Molecular and Cellular Biology of Pathogenic Trypanosomatids E-Book
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- Herausgeber: Bentham Science Publishers
- Kategorie: Fachliteratur
- Serie: Frontiers in Parasitology
- Sprache: Englisch
Every year millions of people living in tropical areas across the globe, are affected by trypanosomatids – the parasites causing Chagas disease, African trypanosomiasis, and leishmaniasis. According to WHO, these diseases are termed as neglected tropical diseases against which there are no effective vaccines and the few available treatments have many side effects, besides posing the risk of emerging drug resistant parasite strains. All these factors represent a significant challenge which imposes a considerable economic burden to public health systems. Many research initiatives have emerged in recent years with the aim to undercover parasitic molecular and cellular biology, parasite-host interactions, mechanisms of disease pathogenesis, molecular mechanisms of drug resistance, all essential for the development of novel anti-parasite treatments and eradication strategies.
This volume highlights discoveries in the field of trypanosomatid molecular and cellular biology. Topics covered include cell organization during development, genome organization and maintenance, control of gene expression, nuclear and kinetoplast DNA replication, mechanisms of DNA damage repair, virulence factors and immune evasion, new methods for molecular diagnosis, new therapeutic tools and recombinant vaccine biology.
This monograph will be of interest to undergraduates (premedical and biologists), graduates (masters and Ph.D. students), the parasitology research community and researchers working in related fields.
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Frontiers in Parasitology
(Volume 1)
Molecular and Cellular Biology of
Pathogenic Trypanosomatids
Edited by:
Marcelo Santos da Silva
Maria Isabel N. Cano
BENTHAM SCIENCE PUBLISHERS LTD.
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FOREWORD
Since 1970, due to important financing programs, Brazilian scientists have been able to practice Biology while studying trypanosomatids, mainly Trypanosoma cruzi and Leishmania spp. The Annual Meeting in Caxambu since 1974, focusing on Trypanosoma cruzi at the beginning, and later extending the interest to other parasites, particularly Leishmania, were the catalysts of a scientific boom in Brazilian biology. Many young and enthusiastic students have been trained by the pioneers in the morphology, physiology, biochemistry, immunology and molecular biology of trypanosomatids, including vectorial transmission and clinical aspects of the diseases. The Caxambu meetings also brought to Brazil renowned scientists of the field who established important and long-lasting connections with Brazilians. After 2005, the field has gained momentum after the publication in Science of the complete genome of three species of the order Kinetoplastida, namely Leishmania major, Trypanosoma cruzi and Trypanosoma brucei. This e-book is a consequence of the Brazilian scientific activities in the field in the last 45 years. Maria Isabel Cano is my scientific grand-daughter and Marcelo Santos da Silva got his Ph.D. under her supervision.
The first chapter deals with the cellular organization of trypanosomatids during the life cycle. This group diverged early in evolution and due to this fact their members conserved certain characteristics not found in other eukaryotes as organelles like glycosomes, reservosomes and acidocalcisomes, among other cell compartments. Differences between species and between the distinct life cycle forms of each organism are comprehensively discussed.
Chapter 2 unveils the fascinating area of the trypanosomatid genomes. Information has been made available by the Tritryps project that provided major insights into the genome structure and organization of these parasites. While T. brucei presents subtelomeric expansion of genes related to antigenic variation, T. cruzi and Leishmania genomes contain species-specific genes related to cellular invasion and survival inside the mammalian host cells. As control of gene expression operates mainly at a post-transcriptional level in trypanosomatids, gene copy number variation is probably an efficient mechanism to enhance gene expression and increase sequence variability. Among the Tritryps, T. cruzi presents the most striking expansion of species-specific multigene families, which could be related to the ability of the parasite to infect any nucleated cell of a broad range of mammals. Chromosomal copy number variation is also well tolerated by these parasites, allowing the expansion of a whole set of genes simultaneously. The functional implications of these chromosomal expansions to the parasite biology are still to be determined.
Chapter 3 clarifies the function of chromosome ends and telomeres in trypanosomatid biology. These structures, in addition to the preservation of chromosomal integrity, play a major role in survival. The telomeric repeat (5'-TTAGGG-3')n is conserved among trypanosomatid species, but adjacent subtelomeric sequences vary between species and chromosomes within the same cell. For example, size and gene content of T. cruzi subtelomeres differ in each chromosome due to differences in the abundance and organization of these genes, whereas in Leishmania spp. subtelomeres show a more conserved organization.
Chapter 4 is a pleasant travel on nuclear and kinetoplast DNA replication in trypanosomatids. These organisms exhibit both conserved and unique non-conserved features in the DNA replication machinery. Curiously, the trypanosomatid pre-replication complex differs from other eukaryotes, having features similar to those of Archaea. The completion of DNA replication, at trypanosomatid telomeres, apparently is similar to other eukaryotes, although the processing of the leading and lagging telomeres required to generate the 3' overhangs, which serve as telomerase substrate, remains unknown. DNA replication in trypanosomatids initiates almost simultaneously in the nucleus and the kinetoplast, suggesting that regulation of DNA synthesis in the two DNA-containing organelles may be coordinated. The kinetoplast DNA consists of mini- and maxi-circles, which are replicated by many proteins with, as yet, unknown mechanisms of action. The complex DNA replication mechanisms, independently acting in both kinetoplast and nucleus, are reviewed.
Chapter 5 discusses mechanisms by which trypanosomatids maintain genome integrity and preserve faithful DNA replication despite multiple environmental aggressions. A growing body of evidence on how trypanosomatids recognize and repair damages is reviewed.
