Trypanosoma cruzi: Morphological and Microtubular Changes Related to Epimastigote Differentiation under Hyperosmotic Stress

Trypanosoma cruzi: Morphological and Microtubular Changes Related to Epimastigote Differentiation under Hyperosmotic Stress Alba Marina Gimenez1* and Estela E. Machado1 1Department of Molecular Biology, Faculty of Exact Sciences, Physical-Chemical and Natural Sciences, National University of Río Cuarto, Río Cuarto (X5800) Córdoba, Argentina SOJ Microbiology & Infectious Diseases Open Access Research Article


Introduction
Infection with the protozoan parasite Trypanosoma cruzi causes Chagas disease, a neglected disease that affects about 6 to 7 million people worldwide, mostly in Latin America (WHO, 2016).This parasite alternates between mammalian hosts and triatomine hematophagous insects, which are responsible for disease transmission.The life cycle of T. cruzi comprises three main forms: amastigotes, epimastigotes, and trypomastigotes, and several intermediate stages between them [1].Due to its importance in the disease transmission, the differentiation from replicative epimastigote to the infective stage of the parasite, trypomastigote (metacyclogenesis process) has been the focus of numerous studies.In many of them the presence

Organisms and growth conditions
Trypanosoma cruzi Tulahuen strain was used.Epimastigote forms were grown at 28ºC in modified Warren medium [9], supplemented with 10% Fetal Bovine Serum (FBS), as described previously [10].Cells in logarithmic growth phase (day 6) were harvested by centrifugation at 1,000 g for 10 min, and washed twice with 25 mM Tris-HCl, pH 7.35, 1.2 mM MgSO 4 , 2.6 mM CaCl 2 , 4.8 mM KCl, 120 mM NaCl, and 100 mM glucose [Krebs-Ringer-Tris (KRT) buffer].Intermediate Forms (IF) were obtained as described previously [8].Briefly, epimastigote forms were grown in the same culture medium but in the presence of 0.5 M NaCl or 1 M mannitol, and the cells were harvested at day 13 by centrifugation at 1,000 g for 10 min and washed twice with KRT buffer.

Morphological studies
Cells were grown as described above and fresh parasite samples were taken and quantified using a Neubauer's hemocytometric chamber.Parasites were fixed with absolute methanol, stained with 10% Giemsa in 0.1 M sodium phosphate buffer, pH 7.2, and observed in a Zeiss Axiolab Microscope with a 100x objective.Parasites were classified as epimastigotes, trypomastigotes, or intermediate forms based on cell shape, size, and Giemsa staining, as described previously [8].

Preparation of membranes
Washed epimastigotes and IF were resuspended in five volumes of 50 mM HEPES (pH 7.4) containing 0.25 M sucrose, 5 mM KCl, 1 mM EDTA, and 4 μg/ ml leupeptin.The suspension was frozen at -180ºC in liquid nitrogen and thawed; this freeze/ thaw cycle was performed three times.Membrane fractions were prepared as described previously [11].Briefly, the homogenate was centrifuged at 100 g for 15 min to remove cell debris, and supernatant was centrifuged at 1,000 g for 15 min and at 105,000 g for 60 min.Pellets from the two centrifugations were washed, re suspended with 50 mM HEPES (pH 7.4), and used as a source of membranes.The protein content of membrane samples was determined by the Bradford method [12], with bovine serum albumin as the standard.

Western blot
50-80 μg of protein from membranes of T. cruzi epimastigotes and IF were solved in 8-12% (w/ v) SDS-polyacrylamide gel electrophoresis and electro transferred to Hybond ECL membranes.The membranes were blocked with 5% (w/ v) nonfat milk suspension in PBS-Tween for 2 h.After 1 h incubation with 1:1,000 dilutions of primary antibodies, detection was carried out by incubating with a 1:2,000 dilution of anti-mouse IgG HRP-conjugated.The latter was developed with the ECL Western Blotting detection system.

Immuno Fluorescence Assay (IFA)
Epimastigotes and IF were harvested and washed twice with KRT buffer as described above.Parasites were quantified using a Neubauer's hemocytometric chamber and aliquots containing 1 x 10 4 parasites were fixed in slides with methanol at -20ºC for 6 min, washed with PBS and incubated with PBS-2% albumin (blocking solution) for 60 min at room temperature.Parasites were then incubated with 1:1,000 dilutions of primary antibodies in PBS for 60 min, washed three times with PBS for 5 min and then incubated for 60 min with 1:2,000 dilutions of secondary antibody FITC-conjugated.Negative controls were incubated just with secondary antibody after blocking solution.The cells were observed in a Zeiss Axiolab Standard Fluorescence Microscope.

