Trypanosoma rangeli: growth in mammalian cells in vitro and action of a repositioned drug (17-AAG) and a natural extract (Artemisia sp. essential oil)

Trypanosoma rangeli and T. cruzi are both parasitic unicellular species that infect humans. Unlike T. cruzi, the causative agent of Chagas disease, T. rangeli is an infective and non-pathogenic parasite for humans, but pathogenic for vectors from the Rhodnius genus. Because both species can coexist in different hosts and overlap their infective cycles but very little is known about the infection of T. rangeli in mammalian cells, we decided to characterize both the development of this parasite in cell culture and the effect of therapeutic agents with potential trypanocidal action on it. We found that T. rangeli exhibits a cycle of infection in Vero cells similar to that for T. cruzi and that the repurposed drug, 17-AAG, and the natural extract Artemisia sp. essential oil produce a toxic effect on epimastigotes showing a trypanocidal action from the fifth day of culture. Both treatments also affected the infection of trypomastigotes and reduced the capacity of replication of amastigotes of T. rangeli. Since T. cruzi / T. rangeli coinfection cases have been reported, the finding of drugs with potential activity against both species could be significant in the future. Furthermore, studies of susceptibility of both species to drugs could also help to know the different mechanisms of pathogenicity in humans displayed by T. cruzi that are absent in T. rangeli. BIOCELL ISSN 1667-5746 (on-line) 2019 43(1): 13-19 ISSN 0327-9545 (printed)


Introduction
Tr y panosoma rangeli is a protozoan parasite that infects mammals and triatomines, causing different levels of pathogenicity in its invertebrate vectors, particularly those from the genus Rhodnius (Ferreira et al., 2018;Grewal, 1957;Añez et al., 1985). Unlike T. cruzi, the causative agent of Chagas disease, T. rangeli infects humans without causing pathologies.
Morphologically corresponds to a trypanosome that measures about 31 µm in length and has a more developed undulating membrane than T. cruzi. Its kinetoplast is subterminal and small, features that allow its morphological differentiation with T. cruzi.
The development of T. rangeli in triatomine insects begins when the trypomastigote forms are ingested with the blood of infected mammals. When these forms reach the midgut of the vector (hemolymph), they differentiate into epimastigotes that become capable of replicating. After 10 to 15 days they invade the salivary glands, where they differentiate into metacyclic trypomastigotes. Rounded forms are also found as well as short and long epimastigotes, which can be transformed into trypomastigotes in the hindgut (D'Alessandro and Hincapie, 1986). After inoculation of man, the parasites enter the circulation (Urdaneta- Morales and Tejero, 1985). Some groups have shown the presence of amastigote nests in tissues (Urdaneta-Morales and Tejero, 1985;Osorio et al., 1995;Eger-Mangrich et al., 2001), while others have not observed them (Tanoura et al., 1999).
The majority of reported human cases of T. rangeli infections correspond to Venezuela, Colombia, Paraná, Guatemala, El Salvador and Brazil. The laboratory procedures used for the diagnosis are based on the direct search, in fresh or stained material, which serves for the morphological study. Its differential diagnosis is important, because T. rangeli and T. cruzi can coexist, producing mixed infections. More specific methods, including immunofluorescence, immunoprecipitation and ELISA techniques, have been used 14 ANA LAURA CIMADOR et al.
to confirm T. rangeli misdiagnosed infections (Hudson et al., 1988) Norte de Santander,Columbia (n = 327. Recently, a conventional PCR and a loop-mediated isothermal amplification (LAMP) assay were developed to differentiate T. rangeli and T. cruzi in samples extracted from the vector bugs (Thekisoe et al., 2010).
The main objective of this work was to characterize the in vitro development of T. rangeli at different times of its life cycle; mainly at the time of infection of trypomastigotes into mammalian cells, and the time of the intracellular replication of amastigotes. Additionaly, we studied the possible effect of potential trypanocidal compounds on the different stages of T. rangeli cycle.
Two classes of treatments were studied: (1) the geldanamycin derivative 17-(allylamino)-17-demethoxygeldanamycin (17-AAG), a drug used in the treatment of certain classes of cancers, and (2) the Artemisia sp. (Asterales, Asteraceae) essential oil. The first one is the less toxic analog of geldanamycin and a potent inhibitor of heat shock protein 90 (Hsp90). It was the first Hsp90 inhibitor to enter clinical trials as an antineoplastic drug due to its significant anti-tumor activity (Jhaveri et al., 2014); 17-AAG was repurposed as a drug against Leishmania spp. (Kinetoplastida, Trypanosomatidae), because of its demonstrated in vitro and in vivo action on this parasite (Petersen et al., 2012), and we thought it could also have trypanocidal activity on T. rangeli. We also explored the action of Artemisia essential oil because there was evidence of its action against T. brucei, T. cruzi and T. congolense (Naß and Efferth, 2018)the safety and efficacy of current synthetic drugs are limited due to the development of drug resistance and adverse side effects. PURPOSE Artemisia annua and artemisinin are not only active against Plasmodia, but also other protozoa. Therefore, we reviewed the literature on species of the genus Artemisia and their phytochemicals regarding their activity against trypanosomes. STUDY DESIGN A PubMed search for \"Artemisia/Artemisinin and Trypanosoma\" has been conducted for literature until December 2017. RESULTS Interestingly, not only A. annua L. and its active principle, artemisinin revealed inhibitory activity towards trypanosomes. Other Artemisia species (A. absinthium, A. abyssinica, A. afra, A. douglasia, A. elegantissima, A. maciverae, A. mexicana, and A. roxburghiana). It has also been proposed as a therapeutic agent in malaria (Pellicer et al., 2018).