Chapter 6 explores one of the most fascinating features that make trypanosomatids unique organisms in the biological world. Despite being eukaryotic organisms they constitutively synthesize polycistronic mRNAs from separate gene clusters. Control of gene expression is not carried out at the DNA level but relies upon post-transcriptional mechanisms. This chapter aptly discusses the common post-transcriptional pathways for most genes, although many regulatory strategies within species of the group differ from each other. It is argued that these complex and diversified regulatory machineries allow rapid responses of these organisms to drastic environmental changes during their life cycle.
Chapters 7 and 8 describe virulence factors and the immune evasion in Leishmania spp and Trypanosoma cruzi, respectively. These parasites co-evolved with their hosts – mammalian and insects – for several millions of years and developed specialized strategies to evade the immune system by overcoming both innate and adaptive immune responses. The role of different species of molecules as virulence factors is discussed.
Diagnosis, chemotherapy, and potential recombinant vaccines are discussed in the ensuing chapters 9, 10 and 11, respectively. The diseases caused by kinetoplastids, called neglected diseases since they are predominant in poorer tropical countries having scarce resources, are responsible for thousands of deaths per year. No vaccines are available for these diseases. Presently, drug therapies are not very effective because the few available drugs are toxic, and treatment is costly. Comprehensively, methods of vector control are insufficient, despite the Southern Cone initiative in the nineties by which transmission of Trypanosoma cruzi in the domicile and outdoors by Triatoma infestans, at least in some countries, has been controlled. Chapter 9, stresses the need for reliable and specific diagnostic tests for epidemiological surveys, surveillance for vectorial transmission, blood screening, screening of pregnant women and their newborns, and in individual patients. The chapter summarizes the most commonly used molecular tools described to date to detect T. cruzi infection and to identify and genotype Leishmania spp. Chapter 10 discusses the present status of the chemotherapy area, emphasizing the screening assays that led to a few drugs reaching the stage of a clinical trial after a selection from millions of molecules tested. A milestone has been set for the year 2020, by the London Declaration: control of Chagas disease and leishmaniasis, and elimination of Human African Trypanosomiasis. Advances to achieve these goals are presented in this chapter. Finally, Chapter 11 deals with the research efforts on the development of recombinant vaccines against trypanosomatids, most of which are at the stage of preclinical experimentation. It is hoped that these efforts can be translated into efficient human vaccines.
This book is being edited in electronic form. In addition to the PDF edition accessible online, it will be aired in open access electronic media. Any reader can also have access to a printed version and also to individual chapters offered by the authors themselves. Thus, it is hoped that biologists, graduate students and post-doctoral researchers benefit with this up-to-date and competent review of the present literature on trypanosomatids.
PREFACE
Among the pathogenic trypanosomatids are the etiological agents of leishmaniasis, African trypanosomiasis and Chaga’s disease, protozoa parasites belonging respectively, to the genus Leishmania and Trypanosoma. The diseases are high prevalent in tropical areas of the globe and according to WHO cause thousands of new cases and deaths every year. It is worth noting though that international migration made Chaga’s disease and leishmaniasis becoming an issue also in many developing countries in Europe, Canada, USA, Australia, and Japan. Although the number of new cases and deaths from Chaga’s disease had decreased in the last decade, the millions of chronically infected persons who are at risk for developing cardiovascular and/or digestive pathology make Chaga’s disease one of the leading causes of cardiovascular morbidity and premature death in Latin America. Human African trypanosomiasis (HAT), also known as sleeping sickness, is endemic in sub-Saharan countries and is caused by T. brucei gambiense and T. b. rhodesiense. The number of HAT cases has been decreasing in the last years due to very active control efforts although according to a recent WHO report, country or regional averages may be misleading since the burden of the disease falls very heavily on some areas. HAT patients require lots of care and disease diagnosis and treatment can be costly and time-consuming. However, in many Africa countries diagnostics and HAT drugs are provided free of charge. Leishmaniasis in its turn is still considered a dangerous menace, principally to the poor, with about 12 million people currently infected. Depending on the species leishmaniasis can be expressed in different clinical forms, with the cutaneous form being the most common, causing self-limiting skin ulcer or a highly disfiguring scar, to the disfiguring and mutilating mucocutaneous form, and the visceral form is the most severe and fatal if not treated. All these diseases are still challenges to overcome since the absence of effective vaccines, and the toxicity of current anti-parasite drugs, in addition to the emergence of drug-resistant parasite strains and HIV co-infections are non-transposable barriers to disease control. Thus, many research initiatives have been direct to understand parasite biology and its interactions with different hosts, as well as the mechanisms of disease pathogenesis, of drug resistance and genome organization and maintenance, with the aim of the development of more efficient diagnostic tools and non-toxic and effective drugs and vaccines.
In this eBook, experts review and explore current knowledge about the molecular and cellular biology of trypanosomatids, highlighting the most important and actual discoveries in each research field. Topics covered include cell organization during development; genome organization and maintenance; control of gene expression; nuclear and kinetoplast DNA replication; mechanisms of DNA damage repair; virulence factors and immune evasion; new methods for molecular diagnosis; new therapeutic tools and recombinant vaccine biology.
We believe that the eBook content will be of keen interest to undergraduates, graduate students and principally to the Parasitology community and researchers working in related fields.
We would like to thank the contributing authors of this book for their time, expertise, and for making this eBook novel, educational, and informative and Professor Walter Colli for written the Forward.