Hydrophobic adhesion assay
The assay was performed as described by Tyler and Engman [13] with some modifications.Microscope slides precoated with amino-alkyl silane were used as hydrophobic surface.Epimastigotes and IF were harvested and washed twice with KRT buffer as described above.Parasites were quantified using a Neubauer's hemocytometric chamber and aliquots containing 1 x 10 4 parasites were spread evenly over a marked 1.5 cm 2 area of the slide.The parasites were allowed to adhere for 10 min in a humid chamber at room temperature.The slide was then washed three times in KRT for 5 min per wash, fixed in methanol at -20°C for 5 min, rehydrated in KRT for 5 min.The slide was stained with 10% Giemsa in 0.1 M sodium phosphate buffer, pH 7.2 and then mounted and examined in a Zeiss Axiolab Microscope.Three independent experiments were carried out in triplicate, and attached parasites were counted in at least 10 fields by slide.

Graphical and statistical analyses
The results are shown as mean ± standard error of the mean (SEM) for two or more independent experiments.Images were obtained and analyzed using Axio Vision 4.8 software.Quantitation and comparison of protein bands was performed using Scion Image software (an arbitrary value of 10 was assigned to the pixels/ mm 2 corresponding to control samples).Graphical and statistical analysis of the data was performed using Sigma Plot 11.0 software.

Morphology of IF
T. cruzi Intermediate Forms (IF) were obtained by growing of epimastigote forms in hyperosmolar media, as described previously [8].The morphological study of these parasites was first carried out by observation (100x objectives) of preparations of Giemsa-stained samples.As shown in Figure 1, IF obtained by addition of NaCl (Figure 1B) or mannitol (Figure 1C) were characterized by having their kinetoplasts adjacent to the nuclei and an increase in the length of the flagellum, when compared to epimastigote forms (Figure 1A) In addition, IF possess either slender (with NaCl addition) or swollen (with mannitol addition) cellular bodies.

Trypanosoma cruzi: Morphological and Microtubular Changes Related to Epimastigote Differentiation under Hyperosmotic Stress
Copyright: © 2016 Gimenez and Machado the proportion of IF increased steadily, and reaching 85-90% of parasite forms at day 13, whereas in the control culture epimastigotes remained the predominant form (65%).The IF generated in hyperosmotic media showed normal viability, a delay in the lag period of the growth curve, increased generation time and a reduction in the maximal growth to ~ 50% of the control value (Figure 1D).A low percentage (2-3%) of trypomastigote forms were obtained at day 13 of growth in hyperosmolar media, similarly to observed when epimastigotes of Y Strain and Berenice Strain were incubated in saline [5].

Binding to a hydrophobic substrate
In the natural life cycle of T. cruzi, epimastigotes interact through its flagellum with triatome rectal walls, where differentiation process occurs.Such interaction, between flagellar proteins and hydrophobic substrates present in triatome rectum, would be crucial for the differentiation to trypomastigotes.Therefore, the IF's ability to interact with hydrophobic substrates was analyzed.
As hydrophobic surface, slides pre-coated with silane were used, as described by Tyler and Engman [13].Epimastigotes and IF were allowed to interact to hydrophobic surface for 10 min and then the slides were washed and Giemsa-stained for better viewing.The samples obtained were observed by optical microscopy (Figure 2).
A total of 60 fields per condition were counted, obtaining an average of 5 ± 3 epimastigotes and 14 ± 3.6 IF adhered per field in a 40x objective (Table 1).
In order to induce complete differentiation, parasites were allowed to adhere to hydrophobic surfaces during 10-60 min, and then were transferred to modified Grace´s medium as previously described [14].In these experimental conditions, no significant increase of trypomastigote forms was determined (not shown).

Flagellar elongation measurement
The flagellar elongation is considered an adaptation of this parasite on adverse conditions, such as nutrient deprivation [13].As shown in the right panels of Figure 2, IF attached to hydrophobic surfaces had elongated flagella, when compared to epimastigotes.Quantitation of this difference (P < 0.05) is summarized in Table 1 (n = 150 epimastigotes and 250 IF).
In summary, differentiation from epimastigotes to IF comprises flagellar elongation that is correlated with an increase in the parasite's ability to interact with hydrophobic substrates.

