Trophozoites of the WB isolate, clone 1267, were obtained from ATCC (American Type Culture Collection catalog number 50582). Cells were cultured in a 16 mL screw-cap tube using TYI-S33 medium supplemented with 10% adult bovine serum and bovine bile and incubated at 37°C (Diamond et al., 1978). Growing trophozoites form a monolayer in the tube, which can be recovered by placing the tubes at 4°C for 30 min, followed by centrifugation at 2,500 rpm for 15 min. The in vitro encystation process was carried out in two stages. In the first stage, the growth medium of the trophozoites cultured to confluence for 24 h was discarded, and the tubes containing trophozoites adhered to their walls were filled with pre-encystation medium (complete growth medium without bovine bile). Finally, this medium was replaced by an encystation medium containing a concentration of 0.45% porcine bile and 0.01% lactic acid, with a pH of 7.8 (Gillin et al., 1989). This medium change induces the differentiation of trophozoites into cysts, which lose their adherence capacity and remain free in the encystation medium.

Wild type trophozoites (WB/1267) were used as hosts for the expression of transgenic genes and as controls. To constitutively express MYST1, MYST2, and GCN5, the plasmid pTubHApac with the tag HA at the C-terminus was employed (Touz et al., 2003). The vector contains a puromycin cassette controlled by the endogenous non-regulated tubulin promoter for cell selection. The tubulin promoter was replaced by a sequence containing the putative endogenous promoter to express the enzymes under their own promoter. Moreover, to inhibit the enzymes’ expression, ORF sequences were amplified and ligated to the pTub-HApac in the opposite direction, resulting in the antisense vector. The primers used are detailed in Table 1. Stable trophozoite transfection was performed as was previously described (Yee and Nash, 1995; Singer et al., 1998). Drug-resistant trophozoites were usually apparent by 7–10 days post-transfection.

Primary antibodies against H3K9ac (1:200; Active Motif, Inc.), H3K27ac (1:200; cell signaling technology), α-Tubulin (1:500; Sigma-Aldrich Co.), and α-Tubulin Acetylated (1:500; Sigma-Aldrich Co.) were used. Cells were fixed with 4% formaldehyde in 1X PBS for 40 min at room temperature, blocked with 3% Bovine Serum Albumin (Sigma-Aldrich Co., USA) and 0.05% Tween (Sigma-Aldrich Co., USA) in 1X PBS. Cells were then incubated with the primary antibody diluted in 1.5% Bovine Serum Albumin (Sigma-Aldrich Co., USA) and 0.05% Tween (Sigma-Aldrich Co., USA) in 1X PBS. After 3 washes with 0.05% Tween in 1X PBS, cells were incubated with Alexa Fluor 488-conjugated secondary antibodies against rabbit or mouse (dilution 1:500, Life Technology) diluted in 1.5% Bovine Serum Albumin (Sigma-Aldrich Co., USA) and 0.05% Tween (Sigma-Aldrich Co., USA) in 1X PBS. Incubation conditions were 1 h at 37°C in a humid chamber. Subsequently, cells were washed three times with 1X PBS and incubated with DAPI (dilution 1:750) for 5 min. Samples were mounted with FluorSave (Merck Group, Darmstadt, Germany) and observed using an Olympus FV1200 confocal microscope. Image analysis was performed using Fiji software² and Adobe Photoshop 8.0 (Adobe System).

The HAT inhibitor garcinol was purchased from Enzo Life Science (USA). Stock solutions of the compound (10 mM) were prepared in dimethyl sulfoxide (DMSO) (Sigma Aldrich Co, St. Louis, USA) and stored at −20°C.

To assess the giardicidal activity of the compound, we performed the MTT assay to measure cell viability by forming formazan crystals due to MTT reduction by viable cells. The compound was dissolved in DMSO (0.5% v/v) to achieve a final concentration of 50 μM. We exposed the drug in a 96-well plate with 5 × 10⁵ cells per well in a final volume of 300 μL of culture medium. Incubation took place for 48 h in a CO₂ chamber at 37°C. After incubation, the plate was centrifuged at 2,000 rpm for 10 min. We performed three washes by centrifugation and the addition of sterile PBS. Trophozoites were resuspended in 200 μL of PBS, and 20 μL of MTT (5 mg/mL in PBS) was added to each well. Incubation was carried out for 4 h at 37°C. The plate was then centrifuged at 2,000 rpm for 10 min. The supernatant was discarded, and the formazan crystals were solubilized in 100 μL of DMSO. Absorbance was measured using a model 680 microplate reader (Bio-Rad, USA) at 570 nm. The percentage of cytotoxicity was determined by comparing with DMSO-treated control cells, which were considered 100% viable as they exhibited behavior like untreated cells. Additionally, we analyzed a range of decreasing concentrations, with a maximum concentration of 50 μM, to determine the half-maximal inhibitory concentration (IC₅₀) using non-linear regression with GraphPad Prism 9.0 software (GraphPad Software, Inc., CA, USA).