Mammalian cells culture
Epithelial cells (Vero cell line) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics at 37°C in an atmosphere of 5% CO 2 .

Trypomastigotes
T. rangeli trypomastigotes (LDG Colombian strain) were obtained by in vitro metacyclogenesis of epimastigotes, as described elsewhere (Contreras et al., 1986), and maintained in a culture of Vero cells in DMEM supplemented with 3% FBS and antibiotics at 37°C in an atmosphere of 5% CO 2 .

AlamarBlue assay
Cell viability was estimated by the AlamarBlue assay (Invitrogen), according to the manufacturer's instructions. Vero cells grown in 96-well plates were washed three times with PBS and incubated in control medium (DMSO) in presence or absence of 0.1 µM 17-AAG and 0.5 μg / ml Artemisia essential oil (stalks, leaves, and flowers, were distilled in water steam stills for extracting the essence) at 37ºC for 24 h. After that, cells were washed and 10% of the AlamarBlue reagent was added to the medium and incubated for 6 h at 37ºC before measurement of absorbance at 540 nm.

Growth of epimastigotes
We started with a solution of 1 × 10 6 epimastigotes per milliliter in control (DMSO), 0.1 µM 17-AAG and 0.5 μg / ml Artemisia essential oil condition. Then, we counted the epimastigotes at different times, using a Neubauer chamber.

T. rangeli infection assays on Vero cells
We collected the trypomastigotes from the infected Vero cell cultures, by centrifugating samples for 15 min at 4000 rpm. The supernatant was discarded and 1 ml of fresh medium was added. Two hous later, we collected the supernatant rich in swimming trypomastigotes. We then treated the trypomastigotes with control medium (DMSO), 0.1 µM 17-AAG and 0.5 μg / ml Artemisia essential oil for 30 min before seeding them on Vero cell monolayers, at a proportion of 10 parasites per cell. After 24 h, the cells were washed with PBS, fixed with 4% paraformaldehyde, quenched with 50 mM ammonium chloride and mounted with Mowiol containing Hoechst. The percentage 17-AAG AND ARTEMisiA ESSENTIAL OIL AFFECT T. RAnGELi INFECTION AND REPLICATION 15 of infected cell was quantified by confocal microscopy using a FV1000 Confocal Olympus microscope.

Growth of amastigotes
Vero cells were infected with T. rangeli tripomastigotes for 24 hours, then they were washed with PBS to eliminate the trypomastigotes that did not infect and fresh medium was placed in control condition (DMSO), 0.1 µM 17-AAG and 0.5 μg / ml Artemisia essential oil for additional 24 h. Then the cells were fixed with 4% paraformaldehyde for 15 min, quenched with 50 mM ammonium chloride for 15 min, treated with albumin saponin for 20 min, and then the amastigotes were detected by immunoluorescence. The number of parasites in each cell was quantified by confocal microscopy using a FV1000 Confocal Olympus microscope.
statistics Multigroup comparisons were made by ANOVA followed by the Tukey test (using Kyplot®).