Finally, we would like to express our gratitude to Mr. Shehzad Naqvi, the Senior Manager Publications from Bentham Science Publishers for his continuous help.
List of Contributors
The Cellular Organization of Trypanosomatids During Life Cycle
Simone Guedes Calderano1,Nilmar Silvio Moretti2,Christiane Araujo3,4,Marcelo S. da Silva3,4,Teresa Cristina Leandro de Jesus3,4,Loyze P. Oliveira de Lima3,4,Mariana de Camargo Lopes3,4,Leonardo da Silva Augusto2,Julia Pinheiro Chagas da Cunha3,4,Maria Carolina Elias3,4,*,Sergio Schenkman2,*Abstract
Trypanosoma cruzi , Trypanosoma brucei and Leishmania spp. are etiological agents of the following neglected diseases: African sleeping sickness (T. brucei ), Chagas’ disease (T. cruzi ) and leishmaniasis (Leishmania spp.). These parasites are eukaryotic cells that diverged early in evolution and therefore harbor modified organelles, such as glycosomes, and present subcellular compartments with unusual characteristics. This chapter aims to overview the most striking features of the structures and functions of these organelles, which ensure the existence of these parasites, and to discuss the differences between species and between the distinct life cycle forms of each organism.
INTRODUCTION
Trypanosomatids are unicellular flagellated eukaryotes that belong to the Kinetoplastida class, the members of which are characterized by the presence of a kinetoplast, which is a structure that contains the mitochondrial genetic material. The Kinetoplastida class includes the Trypanosomatidae family, which comprises human pathogens, such as Trypanosoma cruzi , Trypanosoma brucei and Leishmania spp. These are the etiological agents that cause Chagas’ disease, African sleeping sickness and leishmaniasis, respectively. It is currently estimated that 6.5 million people are infected with T. cruzi, that there are 1.3 million new cases of leishmaniasis and approximately 6,000 cases of sleeping sickness every year (www.who.int).
These protozoans have life cycles stages that possess different morphologies and cellular structures when living in mammalian and insect hosts. The T. cruzi epimastigote is a form that is defined by the lateral exit of the flagellum from the cell body; it is also the non-infective life cycle stage of the parasite. It proliferates via binary fission in the gut of Triatoma infestans insects, which are more commonly known as kissing bugs. In the insect hindgut, epimastigotes transform into the metacyclic-trypomastigotes in which the flagellum is inserted along almost the entire length of the protozoan. When the insect vector ingests blood from a mammalian host, the trypomastigotes are then eliminated with the feces. Released parasites can penetrate the mammalian host through contact with different mucosal tissues. They enter cells by forming a parasitophorous vacuole, which is then disrupted and the parasites transform into amastigotes, which are spherically shaped and have a very short flagellum. Amastigotes proliferate inside the cell cytosol and then transform into non-replicative trypomastigotes, which are released via cell lysis to reach the bloodstream. The life cycle is completed by the ingestion of the blood by insect vectors. The trypomastigotes are able to transform into epimastigotes that replicate inside the insect gut [1].
Leishmania, in contrast, alternates between a promastigote and an amastigote form. Promastigotes are protozoan with a flagellum attached to one extremity that develops in the digestive tract of sand flies. Promastigotes differentiate into metacyclic forms that are eliminated via regurgitation when the insect feeds. The parasites are then phagocytized by macrophages before transforming into amastigotes that divide inside vacuoles. After several rounds of division, the macrophages are disrupted, and the new amastigotes are released to infect adjacent macrophages. Insects ingest the infected macrophages when they feed on mammalian blood. Amastigotes transform into promastigotes in the insect midgut, continuing the life cycle [2].
Unlike what occurs in T. cruzi and Leishmania, the life cycle of T. brucei is entirely extracellular. An infected tsetse fly (Glossina spp.) bites a mammalian host; inoculating metacyclic-trypomastigote forms into the circulatory system. The injected metacyclic-trypomastigotes transform into bloodstream trypo-mastigotes, which then proliferate in the hemolymphatic system as slender trypomastigotes. T. brucei species survive the immune defenses of the host by continuously changing their coat, which is formed by a single variant surface glycoprotein (VSG) through a process known as antigenic variation. When a high density of parasites is achieved in the blood, some of these parasites transform in a non-proliferative, stumpy form. This form is able to differentiate into procyclic trypomastigotes when ingested by new tsetse flies. In the fly midgut, the procyclic form proliferates and then migrates to colonize the salivary glands of the insect, where they transform into epimastigotes that can proliferate by binary fission. After some rounds of duplication, the epimastigotes become metacyclic trypomastigotes, which are then injected into a new mammalian host during a tsetse fly’s bite [1].
These organisms contain organelles that are common to all eukaryotes, but they also harbor unique organelles, such as the kinetoplast, reservosomes, glycosomes and flagellum-related structures, all of which present peculiar features as a consequence of their earlier evolutionary origins and the requirements of adaption (Fig. 1).
This chapter aims to present recent developments that have increased our understanding of how these organelles ensure the survival of the organism in different hosts during parasite life cycles. We have also compared the features that are unique to each trypanosome.
Fig. (1)) Schematic representation of the Trypanosomatid forms found in insects and mammals. The figure on the left illustrates the main replicative forms, which in T. brucei are procyclic-trypomastigotes, in Leishmania are promastigotes, and in T. cruzi are epimastigotes. The right panel shows the corresponding forms that are found in the mammalian hosts. The nucleus is larger than the kinetoplast, and both are shown in blue.NUCLEUS
The nucleus is the largest organelle within the cell, and it stores the genetic material. The first nucleated cell, referred to as the first eukaryotic common ancestor, emerged approximately 2.5 billion years ago and is the ancestor from which all eukaryotic cells, including the Kinetoplastida protozoan parasites, diverged [3, 4].