10 × 10⁶ cells were grown in a 96-well plate for 48 h at 37°C, where the trophozoites were exposed to the IC₅₀ of the compound and a double concentration. DMSO was used as a control. After the incubation, the trophozoites were washed and transferred with cold 1X PBS to a 1.5 mL Eppendorf tube. They were centrifuged at 2,500 rpm for 15 min at 4°C, and the supernatant was discarded. The cells were fixed with a solution containing 4% (v/v) formaldehyde and 2% (v/v) glutaraldehyde in a 0.1 M cacodylate buffer and stored at 4°C. Subsequently, the fixed cells were centrifuged, and the resulting pellets were washed and treated with 1% OsO4 for 1 h. Following dehydration with a graded series of cold acetones, the cells were embedded in Spur resin. Ultrathin sections of 90 nm were prepared after 48 h of polymerization at 60°C using an RMC Power Tome – XL ultramicrotome. Subsequently, the ultrastructural changes were examined using a Hitachi HT 7800 electron microscope (Hitachi, Tokyo, Japan).

To perform this experiment, 5 × 10⁴ trophozoites, suspended in a complete growth medium, were seeded in a 96-well plate. The trophozoites were exposed to the IC₅₀ of the compound and a double concentration of it. Control groups included untreated cells as negative controls and cells treated with metronidazole (MTZ) as positive controls. After a 24 h incubation, apoptotic/necrotic cells were processed using the Dead Cell Apoptosis Kit (Thermo-Fisher Scientific, USA) with Annexin V-Alexa Fluor 488 and propidium iodide (PI), following the manufacturer’s instructions. The cells were centrifuged, washed thrice with 1X PBS, and resuspended in 100 μL of 1X Annexin binding buffer. Subsequently, they were incubated with Annexin V-Alexa 488 and PI for 15 min at room temperature in the dark. Flow cytometry was analyzed using the FACSCanto II instrument (Becton & Dickinson, New Jersey, NY, USA). Annexin V/PI dot plot analysis, divided into quadrants, aided in categorizing cells as live (Annexin V−/PI−), early apoptotic (Annexin V+/PI−), late or secondary apoptotic (Annexin V+/PI+), and necrotic (Annexin V−/PI+).

Cells, both treated and untreated, were detached by incubation in an ice-water bath for 30 min. They were then collected via centrifugation at 2,500 rpm for 10 min at 4°C and washed twice with 1X PBS. The parasites were resuspended in 1 mL of PBS containing 1% v/v and a drop of the suspension was placed on coverslips pre-coated with poly-L-lysine. The coverslips were incubated at 37°C for 30 min to facilitate trophozoite adhesion. Subsequently, the cells were fixed with 4% v/v formaldehyde in PBS for 40 min, followed by two PBS washes of 5 min each. Permeabilization was performed using 0.1% v/v Triton X-100 for 30 min, after which the cells were washed twice again with PBS for 5 min. Finally, the TUNEL (Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling) assay was performed using the In Situ Cell Death Detection Kit, TMR red (Roche), according to the manufacturer’s instructions.

The concentration of reactive oxygen species (ROS) produced by cultured trophozoites (5 × 10⁵ cells) with and without treatment in 96-well plates at 48 h was examined using the Image-iT LIVE Green Reactive Oxygen Species Detection Kit (Invitrogen, MA, USA). The assay is based on a nonfluorescent and cell permeable 5-(and-6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA). The carboxy-H2DCFDA permeates the live cells and is deacetylated by intracellular esterases. The reduced fluorescein compound is oxidized by the cellular ROS and emits bright green fluorescence with excitation/ emission maxima of 495/529 nm. In the assay, after cells treatment, they were collected by centrifugation, washed three times with sterile 1X PBS, and resuspended in 100 μL of Working Buffer (25 μM). After incubating for 30 min at 37°C in the dark, additional washes were carried out with sterile 1X PBS, and the final pellet was resuspended in 100 μL of sterile 1X PBS. The fluorescence intensity was measured using the FACS Canto II flow cytometer (Becton & Dickinson, New Jersey, NY, USA). The data was analyzed using FlowJo software. Furthermore, ROS formation was visualized using confocal microscopy Olympus FV1200 with Hoechst as a nuclear marker, incubating for 5 min at 37°C in the dark. The images were processed using the Fiji Image program.

