Pep5, a Fragment of Cyclin D2, Shows Antiparasitic Effects in Different Stages of the Trypanosoma cruzi Life Cycle and Blocks Parasite Infectivity

Pep5 (WELVVLGKL) is a fragment of cyclin D2 that exhibits a 2-fold increase in the S phase of the HeLa cell cycle. When covalently bound to a cell-penetrating peptide (Pep5-cpp), the nonapeptide induces cell death in several tumor cells, including breast cancer and melanoma cells.

ABSTRACT

Pep5 (WELVVLGKL) is a fragment of cyclin D2 that exhibits a 2-fold increase in the S phase of the HeLa cell cycle. When covalently bound to a cell-penetrating peptide (Pep5-cpp), the nonapeptide induces cell death in several tumor cells, including breast cancer and melanoma cells. Additionally, Pep5-cpp reduces the in vivo tumor volume of rat glioblastoma. Chagas disease, which is caused by the flagellated parasite Trypanosoma cruzi, is a neglected disease that occurs mainly in the Americas, where it is considered an important public health issue. Given that there are only two options for treating the disease, it is exceptionally crucial to search for new molecules with potential pharmacological action against the parasites. In this study, we demonstrate that Pep5-cpp induces cell death in epimastigote, trypomastigote, and amastigote forms of T. cruzi. The Pep5-cpp peptide was also able to decrease the percentage of infected cells without causing any detectable toxic effects in mammalian host cells. The infective, i.e., trypomastigote form of T. cruzi pretreated with Pep5-cpp was unable to infect LLC-MK₂ monkey kidney cells. Also, Pep5-binding proteins were identified by mass spectrometry, including calmodulin-ubiquitin-associated protein, which is related to the virulence and parasitemia of T. cruzi. Taken together, these data suggest that Pep5 can be used as a novel alternative for the treatment of Chagas disease.

INTRODUCTION

The cell cycle in eukaryotes is divided into four specific phases: mitosis, or M phase, in which one nucleus is divided into two identical nuclei; S phase, in which DNA is duplicated; and two gap phases (G₁ and G₂) that separate the M and S phases. Many proteins are involved in cell cycle control, which is mainly regulated by the ubiquitin proteasome system (UPS) (1, 2). After proteasome degradation, peptides of 2 to 21 amino acids are generated (3, 4), though only one peptide derived from each protein escapes from complete proteolytic degradation and is presented at the cell surface as an antigen by major histocompatibility complex class I (MHC-I) (5). It was once believed that the remaining intermediate peptides were entirely degraded into amino acids that could be utilized in the production of new proteins. However, a few years ago, it was demonstrated that some of these peptides can evade complete degradation and remain within cells to participate in signal transduction (6–8). These fragments of proteins generated inside cells by proteasome degradation are named intracellular peptides (6). The first functional intracellular peptide identified was hemopressin, which shows cannabinoid-like inverse agonist effects that regulate food intake in mice and rats (9–11). Some studies have demonstrated that intracellular peptides also have an important function in cell signaling, but only when present inside cells (8). These peptides, which are structurally related to the proteolytic products of proteasomes, were identified by mass spectrometry and then synthesized and reintroduced into cultured cells. Some were able to modulate the signal transduction mediated by angiotensin receptors type 1 (AT1) and adrenergic β2 receptors (7). With proposed effects on cell signaling, others were reported to have effects on protein-protein interactions, intracellular calcium signaling, and antinociceptive activity (12, 13).

Pep5 (WELVVLGKL) is a nonapeptide derived from cyclin D2 that was identified by mass spectrometry as being present during the HeLa cell cycle, with a 2-fold increase in S phase compared to the level in asynchronous cells (8). When coupled with a well-characterized cell-penetrating peptide (cpp; TAT_47–57 [fragment of a protein from amino acid 47 to 57]; YGRKKRRQRRR), Pep5-cpp induced cell death in several tumor cell lines, including breast cancer, melanoma, and thyroid cancer, through a combined apoptotic-necrotic mechanism (8). Moreover, in vivo experiments showed that the cyclin fragment decreased the rat glioblastoma tumor volume by approximately 50% (8). Pep5 interacts with specific intracellular proteins from human MDA-MB-231 breast cancer cells, especially those associated with cytoskeleton and proteasome components, such as plectin (14). Notably, these proteins, such as chloride intracellular channel protein 1 and plectin, exert antiapoptotic and proliferation inhibition effects (14).

