere for 8 h and subsequently treated with the indicated concentrations of PPMP or solvent (control [cntl]) for 16 h. Parasites were then harvested and counted (day 2). Results of a representative experiment are presented as averages of total parasite numbers ± standard errors (n = 4). (B) Parasite cultures were treated with 10 μM PPMP or solvent (cntl) as described above, and parasites in the form of single cells, doublets, and triplets were counted. Results are presented as percentages of total parasite numbers ± standard errors (n = 6). (Inset) Differential interference contrast image of two incompletely divided parasites (doublet). Asterisks, ventral disks. Scale bar, 5 μm.

A qualitative assessment by microscopy analysis of cultures incubated with 10 μM PPMP provided a first indication that the drug compromised cell division, resulting in the accumulation of incompletely divided cells (doublets) containing two sets of properly segregated nuclear pairs and two ventral disks (Fig. 1B, inset).

A quantitative analysis revealed that 35% of PPMP-treated parasites were single cells, whereas 40% were doublets and 25% formed clusters of three connected cells (triplets) (Fig. 1B). In contrast, 85% of untreated parasites were single cells, only 10% were doublets, and 5% formed triplets. These data indicate that PPMP interferes with the completion of cell division, specifically the separation of daughter cells.

Microscopy analysis revealed that doublet cells resulting from PPMP incubation had completed karyokinesis. To confirm this observation, we performed a population-wide analysis of DNA content by flow cytometry. The DNA distributions in untreated trophozoites showed one major peak corresponding to interphase cells (with eight sets of DNA [8N]) (Fig. 2A). However, DNA distributions in PPMP-treated cells presented additional peaks (16N and 24N) mirroring the doublets and triplets observed by microscopic analysis (Fig. 2B). The DNA contents of untreated encysting trophozoites (8N) and cysts (16N) are shown for comparison (Fig. 2C).

FIG. 2.

DNA content distributions in PPMP-treated G. lamblia cells. Freshly inoculated cultures of trophozoites were treated with solvent (control [cntl]) or 10 μM PPMP, as described in Materials and Methods, or allowed to encyst for 24 h. Parasites were then harvested, and nuclear DNA was stained with propidium iodide and analyzed by flow cytometry. T, trophozoites; C, cysts.

PPMP induces the accumulation of G. lamblia intracellular membranes.

To further characterize the PPMP-induced block of cell division, we examined the patterns of cellular membranes and the endoplasmic reticula (ER) in PPMP-treated cells by fluorescence microscopy. Membrane staining with the lipid probe Nile red showed an accumulation of membranes at the caudal area of the parasites, both in single (Fig. 3A) and in incompletely divided (Fig. 3B) cells. Labeling with an antibody against PDI2, a membrane-anchored ER protein, showed that the accumulated membranes belonged to the ER network (Fig. 3C).

FIG. 3.

Effect of PPMP on intracellular membranes. Fluorescence analysis of trophozoites treated with 10 μM PPMP or solvent (control [cntl]) for 16 h. (A and B) Membrane staining with the fluorescent lipid probe Nile red. (C) ER staining with anti-PDI2 serum followed by fluorescein-conjugated secondary antibodies. (Insets) Differential interference contrast images. Scale bars, 3 μm.

Next, we analyzed whether PPMP incubation affected the distribution of mitosomes (mitochondrial remnant organelles) or flagella in G. lamblia by using anti-IscS antibodies for mitosome labeling, Nile red for flagellar membrane labeling, and anti-Flex antibodies for the detection of flagellar exit sites. The typical distribution of mitosomes (27) was not altered in the presence of PPMP, and the central mitosome complex was properly duplicated and segregated also in incompletely divided cells (data not shown). Similarly, the number of flagella and the positions of flagellar exit sites in PPMP-treated cells were unchanged (data not shown). These results indicate that the PPMP-induced arrest in cell division occurs at a late stage of cytokinesis, after organelle segregation into daughter cells.

PPMP inhibits G. lamblia trophozoite adhesion.

