Signaling pathways involved in environmental sensing in Trypanosoma cruzi

Abstract

Trypanosoma cruzi is a unicellular parasite and the etiologic agent of Chagas disease. The parasite has a digenetic life cycle alternating between mammalian and insect hosts, where it faces a variety of environmental conditions to which it must adapt in order to survive. The adaptation to these changes is mediated by signaling pathways that coordinate the cellular responses to the new environmental settings. Major environmental changes include temperature, nutrient availability, ionic composition, pH, osmolarity, oxidative stress, contact with host cells and tissues, host immune response, and intracellular life. Some of the signaling pathways and second messengers potentially involved in the response to these changes have been elucidated in recent years and will be the subject of this review.

Abbreviated Summary.

Trypanosoma cruzi, the etiologic agent of Chagas disease, faces a variety of environmental conditions to which it adapts. We summarize here the signaling pathways and second messengers involved in the response to these changes.

1. INTRODUCTION

Trypanosoma cruzi is the etiologic agent of Chagas disease, a life-threatening sickness affecting 6 to 7 million people in the Americas, where this trypanosomiasis is endemic. Natural transmission occurs when humans come into contact with feces and/or urine of infected blood-sucking triatomine bugs (vector-borne transmission). This slow-progressing infection is considered a major cause of heart disease, although most affected individuals remain undiagnosed and untreated. Understanding T. cruzi biology is a key component to develop alternative strategies to diagnose and treat Chagas disease patients. This parasite has a complex life cycle including four major developmental stages, two of which are replicative, one in the insect vector (epimastigote) and one in the mammalian host (amastigote) and two are non-replicative forms: metacyclic trypomastigote in the insect vector and cell-derived trypomastigote in the mammalian host. Each one of these stages is exposed to very specific microenvironments. The epimastigote replicates in the vector midgut where nutrients from the ingested bloodmeal are still available and differentiates into the infective metacyclic trypomastigote once its environment becomes depleted of nutrients and the parasite adheres to the hindgut epithelium (a process called metacyclogenesis). The metacyclic survives in a poor environment with very harsh conditions, like low pH, and high osmolarity, and suddenly changes its environment when it invades a mammalian host cell, becoming intracellular. Differentiation to amastigotes begins in a temporary parasitophorous vacuole where the parasite is exposed to an acidic environment for a few hours. Released amastigotes replicate in the cytosol of the host cell, an environment of very different ionic and nutrient composition to that of the extracellular medium. After several rounds of replication amastigotes differentiate into cell-derived trypomastigotes, which are released to the bloodstream and invade other cells or are taken up by the insect host with a bloodmeal (Fig. 1). The molecular mechanisms driving developmental transformations during T. cruzi life cycle are in general poorly understood. Here, we will summarize recent advances on the signaling pathways involved in these adaptations and discuss potential approaches for their study.

Figure 1.

Environmental factors encountered by T. cruzi developmental stages throughout its life cycle.

During its life cycle T. cruzi alternates between an insect vector (triatomine bugs) and a mammalian host (including humans). Environmental factors encountered by developmental stages of the parasite (epimastigote, metacyclic trypomastigote, amastigote and cell-derived trypomastigote) are shown in different boxes for each host. Arrows indicate differentiation processes.

2. RELEVANCE OF THE INSECT’S INTESTINAL TRACT ENVIRONMENT IN DIFFERENTIATION

Cell-derived trypomastigotes present in the blood of an infected mammal are ingested by triatomines during their bloodmeal. At this point the parasite faces a dramatic decrease in temperature (usually a decrease from 37 °C to 24–30 °C, depending on the insect’s habitat) and a rapid nutritional transition, as glucose consumption and catabolism of blood meal proteins results in an increase of cations, such as K⁺ and Ca²⁺, amino acids and nitrogen-containing waste metabolites (uric acid and organic anions). When heme, the main product of hemoglobin catabolism, reaches the cytosol of midgut cells, tissues and parasites are subjected to oxidative stress by the generation of reactive oxygen species (ROS) through the Fenton reaction (Melo et al., 2020). Trypomastigotes are exposed to other factors from the insect vector in the intestinal tract, including digestive enzymes, hemolysins, agglutinins, microbiota and especially antimicrobial factors (Garcia et al., 2010). All together, these environmental changes trigger the differentiation of trypomastigotes into replicative epimastigotes in the triatomine’s posterior midgut, by a process known as epimastigogenesis (Ferreira et al., 2016). The signaling pathways mediating this process remain unknown. Further studies using molecular approaches are needed to fill this knowledge gap.