Microtubule organization
Morphological changes observed in the transition from epimastigote to intermediate form (movement of kinetoplast from pre-nuclear to adjacent position and elongation of the flagellum) should be necessarily accompanied by changes in the

Trypanosoma cruzi: Morphological and Microtubular Changes Related to Epimastigote Differentiation under Hyperosmotic Stress
Copyright: © 2016 Gimenez and Machado parasite microtubular network and probably by alterations in tubulin posttranslational modifications [15].
In order to study microtubular reorganization during the transition process from epimastigote to IF, the relative content of different tubulin isotypes in parasite subpellicular membranes was analyzed by Western blot.The recognition of tyrosinated tubulin (Tyr-tubulin) is indicative of microtubule dynamics, being greater the greater the presence of this subtype of tubulin.In an opposite way, stable microtubules would be enriched in acetylated tubulin [15].
Quantitative analyses were performed using Scion Image software, which determines, in pixels / mm 2 , the intensity of the protein bands recognized by antibodies.For graphic plots, an arbitrary value of 10 was assigned to the number of pixels / mm 2 corresponding to the protein bands recognized in epimastigotes membranes.
The presence of tyrosinated tubulin (Tyr-tubulin) was analyzed in both stages, obtaining an increment of 60% in IF membranes, when compared with epimastigotes (Figure 3A).The presence of acetylated tubulin was also compared in both stages.This posttranslational modification occurs in a less extent in IF membranes.There were not significative differences between epimastigotes and IF membranes when total-tubulin was analyzed (Figure 3A).When analyzed by immunofluorescence assay, FITC-fluorescence (anti-acetylated tubulin) was also lower in IF, mainly in the perinuclear area (Figure 3B).As no significative differences were determined by using NaCl or Mannitol for obtaining IF, only the firsts are shown.

Microtubule stability
Taken together, our results indicate that during the differentiation process from epimastigotes to IF a cytoskeletal rearrangement occurs, which makes microtubules from IF less stable than those from epimastigotes.In order to confirm or refute the cytoskeleton lower stability of IF a microtubule depolymerizing agent, Nocodazole, was used.during 15, 30 or 60 minutes, and subsequently membrane fractions from each stage were obtained.In these fractions, the relative amount of α-tubulin was determined by western blot.

Epimastigotes and IF were incubated with 50 mM Nocodazole
As shown in Figure 4, microtubules from IF disassembled more quickly, suggesting less stability than those from epimastigotes.
In the quantification by Scion Image program, an arbitrary value of 10 was assigned to the number of pixels / mm 2 corresponding to the protein bands obtained using both stages, un-incubated with Nocodazole (time: 0).
When epimastigotes and IF were incubated with 10-100 µM Paclitaxel (stabilizing microtubule agent), no significative differences in the relative amount of α-tubulin in membrane fractions were observed (data not shown).

Discussion
The different developmental stages and differentiation processes in the life cycle of T. cruzi have been the focus of numerous studies, most of them emphasizing the metacyclogenesis process, the transition from replicative epimastigotes to infective trypomastigotes.Several factors have been described as involved in triggering this differentiation, such as availability of nutrients, intracellular cAMP levels and amino Differentiation under Hyperosmotic Stress.SOJ Microbiol Infect Dis 4(4): 1-6.