The nuclear environment is delimitated by the nuclear envelope, which is a double bilayer membrane. The inner membrane faces the nuclear lumen, while the outer membrane faces the cytoplasm. The nuclear envelope is mechanically supported by a nuclear lamina underneath the inner membrane and by another filament network that surrounds the outer membrane. Nucleus-cytoplasm communication occurs through nuclear pore complexes (NPCs).
The nucleus of trypanosomes undergoes several morphological changes during the life cycle. For example, while the replicative forms of T. cruzi (epimastigotes and amastigotes) have a rounded nucleus with a clear nucleolus and with heterochromatin at the nuclear periphery, the non-replicative and infective bloodstream trypomastigote and metacyclic trypomastigote forms have an elongated nucleus without nucleolus. Trypomastigotes also lose peripheral heterochromatin [5]. It remains unknown what mechanisms drive these structural changes, but they might be related to modifications in the NUP-1 protein, a protein that is associated with the inner nuclear membrane [6, 7].
Nuclear Organization
Many processes occur in the nuclear space at sites that are non-randomly distributed, meaning that all nuclear events are organized in the space during cell cycle progression. In higher eukaryotes, the formation of heterochromatin and euchromatin are closely linked to the transcriptional status of each DNA region. In addition, the transcriptional status dictates the timing of DNA replication. In other words, while highly transcribed genes are replicated at the beginning of S phase, silent regions and sequences with low transcriptional rates are replicated at the end of S phase. In addition, the location of early and late replication regions varies during S phase, during which discrete replication sites are found throughout the nuclear space at the beginning of S phase, and a more peripheral replication pattern is observed during the final stages of S phase [8].
Trypanosomes display a peculiar nuclear organization. In replicating T. cruzi epimastigotes, RNA polymerase II transcription sites are dispersed throughout the nucleus, with the specific transcriptional site of the spliced leader (SL) genes concentrated in a region adjacent to the nucleolus [9]. SL is transcribed at a high rate because its mRNA serves as the precursor for the first 39 nucleotides that are added to all pre-RNAs. These are transcribed as polycistronic units, and they form mature mRNAs via a trans-splicing reaction. The site of SL transcription in the nucleus is different between T. cruzi, and T. brucei . While in T. cruzi , only one SL transcription site has been observed in the nucleus [10], in T. brucei , two major sites are observed, probably as a consequence of the ploidy of the SL gene repeats in this parasite [11, 12]. This is not observed in Leishmania spp., which, like T. cruzi , has only one SL transcription site. Details about gene transcription in trypanosomatids are described in Chapter 6.
The replication machinery is located in the trypanosome nuclear periphery, revealed by the presence of the sliding clamp of the DNA polymerase machinery and by the incorporation of thymidine analogs that were detected using fluorescence techniques [13, 14]. Moreover, the distribution of chromosomes varies according to the cell cycle stage. At the onset of S phase, T. cruzi chromosomes are dispersed; they then progressively migrate to the nuclear periphery, where replication takes place, and remain there until mitosis is completed. The chromosomes then disperse again throughout the nucleus [14]. It is interesting to note that the same replication organization pattern is not observed in T. brucei and L. donovani. In both parasites, the sliding clamp of the replication machinery is found at several sites distributed throughout the nucleus, indicating a distinct type of nuclear organization [15, 16]. These different localizations of transcription and replication machinery could explain how DNA replication and transcription are maintained separately in the nuclear space, and they may imply that trypanosomes display a rudimentary level of nuclear organization compared to that observed in more complex eukaryotes [17, 18].
Another difference in nuclear organization is the expression of the VSG and procyclin genes, which encode the coat protein of procyclic form in T. brucei . Active VSG genes are located in the telomeric regions of T. brucei chromosomes, and only one gene is expressed at a time. Transcription of this single VSG gene is catalyzed by RNA polymerase I [19] in a locus present in a nuclear region called the expression site body [20]. Procyclin genes, are also transcribed by RNA polymerase I and its transcription occurs at the nucleolus periphery. In contrast, the silent loci for the VSG and procyclin genes remain in the nuclear periphery within the heterochromatin [21, 22]. Differences in telomere distribution have also been observed in L. major, in which telomeres were found to be dispersed in promastigotes but concentrated in the center of the nucleus in amastigotes [23]. These observations collectively indicate the presence of variable nuclear structures and chromosomal localizations, which reflect adaptations in each parasitic stage to the conditions found in the host during their life cycle.
Nuclear Pore Complex
Nuclear pore complexes (NPCs) allow nucleus-cytoplasm communication through the nuclear envelope. In eukaryotes, approximately thirty proteins called nucleoproteins (Nups) participate in the formation of NPCs. The NPC structure consists of a central transport channel, a core scaffold, a cytoplasmic ring, a nuclear ring and eight filaments attached to each ring [24]. The nucleoproteins from the core scaffold in most eukaryotes are arranged in an octagonal framework that surrounds a central transport channel, while the phenylalanine-glycine (FG) repeats from these nucleoproteins occupy the center of the transport channel and are involved in transport and interactions with cargo-carrying transport factors [25]. The nuclear basket proteins associate with the chromosomes influence gene expression by regulating the transport of mRNA and gene regulatory factors. They bind to specific transcription factors and transcriptionally active genes, promoting the assembly and/or maintenance of gene loops, in which the promoter and terminator regions are juxtaposed, accelerating the reactivation of gene transcription [26].