While migrating to the triatomine’s hindgut, T. cruzi epimastigotes face two drastic environmental changes, nutrient deprivation and increased osmolarity (600–1000 mOsm in the final portion of the intestinal tract). Subsequently, epimastigotes adhere to the rectal cuticle before fully differentiating into metacyclic trypomastigotes (Kollien & Schaub, 2000, Garcia et al., 2010). Metacyclogenesis can be induced in vitro by exposing epimastigotes to nutritional stress. A widely used method for in vitro metacyclogenesis consist in incubating stationary phase epimastigotes in triatomine artificial urine (TAU) medium for 2 hours and further incubate them in TAU supplemented with three amino acids and glucose (TAU 3AAG) (Contreras et al., 1985b). During this second step parasites adhere to the flask surface and are subsequently released into the medium upon differentiation into metacyclic trypomastigotes (Hamedi et al., 2015). Using this and other approaches for in vitro metacyclogenesis several studies have shown a role for cAMP in metacyclogenesis (Gonzales-Perdomo et al., 1988, Rangel-Aldao et al., 1988a, Hamedi et al., 2015, Fraidenraich et al., 1993a, Fraidenraich et al., 1993b, Garcia et al., 1995).

Signaling cascades allow the amplification of external stimuli into a large cellular response, such as expression of a specific subset of genes, enzymatic activation and differentiation. 3’,5’-cyclic adenosine monophosphate (cAMP) is a universal second messenger that mediates cell differentiation in T. cruzi. However, this signaling pathway is poorly understood in trypanosomes. Adenylyl cyclases (ACs) catalyze the conversion of ATP to cAMP and at least one of these enzymes (named TczAC) has been reported to be flagellar and calcium-stimulated in T. cruzi (D’Angelo et al., 2002). TcACs are structurally unique, with a single transmembrane domain and a catalytic domain located at the C-terminus of the protein, resembling mammalian calcium-sensitive guanylyl cyclases (Tagoe et al., 2015). These observations suggest that TcACs could link cAMP and calcium signaling pathways. These enzymes conform a multigenic family of receptor-type putative ACs annotated in the T. cruzi genome, but their individual role remains unknown.

Cyclic AMP is enzymatically hydrolyzed by the activity of cyclic nucleotide phosphodiesterases (PDEs), the enzymes that remove intracellular cAMP signals. PDEs finely tune the spatiotemporal distribution of cAMP within the cell, by regulating the amplitude, duration, termination and compartmentalization of the second messenger (Ahmad et al., 2015). There are four different class I PDEs present in the T. cruzi genome, PDEA to PDED. PDEA1 is cAMP-specific, localizes to the cytosol and is not inhibited by canonical PDE inhibitors (Alonso et al., 2007). PDEB1 and PDEB2 are gene tandemly arranged, localize to the flagellum and are also cAMP-specific (D’Angelo et al., 2004, Diaz-Benjumea et al., 2006). PDEC1 is a FYVE-containing enzyme that can use either cAMP or cGMP as substrate (Kunz et al., 2005), while PDEC2 (also a FYVE-containing T. cruzi PDE) is cAMP-specific, localizes to the contractile vacuole complex (CVC) and is involved in osmoregulation (Schoijet et al., 2011), a critical mechanism for T. cruzi differentiation and survival throughout its life cycle, as we will discuss in detail later. There is also a TcPDED whose function remains unexplored. The different subcellular localizations of TcPDEs suggests that their compartmentalization controls local levels of cAMP to trigger specific intracellular responses (Schoijet et al., 2019).

