Types of cell death
Among the pathogenic protozoa, trypanosomatids stand out due to their medical and economic impact, especially for low-income populations in tropical countries. Together, sleeping sickness, Chagas disease and leishmaniasis affect millions of humans and animals worldwide, yet are neglected by the pharmaceutical industry. The current drugs for trypanosomatid infections are limited and unsatisfactory, with severe side effects leading to reduced quality of life and, in several instances, to the abandonment of treatment. An intense search for alternative compounds has been performed, aiming at specific parasite targets by cellular, molecular and biochemical approaches. One interesting strategy could be interference with the protozoan cell death pathways. However, these pathways are poorly understood in unicellular eukaryotes, with the controversial existence and uncertain biological relevance of programmed cell death (PCD). This chapter will discuss apoptosis-like and autophagic cell death and necrosis in Trypanosoma brucei, Trypanosoma cruzi and Leishmania sp. and the possible implications of these pathways for the parasite life cycle and infection persistence. It will also revisit the genomic and proteomic metadata of these trypanosomatids in the literature to rebuild the map of cell death proteins expressed under different conditions. The interaction of leading candidates with parasite-specific molecules, especially with enzymes that regulate key steps in the cell death process, is a rational and attractive alternative for drug development for these neglected diseases.
- Cell death
- Leishmania sp
- T. cruzi
- T. brucei
Neglected tropical diseases (NTDs) are a group of the seventeen mostly life-threatening infections, which affect more than a billion people worldwide. They affect poor populations, often in underdeveloped and developing countries (low-income countries) . Among NTDs, infections caused by the so-called “protozoan” parasites, such as African trypanosomiasis, Chagas disease and leishmaniasis, are responsible for a high annual death toll among the poor populations of tropical countries. New safe and affordable medicines are urgently needed. These diseases all present therapeutic difficulties by developing resistance to existing therapies and/or by toxic side effects.
1.1. Neglected tropical diseases and trypanosomatids
1.1.1. Sleeping sickness
Human African trypanosomiasis (HAT), or sleeping sickness, is caused by extracellular protozoa belonging to the genus
The drug of choice for treatment depends on the infecting species and the stage of infection. In early stages,
1.1.2. Chagas disease
Chagas disease is caused by the intracellular obligatory parasite
The life cycle of
The available chemotherapy for this illness includes two nitroheterocyclic agents, nifurtimox and benznidazole, which are effective against acute infections, but show poor activity in the late chronic phase, with severe collateral effects and limited efficacy against different parasitic isolates. These drawbacks justify the urgent need to identify better drugs to treat chagasic patients, and several new compounds are currently in preclinical development involving
Leishmaniasis, which is caused by different species of
The current drugs are highly toxic, resistance is common and compliance of patients to treatment is low, as the treatment is long and the drug price is high. Although recent initiatives have improved the antileishmanial drug arsenal by combining current medicines or using new formulations of old ones, none are ideal for treatment due to their high toxicity, resistance issues, prohibitive prices, long treatment length and need of intravenous administration [32-34]. Pentavalent antimonials (glucantime and pentostan) are first-line drugs for both VL and CL. However, they present several limitations, including variable efficacy, need for daily injectable administration for approximately one month, and severe side effects. Many patients are unable to complete the treatment, increasing the risk of drug resistance development. Amphotericin B is a systemic antifungal that is used as a second-line drug for VL. It is highly toxic, requiring careful and slow intravenous administration. Lipid formulations of amphotericin B have been developed to improve its bioavailability and pharmacokinetic properties, reducing toxicity . Miltefosine is the most recent antileishmanial drug on the market and the first effective oral treatment against VL . However, it has common gastrointestinal side effects and is also limited by its relatively high cost , potential teratogenicity and growing concerns in relation to increases in clinical isolate susceptibility . Paromomycin is an aminoglycoside antibiotic that is used in topical treatment for CL and as a parenteral drug for VL. Pentamidine was used as a second-line drug in antimony-resistant VL treatment. However, its high toxicity combined with decreased efficacy led to the abandonment of this drug to treat VL in India, but it is valuable for combined therapies .