The ability of trophozoites to attach to a wide variety of surfaces, including intestinal epithelia, by means of their ventral disks is essential for the colonization of the host intestine and is regarded as an indicator of parasite virulence. To analyze the effect of PPMP on parasite adhesion, we counted adherent and nonadherent cells in cultures incubated with 10 μM PPMP. In the presence of the drug, only 50% of the parasites were attached to the culture tubes, in contrast to control cultures, in which 90% of the parasites were attached (data not shown). Thus, these data show that PPMP also compromises the adhesion ability of the parasites.

The PPMP-induced blocking of parasite replication is irreversible.

We next tested whether the effect of PPMP could be reversed after the removal of the drug. Both exponentially replicating and nonreplicating stationary-phase G. lamblia cultures were incubated for 16 h with PPMP and then for 24 h in the absence of the drug. Parasite replication was assessed by counting the cells after the incubation in the presence of PPMP (day 1) and after the subsequent incubation in the absence of PPMP (day 2). The addition of PPMP to exponentially replicating parasites reduced the division rate (day 1), as demonstrated previously. During the incubation in the absence of the drug (day 2), parasites which had been exposed to 5 μM PPMP partially recovered their replication ability, showing a 4.9-fold increase in cell number during day 2 versus a 5.8-fold increase in the number of control cells. However, parasites exposed to 10 μM PPMP did not replicate after 24 h of incubation in the absence of the drug (Fig. 4A). In all samples incubated with PPMP, high percentages of incompletely divided cells and cell clusters were observed after drug removal (data not shown).

FIG. 4.

Reversibility of PPMP-mediated inhibition of G. lamblia replication. (A) PPMP treatment during parasite exponential growth. Freshly inoculated cultures of trophozoites were treated with the indicated PPMP concentrations or solvent (control [cntl]) for 16 h. Parasites were harvested, counted (day 1), and reinoculated for an additional 24-h incubation in the absence of the drug before counting (day 2). (B) PPMP treatment during parasite stationary phase. Confluent cultures of trophozoites were treated with the indicated PPMP concentrations or solvent (cntl) for 24 h, harvested, and counted. Aliquots with equal numbers of parasites (day 1) were inoculated into new cultures for 24 h of incubation in the absence of PPMP before counting (day 2). Results of representative experiments are presented as averages of total parasite numbers ± standard errors (n = 6). Numbers above the bars represent the increases (n-fold) in parasite numbers.

Microscopic examination of the cultures showed no indication that 10 μM PPMP leads directly to cell lysis. This finding suggests that the cytokinesis defect is the main reason for the reduced replication rate. Indeed, when PPMP was added to confluent cultures in stationary phase, cell numbers did not decrease (data not shown). However, when aliquots of these drug-treated parasites were used to start new subcultures in the absence of PPMP, the replication of only those trophozoites previously exposed to 5 μM PPMP was partially restored but the replication of parasites exposed to 10 μM PPMP was completely inhibited (Fig. 4B). Collectively, these results show that the inhibition of parasite replication by PPMP at a 10 μM concentration is irreversible and that inhibition by PPMP only at lower concentrations can be partially reversed. In addition, the incubation of confluent cells with 10 μM PPMP showed that the irreversible blocking of replication is independent of active cell division.

PPMP inhibits G. lamblia encystation.

Encystation is an essential process for the transmission of G. lamblia to a new host and allows parasite survival in the environment. A hallmark of encystation is the expression of cyst wall proteins, which are partitioned into encystation-specific vesicles, from which they are secreted to form the protective cyst wall.

Given the importance of encystation, we tested whether PPMP could affect this process. As that of trophozoites, the treatment of encysting parasites with PPMP caused dose-dependent inhibition of replication compared with the replication of control cells (Fig. 5A). In addition, we analyzed the expression of CWP1 and the production of cysts in the presence of PPMP by immunofluorescence (Fig. 5B). Incubation with the drug inhibited CWP1 expression in a dose-dependent manner, with a 10 μM concentration reducing the number of CWP1-positive cells by 80%. The formation of mature cysts was also inhibited by PPMP, with a 10 μM concentration reducing the number of cysts in the sample by 90%.