Trypanosoma cruzi: Morphological and Microtubular Changes Related to Epimastigote Differentiation under Hyperosmotic Stress
Copyright: © 2016 Gimenez and Machado acid metabolism (reviewed by Goldenberg and Rodrigues Ávila, [16]) and, more recently, redox status [17].Although it is known that metacyclogenesis can be induced in vitro by incubating epimastigotes in TAU3AAG, a chemically defined medium that mimics the urine of triatomine bugs [18], the exact events that induce this process are unknown so far.
This parasite differentiation takes place in the rectum of triatomine bugs, where epimastigotes must face an environment of high osmolarity.In this and in previous work [8] we showed that hyperosmotic conditions in the culture medium led parasites to adopt an intermediate form between epimastigote and trypomastigote, which is characterized by possessing nucleusadjacent kinetoplasts and increased flagellar length.Similarly, other authors reported that epimastigotes incubated in saline solution did not complete the differentiation process, which was interrupted in the same intermediate stage [5].Parasite IF were also described in cultures under the same conditions in which metacyclogenesis is induced, such as nutrient limitation [13], triatomine blood ingestion [19], presence of triatomine urine [5], modification of epimastigotes culture medium [6] and epimastigotes culture aging [7].Disregarding the method of IF generation, they share the morphological characteristics here described; i.e. elongated flagella, altered cellular body, and nucleus-adjacent kinetoplasts.
Both flagellar elongation and parasite's ability to interact with hydrophobic substrates have been considered to be a pre-adaptation of epimastigotes for differentiation to trypomastigotes [13].It is known that the interaction of flagella with an appropriate hydrophobic substrate is a necessary event but not sufficient for differentiation [20,21].It was suggested that the flagellar elongation occurs when epimastigotes bind to substrates similar to those existing in the triatomine rectal walls [22], and other authors also suggested that the cause of the increased hydrophobic interaction would be the limitation on glucose availability, either due to a reduction in the level of glycosylated proteins in the parasite surface or to changes in the expression of specific genes [13].
However, our results support the hypothesis of flagellar elongation as determinant factor for parasite's increased capacity of hydrophobic interaction, since IF generated by growth in high osmolarity (without changes in glucose availability) were able to adhere to hydrophobic substrates in a greater extent than epimastigotes.Several early events triggered by high osmolarity stress were determined in epimastigotes [8,11,14]; however their relationship with the formation of IF remains to be established.
Similarly to T. cruzi, T. brucei pre-metacyclic differentiaton involves the movement of kinetoplast, flagellar elongation and changes in the microtubule network, evidenced by tubulin posttranslational modifications [15,23].The increase in the relative amount of tyrosinated tubulin in T. cruzi IF would indicate a reorganization of cytoskeleton network.This correlation was already described in T. brucei both during flagellum formation and cell division, where new microtubules intermix with pre-existing in the subpelicular cortex [24].In addition, the reduced presence of acetylated tubulin also it involves cytoskeletal reorganization during the transition from epimastigote to IF.This is based on the general principle governing the trypanosomatids cytoskeleton: stable microtubules would be enriched in acetylated and detyrosinated tubulin [15].This is also consistent with the fact of microtubules from IF being less resistant to a destabilizing agent as Nocodazole.Thus, we suggest that this new formation of microtubules in IF would be necessary to keep the parasite in the transitional stage, with its flagellum extended, and thus promoting the interaction with hydrophobic substrates that occurs during differentiation in the rectum of the insect vector.

Figure 1 :
Figure 1: Trypanosoma cruzi developmental stages in axenic cul ture.Parasites were fixed on slides with methanol and Giemsa-stained.Slides were observed by optical microscopy with a 100x objective.Representative images from 3 independent experiments are shown.N: nucleus.K: kinetoplast.(A) Epimastigotes (B) Intermediate forms obtained by growth with NaCl (C) Intermediate forms obtained by growth with mannitol (D) Parasite growth was monitored during 13 days

Figure 2 :
Figure 2: Hydrophobic interaction assay.Parasites were harvested, washed and resuspended in KRT buffer as de scribed in M&M.1x10 4 parasites were spread over a marked 1.5 cm 2 area of microscope slides pre-coated with amino-alkyl silane.Interaction was al lowed during 10 min and slides were washed, fixed with methanol and Giemsa-stained.Slides were examined by DIC microscopy (400x and 1000x).Rigth pannels: magnification of marked areas.(A) Epimastigotes (B) Intermediate forms obtained by growth with NaCl (C) Intermediate forms obtained by growth with mannitol

Figure 3 :
Figure 3: Tubulin posttranslational modifications in T. cruzi epi matigotes and IF. A. Membrane fractions from epimastigotes (E) and Intermediate forms obtained by growth with NaCl (IF-N) or mannitol (IF-M) were analyzed by Western blot with monoclonal antibodies anti-∝-tubulin, anti-acetyltubulin and anti-Tyr-tubulin.Quantitative analyses were performed using Scion Image program.Representative results from two independent experiments are shown.(*) significative differences, P < 0.05; ns = not significative differences.B. Epimastigotes and Intermediate forms obtained by growth with NaCl (IF-NaCl) were fixed on slides and incubated with anti-acetyl-tubulin primary antibody and FITC-conjugated secondary antibody as described in M&M.Negative control was performed by incubating parasites with secondary antibody only.Representative images from two independent experiments are shown.

Figure 4 :
Figure 4: Comparative stability of microtubules from epimatigotes and IF.Membrane fractions from epimastigotes and Intermediate forms incubated with Nocodazole during 15, 30 or 60 minutes were analyzed by Western blot with monoclonal antibody anti-α-tubulin.Quantification was performed with Scion Image program.An arbitrary value of 10 was assigned to the number of pixels / mm 2 corresponding to the protein bands obtained using both stages, un-incubated with Nocodazole (time: 0).

Table 1 :
Morphological differences between T. cruzi epimastigotes, intermediate forms and trypomastigotes.Epimastigotes and IF obtained by growing in hyperosmotic media were morphologically analyzed.N= 60 fields/condition.Mean ± SD of three independent experiments are shown.