Trophozoites (5 × 10⁵ cells) were treated with garcinol in 96-well plates at IC₅₀ and at IC₉₀ concentration, inhibiting ~50% and ~90% of growth within 48 h at 37°C, respectively. Following treatment, plates were centrifuged and washed with PBS. One plate was immediately subjected to an MTT assay to determine initial viability. The second plate was placed in an anaerobic chamber at 37°C for 48 h to allow any surviving parasites to recover without further treatment. Post-recovery, parasite viability was assessed using the MTT assay. DMSO-treated cells were used as control. Each experiment was performed in triplicate. Cytotoxicity percentages were calculated and analyzed using GraphPad Prism software version 9.0. Statistical significance between viability before and after the 48 h recovery period was determined using a t-test.

Results were analyzed for statistical significance using an unpaired, two-sided Student’s t-test with GraphPad Prism 5 Data Analysis Software (GraphPad Software Inc., La Jolla, CA, USA). The mean and standard error of the mean (SEM) were calculated from at least three biologically and technically independent experiments. A p-value of less than 0.05 was considered significant and is indicated by asterisks in the figures.

Based on previous studies by other authors indicating that clear histone deacetylation is necessary for encystation in the parasite, our first objective was to analyze the localization of histone acetylation marks during growth and encystation. We began by studying lysine 9 and 27 of histone H3, potential substrates of the KAT enzyme GCN5 in G. lamblia. Figure 1A shows immunofluorescence images analyzed via confocal microscopy using the anti-H3K9ac and anti-H3K27ac antibodies. Regarding H3K9ac, which is correlated with transcriptional activation, we found a strong mark localized in the nuclei during growth that diminished as encystation progressed. However, it was still present at 48 h p.i. On the other hand, the mark of H3K27ac, also involved in transcriptional activation, was more discrete in the nuclei during growth and disappeared after 24 h p.i. These results confirm previous findings and support that a general deacetylation is needed to allow encystation to progress.

In the genome of Giardia lamblia, there are five putative enzymes described that could act as KATs (Emery-Corbin et al., 2021). To start studying GCN5 (GL50830_10666), we designed primers to overexpress the enzyme under the tubulin promoter (glgcn5-ha transgenic cells) or the endogenous one (glgcn5-en transgenic cells) and to underexpress the expression we used the antisense strategy (glgcn5-as transgenic cells). After performing the transformations in bacteria, the plasmids were purified, the sequences were analyzed, and WB1267 strain trophozoites were transfected, adding puromycin for selection, as usual. Generally, if the cells are transfected 10 days after the addition of puromycin, the cultures begin to grow, forming a monolayer of trophozoites around day 14. We observed that the cells were transfected regardless of whether we performed the overexpression under the endogenous promoter or the tubulin promoter. Still, they remained in a latent state (Figure 1B). There were groups of cells that grew very slowly, but at no point did we detect cells moving or migrating, a usual behavior in trophozoite cultures. We repeated the procedure several times, changing the conditions of the cell/plasmid ratio and incubation times, obtaining similar results. The same occurred using the antisense strategy to reduce the expression of the enzymes. In none of the situations were we able to get a monolayer culture of G. lamblia that would allow us to continue the experiments.

These results prompted us to analyze two other KATs, named MYST1 (GL50803_17263) and MYST2 or ESA1 (GL50803_2851), whose putative substrates are present in histone H3 and histone H4 (Emery-Corbin et al., 2021). We repeated the procedure of designing primers for the three conditions, and after transfection, we found similar results to those with the enzyme GCN5, observing stalled replication. Therefore, overall, we conclude that histone acetyltransferases are indispensable for parasite proliferation in vitro and that any imbalance in enzyme expression induces an arrest in parasite proliferation.

