Frontiers in Parasitology

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.

In 2005, the draft genome sequences of the parasites Trypanosoma cruzi, Trypanosoma brucei and Leishmania major, also known as the Tritryps, were published providing major insights into their genome structure and organization. Even though these parasites diverged around 200 to 500 million years ago, their core genomes are highly syntenic and conserved. These conserved regions are interspersed by retroelements, structural RNAs and species-specific genes related to host-parasite interactions. While T. brucei presents a 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. Duplication events have also shaped the genome architecture of these parasites. 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. These parasites also explore gene conversion mechanisms to generate variants and increase their surface complexity. Among the Tritryps, T. cruzi presents the most striking expansion of species-specific multigene families, which could be related to the parasite’s ability 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 the whole set of genes simultaneously. The functional implications of these chromosomal expansions to the parasite biology are still to be determined.

In this chapter, we focus on the structure and function of telomeres and subtelomeres of human protozoan parasites T. cruzi, T. rangeli and Leishmania spp.. Beyond their role in maintaining the integrity of chromosomes, telomeres and subtelomeres are involved in the survival mechanisms of these single-celled parasites. The telomeric repeat (5'-TTAGGG-3')n is conserved among trypanosomatid species, but adjacent subtelomers vary between species and chromosomes within the same cell. The chromosome ends of T. rangeli, for example, exhibit a simple organization with short subtelomeres whereas T. cruzi subtelomeres are a complex mosaic of genomic fragments including gene/pseudogenes corresponding to large gene families of surface proteins and retrotransposons. Differences in the copy number and organization of these genes determine the variation in the size of subtelomeres on each T. cruzi chromosome. Leishmania subtelomeres, in contrast, lack genes encoding surface antigens; instead they carry conserved repeat sequences referred to as telomereassociated sequences. T. cruzi and T. rangeli chromosomes share a high level of synteny which is lost in the subtelomeric regions. It has been suggested that T. cruzi subtelomeres can serve as recombination hotspots and thus promoting the increase of the repertoire of surface antigens. Many pieces of evidence indicate that telomere maintenance in Kinetoplastids occurs primarily by a telomerase-mediated elongation. The catalytic subunit of telomerase (TERT) is present in all sequenced trypanosomatid species, whereas the RNA component containing a template for telomere repeat extension has recently been identified in T. brucei and Leishmania. Further studies are needed to understanding the regulation of telomere homeostasis and the biology of subtelomeres of trypanosomatids.

Trypanosomatids are among the most primitive eukaryotes and therefore exhibit both conserved and unique non-conserved features in the DNA replication machinery. In eukaryotes, nuclear DNA replication is preceded by the assembly of the pre-replication complex (pre-RC), which is coordinated by the six-subunit origin recognition complex (ORC), which together with the Cdc6 and Cdt1 proteins play a central role in the loading of the hetero-hexamer Mcm2-7. In the domain Archaea there are no Cdt1 protein homologs, Mcm is a homo-hexamer, which is recruited by a protein that shows homology with ORC, and Cdc6 (called Orc/Cdc6). Curiously, trypanosomatid pre-RC differs from others eukaryotes in this context, and it appears more similar to that of Archaea, presenting a homolog of protein Orc/Cdc6 and no homologs of Cdt1, in addition to present Mcm as a hetero-hexamer complex. 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 serves as telomerase substrate, remains unknown. With the generation of overhangs at the ends of the chromosomes, telomeres are frequently extended by the action of telomerase, whose control also remains unknown. It is worth mentioning that 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 (kDNA) consists of mini- and maxicircles, which are replicated by many proteins whose mechanisms of action remain unclear. This chapter aims to review and discuss the complex DNA replication mechanisms that act independently in the kinetoplast and the nucleus, as well as some fascinating peculiarities exclusive to trypanosomatids protozoa group.

The genome is the source of life, providing the information needed to direct all aspects of organismal function. Propagation of life requires copying of the genome and faithful transmission from parent to offspring. Many challenges confront genome propagation, including ensuring the accurate and complete copying of the DNA, circumventing impediments to DNA replication, and maintaining genome integrity in the face of myriad insults and during periods of cellular quiescence. Just as importantly, the genome must be allowed to change, either incrementally through small mutations in sequence or by large-scale rearrangements. Such changes not only drive evolution, but can be integral components of an organism’s life cycle. In this chapter we consider the rapidly growing body of knowledge on how the genomes of kinetoplastid parasites are maintained, by describing the range of genome repair and damage tolerance pathways that operate. We focus on Trypanosoma brucei, Trypanosoma cruzi and Leishmania, three important human and animal pathogens, but we believe the lessons learned from the study of genome maintenance in these genetically tractable parasites are applicable widely, not only to other parasites but throughout biology.

As members of a highly divergent group of eukaryotes, trypanosomatids present peculiar mechanisms of gene expression. These protozoan parasites have transcription and processing machineries that constitutively transcribe clusters of nonrelated genes into polycistronic pre-mRNAs, which are subsequently trans-spliced into monocistronic transcripts. Because of this, control of gene expression relies mainly on post-transcriptional mechanisms that are, for the most part, mediated by RNA binding proteins that control steady-state levels of mRNAs and/or their translation rates. Using primarily Trypanosoma brucei as a model, several groups have begun to elucidate the basic regulatory mechanisms and to define the cellular factors controlling transcription, processing, degradation and translation of mRNAs in trypanosomatids. This chapter describes studies that have been focused on a subset of genes that are differentially expressed during the life cycle of T. brucei, T. cruzi and few species of Leishmania. Although a predominance of regulatory pathways acting at a post-transcriptional level is found for most genes from all three parasites, it is also evident that the regulatory strategies chosen by different trypanosomatid species are not similar. Because of their complex and diversified gene regulatory machinery, T. brucei, T. cruzi and Leishmania spp. are able to respond rapidly to the drastic environmental changes they face during their life cycle, particularly when they move between their different hosts.