2. Cell death: State of art
As used for whole organisms, the term death is employed to describe a sequence of events culminating in the breakdown of all biological functions. However, more than one century after the first citation , cell death still represents a crucial gap in our understanding of cellular physiology. It can be triggered by natural processes or induced by extrinsic factors (exposure to chemicals or physical stresses). The consequent tissue injury usually leads to a state of disease . On the other hand, many studies pointed to cell death playing a fundamental role in the physiology of multicellular organisms, especially in processes such as metamorphosis and embryogenesis . In this context, in 1964, the term programmed cell death (PCD) was created, proposing a sequence of well-controlled steps regulating a non-accidental cell death process in the absence of an inflammatory response . Currently, it is known that distinct death mechanisms and phenotypes participate in PCD, with apoptosis and autophagy being the most prominent .
The apoptotic pathway was first described in the early 1970s as a fundamental step for proper embryo development . This process is crucial during tissue development, especially in immune response regulation and removal of infected or damaged cells [45, 46]. Apoptosis is involved not only in growth regulation in multicellular organisms [47, 48] but also in their defence against viral, bacterial or parasitic infections [49-53] and even against cancer development [54-57]. The removal of non-functional cells by the apoptotic pathway is efficient and prevents the inflammatory response .
During apoptosis in multicellular organisms, the cell activates death machinery that culminates in chromosomal condensation and nuclear DNA fragmentation [59, 60]. Biochemically, apoptosis is orchestrated by the activation of a family of cysteine proteases, named caspases, that are activated by extrinsic and intrinsic factors [45, 46]. The extrinsic pathway is activated by the interaction of death ligands with their respective cell surface receptor (i.e., FasL/Fas, TNF-α/TNFR) [61-63]. Such binding triggers the cleavage of procaspase 8 into active caspase 8, which cleaves procaspase 3. Executioner caspase 3 activates endonuclease G (EndoG), starting the characteristic DNA fragmentation, a distinctive marker of apoptosis [63-65]. On the other hand, the intrinsic pathway can be triggered by two distinct mechanisms with mitochondrion or endoplasmic reticulum (ER) dependency. In the mitochondrial pathway, activation occurs by membrane permeabilization, releasing cytochrome c, apoptosis induction factor (AIF), EndoG and regulators of the B-cell lymphoma 2 (Bcl2) protein family into the cytosol. In the cytosol, the apoptosome is formed by the interaction of released cytochrome c with apoptotic protease activating factor 1 (APAF-1) and procaspase 9, activating caspase 9, which subsequently activates the effector caspase 3 [66-70]. The ER pathway is mainly caspase 12-dependent and occurs in this organelle during stress conditions. Because this pathway was described in the mouse and humans lack functional caspase 12, the relevance of ER-mediated apoptosis is still debatable [71-73].
Undoubtedly, the caspase cascade represents a central point in the apoptotic process. Its regulation is well-controlled by pro- and anti-apoptotic molecules from the Bcl-2 family . The apoptotic morphological and biochemical phenotypes include cell shrinkage, membrane blebbing (formation of apoptotic bodies), chromatin condensation and typical internucleosomal DNA fragmentation, externalization of phosphatidylserine (PS), loss of mitochondrial membrane potential (ΔΨm), and target protein degradation by caspase activation [75-79]. The characterization of apoptosis is experimentally based on the detection of apoptotic markers. The loss of ΔΨm (labelling with rhodamine 123 derivatives, such as TMRE), PS exposure (binding to labelled annexin V), chromatin condensation (DAPI labelling) and DNA fragmentation (TUNEL technique) are usually quantified by fluorescence microscopy or flow cytometry. DNA fragmentation can also be assessed by agarose gel electrophoresis, presenting a laddering pattern that represents internucleosomal cleavage. Analysis of caspase activity using labelled specific substrates and/or inhibitors can be performed by immunotechniques such as ELISA .
In the 1950s, acidic organelles involved in the intracellular degradation of macromolecules were described and termed lysosomes by Dr. Christian de Duve. In a subsequent study , he proposed the term autophagy for a self-degrading process . Currently, the autophagic pathway is considered to be the main cellular mechanism for the degradation of non-functional organelles and/or macromolecules and is fundamental for homeostasis in eukaryotic cells . In other words, autophagy is a housekeeping self-digestion mechanism that is crucial for cellular turnover and recycling and occurs by the engulfment of cytosolic portions containing material that should be degraded. Degradation starts immediately after the fusion of autophagosomes to lysosomes in an organelle named the autophagolysosome [84, 85].