Two possible cAMP effectors have been identified in T. cruzi, protein kinase A (PKA) (Ochatt et al., 1993, Huang et al., 2002, Huang et al., 2006) and cAMP response proteins (CARPs) (Jager et al., 2014). PKAs are serine/threonine kinases considered the major effectors of cAMP in eukaryotic cells. In contrast to what has been demonstrated in T. brucei, where TbPKA is cAMP-independent (Shalaby et al., 2001, Bachmaier et al., 2019), a cAMP-dependent PKA has been identified in T. cruzi (Huang et al., 2002). The holoenzyme consists of two catalytic and two regulatory subunits (Ochatt et al., 1993), both of which have been characterized in this parasite (TcPKAc and TcPKAr) (Huang et al., 2002, Huang et al., 2006). TcPKA substrates have been identified by yeast-two-hybrid experiments in T. cruzi, and include eight proteins that could be involved in environmental sensing and differentiation, two PI3 kinases, a MAPK, a phosphodiesterase (PDEC2), an ATPase, an hexokinase, an aquaporin and a DNA excision repair protein (Bao et al., 2008). In addition, trans-sialidases were found to be TcPKA substrates, which is consistent with a role for cAMP in metacyclogenesis and host cell invasion (Bao et al., 2010, Huang, 2011). Regarding other cAMP effectors, orthologs of Epac (exchange factor directly activated by cAMP) or cAMP-gated channels have not been identified in the genome of T. cruzi. An in silico analysis identified several cyclic nucleotide monophosphate-binding proteins and at least one of them was shown to bind cAMP in vitro (TcCARP1) (Jager et al., 2014). CARPs are kinetoplastid-exclusive proteins and their role in signal transduction has not been investigated. They could play a role in a PKA-independent cAMP signaling pathway. Their absence in mammalian cells suggests they could be explored as drug target candidates.

The role of cAMP in metacyclogenesis was first observed in 1988 (Rangel-Aldao et al., 1988a, Gonzales-Perdomo et al., 1988, Rangel-Aldao et al., 1988b). These studies observed increased levels of cAMP in metacyclic trypomastigotes obtained in vitro, as well as stimulation of T. cruzi metacyclogenesis by cAMP, cAMP analogs, cAMP activators and PDE inhibitors. Later on, a research group was able to induce adenylyl cyclase activity and metacyclogenesis in epimastigotes in the presence of a synthetic peptide corresponding to the amino terminus of chicken alpha D-globin in vitro (Fraidenraich et al., 1993a, Fraidenraich et al., 1993b) and in vivo (Garcia et al., 1995), although the receptor of this peptide has not been identified in the parasite. Subsequently, calcium-sensitive TczAC was shown to form dimers in yeast-two-hybrid assays (D’Angelo et al., 2002). This enzyme also interacts with components of the paraflagellar rod, suggesting it localizes to the flagellum. More recently, a study reported that cAMP levels increase in two phases during in vitro metacyclogenesis, with a first peak occurring upon nutrient deprivation in TAU medium, and a second peak after adhesion of epimastigotes to the inner flask surface while incubated in TAU 3AAG medium, a prerequisite to complete transformation into the infective metacyclic forms (Hamedi et al., 2015).

The presence of different components of the cAMP signaling pathway in the flagellum and the CVC of T. cruzi, and the increase in cAMP levels upon environmental changes in the intestinal tract of the insect host, evidence the importance of this pathway in sensing nutrient deprivation, hyperosmotic stress and parasite adhesion to the insect’s rectal cuticle to trigger the physiological processes that lead to metacyclogenesis. Cyclic AMP signaling is still unexplored throughout T. cruzi life cycle and research in this field should be focused in elucidating the molecular mechanisms underlying this cascade. In this regard the role of the flagellum as a sensory structure is acquiring more relevance in T. cruzi.