The NPC octagonal symmetric framework structure is highly conserved among eukaryotes [27, 28] and differs mainly in its size. In yeast, it is approximately 60 MDa, while in humans, it is 90-120 MDa. However, the proteins forming the complex are not highly conserved among eukaryotes. In T. brucei and Leishmania major , 22 Nups were identified through proteomic analyses [29, 30]. From these, 20 and 16 were localized in the nuclear envelope in T. brucei and L. major, respectively. Similar genes were found in T. cruzi. However, the precise location and function of each protein in the NPC have not been established. TbNup92 and TbNup110 from T. brucei and LmMlp2 from L. major are analogs to the Saccharomyces cerevisiae nuclear basket proteins Mlp1 and Mlp2 that were found to be involved in chromosome segregation during the closed mitosis observed in these organisms [24, 29-31]. It is worth mentioning that TbNup92 is larger than its yeast analog because it contains an extra domain that is typically found in DNA repair and cell cycle checkpoint proteins. These data indicate another possible function of NPCs in trypanosomes.
Nuclear Lamin
Lamins (lamin A, B and C) are intermediate filament proteins that form a meshwork between the nuclear envelope and the nuclear matrix. Lamins associate with peripheral heterochromatin and have diverse functions in Metazoans. They provide support for NPCs and control gene expression during development [32-36]. In non-Metazoan organisms, no lamin orthologues have been identified. For example, lamins are absent in S. cerevisiae [37, 38]. However, T. cruzi and T. brucei contain a coiled coil protein called NUP-1 that resembles lamin and is located in the inner nuclear envelope [6, 7]. This protein lacks similarity with metazoan lamins but has a conserved structure. It has a conserved pattern of coiled-coil domains, suggesting that it is capable of self-assembly, which is characteristic of lamin proteins in metazoans. In T. brucei and T. cruzi, NUP-1 forms a net-like structure at the inner surface of the nuclear envelope and interacts with some chromosomal regions, similar to what has been observed in Metazoan lamins. In T. brucei , NUP-1 also plays a role in telomeric silencing, in the regulation of VSG gene expression and chromatin organization. T. cruzi NUP-1 interacts with some chromosome regions that encode clusters of genes for surface proteins. Therefore, it appears that these interactions are important not only for chromatin organization but also for the control of gene expression in trypanosomes [6, 7, 39].
Nucleolus
The nucleolus is a subcompartment of the nucleus wherein processes including rRNA transcription, pre-rRNA processing and ribosome assembly occur. In addition, the nucleolus is involved in protein sequestration in response to stress, cell cycle control and RNA processing [40-42]. The nucleolus has a tripartite architecture: a fibrillar center and dense fibrillar and granular components. In trypanosomes, a single nucleolus is found in the nucleus, and it has a bipartite fibrillar center and granular component structure [43]. Furthermore, in trypanosomes, the nucleolus is smaller than in most eukaryotes, having a diameter of approximately 1 µm and some peculiar characteristics. In T. cruzi, it is not present in trypomastigote forms, which are a non-dividing stage, while it is present in the T. cruzi replicative epimastigote and amastigote forms. In Leishmania and T. brucei , in contrast, the nucleolus is present throughout the major stages respectively promastigotes and amastigotes, bloodstream and procyclic trypomastigotes. Proteins and mRNA are sequestered in the nucleolus under stress conditions, such as the induction of severe heat-shock in L. major and T. cruzi [44], but this does not occur in T. brucei [45-47].
Chromatin
Chromatin is composed of tightly associated nuclear DNA and proteins. The fundamental unit of chromatin is the nucleosome, in which two copies of each core histone (H4, H3, H2A and H2B) form an octamer of proteins that wraps around every ~154 base pairs (bp) of DNA. This regular array of nucleosomes, which forms 10-nm fibers, has been observed under electron microscopy to form a “beads-on-a-string” structure. A fifth histone called histone H1 (also known as linker histone) associates with the DNA located between two nucleosomes and compacts chromatin into 30-nm fibers known as solenoids. The majority of eukaryotes, however, further compact their chromatin (approximately 15,000 x), as has been observed in fully compacted chromosomes during mitosis. Thus, multiple levels of folding and compaction have been detected in eukaryotes and is used to fit the genomic information into a tiny nucleus (for a recent review, see [48].
Trypanosome chromatin is organized into nucleosome filaments, which form the same 10-nm fibers that have been observed in eukaryotes. However, neither 30-nm fibers nor condensed chromosomes during mitosis have been observed. A possible explanation for this is the lack of a typical N-terminus and the globular domain of histone H1 and/or the absence of a phosphorylation site at H3S10, all of which are associated with condensed chromosomes during mitosis [49]. Trypanosome histones diverged from fungi and other metazoan histones, and a disproportionate amount of this divergence has been observed in their N-terminal domains [50]. The migration patterns of histones in polyacrylamide gels made at acid pH, in urea and in non-ionic detergent, which differentiate histones by their charges and modifications, have also revealed differences between parasites such as T. cruzi and T. brucei [49].