In multicellular organisms, autophagy is involved in many physiological situations, including development, cell growth and cell differentiation. Autophagy sustains cell survival under ‘extracellular stress’, such as nutrient starvation, hypoxia, acidic pH and high temperature. It acts as a housekeeping device under ‘intracellular stress’ by removing damaged or redundant cytoplasmic components, including organelles . Increased autophagic activity is observed in pathological states and in host defences against pathogens [87-92]. Despite the relevant role of autophagy for the maintenance of the regular cell cycle, prolonged starvation periods or other strong autophagic stimuli induce a cellular misbalance and promote autophagic cell death [93, 94].
The autophagic molecular machinery was first assessed in the yeast model
Additionally, there are three types of autophagy: macroautophagy, microautophagy and chaperone-mediated autophagy (CMA). The most common is macroautophagy, a process that involves the engulfment of cytosolic portions by a double membrane structure called the phagophore. The double-membrane vesicle formed from phagophore engulfment is named the autophagosome and is directed to lysosomes for degradation by lysosomal hydrolases. These steps are regulated by Atgs [92, 96-98]. The chronological events related to macroautophagy are (a) autophagic induction; (b) cargo selection; (c) phagophore elongation; (d) autophagosome formation; (e) fusion to lysosomes; and (f) cargo degradation . The early steps in this process depend on the serine/threonine protein kinase TOR (target of rapamycin), which is essential for autophagic regulation. TOR complexes 1 and 2 work as sensors of nutritional availability (especially amino acids). The autophagic enzyme Atg6 (Beclin 1 in mammals) is a phosphatidylinositol 3-kinase (PI-3K) and shares its signalling function with other cellular pathways. For autophagy, these kinases present a critical role for autophagosome formation .
In contrast, there are no autophagosomes in the microautophagic pathway. Invagination of the lysosomal membrane occurs, resulting in a single-membrane small vesicle inside the lysosomes that will be degraded. Interestingly, both macro- and microautophagy could be selective or non-selective processes. Indeed, CMA appears to be the most selective type of autophagy. The proteins that will be degraded contain pentapeptide motifs (KFERQ, QREFK or VDKFQ), the binding sites of a cytosolic chaperone. Such a chaperone-substrate complex binds to a LAMP-2A receptor in the lysosomal membrane, promoting receptor dimerization. A membrane channel is formed, and the specific protein reaches the lysosomal lumen to be degraded [82, 100].
For many years, electron microscopy was the only tool available for the identification of autophagic morphological features, especially the presence of double-membrane vesicles (autophagosomes). In the last 20 years, advances in the molecular description of autophagy allowed the detection, localization and quantification of Atgs by molecular, biochemical and morphological approaches. Currently, the gold-standard method to monitor autophagy is Atg8/LC3 detection by different techniques: (a) Western blotting (presence of two isoforms); (b) confocal or fluorescence microscopy (identification of LC3 puncta); (c) knock down or knock out (deletion and analysis of the phenotype); and (d) pharmacological induction/inhibition (rapamycin and/or PI-3K inhibitors). These techniques can also be employed
Necrosis is a term that is extensively employed as synonymous with cell death. In the Greek aetiology, it signifies the "stage of dying". In this death type, strong cellular damage occurs caused by external stimuli (drugs, infection, mechanical trauma), promoting the random degradation of the whole cell, with plasma membrane disruption. Necrosis is defined as an accidental cell death process, differing from PCD (especially apoptosis) . One of the main differences between apoptosis and necrosis is the induction of the inflammatory response in the latter. The release of intracellular material into the extracellular environment during necrotic cell death triggers intense inflammation in the surrounding cells and tissues . Classical necrotic features are the loss of plasma membrane integrity, cytosolic vacuolization, disruption of calcium homeostasis, general degradation by lysosomal hydrolases and induction of the inflammatory response.