3. INVASION OF HOST CELLS

Three different T. cruzi stages can invade both phagocytic and nonphagocytic mammalian cells: 1) the metacyclic trypomastigote present in the insect vector, which can be obtained in vitro by differentiation of epimastigotes; 2) the bloodstream trypomastigote, which can be obtained in vitro by infection of tissue culture cells; and 3) the amastigote, which is an intracellular form that, because of premature rupture of host cells, can be released to the extracellular medium of tissue culture cells or to the bloodstream of infected animals. Because of low parasitemia in animals and the limited yield of metacyclics from the insect vector, most host cell invasion studies have been done with either tissue culture-derived trypomastigotes and amastigotes, or with metacyclic trypomastigotes obtained in vitro. Studies on amastigotes-host cell interactions have been mainly focused on host cell signaling (Bonfim-Melo et al., 2018). Recent work has also investigated the proteome and phosphoproteome changes in trypomastigotes in contact with the extracellular matrix revealing metabolic changes important for adaptation to host invasion with the potential involvement of protein kinases and phosphatases (Mattos et al., 2019).

It has been indicated that the use of in vitro derived parasites has potential disadvantages as they might not behave as parasites obtained from animals (Moreno & Docampo, 2003). In addition, these stages are obtained after incubation in very rich culture media and some growth factors or compounds not present in the blood or intestine of the hosts can remain attached to the parasites (Scharfstein et al., 2000). The reverse can also be true, factors that are normally attached to the bloodstream forms or metacyclic trypomastigotes in vivo might not be present in in vitro stages. Another complication is the existence of several lineages of T. cruzi that have evolved independently for a long time (Zingales et al, 2000). It has been suggested that their specific attributes are so different as to confer the status of taxonomic species to each group (Zingales, 2000). It is therefore not surprising that apparently conflicting results could be obtained when using tissue culture cell lines of different origin, or different stages or strains of the parasite.

There is evidence of Ca²⁺ signaling in T. cruzi upon their association with host cells. An increase in cytosolic Ca²⁺ occurs in tissue cultured-derived trypomastigotes (Y strain) upon contact with host cells (L₆E₉ myoblast), as detected in Fura-2 AM-loaded parasites. Buffering cytosolic Ca²⁺ in these cells by preloading them with intracellular Ca²⁺ chelators (BAPTA or Quin 2) inhibits host cell invasion (Moreno et al., 1994). Similar inhibition of host cell (rat heart myoblasts) invasion is observed by loading both tissue culture-derived or bloodstream trypomastigotes (Tulahuén strain) with Ca²⁺ chelators (BAPTA or Quin 2), while invasion is stimulated by pretreatment of the parasites with the Ca²⁺-increasing ionophore ionomycin (Yakubu et al., 1994). The signaling mechanism involved in Ca²⁺ increase in bloodstream or cell-derived trypomastigotes is unknown. However, it has been reported that sonicated extracts of HeLa cells and monoclonal antibodies against the surface proteins gp35/50 and gp82 of metacyclic trypomastigotes induce Ca²⁺ responses in Fura 2-loaded parasites of the G and CL strains and it was proposed that the antibodies could be mimicking the host cell receptors for the parasite proteins (Ruiz et al., 1998). In an CL isolate, gp82 has been proposed as the main receptor for the Ca²⁺ response needed for host cell invasion (Ruiz et al., 1998), with the possible participation of a protein tyrosine kinase that phosphorylates a 175-kDa protein (Neira et al., 2002, Favoreto et al., 1998).

Concerning the source of Ca²⁺ increase, there is evidence for the participation of the acidocalcisome. Acidocalcisomes are the most important Ca²⁺ store in trypanosomatids (Docampo & Moreno, 2011) and are characterized by their acidity and high content of polyphosphate bound to different cations, among them Ca²⁺. The inositol 1,4,5-trisphosphate receptor (IP₃R) localizes to the acidocalcisome of T. cruzi (Lander et al., 2016) and ablation of its gene inhibits host cell invasion by cell-derived trypomastigotes (Chiurillo et al., 2020). In this regard, early work (Neira et al., 2002) showed that inhibitors of phospholipase C, which converts phosphatidylinositol 4,5-bisphosphate (PIP₂) into diacylglycerol and IP₃, diminishes the infectivity of CL but not G strain metacyclic trypomastigotes, and treatment of parasites with a combination of ionomycin plus NH₄Cl or nigericin, which are known to deplete acidocalcisome Ca²⁺ (Docampo et al., 1995) reduces the infectivity of metacyclic forms of G but not CL strain (Neira et al., 2002).