T. rangeli infection course in Vero cells
As mentioned above, we wanted to study the capacity of T. rangeli to infect and to develop into mammalian cells. To asses this, we performed an in vitro assay using Vero cells as host cells. These kidney-derived epithelial cells are broadly used for growing viruses and eukaryotic parasites, especially the trypanosomatids. We also used these cells to generate the parasites used for infection assays. In the experiment, cells were incubated in the presence of trypomastigotes of T. rangeli (MOI = 10) for 24 h (interaction period) and, after washing, they were fixed and stained with the DNA marker Hoechst 33342 for microscopy analysis. Other samples were washed and incubated in medium without parasites for an additional time of 24 h before fixation (chase period) and then processed as described before (Fig. 1A). Microscopy studies showed the presence of parasite nuclei and kinetoplasts inside Vero cells after 24 h incubation (Fig. 1B). Interestingly, samples maintained for 48 h showed a higher number of amastigote-like parasites surrounding Vero cell nuclei (Fig. 1C), denoting that parasites can replicate inside the cells. Quantitative data showed that more than 50% of cells became infected after 24 h of interaction (Fig. 3D) and approximately 10 amastigote-like parasites/cell developed at 48 h (Fig. 4C). Seven to 10 days after interaction, new trypomastigotes were released into the culture medium (data not shown), indicating that T. rangeli had completed its intracellular cycle by this time. Taking together, these results revealed that T. rangeli can infect and replicate in mammalian cells and that its intracellular cycle can be studied in vitro.

FIGURE 1. T. rangeli infection and replication in epithelial cells in vitro.
Interaction of trypomastigotes of T. rangeli with Vero cells were evaluated by adding trypomastigotes (MOI = 10) to cell monolayers for 24 h (interaction period) before washing and fixation. Other samples were washed and left for additional 24 h to evaluate the replication of amastigotes (chase period). After fixation, both samples were stained with Hoechst 33342 to label kinetoplast DNA from parasites and nuclear DNA from parasites and host cells.

FIGURE 2. Effect of drugs on the growth of epimastigotes of T. rangeli.
We started with a culture of 1 × 10 6 T. rangeli epimastigotes /ml in Diamond medium under control conditions (with DMSO), 0.1 μM 17-AAG and 0.5 μg / ml Artemisia essential oil. A small aliquot of each sample was taken on days 3, 5, and 10 to quantify the number of parasites by counting them in a Neubauer chamber. The graph shows the mean ± SE of each treatment at the different times.

FIGURE 3. Effect of drugs on T. rangeli infection of epithelial cells.
First, we evaluated the cell viability by the AlamarBlue test. Vero cells grown in 96-well plates were incubated in control medium in the presence or absence of 0.1 μM 17-AAG and 0.5 μg / ml Artemisia essential oil at 37°C for 24 h. After washing, the AlamarBlue reagent was added to each sample and incubated for 6 h at 37°C before absorbance measurement at 540 nm. Cell growth, based on the detection of cell metabolic activity, was proportional to these value. In other set of experiments, trypomastigotes were incubated in control condition (DMSO), 0.1 μM 17-AAG and 0.5 μg / ml Artemisia essential oil for 30 min at 37°C. After that, parasites were placed on Vero cell monolayers for 24 h before washing and fixation.

17-AAG and Artemisia essential oil affected the growth and caused death on epimastigotes of T. rangeli
Next we studied the effect of the two potential trypanocidal treatments on the different stages of the T. rangeli life cycle. Initially, we analyzed the action of treatments on axenic epimastigote cultures, by incubating the parasites in Diamond medium alone (control) or supplemented with 0.1 µM 17-AAG and 0.5 µg / ml Artemisia essential oil for 10 days. We selected these concentrations from previously published works, which have shown the effect of these compounds on other trypanosomatids (Santos et al., 2014;Pellicer et al., 2018). The number of parasites counted at different times of incubation showed that epimastigotes increased exponentially in the control condition from day 0 to 10 (Fig. 2). No significant differences were observed in the 17-AAG and Artemisia essential oil conditions at the initial times of growth compared to controls. Interestingly, from the fifth day both 17-AAG and Artemisia essential oil curves decreased and reached to value 0 on day 10. These data suggest a trypanocidal action of both treatments from this day onwards.