Necrosis can also be a regulated process. Necroptosis is a programmed and non-accidental death pathway. Surprisingly, the activation of this pathway can occur by TNF-α or FasL, classical apoptotic ligands. Necroptosis depends on the participation of the receptor-interacting protein kinases 1 and 3 (RIPK1 and RIPK3), which are kinases that regulate this pathway. RIPK1 is pharmacologically inhibited by a small molecule named necrostatin-1 (Nec-1) [104-106].
In addition to apoptosis, autophagy and necrosis (accidental or not), other non-canonical death styles can take place in eukaryotic cells. In an inflammatory context, pyroptosis and NETosis are prominent. Pyroptosis, primarily observed in macrophages after bacterial infection, is caspase 1-dependent. This caspase promotes an increase in the inflammatory cytokine levels (IL-1β and IL-18) and the formation of plasma membrane pores, leading to the release of cellular material to the extracellular matrix. The main difference between pyroptosis and apoptosis is the participation of caspase 1, which is only involved in the pyroptotic death pathway, a proinflammatory PCD [106-108]. Another type of cell death that plays a crucial role in the innate immune response is the neutrophil extracellular trap (NETosis), where neutrophilic death leads to the release of a neutrophil DNA network coated with histones and elastase to the extracellular environment to capture pathogens. However, the direct antimicrobial effect of the NETs is still controversial [109, 110]. Currently, DNA release has also been described in other immune cells, such as eosinophils, basophils, macrophages and mast cells, but its precise role deserves further analysis [110-114].
Other cell death types not involved in inflammation have been characterized. Ferroptosis is iron-dependent cell death that has been identified in some mammalian cells and involves oxidative stress induced by a small molecule named erastin, which is inhibited by ferrostatin 1. Despite that lack of complete understanding of the erastin mechanism, the XC−Cys/Glu antiporter system is inhibited in ferroptosis, leading to a misbalance of these amino acids inside the cell [106, 115]. Additionally, there is another non-canonical cell death pathway in cancer cells (
externalization of PS
activity of caspases
regulation by Bcl-2 family proteins
loss of ΔΨ
release of cytochrome c
no inflammatory response
|[44, 76, 78]|
|autophagy||presence of autophagosomes
participation of Atgs
regulation by PI-3K and TORC
degradation by lysosomes
presence of KFERQ, QREFK or VDKFQ motifs in the protein to be degraded (only in CMA)
|necrosis||disruption of plasma membrane
imbalance of Ca2+ homeostasis
release of lysosomal enzymes
induction of inflammatory response
|necroptosis||participation of RIP1 and RIP3
inhibition by Nec-1
|pyroptosis||participation of caspase 1
increase in IL-1β and IL-18 levels
induction of inflammatory response
|NETosis||formation of NETs
participation of elastase and histones
occurrence in neutrophils, macrophages, mast cells, eosinophils and basophils
|ferroptosis||participation of iron
presence of oxidative stress
induction by erastin
blockage of XC−Cys/Glu antiporter system
inhibition by ferrostatin 1
random DNA fragmentation
extrusion of large cytosolic portions (without organelles)
degradation of cellular components
increase in the AV-/PI+ population
3. Cell death in trypanosomatids: An overview
The term PCD was employed for decades to exclusively describe cell death in metazoans and its involvement in embryogenesis and maintenance of homeostasis. Indeed, the relevance of PCD for lower eukaryotes is unclear. In an evolutionary scenario, these regulated processes could allow clonal selection in the parasite population, guaranteeing the propagation of identical genetic information even in adverse environmental conditions. However, differences in the cell death mechanisms observed between metazoans and protozoans must be considered [78, 118]. In the following sections, we will discuss the role of different death styles described in pathogenic trypanosomatids.
In trypanosomatids, the first PCD report was published in 1995 by Ameisen and coworkers describing apoptotic characteristics (DNA fragmentation and cytoplasmic and nuclear morphological alterations) in
As in other pathogenic trypanosomatids, apoptotic features were also identified in
In unicellular organisms, the mitochondrion is a central organelle in cell death pathways, leading to ROS production . In
|Elongation factor 1 ∝||
||[147, 162, 163]|
||[145, 147, 162]|
||[145, 150, 153, 162]|
||[145, 150, 162, 165]|
|Metacaspase Z-DEVD-FMK -sensitive||
||[124, 134, 136]|
||[132, 158, 166]|
The participation of EndoG-like in mitochondrial-mediated cell death has been reported, but the process is metacaspase-independent (Table 2) [132, 158, 159].
