Laboratory of Insect Biochemistry and Physiology, Oswaldo Cruz Institute/Fiocruz
Eloi S. Garcia
Laboratory of Insect Biochemistry and Physiology, Oswaldo Cruz Institute/Fiocruz
In natural conditions, Trypanosoma cruzi infects more than 100 species of mammals of different orders. In nature, the parasite exists in different populations of vertebrate hosts, such as humans, wild or domestic animals, and of invertebrates, such as vector insects. T. cruzi has morphological and functional variations, alternating between phases of binary division and the non-replicating, infecting forms. Replicating forms include epimastigotes present in the digestive tract of the vector insect and the amastigotes observed inside mammal cells. Non-replicant infecting forms, called metacyclic trypomastigotes, can be found in the feces and urine of the vector insect, while trypomastigotes circulate in the blood of mammals.
During the phase of the cycle in the invertebrate host, T. cruzi differentiates into epimastigotes. Then, in the hindgut, these differentiate into metacyclic trypomastigotes (a process known as metacyclogenesis), which are eliminated through the stools and urine of the vector insect and can then infect the vertebrate host. The parasite cannot penetrate intact skin, and can only infect the host via mucosa or skin lesions. In mammals, parasites develop within the cells and are released into the bloodstream after the host’s cells break. Many factors can influence the development of T. cruzi in the vector insect. Some of them were first described by Carlos Chagas (1909) and Emanuel Dias (1934). However, the T. cruzi–vector species interaction has only been reviewed more recently. As the insect feeds, the trypomastigotes found in the blood of the infected vertebrate host are ingested by the insect. A few days after the insect has fed, the parasites turn into epimastigotes and spheromastigotes. Once the infection is established, the epimastigotes within the stomach of the vector insect begin dividing repeatedly through binary division, and can adhere to the perimicrovillar membranes of the intestinal cells. In large numbers, the epimastigotes attach themselves to the rectal cuticle, differentiate themselves into metacyclic trypomastigotes and now both forms, whether differentiated or not, can be eliminated through feces and urine (Figure.
The establishment of the infection by T. cruzi in the digestive tube of the vector insect is subject to and regulated by physiological and biochemical factors of the vector insect. Bio assays were used to evaluate a variety of factors that influence the development of the parasite, such as (i) ultrastructural alterations of the epithelial cells of the midgut, (ii) litic factors, (iii)digestive enzymes and (iv) hormonal development physiology of the insect, as recently reviewed by Garcia and collaborators.
After the ingestion of infected blood, in the anterior and posterior regions of the midgut (stomach and intestine, respectively), the parasites come across components that exist in these digestive compartments. These include hemolitic factors, peptides derived from the α D-globin chain, defensins, prophenoloxidase, NO and lectins, which can modulate the dynamic of the multiplication and differentiation of T. cruzi in the digestive tube of the vector insect, illustrating the complexity of the mechanisms involved in the interaction with T. cruzi .
Pereira and collaborators reported the presence of lectins in the digestive tube and hemolymph of R. prolixus, suggesting that these molecules are involved in the development of T. cruzi in the vector insect. Mello and collaborators demonstrated differences in the development of three strains of T. cruzi in the gut of R. prolixus and linked the infectivity of these parasites to the hability of the digestive tube extract to agglutinate the flagellates in vitro. Thus, lectins aglutinated clone Dm28c of T. cruzi , which presented the maximum level of infectivity, while, on the contrary, the Y strain of the parasite, not agglutinated, was lysed in the digestive tube of the vector insect.