How Ca²⁺ signaling modulates the ability of the parasite to invade host cells is still unknown. However, attachment and penetration into host cells are active processes that require energy (Schenkman et al., 1991) and it has been shown that IP₃-mediated Ca²⁺ release from acidocalcisomes is important for mitochondrial Ca²⁺ uptake and generation of ATP (Chiurillo et al., 2020). Knockout of the genes involved in mitochondrial Ca²⁺ uptake (Bertolini et al., 2019, Chiurillo et al., 2019) or in mitochondrial activation of bioenergetic metabolism (Lander et al., 2018), inhibit host cell invasion.

Other signaling pathways involving adenylyl cyclase, phosphatidylinositol 3-kinase, and protein kinase C present in metacyclic trypomastigotes (Neira et al., 2002, Maeda et al., 2012) or culture-derived trypomastigotes (Mattos et al., 2019) have also been suggested to participate in host cell invasion. However, some of these suggestions were based on the effects of activators and inhibitors, and no molecular evidence has yet been presented.

4. INTRACELLULAR REPLICATION

After active penetration of host cells, trypomastigotes remain in a lysosome-acidified parasitophorous vacuole for a few hours. There they start their differentiation (a process known as amastigogenesis) and escape to the cytosol to proliferate as amastigotes for 4–5 days. After this replication, amastigotes differentiate back into trypomastigotes, which escape to the extracellular medium upon host cell lysis. Amastigogenesis can be induced in vitro by incubating tissue culture-derived trypomastigotes in low pH medium (pH 5.0) at 37 °C (Tomlinson et al., 1995).

Amastigotes survive and replicate within the cytosol of the host, which is an environment considerably different from the extracellular medium. The cytosolic concentrations of Ca²⁺ (~100 nM), Mg²⁺ (~0.8 mM), K⁺ (~140 mM), Na⁺ (~12 mM), and Cl⁻ (~4 mM), are very different from their surrounding fluid levels, like in blood (~1.8 mM, ~1.5 mM, ~4 mM, ~145 mM, ~116 mM, respectively) (Lodish H. et al., 2000). The differences in Ca²⁺ concentrations are extreme and about 10,000–20.000-fold higher in the extracellular medium. If the cytosolic Ca²⁺ levels are further reduced by loading cells with Ca²⁺ chelators, amastigote replication is greatly affected (Schettino et al., 1995). There are also marked differences in the abundance of intracellular metabolites, and some, like amino acids, are abundant in the host cytosol. Glucose has been considered to be low since it is rapidly phosphorylated to glucose 6-phosphate upon entry (Maugeri et al., 2011). It has been proposed that amastigotes obtain their energy from amino acids and fatty acids (Silber et al., 2009, Atwood et al., 2005) although glucose has also been suggested as a potential energy source (Shah-Simpson et al., 2017).

Differentiation programs in mammalian cells usually involve the activation of transcription factors. However, current knowledge indicates that trypanosomes lack precise transcriptional control because no classical promoters have been identified (Clayton, 2002, Palenchar & Bellofatto, 2006). Genes coding for proteins of unrelated functions are transcribed in large polycistrons, and these polycistrons are co-transcriptionally processed by trans-splicing and polyadenylation to produce monocistronic transcripts. Regulation of gene expression is mainly at the post-transcriptional level, and in T. cruzi it has been proposed to occur through pre-mRNA processing, RNA degradation (D’Orso et al., 2003, Jager et al., 2007), or translational repression (Nardelli et al., 2007, Cassola et al., 2007, Smircich et al., 2015). It has been reported that multiple mRNAs can be regulated by one or more sequence-specific mRNA binding proteins (RBP) that organize their splicing, export, stability, localization, and translation, acting as post-transcriptional regulons (Keene, 2007). In this regard, a 43-nucleotide U-rich RNA element located in the 3’-UTR of more than 900 mRNAs of large gene families (trans-sialidase, MASP, mucin surface protease GP63, and protein kinase), as well as of single and low copy number genes, is predominantly detected in intracellular amastigotes and interacts with RBP TcUBP1 (Li et al., 2012). The results suggests that this RBP organizes these RNA transcripts to facilitate their translation and form a functional post-transcriptional regulon (Li et al., 2012). It has been proposed that these regulons could be very important for trypomastigote to amastigote differentiation in the mammalian host, a process that occurs in only a few hours but that involves drastic morphological and metabolic changes (Li et al., 2012). Interestingly, incubation of trypomastigotes in medium at pH 5.0 for 2 hours, a treatment that resembles their environment in the parasitophorous vacuole of host cells, is sufficient to trigger their transformation into amastigotes (Tomlinson et al., 1995). Which signaling pathways are activated by these external pH changes and how these signals are transmitted to the RBP that organize these and other regulons is unknown. Quantitative proteomic and phosphoproteomic analysis of T. cruzi amastigogenesis in vitro has revealed upregulation and downregulation of membrane proteins, and phosphorylation changes that could be involved in these processes (Queiroz et al., 2014).