Histone H1 is the most divergent histone. It comprises only the C-terminal domain of the eukaryotic histone H1. In T. cruzi , histone H1 is constitutively synthesized throughout the cell cycle but at an increased rate during S-phase, while the other histones are synthesized concomitant with DNA replication [51]. In contrast, the expression of histone H1 is related to the progression of the cell cycle and cellular differentiation in L. major [52], and histone H1 is not essential to T. brucei growth in vitro. T. brucei lacking H1 live longer than wild-type T. brucei in infected animals, probably because H1 depletion resulted in significant changes in the expression of RNA-Pol I genes (VSG and ESAGs). There is evidence showing that trypanosome histone H1 may also play a role in DNA repair because H1-depleted clones were clearly more resistant than the wild-type clones to drugs that induce DNA damage [53, 54].
In addition to the canonical histones (H2A, H2B H3 and H4), variant forms of these histones are present in trypanosomes. These variants are present in reduced amounts but may have unique functions that affect gene expression, centromere function and DNA damage/repair [55]. They include the histone variants H2A, H2B and H3. T. cruzi and T. brucei also encode H4 ‘orphans’ that have 85% and 96% identity to canonical H4, respectively [56]. Despite this finding, trypanosomes do not contain homologs for H3.3, a variant that replaces histone H3 at highly transcribed sites, or CenH3, a variant that is found at the centromeres of eukaryotes. T. brucei H3v shares some similarity with CenH3, but it localizes to telomeres and is not required for viability or chromosome segregation [57].
T. cruzi and other trypanosomes do contain H2AZ and a new variant, H2B (H2ABv), which is associated with transcriptional activation, gene silencing and the avoidance of heterochromatin formation in euchromatin regions [55]. Studies performed on T. brucei have shown that H2Bv, which has been identified in few organisms and only under specific situations [58], dimerizes with H2AZ and is essential for cell viability. Interestingly, these dimers are absent from highly transcribed sites [59].
Histone Post-Translational Modifications (PTM) in Trypanosomes
Histones can, in general, be chemically modified both at the tail, which extends from the nucleosome, and inside the octamer. Modifications, such as phos-phorylation, acetylation, methylation (mono, di or tri), and sumoylation, have been identified in histones and have, more importantly, been associated with gene expression, DNA repair and replication, chromosome condensation and other important regulatory processes in cells. Although trypanosome histones are highly divergent from the histones identified in other eukaryotes, their sequences, including their tails, are conserved among trypanosomatids, and specific (as well as unusual) PTMs have been identified.
T. cruzi histone H1 is phosphorylated in a cell cycle-dependent manner at a typical cyclin-dependent kinase site (12SpPKK), most probably by TzCRK3 (a T. cruzi CDK-like protein), as has been observed in higher eukaryotes, mainly from S to mitosis [60]. Phosphorylated histone H1 is homogenously distributed in foci in T. cruzi epimastigote nuclei, which appear in the G2 phase of the cell cycle, increase until mitosis and disappear during cytokinesis. Interestingly, the majority of non-phosphorylated histone H1 is located in the central regions of the nucleus, but when cells progress to G2 phase, it becomes phosphorylated and begins to diffuse in the nuclear space [61, 62].
PTMs at histone H4 of T. cruzi were identified using mass spectrometry (MS). Lysines (K) 4, 10, 14 and 57 are acetylated (ac); K18 is mono-methylated (me1), and arginine (R) at position 53 is dimethylated (me2) [63]. In epimastigotes, the majority of histone H4 is acetylated at K4, while less than 5% of H4ac proteins are acetylated at K10 and K14, suggesting that these modifications play a regulatory role [64]. H4K4ac decreases following DNA damage and in non-proliferative forms. In contrast, H4K10ac and H4K14ac increase at these times. Histone acetylation is well known for its role in transcription. However, no colocalization was observed between acetylations of histone H4 and the major RNA Pol II labeling site in T. cruzi , which corresponds to SL transcription. Nevertheless, H4K10ac and H4K14ac were found at eu- and heterochromatin boundaries, which are involved in events related to the active metabolism of DNA. No MS analyses have been performed on the remaining histones of T. cruzi , although radiolabeling experiments in parasite cultures have indicated that histone H2A is mainly acetylated, while H3 and H2B are mainly methylated [62, 63].
A complete scenario for histone PTMs has been described for T. brucei . Electrophoresis analyses indicated that H3 and H2A are highly modified compared to H4 and H2B [65-67]. For example, the methylation states of H3K76 in T. brucei depend on two methyltransferases called DOT (Disruptor Of Telomeric silencing, DOT1A and DOT1B), which is unlike to other eukaryotes that have a single DOT1 enzyme. The methylation of H3K76 appears to be analogous to the methylation of H3K79 observed in other eukaryotes, which has been found to be associated with the prevention of heterochromatin formation [68]. H3K76me2 is regulated by the cell cycle and occurs in T. brucei during mitosis, while K76me3 is present throughout the cell cycle. Furthermore, DOT1A is essential for viability, while DOT1B is not. However, DOT1B is necessary for differentiation during the transition to procyclic forms and for VSG switching and silencing [69]. In contrast to other organisms, the T. brucei histone H2A and H2B N-terminal domains show few modifications, while the C-terminal domain of H2A is highly acetylated. To date, no function has been attributed to these PTMs.
One of the earliest markers of DNA damage is the phosphorylation of the H2AX histone. In mammals, serine 139 is phosphorylated in the histone variant H2AX, while the replication-dependent H2A histone is phosphorylated in yeast. In T. brucei , it has been shown that the majority of gamma H2A becomes phosphorylated at threonine 130 in response to DNA damage [70]. This phosphorylation could play a role in signaling the presence of damaged DNA. The region surrounding the phosphorylated residue is highly conserved in trypanosomes, suggesting that they might also be modified in other species.