Frainderaich and collaborators demonstrated that the metacyclogenesis of T. cruzi was induced in vitro by peptides derived from α-D-globin. Garcia and collaborators studied, in R. prolixus in vivo, the effects of hemoglobin and synthetic peptides corresponding to the fragmented regions of the α-D-globin sequence on the development and transformation of epimastigotes of T. cruzi into metacyclic trypomastigotes. This differentiation in the digestive tube of the insect occurred when hemoglobin and the synthetic peptides corresponding to residues 30-49 and 35-73 of α-D-globin were added tto the plasma diet. However, synthetic peptide 41-73 did not induce the differentiation of epimastigotes into metacyclic trypomastigotes, even in the presence of the first two peptides. Hemoglobin proved to be an important blood component for the development of parasites. These results identified an uncommon molecular mechanism that modulates the dynamics of the transformation from epimastigotes into metacyclic trypomastigotes in the digestive tube of the vector insect.
As for digestive enzymes, recent experiments showed that insects infected with T. cruzi had increased cathepsin d activity. However, feeding R. prolixus with SH-dependent proteinase inhibitor had no effect on the infection of the vector insect. Urisc-Bedoya and Lowerngerger revealed that the level of transcription of cathepsin B did not vary when comparing infected and non-infected vector insects.
One of the important processes of the physiological interaction of T. cruzi and its vector insect is parasite adherence to the surface of intestinal cells. Nogueira and collaborators, using video microscope analysis, showed that the epimastigote and trypomastigote forms of T. cruzi move in the direction of the surface of intestinal epithelial cells. However, while trypomastigote forms do not adhere to the epithelium, epimastigotes adhere to the stomach and to the gut. This recognition process apparently involves molecules of glycoinositol-phospholipids (GIPLs), found in abundance on the surface of epimastigotes. The attachment of purified GIPLs to the surface of hindgut cells visible through exposure to immunofluorescence indicates that these molecules are one of the components involved in the adhesion of T. cruzi to the insect’s gut, as reviewed by Nogueira and collaborators. The same group demonstrated that hydrophobic proteins located on the surface of epimastigotes bind themselves to the glycoproteins of intestinal perimicrovillar membranes. The inhibition of a gene involved in the synthesis of glycoproteins of T. cruzi resulted in the non-adherence of the parasite’s flagellum to the intestinal epithelium, diminishing the population of T. cruzi in the vector insect.
One approach to inhibit interactions between T. cruzi and the vector insect was the use of a transgenic symbiotic bacterium that attacked the parasite directly. Rhodococcus rhodnii, a symbiont of R. prolixus, can be genetically modified by inserting the gene of cecropin A, an antimicrobial peptide induced by the humoral immune response of Hyalophora cecropia, and which can destroy both Gram-positive and Gram-negative bacteria. This compound produced by the transformed symbiont drastically reduced the number of T. cruzi in the digestive tube of the vector insect and therefore has huge potential to reduce vector competence.
Recently, Azambuja and collaborators demonstrated that bacteria present in the digestive tube of R. prolixus can lyse erythrocytes and also reduce infection by T. cruzi. The authors isolated the bacterium and identified it as Serratia marcescensbiotype A1a, producer of the prodigiosin pigment. Apparently, the lyse of the parasite depends on mannose-dependent receptors. Investigation of the bacteria present in the digestive tract of the vector insect may be able to identify tools to block the development of the parasite in the vector insect.
Interesting results of the T. cruzi–vector insect interaction were obtained from investigations using azadirachtin, an insect development inhibitor obtained from the plant Azadirachta indica, and which strongly interferes with the insect’s neuroendocrine regulation. This drug, administered orally before, during or even after infection by T. cruzi, not only interfers with the development of the insect, but also with the establishing of a parasite infection in the digestive tube of different triatomine species.
Parasites in their epimastigote form did not adhere to the epithelium of the insect’s midgut when larvae of R. prolixus were beheaded or treated with azadirachtin. Treating the insect with ecdione and implanting normal heads into insects treated with azadirachtin reverted the adhesion process and the development of the insects, as reviewed by Garcia and collaborators. The insects that were given the drug or were decapitated showed a disorganization of the perimicrovillar membranes of intestinal epithelial cells, indicating that the pathways of the prothoracicotropic hormone-stimulator of ecdisone synthesis is involved in the development process of T. cruzi in its vector insect.