Enzyme inhibitors have been used to analyze the role of post-translational modifications of enzymes indirectly. Considering the essential role of acetylation for G. lamblia viability, we sought an acetylation inhibitor that could be used in G. lamblia. We selected a natural compound called garcinol, a polyisoprenylated benzophenone isolated from the dried rind of the edible fruit kokum (Garcinia indica), a plant native to southern India, which is a potent histone acetyltransferase inhibitor (Balasubramanyam et al., 2004). This compound has already been tested in other parasites, showing a decrease in global lysine acetylation in T. gondii and an arrest of parasite proliferation in both T. gondii and P. falciparum (Jeffers et al., 2016). We used concentrations of garcinol ranging from 0 to 100 μM, and we performed cell viability assays using the quantitative colorimetric technique with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, or MTT. We determined that the IC₅₀ of garcinol is 21.32 ± 0.95 μM. As a control, we always incubated the cells with the vehicle (DMSO) (Figure 2A). Based on these results, we decided to perform the following experiments during a 48 h treatment period using two different concentrations of garcinol: 20 μM (IC₅₀) and 40 μM (2 × IC₅₀) and DMSO in control cells, unless specified in a particular experiment.

Morphological analysis using DIC revealed that the cell morphology did not change significantly after treatment with 20 μM of garcinol. However, some trophozoites displayed round-shaped elevations inside the cells, with a few of these structures found in the extracellular space (Figure 2B). These features were more prominent with 40 μM of garcinol. The size of the protrusions increased considerably (Figure 2C). At this concentration, morphological changes indicative of cellular deterioration were observed.

Continuing our study, we delved into the ultrastructural elements of the cells using transmission electron microscopy. We observed projections on the ventral surface of trophozoites when treated with 20 μM garcinol (Figure 3B, asterisks). Additionally, in some trophozoites, lamellar structures were observed within the cytoplasm (arrowhead). In the extracellular space, we found apoptotic-like bodies containing cellular material (black arrows). When the cells were exposed to 40 μM garcinol, the morphological changes were more evident, with a greater number of vacuoles containing lamellae observed inside the cells, which also were larger in size (Figure 3C, arrowheads). No significant changes were observed in the peripheral vesicles or in the structure of the ventral disc, nor were any changes detected in chromatin condensation within the nuclei. In panels 3 and 5, cells show pronounced signs of cell death, such as disruption of the plasma membrane and the ventral disc, as well as disorganization of cytoplasmic structures. In the extracellular medium, large structures (4) that seem to contain granules were observed, which appear to hold remnants of cellular content.

It was previously shown that the histone acetyltransferase inhibitor garcinol induces apoptosis and DNA fragmentation in HeLa cells at 30, 70, and 100 μM (Balasubramanyam et al., 2004). To analyze whether G. lamblia trophozoites suffered apoptosis/necrosis under garcinol treatment, we performed flow cytometry assays using PI, a membrane-impermeable nucleic acid-specific dye, that reflect cell death (late apoptosis/necrosis), and Annexin V that detect phosphatidylserine when it is exposed to the plasma membrane of the cells during early apoptosis (Figure 4A). After 24 h of treatment, we observed that the percentage of cells in early apoptosis rose in correlation with the increasing concentration of garcinol, with almost 60% of cells in early apoptosis under 40 μM of garcinol. No differences were observed in the proportion of necrotic cells under treatment (Q1). Also, cells in late apoptosis/necrosis increase, with 5% of cells in this stage under the highest concentration of garcinol (Figure 4B).

During apoptosis, biochemical and morphological events lead to programmed cell death. One of these events is the activation of specific endonucleases, which are enzymes that cut DNA into fragments from 50 to 300 kb and are further degraded to low molecular weight oligonucleoside-sized fragments (Di Filippo and Bernardi, 2009). The TUNEL assay was performed on cells treated with DMSO, garcinol 20 μM, and 40 μM; however, no TUNEL-positive cells were found under any condition (Figure 4C). Since the entire apoptotic machinery seems absent in Giardia, garcinol probably induces apoptotic-like cell death through a non-classical pathway.