Almost forty years ago, the first morphological autophagic evidence was described in trypanosomatids by electron microscopy of
loss of the ΔΨm
release of cytochrome c
||[76, 118, 119, 130]|
|autophagy||presence of autophagosomes-like
Golgi and/or ER profiles surrounding organelles
detection of Atg8 and Atg4
||[82, 92, 167]|
plasma membrane disruption
||[149, 168, 169]|
In the last few years, a functional autophagic pathway was characterized in trypanosomatids and ATG homologues were identified. However, almost half of the yeast Atgs are lacking in these protozoa [167, 179-181]. Currently, in trypanosomatids, twenty autophagic genes have been found to be involved in all of the steps, from vesicle expansion and completion to degradation (Figure 2) . Bioinformatic approaches revealed all four genes of the Atg8 conjugation system (Atg3, Atg4, Atg7 and Atg8). Atg8 is well-characterized in
|Atg5, Atg10, Atg12||
|Atg8A, Atg8B, Atg8.2||
|Atg8, Atg8A, Atg8B, Atg8C||
||[186, 198, 199]|
In addition to the recycling function, autophagy plays a fundamental role in parasite differentiation and survival, mitochondrial function and homeostasis of phospholipids [92, 182, 189, 190]. In metacyclogenesis, the autophagic pathway is triggered by nutritional deprivation, playing an important function in both the infectivity and virulence to the vertebrate host . During the
A peculiar role for autophagy was observed in
Autophagic cell death occurs when the homeostatic balance is broken . To evaluate whether autophagy participates in the cell death process, the use of the PI-3K inhibitors wortmannin and 3-methyladenine (3-MA) before the autophagic stimulus is provided is an interesting experimental approach. Pre-treatment with these inhibitors totally abolished the trypanocidal activity of naphthoimidazoles in
As described for higher eukaryotes, necrosis is poorly studied in protozoa, especially due to its conception as an accidental and uncontrolled process. The most typical necrotic feature is the plasma membrane rupture that leads to the loss of cellular homeostasis and consequent cell lysis as the consequence of a mechanical or chemical stimulus . Necrosis is always the cell death endpoint, culminating in the generation of cellular debris. Thus, independent of the cell death mechanism that is induced, all parasites will lyse in a system without phagocytic cells to clean the microenvironment. In this context, a high percentage of anti-trypanosomatid natural or synthetic drugs present a mechanism of action with a lytic effect [29, 149, 202-205] (Table 3).
Another crucial stress condition that induces trypanosomatid disruption is the activation of the complement pathway. This cascade can be triggered by the binding of lectins to lipophosphoglycans presented on the surface of
Curiously, no studies have been reported about non-canonical PCD pathways in trypanosomatids. Pyroptosis and NETosis are processes that are characterized exclusively in mammalian cells, specifically during an inflammatory response. Such pathways involve the death of immune cells to block the progression of any infection by a well-regulated mechanism [106, 110]. The absence of these PCD types in unicellular organisms is not strange. On the other hand, the existence of specific oxidative stress-related cell death types in trypanosomatids would be reasonable. Continuous exposure of these parasites to ROS under distinct environmental conditions during their life cycles indicates the important role of oxidative stress in the control of protozoa populations. ROS involvement in trypanosomatid apoptosis-like processes and autophagy has been described in different experimental conditions [130, 155, 156, 211, 212], but ferroptosis has not yet been investigated. Further studies about the effect of erastin as well as the inhibition by ferrostatin 1 should be performed in these parasites. Autoschizis was only observed in cancer cells under very specific conditions, but interestingly, an autoschizic phenotype (high percentages of AV-/PI+ cells) was detected in
3.5. Cell death and evasion of host immune response
Trypanosomatids presented a highly sophisticated repertoire to evade mammalian immune systems, including the capacity to prevent the cell death pathways of the infected host cells . This efficient strategy allows host PCD modulation by the parasites to establish the infection. Depending on the protozoan species and the host cell type, PCD exacerbation or inhibition fluctuates. For example, the induction of apoptosis in immune cells increases the parasite persistence and survival in immunocompetent hosts . In
Host autophagy also represents a valuable mechanism for both innate and adaptive responses to stop the infection. Its blockage is a crucial tactic for pathogenic trypanosomatids to evade host defences. Autophagy uses a process to eliminate pathogens, called xenophagy, directing microorganisms to be digested in lysosomes. This strategy is usually employed by protozoa living inside parasitophorous vacuoles to use the autophagic machinery to provide nutrients . However, protozoa, such as