17-AAG and Artemisia essential oil reduce the infective capacity of trypomastigotes of T. rangeli
Since those results were encouraging, we decided to test the effect of both treatments on the infective forms of T. rangeli. To control for a possible toxic effect of compounds on Vero cells, we subjected cells to the treatments for 24 h at 37°C and then analyzed cell viability using the AlamarBlue assay. Both 17-AAG and Artemisia essential oil did not affect cell viability (Fig. 3A). Next, we tested the effects of these compounds on trypomastigote-infected Vero cells. For such purpose, T. rangeli trypomastigotes were exposed to control medium (with DMSO), 0.1 μM 17-AAG or 0.5 μg / ml Artemisia essential oil for 30 min and then placed on Vero cell monolayers for 24 h in the same conditions (Fig. 3B). After fixation, we stained with the fluorescent DNA dye, Hoechst, as explained above, to evaluate the level of host cell infection. As shown in the images depicted in Figure 3C the amount of parasites in cells was lower under both treatments than in control cells., The percentage of infected cells was, in consequence, significantly reduced at these conditions compared to cells under control medium (Fig. 3D). We concluded that both drugs significantly reduced the infective capacity of trypomastigotes of T. rangeli, having 17-AAG a more marked effect.

17-AAG and Artemisia essential oil affect replication of amastigotes of T. rangeli long after infection
Next we analyzed the effect of treatments on amastigote replication. Vero cell monolayers were infected with trypomastigotes of T. rangeli (MOI = 10) for 24 h. Then they were washed to eliminate free trypomastigotes and incubated in control (DMSO), 0.1 μM 17-AAG and 0.5 μg / ml Artemisia essential oil conditions for a chase period of 24 h (Fig. 4A). After fixation, samples were subjected to an indirect immunofluorescence method to detect the parasites. Remarkably, we observed that samples treated with 17-AAG and Artemisia essential oil displayed a fewer number of amastigote-like forms than that in controls (Fig. 4B). Further quantification showed an important reduction in the number of amastigotes per cell as compared to controls, 17-AAG AND ARTEMisiA ESSENTIAL OIL AFFECT T. RAnGELi INFECTION AND REPLICATION 17 indicating that both treatments affect amastigote replication of T. rangeli (Fig. 4C).