To delve deep into the mechanism that finally induces cell death in G. lamblia under garcinol treatment, reactive oxygen species (ROS) were analyzed using flow cytometry and immunofluorescence assays. We found no differences in the percentage of ROS generation between control and trophozoites treated with 20 μM of garcinol (Figures 5A,B). At 40 μM, the rate of positive cells doubled compared to the control. Through fluorescence assays, we analyzed the ROS-positive cells. Considering that this assay is performed on live cells, in Figure 5C we show examples of ROS-positive cells, even though the majority were negative. We observed that as the garcinol dose increased, the distribution of ROS in the cell cytoplasm also increased. Additionally, the presence of ROS can be observed in apoptotic-like bodies (black arrow). After a thorough analysis of these results, we can conclude that ROS production is not the primary mechanism by which garcinol induces cell death, considering the low percentage of ROS-positive cells, but the presence of ROS in the round-shape structures reinforces the idea that there is a fragmentation of the cytoplasm of the cells, that finally produces apoptotic-like bodies.

Having observed the morphological changes, we explored whether garcinol also affected the localization of tubulin and acetylated tubulin in trophozoites treated using fluorescence microscopy. We found that the classical localization of tubulin in the median body and flagella changed after garcinol treatment (Figure 6A). When the cells were treated with 20 μM garcinol, we observed that in most cells, tubulin remained localized in the flagella. However, no staining was observed in the median body, which was also not evident in the DIC images. When the cells were treated with 40 μM garcinol, tubulin was observed distributed throughout the cytoplasm, with some staining retained in the flagella. No localization in the median body was observed in any of the cells, and the large vesicles or membrane blebs, both localized intracellular and extracellular (asterisk), were marked with tubulin.

Like many protozoan parasites, most of the tubulin in G. lamblia is acetylated, showing flagella, median body, and parts of the ventral disc marked with the antibody. We observed similarities to what was seen with tubulin, with a loss of marking in the median body at lower garcinol concentrations and a clear cytoplasmic distribution at higher concentrations (Figure 6B). Again, we observed those large vesicles or membrane blebs, which contained acetylated tubulin inside or emerging from the cell (asterisk). Overall, there seems to be a redistribution of tubulin, which may be associated with profound changes in the parasite’s ultrastructure but no decrease in tubulin acetylation.

Garcinol is a potent inhibitor of histone acetyltransferases p300 and PCAF. It also inhibits histone acetylation in vivo but does not affect the deacetylation of histones (Balasubramanyam et al., 2004). To analyze whether changes in acetylation of lysine residues 9 and 27 on histone H3 were observed, we incubated WB1267 cells in the presence of garcinol at 20 μM and 40 μM for 48 h. Following the treatment, we performed immunofluorescence assays and confocal microscopy. Regarding the acetylated lysine 9 mark on histone H3, we observed that even at the IC₅₀ concentration, there was a change in the acetylation pattern, showing reduced fluorescence intensity within the nuclei. The acetylation mark was sometimes observed in a perinuclear location (Figure 7A). The effect was more evident when treating the cells with 40 μM garcinol, where we no longer observed the mark within the nuclei but rather at the periphery. Notably, in some cells, the mark was present in only one of the nuclei. Finally, we found cells without the H3K9ac mark within the nuclei.

In cells treated with DMSO, the acetylation mark of lysine 27 on histone H3 was similar to that observed in untreated trophozoites, where the nuclear mark was discrete (Figure 7B). Upon treatment with 20 μM garcinol, the nuclear localization was lost and a clear perinuclear pattern was evident, in both or only one of the nuclei. The mark is barely observable at 40 μM garcinol treatment and in some trophozoites. These results indicate that garcinol profoundly affects the acetylation of both lysine 9 and lysine 27 on histone H3 but differentially compared to a physiological situation such as encystation.

Having observed the profound effect of garcinol on Giardia lamblia viability, we analyzed if garcinol induced a giardiacidal versus giardiastatic effect. Therefore, recrudescence was evaluated by testing the parasite’s viability when treated with the compounds IC₅₀ (20 μM) and IC₉₀ (60 μM) before and after 48 h of drug removal to determine if the surviving cells could proliferate in the absence of the drug. (Figure 8). Recrudescence was defined as viability above 10% post-recovery, significantly different (p < 0.05) from viability observed after 48 h treatment with garcinol alone. We found that garcinol effectively prevented recrudescence, suggesting a giardiacidal effect ideal for treating parasitic diseases and preventing recurrent infections caused by dormant parasites. This result highlights the potential of garcinol as a promising drug for the treatment of giardiasis.