Trypanosomatids, including Trypanosoma cruzi, Trypanosoma brucei, and Leishmania spp., which are the etiologic agents of Chagas disease, sleeping sickness, and leishmaniasis, respectively, exhibit a life cycle that involves replicative and nonreplicative forms in both invertebrate and mammalian hosts (15). According to the World Health Organization (WHO), these three illnesses are among the 20 parasitic infections that affect people living in developing countries (16) and are classified as neglected diseases. Specifically, according to the number of infected individuals, followed by death, socioeconomic impact, and geographical distribution, Chagas disease represents one of the most important public health issues in the Americas. Although the number of new infections has diminished in Brazil and in other countries due to vector control programs, approximately 8 million people are still infected (17). Additionally, the compounds used in the treatment of Chagas disease, namely, nifurtimox (NFX) {(R,S)-3-methyl-N-[(1E)-(5-nitro-2-furyl)methylene]thiomorpholin-4-amine-1,1-dioxide} and benznidazole (BZ) (N-benzyl-2-nitroimidazolylacetamide), have several side effects, such as weight loss, anorexia, sleepiness, digestive system alterations, dermatitis, fever, and muscular pain. These drugs also appear to be inefficient during the chronic phase of the disease (18–21). Considering that available options for treating these neglected diseases are limited and extremely unsatisfactory, it is important to expand the portfolio of new drugs that can be applied. In the present study, we report the trypanocidal effect of Pep5-cpp. We show its effect on epimastigotes, trypomastigotes, and extracellular amastigotes of T. cruzi and on infected LLC-MK₂ monkey kidney cells. These exciting data suggest new perspectives for the treatment of Chagas disease.

RESULTS

Pep5-cpp induces cell death of the T. cruzi epimastigote form.

We initially verified that Pep5-cpp was found inside parasites, and we were interested in evaluating whether it localizes to a specific subcellular compartment after uptake. Using a fluorescent derivative (Pep5-cpp-Dye555), it was possible to observe Pep5-cpp inside epimastigotes near the nuclear region, similar to what was observed in mammalian cells (Fig. 1A). Next, we evaluated the effect of Pep5-cpp on replicative epimastigotes. Due to the widely reported genetic and phenotypic variability in T. cruzi (22), we chose to initially use two different strains: CL Brener and Y. Both strains were cultured in the presence of Pep5-cpp at different concentrations, and the effects of the peptide on cell cycle/cell death features were analyzed by flow cytometry. We observed an increase in the percentage of cell death in both strains when we treated T. cruzi epimastigotes with Pep5-cpp (Fig. 1B and C), with 50% effective concentration (EC₅₀) values of 25.16 μM and 24.92 μM for strains CL Brener and Y, respectively. No significant effects on the cell cycle or cell death features of the groups treated with cpp or Pep5, as controls, were observed (Fig. 1B). Therefore, we decided to perform all experiments using only the Y strain, which is regularly used in infection assays in our laboratory.

FIG 1.

Pep5-cpp effect on T. cruzi epimastigotes. (A) T. cruzi epimastigotes (strain Y) were incubated with Pep5-cpp-Dye555 for 15 min and then analyzed by fluorescence microscopy. Shown are representative images of Pep5-cpp accumulated inside parasites near the nuclei. Subsequently, epimastigotes were treated with Pep5-cpp for 3 h; after incubation, the parasites were analyzed by flow cytometry. DIC, differential interference contrast. (B and C) The graphs show the percentage of cell death for each strain compared to the nontreated group (NT) for strains CL Brener (B) and Y (C). To characterize cell death, epimastigotes (strain Y) were treated with Pep5-cpp for 3 h and then subjected to cell death assays. (D) Parasites treated with Pep5-cpp showed increased peroxidase activity compared to the nontreated group. (E) Intracellular calcium measurement after Pep5-cpp induction. Fluorescence was measured using a FlexStation 3 multimode microplate reader (Molecular Devices). (F) PS exposure assay in nontreated parasites (left) and those treated with Pep5-cpp (right) by flow cytometry. R1, PI positive; R2, PI and annexin V positive; R3, viable parasites; R4, annexin V positive. A total of 50,000 events were analyzed per replicate. The graphs represent the means and SEM of the results of at least two biological experiments performed in triplicate. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

To characterize the type of cell death caused by Pep5-cpp, we assessed whether treatment of parasites with Pep5-cpp induces activation of signals typically associated with programmed cell death (PCD). First, epimastigotes were treated or not with Pep5-cpp (25 μM; 3 h), and the levels of H₂O₂ were measured. Pep5-cpp-treated cells showed higher production of reactive oxygen species (ROS) than untreated cells (Fig. 1D). Subsequently, the concentration of intracellular calcium was also evaluated in treated and untreated parasites, and the results suggested increased cytosolic calcium release in the presence of Pep5-cpp compared to untreated parasites (Fig. 1E). When we evaluated exposure of phosphatidylserine (PS) in the membrane, the treated parasites increased annexin V (1.6%) compared with the control (0.2%). Also, approximately 12% of the parasites treated with Pep5-cpp showed double staining (propidium iodide [PI]-annexin V) compared to less than 1% in the nontreated group (Fig. 1F). Similar results were observed after 30 min of treatment (see Fig. S1 in the supplemental material). These data suggested that Pep5-cpp triggered parasite cell death with characteristics of programmed cell death.

Pep5-cpp induces trypomastigote cell death, inhibiting infection.

Trypomastigotes were also subjected to treatment with Pep5-cpp (25 μM). Induction of cell death was observed only in cells treated with Pep5-cpp (28.6% ± 2.8% death in cells treated with Pep5-cpp compared to 5.9% ± 0.8% in nontreated cells), indicating that trypomastigotes are less sensitive to Pep5-cpp than are epimastigotes at the same concentration (Fig. 2A). To evaluate which type of cell death was induced in trypomastigotes, exposure of PS on the membrane surface was also analyzed. After treatment with Pep5-cpp, 37.52% ± 3.52% of the parasites showed an increase of green fluorescence, which corresponded to PS exposure at the cell membrane (Fig. 2C). These data suggest that Pep5-cpp increases PS exposure in trypomastigote membranes and supports PCD activation.