FIG. 5.

PPMP inhibits G. lamblia encystation. (A) Encysting G. lamblia cultures were treated with the indicated concentrations of PPMP or solvent (control [cntl]) as described in Materials and Methods. Parasites were then harvested and counted. Results of a representative experiment are presented as percentages of numbers of untreated control parasites ± standard errors (n = 6). (B) Encysting parasites were treated with PPMP as described above, harvested, and stained with anti-CWP1 antibody for immunofluorescence analysis. The number of parasites expressing CWP1 or forming cysts was normalized by the total number of parasites, assessed by nuclear staining, and expressed as a percentage of the number of untreated control cells (cntl) ± the standard error (n = 6).

These results indicate that PPMP inhibits the replication of encysting parasites, as well as CWP1 expression and cyst formation.

PPMP effect on mammalian cells.

In host organisms, Giardia parasites grow in close contact with intestinal epithelial cells, to which they attach with a specialized organelle, the ventral disk. PPMP has been used in vivo in a mouse model at concentrations inhibitory for G. lamblia in vitro, and the drug was reported to be well tolerated by the animals (39). However, PPMP has been shown in vitro to induce apoptosis of a variety of eukaryotic cells, likely via ceramide buildup (4, 18). To analyze whether PPMP was detrimental to the mammalian host cells at concentrations inhibitory for G. lamblia, we tested the metabolic activity (as a measure of cell viability) of different intestinal and kidney cell lines exposed to the drug. As shown in Fig. 6, cell metabolic activity was unaffected by 24 h of treatment with a PPMP concentration causing G. lamblia lysis (20 μM). In addition, the IC₅₀ for the three cell lines tested was above 55 μM, more than 10-fold higher than the IC₅₀ for the parasite (3.5 μM). PPMP treatment of host cells was performed for up to 3 days: while 50 μM PPMP induced apoptotic nuclear condensation events in the treated cells, neither decreases in metabolic activity and cell number nor an apoptotic phenotype was observed among cells treated with PPMP concentrations inhibitory for the parasite (data not shown). These results show that PPMP at concentrations inhibitory for G. lamblia does not reduce the viability of mammalian cells.

FIG. 6.

The metabolic activity of host cells treated for 24 h with solvent (control [cntl]) or PPMP at the indicated concentrations was assessed by measuring Alamar blue (Biosource) reduction. Results of a representative experiment are presented as percentages of the activity of the control cells ± standard errors (n = 6).

DISCUSSION

Sphingolipid biosynthesis is revealing its potential as a source of drug targets against a variety of pathogenic organisms, including fungi (28), nematodes (19), and parasites (15-17, 30, 32). In the present work, we show that PPMP, a known modulator of sphingolipid biosynthesis, is a strong inhibitor of the replication and differentiation of cells of the primitive eukaryote G. lamblia. Most importantly, despite the presence of the PPMP target in host cells, our data indicate that the inhibitory effect of the drug on G. lamblia is much greater than that on the mammalian cells, highlighting the practical feasibility of using sphingolipid inhibitors as a treatment for parasite infection.

Our data show that micromolar concentrations of PPMP inhibit three different biological processes of G. lamblia, namely, parasite replication, adhesion, and differentiation into cysts. Firstly, the inhibition of replication was dose dependent and was characterized by a precise arrest in late cytokinesis, after the nuclei had already successfully duplicated and segregated. Importantly, the irreversibility of the phenotype after the removal of the drug indicates that structural and/or signaling elements crucial for the proper accomplishment of the cell cycle were severely compromised. The inhibition of cytokinesis in yeast and mammalian cells after exposure to sphingolipid inhibitors with different targets from that of PPMP has been observed previously (8, 14, 22, 31); however, to our knowledge, a similar PPMP-mediated impairment of cell division in other cell systems has not been reported.