4. Concluding remarks
In spite of the variety of studies about cell death in protozoans, including trypanosomatids, and the evidence of PCD, the detailed aspects of the molecular mechanisms and regulation remain unclear. The absence of key molecules together with the lack of an obvious role for this process in unicellular organisms makes the existence of PCD in these cells a debatable point, and the term “apoptosis-like” is more convenient [130, 172, 229]. In this context, the lack of a strong correlation between the proteolytic properties of caspases and their role in PCD should be highlighted. Currently, there is no description of the participation of trypanosomatid metacaspases in cell death processes, but these proteases have been postulated to function in proliferation and differentiation, which are important events for parasite survival [145, 148, 149, 153, 230]. In the post-genomic era, a rigorous search should be performed in proteomic databases of pathogenic trypanosomatids to correct misannotations in cell death proteins, validating the real role of these molecules for PCD processes.
Nevertheless, PCD was conserved during evolution, suggesting its essential function for the survival and maintenance of these species. However, it has been proposed that these pathways appeared in the phylogenetic tree in the multicellular organism branches, suggesting that the death molecular mechanisms identified in unicellular parasites came from a divergent evolutionary event . This idea is supported by the replacement or complete absence of some PCD molecules, justifying the differences observed in protozoa mechanisms . In addition to being an interesting evolutionary model for PCD, its physiological relevance for protozoa is still the most attractive question.
An altruistic hypothesis has been raised for unicellular organisms, especially for pathogenic trypanosomatids . It was associated with the control of parasite populations, including protozoa density regulation, clonal selection and immune host system evasion, events related to the success of the infection [7, 76, 82, 136, 231]. Trypanosomatid cell death limits parasite colonization in insects in response to scarce resources of nutrients, avoiding invertebrate death [118, 130, 134]. On the other hand, PCD of
Autophagic cell death has been proposed as a PCD pathway, suggesting an active role of autophagy in death processes, but the precise mechanisms of regulation are not yet clear [174, 178, 232]. The majority of the autophagic studies were performed in yeast and mammal models. However, little is known about protozoan pathways. Autophagy is a regulated process that is directly involved in the preservation of cellular homeostasis and survival. Several hypotheses have been raised about the participation of this pathway in cell death in dying cells. The selective autophagic degradation of essential cellular factors, such as cell death regulators, triggers death events, including caspase activation [232, 233]. Another hypothesis suggested that autophagy is not a specific and regulated cell death process but is a consequence of extensive injury. Once such an injury compromises cellular physiology, the damaged structure needs to be degraded for cell survival. This hypothesis also explains the presence of similar phenotypes in parasites after treatment with different compounds with distinct mechanisms of action. Such autophagic phenotypes, detected independent of the stimuli, reinforced this pathway as a desperate attempt of the cells to stay alive [168, 212, 232]. The determination of the connection between the autophagic cell death of pathogens, such as trypanosomatids, could have crucial implications for human health, but further mechanistic studies should be addressed in this field.
The existence of cross-talk among different cell death pathways, especially autophagy and apoptosis, has been proposed (Figure 3) [93, 234]. In unicellular parasites, different cell death types have been described to be induced by physical and/or chemical stress conditions (drugs, heat shock, and nutritional deprivation, among others), resulting in a non-classical cell death phenotype. The total absence of commercial typical PCD markers, such as antibodies and enzyme activity kits, for protozoa and of key autophagic and apoptotic-like molecules reinforce the hypothesis of an interplay of distinct death mechanisms, suggesting their convergence, leading to necrosis. Likewise, the possibility of the occurrence of other PCD forms cannot be excluded [74, 78, 168, 178]. A better molecular characterization of cell death in pathogenic trypanosomatids is essential for advances in novel alternatives for therapeutic intervention.
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