FIG 2.

T. cruzi trypomastigotes (strain Y) were treated with Pep5-cpp for 3 h. (A) Percentages of cell death in trypomastigotes after PI staining plotted as the means and SEM of the results of three independent experiments performed in triplicate. NT, nontreated. (B and C) Analysis of PS residue exposure in trypomastigotes not treated (B) or treated (C) with Pep5-cpp. Flow cytometry analysis through annexin-V/PI staining was performed in trypomastigotes treated with Pep5-cpp for 3 h. The histogram shows parasites with increased PS exposure (R4) and the percentages of only PI-positive cells (R1) and cells positive for both effects (R2). Viable cells are found in the R3 quadrant. A total of 50,000 events were analyzed per replicate. The data are representative of the results of two biological experiments performed in triplicate. *, P < 0.1.

After evaluating the effects of Pep5-cpp in epimastigotes and trypomastigotes, we sought to examine the capacity of the peptide to inhibit mammalian host cell infection in vitro. Because Pep5-cpp induces cell death in both host cells and parasites, we estimated the concentration of Pep5-cpp that induces cell death only in parasites and not in mammalian cells. First, LLC-MK₂ cells were incubated in the presence of different concentrations of Pep5-cpp for approximately 20 h. After ethanol fixation and propidium iodide staining, the cells were analyzed by flow cytometry, and the percentage of dead cells was obtained. Pep5-cpp (EC₅₀, 62.28 μM) increased cell death only at concentrations above 50 μM (Fig. 3), demonstrating the low level of toxicity of Pep5-cpp in LLC-MK₂ cells compared to parasites. Based on these results, we evaluated the effect of Pep5-cpp on the number of infected cells using a concentration of 25 μM (the EC₅₀ calculated for epimastigotes and used to treat isolated trypomastigotes), which had no significant impact on cell death or the cell cycle of LLC-MK₂ mammalian cells but was effective in inducing cell death in parasites (selectivity index, 2.49).

FIG 3.

LLC-MK₂ cells are resistant to low concentrations of Pep5-cpp. LLC- MK₂ cells were treated with Pep5-cpp for approximately 20 h. After treatment, the cells were washed in PBS, fixed in 70% ethanol, stained with a propidium iodide solution for 30 min, and analyzed by flow cytometry. The percentage of cell death is plotted as the means and SEM of the results of three biological experiments performed in triplicate. ***, P < 0.001.

As mentioned above, the effect of Pep5-cpp was also investigated during infection of LLC-MK₂ cells (Fig. 4). When Pep5-cpp (25 μM) was added simultaneously with trypomastigotes to a culture containing host cells for a period of 24 h (Fig. 4C), the percentage of infection and the number of parasites per infected cells were drastically reduced 48 h after infection (Fig. 4A to F). These data suggest that Pep5-cpp has a pronounced inhibitory effect during in vitro infection by T. cruzi. Although cell death induction in trypomastigotes (Fig. 2A) was only 28%, the blockage of infection presented in this study was approximately 90% (Fig. 4D). Nevertheless, as both host cells and parasites were subjected to treatment with Pep5-cpp during infection, the inhibitory result may have been a consequence of a synergic effect given by Pep5-cpp in both organisms. Therefore, we performed isolated treatment of trypomastigotes prior to the infection assay; infection was drastically decreased compared to the nontreated group even when the parasites were pretreated with Pep5-cpp for 3 h and extensively washed with phosphate-buffered saline (PBS) before the mammalian cells were infected, without any contact with Pep5-cpp (treated/untreated ratio of infected cells, 0.024; ratio of the number of amastigotes, 0.32) (Fig. 4G to J). Interestingly, when the mammalian cells were pretreated with Pep5-cpp and then infected with Pep5-cpp-free trypomastigotes, the numbers of infected cells (ratio, 0.83) and amastigotes (ratio, 0.44) inside them were also diminished (Fig. 4K and L).

FIG 4.

Pep5-cpp effects during infection. In all experiments, LLC- MK₂ cells were infected and stained with eosin-methylene blue, and the infected cells and amastigotes (strain Y) were counted under a microscope. (A) Nontreated cells showing parasites in the cytoplasm. Bar, 20 μm. (B) Infected cells treated with Pep5-cpp. (C) Cells were infected and treated simultaneously with Pep5-cpp for 24 h, after which the medium was removed and fresh medium free of Pep5-cpp and trypomastigotes was added for an additional 24 h. (D) Numbers of infected cells. (E) Numbers of amastigotes per cell. (F) Total numbers of amastigotes. (G) Trypomastigotes (strain Y) were pretreated with Pep5-cpp for 3 h, extensively washed in PBS, and then used for infection. The infected cells and intracellular parasites were counted 48 h later. (H) Numbers of infected cells. (I) Numbers of amastigotes per cell. (J) Total numbers of amastigotes. (K and L) In order to check the potential impact of Pep5-cpp directly on mammalian host cells, LLC-MK₂ cells were treated with Pep5-cpp for 3 h, washed with PBS, and then infected with trypomastigotes. (K) Numbers of infected cells. (L) Numbers of amastigotes per infected cell. In all the assays, a total of 100 cells were counted per replicate. The graphs represent the means and SEM of the results of at least two biological experiments performed in triplicate. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Another approach frequently used as a tool for measuring the effect of a drug on in vitro parasite growth is manual microscope counting (23–25). Thus, we recorded the number of trypomastigotes in the supernatants of the infected cell cultures after treatment (or not) with Pep5-cpp (25 μM; 24 h) (Fig. 5A). Incubation with Pep5-cpp for only the first day of the assay was sufficient to decrease the number of trypomastigotes released by host cells until the end of the experiment, which corresponded to a period of 7 days (Fig. 5B). Additionally, cpp was used as a control, showing that the effect of this inhibition was promoted by Pep5-cpp and not due to the carrier itself, which confirmed previously published data (8).