Wanderley de Souza and Emile S. Barrias
Hertha Meyer Cellular Ultrastructure Laboratory, Carlos Chagas Filho Biophysics Institute, Federal University of Rio de Janeiro
The cycle in the vertebrate host may occur in different mammal species. Humans are the most relevant definitive hosts. There is currently evidence that dogs, cats, commensal rodents and domesticated Guinea pigs are capable of maintaining T. cruzi in the absence of any other host species. They play crucial roles as amplifiers of hosts and sources of T. cruzi in many peridomestic transmission cycles, covering a wide diversity of ecoregions, ecotopes and triatomine species. No other domestic animal plays this same role. Dogs have all the desirable attributes of natural sentinels and sometimes can be an entrance point for strains of wild parasites (Gurtler and Cardinal, 2015). The first contact between the vertebrate host and T. cruzi in a vectorial infection occurs with the metacyclic trypomastigot that are eliminated by the vector insect and come into contact with the injured mucosas or skin areas of these hosts. These forms are highly infective and can invade the first cell types they come across, whether they are macrophages, fibroblasts or epithelial cells, among others. As they invade these cells using the mechanisms described in the items below, intracellular proliferation occurs and trypomastigote forms are released, together with some intermediary and amastigote forms (the latter in a smaller amount) into the intercellular space. These forms can invade new cells located at the infection site, but they can also reach the bloodstream and therefore all of the host’s tissues, where they can invade the most different cell types. One should not expect any homogeneous behavior from Trypanosoma cruzi. In fact, the kinetics of the process described above, as well as which tissues are most infected, will vary according to the strain of T. cruzi and to the infected animal. In some cases, infection is quick. In others, it can only be detected long after the inicial infection. This is because the population of T. cruzi that occurs naturally consists of a highly heterogeneous group of strains with marked intra-species variations and with wide-encompassing biological characteristics, such as distinct morphology, growth rate, parasitemia curves, virulence, sensitivity to drugs, antigenic profile, differentiation (metacyclogenesis) and tissue tropism. This variability can explain the wide pathogenesis observed in infections by T. cruzi (Buscaglia and DiNoia, 2003).
The observation of trypomastigote forms in the blood of infected animals showed that not all are identical, which indicates the existance of dimorphism, or even polymorphism, in T. cruzi, characterized by the presence of stumpy, slender and intermediary forms, as was described for African trypanosomes. There have been many different interpretations for this fact, including it being an indication of sexual dimorphism. Some strains have predominantly slender shapes, such as the Y strain. Others, such as the CL strain, have mostly stumpy forms. Brener and collaborators carried out many studies to clarify the possible differences between the two forms. For instance, they noticed that in some populations there was a prevalence of slender forms throughout the course of the experimental infection in mice. In some strains, as the infection proceeded, stumpy shapes began to be more predominant. A set of observations led this group to consider that the slender forms disappear rapidly from the bloodstream to continue on in their intracellular cycle, while stumpy shapes remain in the bloodstream. These forms would apparently be less infective and more adaptive to development within the vector. A significant number of studies was carried out comparing trypomastigotes of Y and CL strains as for in vivo and in vitro infectivity, in vitro differentiation capacity, susceptibility to lyse mediated by complement, and presence of surface antibodies. Recently, the transcriptome analysis of an infective strain (CL Brener) and a non-infectious strain in in vivo models (CL-14) showed a big difference in the genic expression between trypomastigotes and intracellular amastigotes. This is related to the parasite’s adaptation to different environments while they are infecting mammal cells, including changes in energy sources, oxidative stress response, cell cycle control, and cellular surface components. It is important to mention that the remodelling of gene expression of CL-14 may be caused, at least in part, by a reduced or delayed expression of genes that codify surface proteins associated to the transition from amastigotes to trypomastigotes, an essential step to establish infection in the mammal host, explaining the non-infectivity of the strain (Bellew et al, 2017).