5. CHANGES IS OSMOLARITY

The development of T. cruzi different stages in their insect and mammalian hosts expose them to drastic changes in osmolarity. Epimastigotes convert into metacyclic trypomastigotes and during this conversion are exposed to increasing osmolarities that reach values above 1,000 mOsm/kg when metacyclic reach the feces and urine of the insect vector (Kollien et al., 2001). Bloodstream trypomastigotes circulate through the kidney of the mammalian host where they are exposed to osmolarities of up to 1,300–1,400 mOsm/kg (Lang, 2007). The osmolarity to which both metacyclic and bloodstream trypomastigotes are exposed, suddenly changes to isosmotic conditions (300 mOsm/kg) (Lang, 2007) when they leave the kidney circulation or invade host tissues and cells, respectively. In addition, it has been shown that some organs invaded by T. cruzi, like liver, spleen, and lymphoid tissues, have higher osmolarity than serum (330 mOsm/kg) (Go et al., 2004). On the other hand, like all other cells, T. cruzi needs to regulate its volume continuously (Lang, 2007). The responses of this parasite to hypoosmotic and hyperosmotic stresses have been studied in some detail, and two organelles, the acidocalcisome, and the contractile vacuole, have an important role in these processes (Docampo et al., 2013).

When exposed to hypoosmotic stress cells swell and then recover their volume through a process denominated regulatory volume decrease (RVD). An RVD mechanism is present in epimastigotes, tissue culture-derived trypomastigotes, and amastigotes (Rohloff et al., 2003). Two mechanisms are involved in the RVD, which is complete in less than 5 min. One is the release of uncharged or acidic amino acids (mainly glutamic acid, glycine, proline, and alanine), which accounts for 50% of the RVD, and a small contribution of K⁺ efflux, which accounts for only 7% of the RVD (Rohloff et al., 2003). A rise in intracellular Ca²⁺ appears to be important to modulate the early phase of amino acid release. The second mechanism is the water elimination through the contractile vacuole complex (CVC), an organelle present in all stages of the parasite (Rohloff et al., 2004). Cyclic AMP is involved in this mechanism as its level increases when epimastigotes are subjected to hypoosmotic stress. A model was proposed (Rohloff et al., 2004, Docampo et al., 2013) in which the stimulus of cell swelling by a mechanosensitive mechanism activates an adenylyl cyclase causing a spike in intracellular cAMP that results in a microtubule-dependent fusion of acidocalcisomes with the contractile vacuole and translocation of an aquaporin (TcAQP1) to this organelle (Montalvetti et al., 2004). Concomitantly, a rise in ammonia (NH₃) and its sequestration in acidocalcisomes as NH₄⁺ would activate an exopolyphosphatase in the organelles releasing Pi and inorganic and organic cations bound to polyphosphate, resulting in an osmotic gradient that would favor water uptake by TcAQP1. Water alone would be ejected into the flagellar pocket and a cAMP phosphodiesterase would terminate the reaction by hydrolyzing cAMP to 5’-AMP. A scheme showing these reactions has been published (Docampo et al., 2013).