FIG 5.

Pep5-cpp effect on trypomastigote release by infected cells. LLC-MK₂ cells were infected with trypomastigotes (strain Y) and treated with Pep5-cpp or cpp for 24 h. The next day, the parasites and peptide were removed, and fresh medium without peptide was added daily until the end of the experiment. The trypomastigotes in the supernatants of infected cells were counted on days 4, 5, 6, and 7 after infection (days 4, 5, 6, and 7 correspond to the period in which trypomastigotes are released by cells). The means ± SEM of the results of two biological experiments performed in triplicate are plotted in the graph. **, P < 0.01; ***, P < 0.001.

Extracellular amastigotes are susceptible to cell death after treatment with Pep5-cpp.

As mentioned above, the nonapeptide derived from cyclin D2 was able to block infection caused by trypomastigotes. Subsequently, the effect of the peptide was evaluated directly on the intracellular amastigotes. When infected cells (after 24 h of infection with trypomastigotes [Fig. 6A]) were treated with Pep5-cpp, no effect on the number of intracellular parasites or infected cells was observed (Fig. 6A to D). To assess the location of Pep5-cpp in infected cells, LLC-MK₂ cells were infected with T. cruzi trypomastigotes for 24 h and incubated with the fluorescent version of Pep5-cpp (Pep5-cpp-Dye555) for 15 min. Figure 6E shows that Pep5-cpp (red fluorescence) was located close to the nucleus in mammalian cells, without evidence of contact with any intracellular amastigotes, in all the cells analyzed (Fig. 6E). This result supports the idea that the tested peptide does not kill the parasites once they are already inside host cells as intracellular amastigotes (Fig. 6A to D) because of the location of Pep5-cpp and its preference for the mammalian nucleus. To confirm this, we induced the transformation of trypomastigotes, found in the supernatant of infected cells, to amastigote forms and submitted them to treatment with the Pep5-cpp. Remarkably, when the extracellular amastigotes were treated with Pep5-cpp, cell death induction occurred quite well (Fig. 6F and G), similar to the results for trypomastigotes and epimastigotes.

FIG 6.

Effect of Pep5-cpp on amastigote forms. (A) Cells were infected with trypomastigotes (strain Y); the medium was changed 24 h later; and fresh medium, free of parasites, was added to the cells in the presence or absence of Pep5-cpp for an additional 24 h. After this time, the cells were washed in PBS, fixed, stained with eosin-methylene blue, and analyzed with fluorescence microscopy. A total of 100 cells were counted per replicate. (B to D) The graphs represent the means and SEM of the results of two biological experiments performed in triplicate. (B) Numbers of infected cells. (C) Numbers of amastigotes per cell. (D) Total numbers of amastigotes. (E) Pep5-cpp locations in infected cells. LLC- MK₂ cells were infected with T. cruzi trypomastigotes (strain Y) and then treated with fluorescent Pep5-cpp (Pep5-cpp-Dye555) for 30 min. After the treatment, the cells were extensively washed with PBS, fixed in 4% PFA, and mounted with Vectashield-DAPI. N, nucleus of the host cell; white arrows, intracellular amastigotes; yellow arrows, Pep5-cpp locations. In total, 100 cells were analyzed by fluorescence microscopy, and the images are representative of three different fields. Bar, 20 μm. (F and G) Next, Pep5-cpp action was also investigated in extracellular amastigotes. Amastigotes (strain Y), obtained by transformation of trypomastigotes found in the supernatants of cells, were treated with the cyclin D2 fragment for 3 h and then fixed, incubated with propidium iodide, and analyzed by flow cytometry. A total of 50,000 events were analyzed per replicate. The histograms are representative of the results of two experiments performed in triplicate. (F) Nontreated cells. (G) After Pep5-cpp treatment.

Pep5 interacts with specific proteins from T. cruzi.

Finally, to obtain new insight into the molecular mechanism of Pep5 in T. cruzi, we performed global analysis of the proteins that bind to the nonapeptide in vitro. Extracts from epimastigotes were incubated with a column containing covalently immobilized Pep5 (cpp immobilized to another column was used as a control). All the proteins that bound to Pep5 or cpp were eluted and identified by mass spectrometry (Table 1; see Fig. S2 in the supplemental material). Among the known proteins that interact with Pep5 are calmodulin-ubiquitin-associated protein, GTPase-activating protein, and a protein kinase.