Técia Ulisses de Carvalho and Emile S. Barrias
Biophysics Program, Carlos Chagas Filho Biophysics Institute, Federal University of Rio de Janeiro
Trypanosoma cruzi infects a great variety of nucleated cells, both in vitro and in vivo, although it has tropism for certain cell types, such as muscle cells and macrophages. These differences in cellular tropism are related to the genetic diversity that exists between different strains of T. cruzi. (Andrade et al, 1999). As one of the most important segments of the life cycle of the T. cruzi is the intracellular cycle, eukaryotic cell culture has been very commonly used in scientific research, in an attempt to understand the molecular mechanisms of the process of recognizing, signalling, and invasion (and/or phagocytosis) of trypomastigote and amastigote forms of T. cruzi. Great contributions in this field were made by the pioneer research of Dr. Hertha Meyer at the Carlos Chagas Filho Biophysics Institute of the Federal University of Rio de Janeiro, in Brazil. Today different cell types are used by research groups in an attempt to reproduce the intracellular cycle in vertebrate hosts. Among the most commonly used cell types are: lineages of epithelial cells, as the parasite’s first contact takes place with cells of this type; macrophages (primary human culture or murine cells, from established lineages), as these cells are the first ones of the immune system with which the parasite comes into contact; and cardiomyocytes (primary culture or established lineages), as these parasites have tropism for these cell types as they establish infection.
In general, cells are cultured in a aseptyc (sterile) environment, in Petri dishes, bottles or slides (sterile) and made to interact with T. cruzi. The cell growth support used (bottles, dishes or slides) will depend on the type of experiment to be carried out. Cell culture uses culture media that are specific for the cell type used, supplemented with bovine fetal serum. This ensemble (culture medium and bovine fetal serum) is responsible for providing amino acids, vitamins, minerals and growth factors that are essential for cellular maintenance and multiplication. After the cell culture (host cell), the interaction process with infective forms of T. cruzi can be done for short periods (a few minutes or hours) to understand the parasite’s early recognition and invasion processes, or longer periods (days) for studies of the parasite’s intracellular behavior. Trypomastigotes can be obtained from cell cultures (culture trypomastigotes, in which cells are previously infected with trypomastigotes and the pool of parasites is collected after breaking the cells), from the blood of previously infected animals (blood trypomastigotes) or through a process known as metacyclogenesis (differentiation of epimastigotes into trypomastigotes, which can be achieved in the laboratory, under specific conditions). Cultures are maintained in the stove, at 37º C, with an atmosphere containing 5% of CO2. These cells, recently infected or that have been infected for a long time, can be analyzed in different manners: (i) fixated with Bouin and stained with Giemsa; (ii) video microscopy of live cells (using phase contrast or interference microscopy); (iii)through immunofluorescence; (iv) via scanner and/or transmission electron microscopy. The use of cell culture makes it possible to reproduce, in vitro, the life cycle of the parasite. In the cycle, trypomastigotes interact with the cell through a relatively complex entry process that involves different receptors/binders in the two cells involved. After cellular recognition, different signalling pathways are triggered and culminate in the internalization of the parasite by the host cell, forming a structure known as parasitophorous vacuole (which contains the parasite). This vacuole then merges with acid organelles called lysosomes. Lysosomes are responsible not only for acidifying the parasitophorous vacuole, but also for donating membrane for the formation of the vacuole. In this acid environment, trypomastigote forms secrete enzymes that will act on the membrane of the phagolysosome (or parasitophorous vacuole), creating small pores that will result in the degradation of this membrane. During this process, the trypomastigote form begins a differentiation process into an amastigote form, being then released into the cytoplasm of the host cell, where it multiplies several times (through binary asexual division). When the cytoplasm of the host cell becomes full of amastigote forms, the new differentiation begins, now from amastigote to trypomastigote. The trypomastigote form is very mobile and secretes an enzyme that will act on the plasma membrane of the host cell. These two factors lead to the rupture of this cell, releasing many trypomastigote forms into the extracellular environment. Although the trypomastigote form is acknowledged as the main infective form of T. cruzi, studies have been increasingly often demonstrating that amastigotes also have the ability to infect nucleated host cells. As a consequence, several groups have been using the cell culture system described above for infection studies using amastigotes. Infection is therefore made with this form, instead of with trypomastigotes. In general, there is also a recognition of amastigotes with host cells, trigger of signalling pathways, formation of parasitophorous vacuole with lysosome fusion, degradation of vacuole membrane, intracellular multiplication, and differentiation into trypomastigotes before cellular rupture. In this case, the only phase of the cellular cycle that fails to take place is the differentiation from trypomastigotes into amastigotes (which becomes unnecessary). The amastigotes used for the infection can come from amastigotes released during natural cellular rupture (as not all amastigotes finish their differentiation before cellular rupture), from cellular rupture before the differentiation into trypomastigotes (experimental mechanical and/or chemical methods), or through a process known as amastigogenesis.