Evidences for this model are: a) the presence of a mechanosensitive channel in the CVC (Dave et al., 2019) that could be the sensing protein; b) the increase in cAMP levels detected upon hypoosmotic stress (Rohloff et al., 2004); c) video microscopy (Rohloff et al., 2004) and cryo-electron microscopy (Niyogi et al., 2015) evidence of acidocalcisome translocation and fusion with the CVC (Fig. 2), and fluorescence microscopy evidence for the translocation of TcAQP-GFP to the CVC (Rohloff et al., 2004); d) stimulation of this translocation by cAMP analogs (dibutyryl AMP) and its inhibition by adenylyl cyclase inhibitors (dipyramidole, 3-isobutyl-1-methylxanthine, 2-deoxy-AMP) and microtubule inhibitors (chloranin, trifuralin) (Rohloff et al., 2004); e) inhibition of the RVD by TcAQP1 inhibitors (HgCl₂, and AgNO₃) (Rohloff et al., 2004); f) increase in NH₃ levels is epimastigotes submitted to hypoosmotic stress with alkalization of acidocalcisomes (Rohloff & Docampo, 2006); g) evidence of polyphosphate hydrolysis in acidocalcisomes alkalinized by NH₄Cl treatment (Ruiz et al., 2001); h) localization of a cAMP phosphodiesterase C in the CVC (spongiome) (Schoijet et al., 2011), and that inhibition of this enzyme caused inhibition of the RVD (King-Keller et al., 2010).

Figure 2.

Fusion of acidocalcisomes to the contractile vacuole complex.

(A-C) Virtual section (1-nm thickness) sequence of a tomogram showing the anterior region of the parasite. The CVC is represented by the central vacuole or bladder (CV) and the spongiome (Sp). Acidocalcisomes (Ac) in the neighboring region are in close contact with the CVC. In the left lower corner, the section number is shown. In C, it is possible to observe a close apposition between an acidocalcisome and the CVC, which is suggestive of a fusion event (arrow) between the two organelles.

(D-F) 3D models of the CVC (blue) and the close contact with an acidocalcisome (orange).

F. Tilted view of the 3D model at 45° around the X axis. Spongione (Sp) and flagellum (F) are shown. Scale bar = 200nm.

Reproduced with kind permission from figure 4 of Niyogi et al. (2015).

Other proteins probably contributing to the RVD include a class III phosphatidylinositol 3-kinase (TcIP3K), orthologue to the yeast vacuolar protein sorting 34 (Vps34p), whose overexpression affects the RVD (Schoijet et al., 2008), and a phosphate/sodium symporter that localizes to the CVC (Jimenez & Docampo, 2015) and could be involved in recycling phosphate produced by hydrolysis of polyphosphate during RVD.

When epimastigotes are submitted to hyperosmotic stress they shrink within a few seconds but in contrast to what occurs with mammalian cells that rapidly regain their volume by a regulatory volume increase (RVI) (Lang et al., 1998, McManus et al., 1995), they do not rapidly recover their volume (Li et al., 2011). This suggests that there is no immediate inorganic ions and water uptake as in mammalian cells. The CVC initially swells. Knockdown of TAQP1 or treatment with the TcAQP1 inhibitor HgCl₂, reduces the intensity of shrinkage suggesting that the CVC mediates the initial water efflux (Li et al., 2011). The cells resist well these changes and their motility is normal. Within minutes of hyperosmotic stress, there is a decrease in ammonium production and accumulation of amino acids. Protein content decreases within 3 hours indicating protein degradation to increase the amino acid pool. These amino acids would replace inorganic ions as compatible osmolytes (Li et al., 2011). Polyphosphate synthesis is stimulated within minutes suggesting that this is a mechanism to sequester cations to reduce the ionic strength of the cells and prevent damage (Li et al., 2011). A cation channel (TcCAT) is translocated to the plasma membrane upon hyperosmotic stress and probably contributes to cation elimination as inhibitors of this channel (BaCl₂, 4-aminopyridine) inhibit shrinking (Jimenez & Docampo, 2012). In a second phase of recovery there is an induction of amino acid transporters that would contribute to replace the inorganic ions sequestered with polyphosphate in acidocalcisomes (Li et al., 2011). In conclusion, the response to hyperosmotic stress includes an early shrinkage mediated by TcAQP1 located in the CVC, with no early RVI, an increase in the cytosolic ionic strength counteracted by early synthesis of polyphosphate with sequestration of inorganic ions in acidocalcisomes, and replacement of these inorganic ions by amino acids produced by reduction of their catabolism, increased protein degradation, and in a second phase, uptake by protein transporters (Docampo et al., 2013). A scheme showing these reactions has been published (Docampo et al., 2013).