TABLE 1.

Proteins from T. cruzi epimastigote extracts (strain Y) that interact with Pep5 or cpp

Protein namea	Accession no.	Protein scoreb
Pep5
Uncharacterized protein	K4EAD5	33
Calmodulin-ubiquitin-associated protein	H2DQH1	29
Uncharacterized protein	K2N4U0	28
GTPase-activating protein, putative	K2MZ21	23
Protein kinase, putative	K2NF31	17
TAT (cpp)
Heat shock-like 85-kDa protein HSP85	K4DIK9	60
Uncharacterized protein	Q4CKB9	31
Histone H2A	K2LVU2	28
Uncharacterized protein	Q4CP04	26
Histone H4	Q4DEL1	25
Uncharacterized protein	Q4DBP1	24
Uncharacterized protein	V5D279	24
Uncharacterized protein	K2N912	23
Uncharacterized protein	Q4DG62	20
Tryparedoxin peroxidase, putative	Q4CM56	20
Aldehyde dehydrogenase	K2MRC3	20
Uncharacterized protein	K2NNS0	18
Sf3b complex subunit 3	Q5EFC9	17
Uncharacterized protein	K4E508	16
Uncharacterized protein	K4E1X1	13

^aProtein extracts derived from exponential growth of T. cruzi epimastigotes (strain Y) were incubated with Pep5 or TAT (cpp) peptide that had been covalently immobilized on HiTrap columns. Proteins interacting with these peptides were eluted and identified by mass spectrometry. Characterized proteins that interacted with Pep5 are in boldface.

^bThe protein score in the result report from an MS/MS search is derived from the ion scores.

DISCUSSION

The search for new drugs for treating Chagas disease with higher efficiency and less toxicity remains a challenge because the only two compounds licensed for treatment are far from ideal in terms of safety and efficacy. Many adverse effects have been reported, including neurological disorders, anorexia, weight loss, nausea, vomiting, fever, rash, headache, and sleep disorders (19, 26, 27). Based on our results from this study, we propose Pep5, a nonapeptide derived from human cyclin D2, as a possible trypanocidal/trypanostatic drug. Pep5 is able to induce cell death in both replicative and nonreplicative forms of T. cruzi parasites at concentrations that do not have significant effects on host cell viability. Some effects of the peptide on tumor cells were found in a previous study (8), and it is known to kill cells that replicate continuously. In addition, the compound exhibits increased action in cells synchronized at S phase of the cell cycle (14).

The antiparasitic activity of Pep5-cpp was initially demonstrated in epimastigote forms of two different T. cruzi strains. Both strains (Y and CL Brener) were highly sensitive to the effect of this fragment of cyclin, with an increased percentage of cell death, even at concentrations as low as 10 and 25 μM, and only when the peptide was inside the parasites. These data were confirmed by treatment of parasites with cpp alone or Pep5 without the carrier, showing that the effect caused by the cyclin D2 fragment is not due to extracellular activation, a phenomenon already observed in tumor cells (8). Other compounds tested in T. cruzi, such as memantine, MK-801, and amantadine, are effective only at high concentrations, with 50% inhibitory concentration (IC₅₀) values of 172.6 μM, 300 μM, and 451.2 μM, respectively (28). Although the molecular mechanisms involved in the activation of cell death promoted by Pep5-cpp in T. cruzi parasites still need to be further investigated, we showed that Pep5-cpp increased intracellular ROS production and intracellular calcium concentrations in epimastigotes, which are signals that frequently correlate with the activation of PCD in T. cruzi (28). Additionally, when treated with Pep5-cpp, trypomastigotes exhibited a significant increase in cell death in a process with characteristics of PCD activation. A significant number of trypomastigotes displayed increased amounts of phosphatidylserine in the outer membrane, an assay regularly used to analyze cells that have died due to type I PCD (29). Although there is skepticism about the existence of PCD in unicellular organisms, there is an increasing body of evidence demonstrating that different protist species live and behave as a complex community and, when necessary, trigger a signaling process of individual self-elimination in a programmed way without any damage to other individuals in the population (30–32). Several morphological and biochemical features involved in controlled cell death have been observed in all members of the trypanosomatid group (28, 33). The literature shows events related to PCD in T. cruzi, such as DNA condensation and fragmentation, PS exposure, mitochondrion swelling and kinetoplast disorganization, and activation of caspase-like proteins (28, 32, 34, 35). Based on the data presented in this study, we propose that the cell death induced by Pep5-cpp occurs, at least in part, through a controlled PCD event.

Moreover, Pep5-cpp appears to block the infective ability of previously Pep5-cpp-treated trypomastigotes that are still viable. Although Pep5-cpp induced cell death in the infective form, it was limited to ∼28%. However, we did observe a greater than 90% reduction in the numbers of infected cells and of amastigotes inside these cells, meaning that the trypomastigotes that did not die in the presence of Pep5-cpp were no longer able to invade host cells. This effect was maintained for 7 days after treatment and infection, even when the treatment was performed for only a few hours at the beginning of the experiment. These results suggest that Pep5-cpp promotes a barrier that blocks the capacity of trypomastigotes to infect cells. Interestingly, we also observed a protective effect produced by Pep5-cpp on the host cells, showing a synergistic effect produced by the cyclin D2 fragment during the T. cruzi infection, although this effect was not as effective as that on trypomastigotes. Therefore, we conclude that the diminished number of infected cells was more likely due to the action of Pep5-cpp against trypomastigote parasites than to the effect on host cells, though this protective effect does exist and needs to be further investigated. In summary, Pep5-cpp appears to have a potential effect in blocking infection at a concentration that does not interfere with the cell cycle/death features of infected host cells.