Protozoan parasites of the genus Leishmania cause a group of diseases, known as leishmaniasis, affecting humans and also household pets, mainly canids. In the human host, different pathological outcomes ranging from self-healing cutaneous lesions to systemic visceral leishmaniasis are produced by these parasites; these diseases affect millions of people worldwide. Similar to a virus, bacteria and other parasites, Leishmania need to evade immune destruction with the aim of completing their life cycle in their mammalian hosts. Moreover, the long co-evolutionary history between parasites of the genus Leishmania and their hosts for several millions of years has led to a balanced relationship. To avoid the powerful immune system of mammals, the parasite has developed a set of sophisticated mechanisms to persist, replicate, and spread.

Among the pathogens that have developed a variety of strategies to overcome the host immune system, is the causative agent of Chagas disease, Trypanosoma cruzi. During a long co-evolution process, the parasite has learned how to live in many different environments, including vertebrate and invertebrate hosts. The parasite has also evolved many invasive strategies, including several different ways to enter the host and also the capacity to target different host tissues. An acute systemic response arises in the host after the rapid parasite colonization, interfering with both innate and adaptive immunity. The capacity of T. cruzi to interfere with humoral and cellular immune responses is demonstrated by the expression of different sets of molecules called virulence factors. Among them, the role of antioxidant enzymes, cruzipain, the Tc85/transialidase superfamily, mucins, MASPs, GPI anchors, complement regulatory proteins and others are discussed in this chapter. The expression of parasite-specific virulence factors allows T. cruzi to overcome host immunity successfully and also to invade and disseminate in many different mammalian hosts. However, the picture that has emerged suggests that the basis and mechanisms of parasite virulence could be more complex than expected. Different aspects such as parasite genetic diversity, the effects of polyparasitism and the potential effects that vertebrate and invertebrate hosts have on parasite virulence and the outcome of natural or experimental infection by T. cruzi should be taken into account in futures studies to understand T. cruzi virulence.

Chagas disease and leishmaniasis, caused by the kinetoplastid protozoans Trypanosoma cruzi and Leishmania spp., respectively, affect millions of people worldwide, most of them belonging to neglected populations. Diagnostic tests for Chagas disease are employed during epidemiological surveys of vectorial and oral transmission, blood bank screening, analysis of pregnant women and their newborns, and in individual cases. However, the currently available assays need improvement. The different phases of the disease, the transmission mode and the high genetic variability of the parasite increase the difficulties of making diagnostic kits with different markers suitable for the diverse scenarios of T. cruzi infection. Different Leishmania species cause diverse clinical features and sequelae and require different clinical management. In contrast to Chagas disease diagnosis, molecular diagnosis for leishmaniasis requires not only confirmation of the infection but also the genotyping of complexes, species or subspecies. Precise diagnosis and rapid species identification can facilitate decision-making respect to treatment and follow-up of parasite spread. The aim of this chapter is to summarize the most commonly used molecular tools described to date to detect T. cruzi infection and to detect and genotype Leishmania spp.

Chagas disease is caused by the protozoan parasite Trypanosoma cruzi affecting mostly the American continent eventually leading to chronic cardiomyopathy or digestive syndromes. Human African Trypanosomiasis is caused by the related parasite T. brucei, endemic in the African continent and being characterized by invasion and damage in the central nervous system. Leishmaniasis is caused by a number of species from the genera Leishmania and can manifest with different clinical outcomes including skin ulcers and visceral organ damage, being endemic in 88 countries in tropical areas of the globe. Despite having different geographical distribution and unique clinical symptoms, these diseases are all caused by related protozoan parasites from the order Kinetoplastida. Another aspect shared by these diseases is related to the treatment options currently available, unfortunately, all inadequate. Serious problems are toxicity and inefficacy due parasite acquired resistance or lack of natural susceptibility. The population affected by these three diseases does not represent an attractive economic market, reflecting on little pharmaceutical industry interest in developing better chemotherapies. However, in the last decade, the situation has dramatically improved, with the active engagement of philanthropic financial support, national government organizations, research centers, the pharmaceutical industry and the Drugs for Neglected Diseases Initiative (DNDi). After the development of screening assays, millions of molecules have been tested, and some have reached Clinical Trials stage. 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 and progress to achieve these goals are presented in this chapter.

The Leishmania spp, Trypanosoma cruzi and Trypanosoma brucei spp are the causative agents of tropical infections, and over 20 million people worldwide suffer from these neglected diseases. During the last century, vaccine development has had an undeniable impact on public health and may offer some alternatives for the control of parasitic diseases. Immune protection against experimental infection with these parasites has been studied and many types of immunogens have been used. Use of new technologies has allowed the development of recombinant proteins and DNA-based vaccines against those protozoans, aiming to generate both humoral and cellular protective responses. A large amount of data have been obtained from preclinical model systems which gave us promising results. The main challenge at the present is to translate what has been succeeded in these models into efficient human vaccines. The objective of this review is to summarize the efforts of the science community about the development of recombinant vaccines against trypanosomatids.