6. OXIDATIVE STRESS

Epimastigotes replicate in the intestine of the insect vector, which is a low O₂ environment, and although they express several antioxidant enzymes (Fig. 3), their activities are low when compared with the equivalent host enzymes (Docampo & Moreno, 2017). It has been indicated that epimastigotes might be protected for dealing with a slow endogenous generation rate of oxidants but they would be quite sensitive to increased steady state concentrations (Docampo & Moreno, 2017), produced for example, by phagocytic cells or drugs. In contrast, metacyclic trypomastigotes have higher protein expression levels of antioxidant enzymes than epimastigotes (Atwood et al., 2005). Ascorbate peroxidase, mitochondrial tryparedoxin peroxidase, tryparedoxin, trypanothione synthase, iron superoxide dismutase, and the pentose-phosphate pathway enzymes that provide NADPH for reduction of trypanothione have all higher expression levels (Atwood et al., 2005). These differences suggest a preadaptation of these stages to withstand the more aerobic metabolism of intracellular life forms (Silber et al., 2009) and the more aerobic environment of the mammalian host. It is not yet clear whether this preadaptation depends on the activation of a signaling pathway and whether the expression of these antioxidant enzymes depends on a specific regulon organized by a particular RBP.

Figure 3.

Metabolism of reactive oxygen species (ROS) in T. cruzi. Reduction of trypanothione (T(S)₂) to dihydrotrypanothione (T(SH)₂) is catalyzed by trypanothione reductase with conversion of NADPH to NADP⁺. Oxidation of dihydrotrypanothione to trypanothione is coupled to the reduction of tryparedoxin (Try(S)₂), oxidized glutathione (GSSG), and dehydroascorbate (dhASC), and these compounds are generated by the action of peroxidases catalyzing the decomposition of H₂O₂ (ascorbate peroxidase and tryparedoxin peroxidases) or hyroperoxides (ROOH) (GSH peroxidases I and II, and tryparedoxin peroxidases). Ascorbate peroxidase, mitochondrial TryX peroxidase, and TryX are more highly expressed in metacyclic trypomastigotes.

5. FUTURE PERSPECTIVES

Coordination of adaptation changes to new environments in T. cruzi appears to depend on post-transcriptional mRNA regulons organized by RBPs, and some examples have recently been described (Li et al., 2012, Sabalette et al., 2019). However, the stimuli and the potential second messengers involved in the stimulation of these RBPs have been difficult to identify. Nutrient deprivation, hyperosmotic stress and parasite adhesion to the insect’s rectal cuticle are potential stimulus for epimastigote to metacyclic trypomastigote transition mediated through cAMP (Contreras et al., 1985a, Hamedi et al., 2015). Acidic pH is important for trypomastigote to amastigote conversion (Tomlinson et al., 1995), and mechanical stimulation is relevant for osmotic adaptations (Li et al., 2011, Rohloff et al., 2004, Rohloff et al., 2003), and host cell invasion (Moreno et al., 1994), respectively, but for some transitions (amastigote to trypomastigote transformation and trypomastigote to epimastigote) the stimulus is more difficult to identify. The cAMP pathway is involved in differentiation and osmoregulation under hypoosmotic conditions (Rohloff et al., 2004) while Ca²⁺ is a second messenger involved in host cell invasion (Moreno et al., 1994). How these second messenger signals are translated into physiological changes is starting to be studied. Protein kinases and phosphatases have been suggested to be involved in some of these changes but studies have been limited to transcriptomic (Li et al., 2016) and proteomic (Mattos et al., 2019) approaches or the use of inhibitors and activators (Neira et al., 2002, Maeda et al., 2012). Further work is also needed to investigate signaling events acting at other levels, such as transcription, translation, cell cycle, and DNA damage response. The development of gene editing tools for the study of T. cruzi (Lander & Chiurillo, 2019, Lander et al., 2019) will have an important role in the elucidation of these pathways.