Wanderley de Souza and Juliana Vidal
Carlos Chagas Filho Biophysics Institute, Federal University of Rio de Janeiro
The epimastigote form of Trypanosoma cruzi can easily be cultivated in axenic media. This has made it possible to carry out several studies that contribute to the better ultrastructural, biochemical and physiological understanding of this form. Recent studies show that we can consider two types of epimastigotes. One corresponds to those maintained after many passages in culture. A second type is obtained directly from trypomastigote forms, and are infective. It was later observed that depending on culture conditions, a certain part of epimastigotes turns into trypomastigotes. This fact also facilitated many studies of this infective form.
Initially, epimastigote cultures used biphasic media, of the blood agar type, with a liquid phase containing bovine fetal serum. T. cruzi and other trypanosomatids grow very well in this type of medium. Biphasic media are often used to isolate new strains and new recently isolated trypanosomatids. In spite of that, biphasic media are inadequate for biochemical studies due to the presence of countless macro molecules. In addition, parasites frequently grow in an aggregated fashion in these media, making cellular growth quantitative studies more difficult.
The development of monophasic media, in which epimastigote forms grow freely, has opened the possibility of quantitative studies, including with the use of electronic counters to evaluate cellular growth. This type of culture is suitable for studying the effect of drugs on cellular growth, in the process of cellular transformation etc. There are many media used on a regular basis to grow epimastigote forms of T. cruzi. Worthy of mention among these are the LIT and Warren media. The LIT medium (“liver infusion tryptose”), currently the most commonly used, was initially idealized by Yager and made popular by Camargo, who carried out basic studies on the growth and differentiation of T. cruzi. Significant breakthroughs occurred in the cultivation of non-pathogenic trypanosomatids, up to the development of chemically defined media, which is important for the study of certain metabolic pathways, to get to know the nutritional demands of these protozoa, among others. Another medium that is commonly used is the Roitman defined medium. In general, this medium contains glucose, aminoacids, vitamins, a source of purin, and hemin. In the case of digenetic trypanosomatides, various attempts were made to simplify the medium, eliminating macro molecules until Cross and Manning obtained a medium that made it possible to grow T. brucei. Later, many authors used this medium successfully, with small variations, to grow T. cruzi. However, growth is relatively small, which has limited its use. New studies are certainly necessary in this area.
Even from the first cultures using biphasic media, such as NNN (Neal, Novy, Nicolle), it was observed that trypomastigote forms appeared in the stationary phase of the culture, and that its percentage increased as the culture aged. These trypomastigotes were also named metacyclic trypomastigotes, as they originate from the transformation from epimastigote forms in media that mimic the environment found within the digestive tract of the vector insect. This transformation process was named metacyclogenesis. Camargo was the first to systematically analyze the transformation process of epimastigotes into tryptomastigotes using the LIT medium. It was shown that after the fourth day, the culture reaches its stationary phase, after which the transformation process begins.