Chromatin Regulation at Specific Genomic Regions
Trypanosomatid genes are distributed in large co-directional clusters that are expressed as polycistronic transcripts. These clusters, which may be located in different DNA strands or in the regions between clusters, have been proposed as transcription initiation sites [71, 72]. A TATA-binding protein and a Small Nucleolar-activating protein complex (SNP50) that is involved in initiating the transcription of small nuclear RNA were found to be associated with tRNA, snRNA, and SL RNA gene promoter regions [73]. Whether and how these factors are involved in the initiation of transcription in trypanosomatids is unknown. Furthermore, non-canonical DNA regulatory sequences have been identified in trypanosomes, suggesting that their chromatin structure and the presence of histone modifications may play a prominent role in the initiation of transcription [74]. Chromatin immunoprecipitation (ChIP) assays performed in T. cruzi showed enrichment in H3K4me3, acH3 and acH4 at divergent DNA strand regions [75]. A similar scenario was observed in a genome-wide ChIP-chip analysis of L. major promastigotes that showed that the majority of the acetylated H3-enriched regions are at divergent strand-switch regions [73]. The probable transcription start sites of T. brucei are enriched with H4K10ac and H3K4me3. Interestingly, H4K10ac represents only 10% of the total H4. Moreover, H2AZ, H2BV, and the bromodomain factor BDF3 are enriched up to 300-fold in the same regions. In contrast, the histone variants H3V and H4V are enriched at probable transcription termination sites. No acetylated histones H3 and H4 were detected in the promoters of highly expressed genes, such as 18S rRNA and SL RNA, at which the depletion of nucleosomes was detected. A similar observation was made in L. tarentolae, in which the SL promoter lacks nucleosomes [10]. Supporting its potential presence at the heterochromatin, the highly repetitive satellite DNA was found to be depleted of H4ac [75]. These results suggest that chromatin structure might regulate the transcription of polycistronic genes by RNA Pol II, as indicated by the presence of special PTMs and variants at its initiation and termination sites [76, 77].
DNA Modifications
Trypanosome DNA is uniquely modified by the addition of glucopyranosyl residues to nucleotides, resulting in the formation of β-d-glucopyranosyl-oxymethyluracil. This modification was called ‘base J’, and it was discovered in T. brucei [78, 79] because silenced telomeric VSG sequences were found to be resistant to some nucleases. J-base predominates in the telomeric repeats but is also present within sequences flanking the polycistronic units. The exact role of this modification remains unknown. Decreased levels of base J increase transcription by RNA Polymerase II, resulting in genome-wide increases in global gene expression [80, 81]. Similarly, 99% of DNA base J is found at telomeric sites in Leishmania, and the remainder is located at RNA Pol II termination sites. Knockouts of JBP2 (J Binding Protein 2) in Leishmania, one of the enzymes responsible for catalyzing the formation of base J, reduced the level of internal J and generated a massive read-through of transcriptional termination sites. Therefore, inducing the reduction of J in L. major resulted in genome-wide defects, likely as a consequence of the generation of antisense RNAs by avoiding transcriptional termination at the end of polycistronic gene clusters. In T. brucei , the loss of J base also affected the termination of gene expression at specific sites within polycistronic gene clusters, but the termination is only affected by depletion of the histone H3V [82].
CYTOSKELETON
The cytoskeleton of trypanosomes is quite simple compared to that of free-living flagellates. A subpellicular corset of microtubules cross-linked to each other underneath the plasma membrane maintains the peculiar cell shape of different trypanosome stages and forms. This cytoskeleton forms a dynamic matrix that also includes the mitotic spindle and encloses some specific regions of the trypanosomatid cell, such as the flagellum attachment zone, the paraflagellar rod, the flagellum, and basal bodies [83-85]. Trypanosome microtubules have an intrinsic polarity and are formed mainly by α and β-tubulin heterodimers, which undergoes several post-translational modifications [83, 84]. These organisms also contain structures similar to microfilaments, which are composed mainly of actin and are distributed throughout the cytoplasm as rounded and punctuated structures [86]. Actin is present along the flagellum but is more concentrated at the base and the tip [87]. In Leishmania species, actin is also associated with the nucleus, kinetoplast, plasma membrane, and flagellum, and it is found associated with subpellicular microtubules [88]. Knockdown of actin genes in T. brucei affected endocytosis, indicating a role for microfilaments in trypanosome membrane trafficking [89].
Trypanosomes display mitotic spindles, which are composed mainly of microtubules. Their spindles include longer ‘pole-to-pole’ microtubules and a classical kinetochore, which is a protein structure that is associated with chromosomes, to which the fibers of the mitotic spindle attach during cell division [83]. Unlike other eukaryotes, the microtubule organizing centers associated with spindle poles are often anchored in the nucleoplasm rather than to the internal face of the persistent nuclear envelope [83]. During mitosis, the nucleolar material persists and is stretched along spindle microtubules, as can be visualized using indirect immunofluorescence with an antibody against tubulin [83, 90].
Trypanosomes contain at least five different centrins, which are calcium-binding proteins that are associated with basal bodies. Centrins are involved in organelle duplication and segregation. Depletion of centrins 2 and 4 in T. brucei caused abnormal cell division, which resulted in enlarged cells that contained duplicated basal bodies and multiple nuclei and displayed new flagella that were detached along the length of the cell body [91]. T. brucei centrin 3 was detected in the flagellum, and its depletion affected motility by disrupting its interactions with flagellar dynein [92]. Furthermore, centrins participate in the formation of a unique structure called a bilobe, which is associated with the flagellum and is probably involved in the formation of the flagellar pocket [93].