Discussion
The main interest in the study of Trypanosoma rangeli, a trypanosomatid initially described by Tejera in 1920 (Borzone et al., 1950), is that it has the same geographical distribution, is frequently transmitted by the same vectors, and infects the same vertebrates as T. cruzi, the etiologic agent of Chagas disease. This explains the number of morphological, biochemical and molecular studies pursuing its differentiation from T. cruzi, which have been based in the morphology and DNA of kinetoplasts, the electron microscopy study of epimastigotes in many trypanosomatid species under standardized conditions (Mühlpfordt, 1975), the identification of carbohydrates as beta-D-galactose and alpha or beta-N-acetyl-D-galactosamine on the cell surface (Marinkelle et al., 1986;Chung et al., 2003;Bretting and Schottelius, 1978;González et al., 1996;de Miranda Santos and Pereira, 1984), and the isoenzyme patterns (Kreutzer and Sousa, 1981;Tibayrenc and Le Ray, 1984). The use of restriction enzymes that divide the mini circles of DNA of the kinetoplast in its variable region (Frasch et al., 1981) and the amplification of the mini-exon gene repeated in tandem (Murthy et al., 1992) or DNA fingerprints (Pires et al., 2008) are also some of the molecular methods that make possible the differentiation of T. cruzi from T. rangeli. Some metabolic differences between both species have also been observed (Avila et al., 1981;Nosei and Avila, 1985;Holguín et al., 1987), as well as the resistance of epimastigotes to lysis by serum (Schottelius, 1982;Marinkelle et al., 1986). Regarding infection and intracellular cycle in mammalian cells, this study showed that T. rangeli shows a behavior similar to T. cruzi. We observed that trypomastigotes of T. rangeli can infect cells, replicate intracellularly as amastigotelike forms and exit the cells as trypomastigotes. Although we have not studied the complete intracellular cycle displayed by T. rangeli, our observations suggest that this parasite can differentiate from trypomastigote to amastigote-like forms and viceversa. More experiments will be needed to elucidate the mechanisms implicated in these processes.
There is controversy about the course of infection of T. rangeli in vertebrate hosts. The first case of human infection was discovered in Brazil (de Lucena and Marques, 1954). In white male NMRI mice, a high (up to 7 times the original inoculum in the peak) and persistent parasitemia (for up to 2 weeks) have been observed (Urdaneta- Morales and Tejero, 1985). The parasites disappeared completely from the circulation after 20-25 days. Using a similar infection model, these and other authors have also observed numerous nests or intracellular pseudocysts containing amastigotes and trypomastigotes in the heart, liver, and spleen ( Scorza et al., 1986;Urdaneta-Morales and Tejero, 1985;Osorio et al., 1995;Eger-Mangrich et al., 2001). The above characteristics, as well as the location of the pseudocysts in tissues, are similar to those of T. cruzi. However, there are other works in which the histological examination could not detect any form of T. rangeli in several organs of mice (Tanoura et al., 1999). Our in vitro results tend to support the findings of the first group of authors, because of the intracellular formation of amastigote nests in cultured cells.
It is interesting to observe that, among the trypanosomes that can infect humans, T. cruzi seems to be pathogenic for vertebrates but not for invertebrates (Chagas, 1909), whereas on the contrary, T. rangeli seems to be pathogenic for invertebrates but not for vertebrates (Tejera, 1920). Moreover, it has been demonstrated that both trypanosomatid species can coexists in the host (Araújo et al., 2013) mixed infections and their consequences for the host's health and parasite transmission are still a poorly known phenomenon. The mini-exon multiplex PCR characterization detected the infection by T. rangeli and T. cruzi (TcI genotype and even this coinfection could be benefitial for them. New investigations are needed to clarify these aspects. Considering the similar intracellular cycle of T. rangeli as compared with that of T. cruzi, as well as the nonpathogenic profile of this parasite, we next focused our study to the analysis of the effect of two new treatments against T. rangeli that could be tested against T. cruzi in the future. We used 17-AAG, a repurposed drug that were fistly used in the therapy against cancer (Menden et al., 2018), and with good results in the treatment of Leishmania infections (Petersen et al., 2018;Santos et al., 2014;Petersen et al., 2012). Our data showed that 17-AAG was effective to reduce the infection of trypomastigotes on Vero cells and the replication of epimastigotes and amastigotes. Similar results were obtained with the Artemisia essential oil, although this extract could show toxic effects at large doses and after prolonged periods of treatment (Ribnicky et al., 2004) a common medicinal and culinary herb with centuries of use. Artemisia dracunculus is a close relative of the French or cooking tarragon and contains components common to many herbs that are routinely consumed without reported adverse effects. Since safety information of Artemisia dracunculus and its extract is limited to historical use, TARRALIN was examined in a series of toxicological studies. Complete Ames analysis did not reveal any mutagenic activity either with or without metabolic activation. TARRALIN was tested in an acute limit test at 5000 mg/kg with no signs of toxicity noted. In a 14 day repeated dose oral toxicity study, rats appeared to well tolerate 1000 mg/kg/day. Subsequently, TARRALIN was tested in an oral subchronic 90-day toxicity study (rat. This is why we are interested in testing the effect of the specific component, artemisinin, which is effective in the treatment of malaria and is not toxic (Desrosiers and Weathers, 2016).
We consider that our work makes a contribution in the field of therapies against trypanosomatid parasites. Future experiments will be conducted with T. cruzi to confirm the possible antichagasic action of these treatments. Furthermore, the comparative study of these therapies on both species could be useful to elucidate the mechanisms for the different pathogenicity of them in vertebrate and invertebrate hosts.