Metacyclogenesis can be mimicked by stimulating some of the factors involved in the differentiation process. The factors that have best been defined so far are depletion or lack of certain nutrients, and pH. The metabolism of T. cruzi is organized based on rapid glucose consumption, but glucose is not completely metabolized in this process. As a consequence of the depletion of carbohydrates, parasites meet their energetic needs by consuming amino acids. Based on this, the protocol most commonly used to make metacyclogenesis in vitro for T. cruzi is the one developed by Samuel Goldenberg and collaborators. This protocol uses clone Dm28c maintained as epimastigote in the LIT medium, and is then moved to a poor medium that partially reproduces the composition of triatomine urine, therefore named TAU (Triatomine Artificial Urine). When this system is used, initially it is possible to observe an adhesion of the epimastigote forms to the substrate and, later, their detachment and transformation into trypomastigotes, mimicking what takes place in the invertebrate host. Metacyclogenesis rates of 90% have been described with this medium, but only for clone Dm28c. However, to maintain reasonable rates of metacyclogenesis the strain must be periodically “recycled” by passing through the vector insect. On the other hand, this system does not offer satisfactory results for other strains of T. cruzi.The metacyclogenesis process involves morphological, genetic and metabolic alterations in the parasite. In addition, it promotes alterations in cellular structures, such as the DNA arrangement of the kinetoplast and in the shape of the nucleus (Figure 1). Physiological changes, such as infectivity, often precede the full morphological transformation. As a consequence, what we can consider intermediary forms appear, often with typical characteristics of the trypomastigote form. Some of the modifications that occur on the surface level can be used to separate the epimastigote forms from the trypomastigotes. Epimastigote forms are lysed by the complement system (in general, fresh Guinea pig serum is used), while trypomastigotes are more resistant. The intact forms, the trypomastigotes, can easily be separated from cellular remains of the lysed forms through gradient centrifugation. Another property that changes during the epimastigote-trypomastigote transformation is the surface load, allowing for the separation of these two forms using ionic exchanges with DEAE cellulose columns. Today it is also possible to revert the metacylclogenesis process by innoculating trypomastigote forms obtained as described above in LIT medium. Using this methodology, a recent study showed that recently differentiated epimastigote forms (obtained from culture trypomastigotes or metacyclical trypomastigotes) are capable of infecting mammal cells in vitro.
The amastigote form of T. cruzi can also be obtained from axenic culture. Right from the very first observations of T. cruzi in culture media it was observed that the incubation of blood trypomastigotes in culture at room temperature led to the formation of rounded forms. Very often, as shown by Brener and Chiari, rounded masses formed which were different to dissociate. Later, Pan developed some media in which amastigotes could be cultivated at 35.5 ºC. Rondinelli and collaborators showed, using the CL strain, that when epimastigotes growing in the traditional LIT medium are transferred to a slightly modified medium, which Chiari called M16, they turn into trypomastigotes. Treating this population wish fresh human serum and then moving it to the LIT medium gives origin to round-shaped aggregates with characteristics of amastigote or spheromastigote forms. These forms are resistant to lyse by the complement system. When these forms are maintained in a medium containing high concentration of serum (55%) at 37 ºC, trypomastigote forms appear, morphologically similar to blood forms. If the cultures are aged or kept at the same temperature, but in the presence of a low concentration of serum (10%), epimastigote forms are observed. With this system it is possible to obtain a significant number of the three evolutionary forms (108 cells/ml) for biochemistry studies.
The axenic cultivation of epimastigote forms, as well as the media that make it possible to differentiate trypomastigotes and amastigotes, have made it possible to carry out several studies that add relevant information on the cellular biology of this human parasitic protozoan. These data will help unravel essential mechanisms for the biology of this protozoan and may provide support to research lines that aim at elaborating strategies for efficient therapeutic interventions against T. cruzi.