It is possible that the mechanisms by which the different trypanosomes have adapted to obtain different nutrients have shaped the evolution of these various structures. For example, T. cruzi, unlike other trypanosomes, has retained an additional feeding apparatus called a cytostome that is located, outside the flagellar pocket [94]. The structural features of this element and the molecular basis of its retention should be further investigated in T. cruzi.
FLAGELLUM
Phylogenetic evidence indicates that it is likely that the last common ancestor of eukaryotes had a flagellum that provided motility and sensory information [95, 96]. This flagellum may have been set as a tail-like structure that emerged from the cell. Although motility and sensory perception functions have already been established for trypanosomatids flagella, recent functional studies have shown that they also have other roles, for example, in cell signaling, cellular morphogenesis, the direction of cytokinesis during cell division, and immune evasion [97-101]. Therefore, the trypanosome flagellum is considered an essential and multi-functional organelle that is typical of unicellular organisms [98, 101, 102].
Trypanosomes present a single flagellum that normally emerges from a specialized invaginated region of the plasma membrane that is called the flagellar pocket [103, 104]. Nine outer ring microtubule doublets and a central pair of microtubules form the inner core of the flagellum, and these are collectively called the axoneme [98, 103]. The axoneme is assembled on the basal body, which is itself attached to the kinetoplast [103, 105]. The basal body is an organelle that corresponds to the centriole of higher organisms and that acts as a microtubule organizing center that forms the flagellum axoneme. Each cell contains two basal bodies, but only one gives rise to the flagellum [103, 104] (for details regarding the flagellar structure and its composition, see the figures in the review [98]). In trypanosomes, the flagellum contains a large paracrystalline filament structure known as the paraflagellar rod (PFR) [106]. The PFR is a stable lattice-like structure that is formed mainly by two major proteins (PFR1 and PFR2) and several minor proteins [104, 106, 107]. Some studies have suggested that the PFR provides a scaffold for the assembly of the regulators of axoneme motility [98, 108, 109]. The flagellum plasma membrane is covered by specialized domains and has distinct biophysical properties compared to the plasma membrane of the rest of the cell body [110-112].
The flagella of organisms within the genus Trypanosoma are the most well-studied and well-characterized among the trypanosomatids parasites [113-116]. T. cruzi epimastigote and trypomastigote flagella are long but become reduced in size in amastigotes, which are adapted to survive inside host cells. The paraflagellar rod is absent in amastigote flagella [83, 105]. The small amastigote flagellum may be involved in sensory perception and cellular organization, which are critical for intracellular survival during the intracellular parasite stages within host cells [100, 104, 105].
Unlike T. cruzi , all T. brucei stages possess a long flagellum [117]. By using genetic modifications, mainly in T. brucei , flagella have been shown to be a key structure in the coordination of trypanosome morphogenesis [101, 118], host-parasite interactions [101, 119], mitochondrial DNA segregation [120], virulence [101] and cell division [101, 118, 121]. Furthermore, knockout and knockdown of some paraflagellar rod components showed that the paraflagellar rod is a key element in flagellar motility [108, 109, 122], which is necessary for parasite survival in the host [102]. Proteins located at the base of the flagellar axoneme are also involved in functions related to virulence and cytokinesis [123]. Therefore, the trypanosome flagellum can be considered a critical interface between the parasite and its hosts [111, 124].
MITOCHONDRION
Mitochondria were discovered as a collection of granules that form threads inside the cell. Mitochondria house the machinery that produces energy and synthesizes specific metabolites and iron-sulfur clusters. These functions make it one of the most important organelles in eukaryotic cells. This importance is reflected in their wide distribution, their number and their sizes in different organisms [125]. Multicellular organisms typically contain a vast number of mitochondria, reaching approximately 2,000 mitochondria per cell in the liver. Single-celled organisms, such as Apicomplexa and Kinetoplastida, possess a single ramified mitochondrion that has peculiar ultrastructural aspects that are different from those of multicellular organisms, especially in the density of its matrix and the number and shape of its cristae [114].
Paulin confirmed the unitary nature of the mitochondrion in trypanosomatids in a series of papers based on 3D reconstructions [126, 127]. The models generated by Paulin have a fidelity and a resolution that remains impressive, even today. In general, the mitochondrion of the trypanosomatids has a dense and sparse matrix that is distributed in branches under the subpellicular microtubules; but this structure can vary depending on the species and strain of the trypanosome. Moreover, changes in environmental and nutritional conditions can affect the size of the mitochondrion, which, depending on the situation can fill up to 12% of the volume of the parasite [128].
The mitochondrial structure of T. cruzi varies during its life cycle and assumes different shapes depending on its form. In epimastigotes, the mitochondrion has the shape of an inverted triangle. The base of the triangle encloses the nucleoid of the kinetoplast [126]. Two closely appressed finger-like projections extend from this base into the lancet-like apex of the cell. The mitochondrion of the trypomastigote form of T. cruzi is unbranched, appearing as a sausage-shaped structure with a smooth outer surface. The kinetoplast nucleoid, which contains the mitochondrial DNA, is found at the posterior end of the mitochondrion within a bulbous swelling. A short, finger-like projection of the chondriome pervades the truncated posterior pole of the cell, with the major portion coursing distally through about two-thirds of the cell body, anterior to the kinetoplast [129]. Amastigote mitochondrion is morphologically dissimilar to the structure found in the trypomastigotes (sausage-shaped) or epimastigotes (reticulated), instead presenting with a circular or horseshoe-like shape [127