Thus, sphingolipids are likely to play an important role in cell division, although the molecular mechanism is not yet completely elucidated. A possible explanation for the PPMP-induced blocking of cytokinesis observed in our study is suggested by the recent investigation of dividing sea urchin eggs showing that membrane domains enriched with sphingolipids and cholesterol move to the cleavage furrow separating daughter cells (24). Interestingly, the isolation of these membrane domains revealed the presence of signaling molecules, such as Src and phospholipase C-γ, whose activation via phosphorylation is required for furrow progression and actin assembly (24). Moreover, analyses of the furrow membrane origin showed highly dynamic membrane trafficking and the fusion of vesicles derived mainly from the Golgi apparatus (reviewed in reference 2). Thus, it is tempting to speculate that the addition of new membrane material, possibly composed of newly synthesized lipids, to the furrow creates signal platforms that help to orchestrate the cell division process. In this context, the inhibition of lipid synthesis, at a time when lipid demand increases steeply, may compromise cytokinesis at both the structural and the signaling levels.

Alternative explanations for the compromised cell division observed in G. lamblia are alterations at the level of the actin cytoskeleton, as reported for other cell systems after incubation with the different sphingolipid inhibitors mentioned above (8, 22). Although anomalies in actin assembly cannot be excluded, we did not observe the rounding up of cells, flagellar displacement, or aberrant flagellum numbers, as were reported following the treatment of G. lamblia with actin inhibitors (5), suggesting that actin alteration is unlikely to play a major role in the PPMP-induced inhibition of cell division. In addition, the observed accumulation of membranes preferentially in the caudal area of PPMP-treated parasites, where cell division is blocked, supports a lipid-mediated effect. We are currently characterizing the target of PPMP in G. lamblia. Two lines of evidence support GlcCer synthase as a target in this parasite: (i) a putative gene coding for GlcCer synthase is annotated in the parasite genome (Giardia Genome Database open reading frame 11642), and (ii) initial experiments indicate an increase in ceramide levels in the parasite after PPMP treatment, a known result of the PPMP-mediated inhibition of GlcCer synthase in mammalian cells (S. Sonda and A. B. Hehl, unpublished data). Hence, our findings suggest that, even with the high degree of evolutionary divergence between G. lamblia and higher eukaryotes, key enzymes of sphingolipid biosynthesis may be highly conserved in this primordial organism.

Secondly, PPMP inhibited not only parasite replication, but also adhesion. Since attachment is transiently lost during cell division (34), the most likely explanation for the reduced parasite adherence is incomplete cell division and cluster formation upon PPMP incubation, although direct damage of the ventral disk responsible for parasite adhesion cannot be ruled out. The ability to tightly adhere to the intestinal mucosa is necessary for G. lamblia to colonize the small intestine, and it is also likely to be responsible for the damage to the intestinal epithelium, including the disruption of tight junctions, cytoskeleton rearrangement, and the apoptosis of host cells (23, 33). Thus, given the role of parasite adherence in the pathogenesis of the infection, it is tempting to speculate that a therapeutic compound able to reduce parasite adhesion, like PPMP, will limit not only the parasite burden, by the premature excretion of trophozoites, but also the intensity of the disease symptoms.

Finally, PPMP reduced G. lamblia encystation levels to 10%, implying that the inhibitor targets a cellular process required for parasite differentiation. Given the essential role of cyst formation in both parasite survival in the environment and transmission to a new host, a pharmacological compound able to compromise parasite encystation would be extremely valuable to contain the spreading of G. lamblia.

In summary, our study demonstrates that PPMP is a potent anti-G. lamblia drug in vitro. We showed that PPMP acts at three different levels in the physiology of G. lamblia, leading to dramatic reductions in parasite replication, adhesion, and encystation. Importantly, these effects occur at concentrations and incubation times which do not affect the viability of host-derived cells. PPMP irreversibly inhibits cellular processes that are essential for both parasite survival in a given host and parasite transmission to a new host via encystation and excystation. This synergistic effect further adds to the potential of PPMP as an alternative drug to combat both acute giardiasis and disease transmission.