Although Pep5-cpp had no significant effect on amastigotes when they were already inside host cells, we suggest that this was due to the preference of cpp (TAT_47–57) to transport cargo to the nuclei of mammalian cells. This synthetic peptide is a fragment of the HIV TAT protein, which is responsible for promoting its entrance into host cells. Some studies have demonstrated that this specific cpp has great affinity for the nuclear compartment and that it is quickly carried there through the cellular membrane, escaping the endocytic pathway (14, 36, 37). Thus, Pep5-cpp is rapidly localized to the nuclei of LLC-MK₂ cells and is unavailable for distribution in the cytoplasm and for contact with amastigotes (Fig. 6E). It was clearly confirmed that extracellular amastigotes treated with Pep5-cpp underwent increased cell death.

Although Pep5-cpp showed trypanocidal activity toward trypomastigotes and amastigotes in the micromolar range (EC₅₀ > 25 μM), we emphasize, as discussed above, that treated trypomastigotes were no longer capable of infecting host cells. Moreover, our work offers a new possibility for optimizing Pep5 activity by improving the delivery system to enhance its efficacy against the intracellular forms of the parasites. In fact, some research has already demonstrated that new advances in the development of modifier cpps used for cancer treatment are efficient in reducing cytotoxicity in normal cells and in increasing target selectivity (38). In summary, the discoveries presented here open a wide range of possibilities for developing this peptide as a strategy for the treatment of Chagas disease.

In a search for targets of Pep5 inside parasites, we observed that this fragment of cyclin D2 bound to specific intracellular proteins. All of these proteins were previously shown to have important functions in cell signaling and survival and in stimulating proteins involved in the control of cell growth and differentiation processes in eukaryotic cells, and all are considered important drug targets from a therapeutic perspective. Among the proteins identified as binding to Pep5 are GAP and a protein kinase. GAP proteins are involved in several processes in eukaryotes, such as cell growth, cell survival, apoptosis, differentiation, vesicle trafficking, and migration, and some members of this family are involved in cytokinesis and are transcriptionally upregulated in several types of cancer. The proteins control the activities of a variety of small GTPases by increasing their GTPase activities and contributing residues to the active site; in addition, the conformational change promoted by this activation alters the ability of the protein to bind to downstream effectors (39). Certain inhibitors of GAP family members have been found to cause reductions in cell division and cell cycle arrest, as well as impaired cytokinesis and cell death (40–42). Several proteins from the GTPase superfamily are present in parasites, such as T. brucei, T. cruzi, and Leishmania (43, 44). Additionally, protein kinases are essential for cell signaling and transduction and are responsible for phosphorylating many diverse substrates; in human cells, they are essential targets for drug discovery (45–47). As observed for GAP proteins, the use of kinase inhibitors causes cell death in trypanosomatids, such as T. brucei (47, 48). According to the data presented in this study, Pep5 interacts with these two proteins, which may contribute to the eventual activation of cell death in T. cruzi parasites.

Pep5 also interacts with calmodulin-ubiquitin-associated protein, which is particularly exciting. Some studies have demonstrated that the calmodulin-ubiquitin (CUB) gene in T. cruzi plays a crucial role in the parasite life cycle (49). This single-copy gene encodes a 208-amino-acid polypeptide that contains a calcium-binding domain linked to a downstream ubiquitin domain (50). When associated with calcium, calmodulin participates in activating more than 20 different enzymes and has regulatory roles in cell proliferation (51). Ubiquitin binds to many proteins involved in several events and alters their stability, localization, or activity (52). Based on the importance of these indispensable cellular events, fusion of the two genes in T. cruzi has led some authors to propose that it may have an important role in parasite survival (53). In fact, when one allele of the gene was deleted in trypomastigotes, the clone TulCub8 showed loss of virulence in mice, and the parasitemia of the mutant was 68-fold lower than that of the wild-type parasite (53). In the present study, we demonstrated that Pep5 is able to bind to a calmodulin-ubiquitin-associated protein (CUB2.65). Due to the importance of this gene to T. cruzi trypomastigote infectivity, it is possible that the lower infectivity observed in our experiments was related to an inhibitory effect of Pep5 on calmodulin-ubiquitin-associated protein. In summary, we suggest that Pep5 interacts with the proteins identified in this study to exert its beneficial effects, reducing the ability of trypomastigotes to infect host cells and/or contributing to the cell death induction of all the forms treated with the peptide.

Altogether, the data presented in this study support our hypothesis that Pep5 provides a novel and interesting strategy for the development of new tools to control T. cruzi infection and Chagas disease.

MATERIALS AND METHODS

Peptides.

Pep5 (WELVVLGKL) was synthesized coupled (Pep5-cpp) or not (Pep5) to a cell-penetrating peptide (cpp: YGRKKRRQRRR; TAT_47–57) at its C terminus (8, 36) and also as a fluorescent derivative, Pep5-cpp-Dye555 (Dye555-WELVVL-YGRKKRRQRRR). All the peptides had a purity of >95%, as confirmed by mass spectrometry and high-performance liquid chromatography (HPLC), and were purchased from Proteimax Biotecnologia LTDA, São Paulo, Brazil. The peptides were diluted in Milli-Q water and stored at −20°C. Tests were performed in fetal bovine serum (FBS)-free medium at concentrations ranging from 1 to 150 μM.

Parasites.

T. cruzi epimastigotes, including strains Y and CL Brener, were cultured at 28°C in liver infusion tryptose (LIT) medium with 10% FBS. Trypomastigotes (strain Y) were obtained from the supernatant of infected LLC-MK₂ monkey kidney cells. Briefly, LLC-MK₂ cells were maintained in Dulbecco's modified Eagle’s medium (DMEM) (Life Technologies, Carlsbad, CA, USA)-10% FBS and used as host cells for infection with trypomastigote forms in vitro. The day before infection, the LLC-MK₂ cells were reseeded, and then infection was done with trypomastigote forms from strain Y (multiplicity of infection [MOI], 1:20). After 24 h of infection, the cells were washed with PBS to remove the remaining parasites, and fresh DMEM-10% FBS was added. Seven days after infection, trypomastigotes from the supernatant were collected, centrifuged (500 × g; 10 min), and used for peptide experiments. Amastigote forms were obtained by differentiation from trypomastigotes collected in the supernatant of infected cells (tissue culture-derived trypomastigotes). To do that, trypomastigotes were incubated in DMEM-10% FBS, pH 5, overnight to differentiate to amastigotes, as described by Tomlinson et al. (54). These amastigotes (i.e., extracellular amastigotes) were then washed in PBS and subjected to peptide treatment on fresh medium.

Cell death assay.

Cell death, based on DNA content (55, 56), was analyzed by flow cytometry using an Attune acoustic focusing cytometer (Applied Biosystems). LLC-MK₂ cells (2 × 10⁴) or parasites (including epimastigotes, trypomastigotes, and extracellular amastigotes; 3 × 10⁶ parasites per sample; strain Y) were treated with different concentrations of Pep5-cpp for 3 h, centrifuged (2,500 rpm for 5 min), and washed twice in PBS; the samples were then fixed on ice in 70% ethanol diluted in PBS overnight at −20°C. The pellet was resuspended in 500 μl of PBS containing 2 μl of propidium iodide (1 mg/ml) and 10 μl RNase (10 mg/ml) and maintained on ice for 30 min. A total of at least 10,000 events were analyzed per sample.

Peroxidase assay.

Levels of hydrogen peroxide (H₂O₂) were measured in T. cruzi epimastigotes (strain Y) cultured in the presence or absence of Pep5 bound to cpp for 3 h. After incubation, the parasites (1 × 10⁷/ml) were washed twice in 1× PBS and resuspended in 100 μl of PBS-succinic acid (5 mM), after which 12 μM Amplex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine; ThermoFisher) and 0.05 U/ml horseradish peroxidase (HRP) were added to each sample according to the manufacturer’s instructions. Fluorescence was measured using a 96-well fluorescence microplate reader (FlexStation 3 Multi-Mode; Molecular Devices) with excitation at 563 nm and emission at 587 nm.

Intracellular Ca²⁺ levels.

Calcium release was measured using a Fluo-4-AM kit (Invitrogen). Parasites (2.5 × 10⁷ epimastigotes/ml; strain Y) were incubated in the presence of 5 μM Fluo-4-AM for 1 h at 28°C after treatment with Pep5-cpp (25 μM; 3 h). Following incubation, the parasites were washed twice with HEPES-glucose buffer (50 mM HEPES, pH 7.4, 116 mM NaCl, 5.4 mM KCl, 0.8 mM MgSO₄, 5.5 mM glucose, 2 mM CaCl₂), resuspended in the same buffer, and distributed in a 96-well microplate. Ca²⁺ fluorescence was estimated using a FlexStation 3 multimode reader (Molecular Devices) at an excitation wavelength of 490 nm and an emission wavelength of 518 nm.

Phosphatidylserine residue exposure.

Epimastigotes and trypomastigotes (1 × 10⁶ parasites; strain Y) were treated with Pep5-cpp (25 μM) for 3 h and analyzed by flow cytometry. After incubation, the samples were washed in cold PBS and diluted in 500 μl of 1× annexin-binding buffer (annexin V-fluorescein isothiocyanate [FITC] apoptosis detection kit; Sigma). Following the manufacturer’s instructions, 5 μl of annexin V-FITC and 10 μl of 100 μg/ml PI working solution were added to the cell suspension. The cells were incubated at room temperature for 10 min. The samples were analyzed by flow cytometry to measure fluorescence emission at 530 nm (BL1) and >575 nm (BL2). The population was separated into four groups: live cells showing only a low level of fluorescence, cells with PS residue exposure showing green fluorescence, dead cells showing both red and green fluorescence, and PI-positive cells showing only red fluorescence.

LLC-MK₂ cell resistance to Pep5-cpp treatment.

In order to evaluate the viability of the LLC-MK₂ host cells in the presence of Pep5-cpp, the cells were treated with different concentrations of Pep5-cpp (10 to 150 μM) for 20 h. After treatment, the cells were washed in PBS, fixed in 70% ethanol, and stained with a propidium iodide solution, and their viability was analyzed by flow cytometry, as described above.

Pep5-cpp effects during the infection assay.

In order to analyze the effect of Pep5-cpp during T. cruzi infection, approximately 2 × 10⁴ LLC-MK₂ cells were cultured in glass bottom microwell dishes and infected with 10⁶ parasites (strain Y) in the presence of Pep5-cpp (25 μM) for 24 h. After this time, the medium containing the remaining parasites and peptide was removed, the cells were washed with PBS, and fresh Pep5-cpp-free DMEM-10% FBS was added. The number of infected cells was analyzed 48 h after infection. The cells were fixed in 4% paraformaldehyde (PFA) for 30 min, washed several times with PBS, and stained with eosin and methylene blue for 20 min and then dehydrated in acetone, followed by a gradient of acetone-xylol. The samples were mounted with Entellan rapid mounting medium for microscopy (Sigma-Aldrich) and analyzed under a BX51 microscope (Olympus) to evaluate the numbers of infected cells and of amastigotes per infected host cell in each group.

Pep5-cpp effects on trypomastigotes before infection.

To evaluate the effect of the pretreatment of trypomastigotes (strain Y) with Pep5-cpp on infection, the parasites were incubated with the peptide (25 μM) for 3 h, washed three times in PBS, and used to infect LLC-MK₂ cells. The pretreated parasites were incubated with the host cells for 24 h, after which the remaining parasites were removed and fresh medium was added. The numbers of infected cells and intracellular parasites were analyzed 48 h after infection, as described above.

Effect of Pep5-cpp on LLC-MK₂ cells before infection by trypomastigotes.

In order to evaluate the impact of Pep5-cpp on host cells before infection, LLC-MK₂ cells were pretreated with Pep5-cpp (25 μM; 3 h), washed several times, and infected with trypomastigotes (strain Y) for 24 h. Then, the remaining parasites were removed, the cells were washed with PBS, and Pep5-cpp-free medium was added. The number of infected cells was analyzed 48 h after infection.

Pep5-cpp effects on intracellular amastigotes.

For experiments examining the effect of Pep5-cpp on the intracellular amastigote forms (strain Y), cells were preinfected with T. cruzi trypomastigotes (strain Y). After 24 h, the medium containing the trypomastigotes was removed, and fresh medium containing Pep5-cpp was added for an additional 24 h. The numbers of infected cells and of amastigotes per cell were analyzed as described above 48 h after infection.

Immunofluorescence assay.

Fluorescent Pep5-cpp-Dye555 (Dye555-WELVVL-YGRKKRRQRRR) was used to observe the location of Pep5 inside infected host cells and in isolated parasites. Pep5-cpp-Dye555 was added to cultures at concentrations of 10 to 15 μM for 15 to 30 min. After incubation, parasites (3 × 10⁶ epimastigotes; strain Y) or infected cells (2 × 10⁴ LLC-MK₂ cells infected with 10⁶ trypomastigotes, strain Y) were extensively washed in PBS, fixed in 4% paraformaldehyde for 20 min, and blocked in PBS with 3% bovine serum albumin. The slides were mounted with Vectashield antifade mounting medium with DAPI (4′,6-diamidino-2-phenylindole) (Vector Laboratories) and analyzed under a microscope (BX51; Olympus). Images were acquired at a magnification of ×1,000.

Affinity chromatography and mass spectrometry.

To identify possible intracellular targets of Pep5 in T. cruzi parasites, peptide-linked affinity chromatography columns were obtained. Pep5 or cpp (TAT_47–57) was covalently bound through the NH₂ terminus to the gel of an N-hydroxysuccinimide (NHS)-activated HiTrap HP column (Amersham Biosciences, Piscataway, NJ, USA). Protein extracts derived from 10⁸ epimastigotes (strain Y) were incubated with the columns for 1.5 h at 4°C; the columns were then washed with PBS containing protease inhibitors and eluted with 10 ml of 0.1 M glycine. To avoid nonspecific and low-affinity protein binding, the first milliliter eluted was discarded, and the other 9 ml (subjected to buffer exchange in 10 mM Tris-HCl, pH 7.4) was concentrated using a Millipore (Darmstadt, Germany) centrifugal filter device with a cutoff value of 5,000 Da. All proteins (after in‐solution digestion) that bound to Pep5 or cpp were identified by mass spectrometry using an Easy-nLC nanoflow liquid chromatograph (Thermo Scientific, Waltham, MA, USA), along with an LTQ-Orbitrap Velos mass spectrometer (Thermo Scientific), as previously reported (14).

Statistics.

Values are expressed as means and standard errors of the mean (SEM) of the results of at least two independent experiments performed in triplicate. All analyses were conducted using analysis of variance followed by Tukey’s or Bonferroni’s test to compare more than two groups in GraphPad Prism version 5.0; P values of ≤0.05 were considered significant.