Trypanosomatid comparative genomics: Contributions to the study of parasite biology and different parasitic diseases

Abstract

In 2005, draft sequences of the genomes of Trypanosoma brucei, Trypanosoma cruzi and Leishmania major, also known as the Tri-Tryp genomes, were published. These protozoan parasites are the causative agents of three distinct insect-borne diseases, namely sleeping sickness, Chagas disease and leishmaniasis, all with a worldwide distribution. Despite the large estimated evolutionary distance among them, a conserved core of ~6,200 trypanosomatid genes was found among the Tri-Tryp genomes. Extensive analysis of these genomic sequences has greatly increased our understanding of the biology of these parasites and their host-parasite interactions. In this article, we review the recent advances in the comparative genomics of these three species. This analysis also includes data on additional sequences derived from other trypanosmatid species, as well as recent data on gene expression and functional genomics. In addition to facilitating the identification of key parasite molecules that may provide a better understanding of these complex diseases, genome studies offer a rich source of new information that can be used to define potential new drug targets and vaccine candidates for controlling these parasitic infections.

Keywords: Leishmania major; RNAseq; Trypanosoma brucei; Trypanosoma cruzi; genome.

Figure 1

The Tri-Tryp life cycles. Representation of the life cycles of Leishmania major, Trypanosoma cruzi and T. brucei, the etiological agents of leishmaniasis, Chagas disease and sleeping sickness, respectively, are shown, with the parasitic forms that are present in the insect vectors and the mammalian hosts. Leishmania major proliferates as promastigotes (P) in the sand fly midgut. The parasite is transmitted during bites by this fly and invades mammalian macrophages in the metacyclic promastigote (M) form. Inside the cell, the M form is converted into amastigotes (A) and divides before been released during cell lysis. Trypanosoma cruzi replicates as epimastigotes (E) in the reduviid bug midgut and develops into infective metacyclic trypomastigotes that are excreted in the feces (M) and invade different cell types when in contact with the mammalian host. After differentiation into proliferative amastigotes (A), these are transformed into bloodstream trypomastigotes (T) that cause cell lysis and invade new cells. Trypanosoma brucei differentiates from procyclic (P) to epimastigote (E) proliferative forms in the tsetse fly before being transformed into infective, metacyclic forms (M) in the salivary glands. After being injected into the host during a blood meal, M forms differentiate into long slender forms (L) that proliferate in the bloodstream and can reach the central nervous system. After increase of parasite numbers these last forms are replaced by non-proliferative stumpy forms (S).

Figure 2

Gene organization in the Tri-Tryp genome. Panel A shows the gene distribution in a 0.8 Mb region of T. brucei chromosome V with eight large polycistronic transcription units (blue arrows: plus strand encoded open reading frames or ORFs; red arrows: minus strand encoded ORFs). In panel B, a genomic region at around 960 kb is magnified to show the gene synteny in the genomes of various trypanosomatids (blue and red boxes correspond to + and − strand-encoded ORFs, respectively). The orange line in both panels corresponds to the chromosome position. Sequence information used to draw panel A and the graphic representation in panel B were obtained from the Tri-Tryp database (Aslett et al., 2010).

Figure 3

Gene expression in trypanosomatids. Large clusters of unrelated genes (arrow boxes) are organized as polycistronic transcription units (PTUs) that are separated by divergent or convergent strand-switch regions. RNA Pol II transcription start sites (TSS) are usually located upstream of the first gene of the PTU (Martínez-Cavillo et al., 2004) or can be located as an internal TSS (Kolev et al., 2010). At the TSS (large bent arrow), the histone variants H2AZ and H2BV (Siegel et al., 2009), modified histones [K9/K14 acetylated and K4 tri-methylated histone (Respuela et al., 2008; Thomas et al., 2009; Wright et al., 2010) and K10 acetylated histone H4 (Siegel et al., 2009)], bromodomain factor BDF3 (Siegel et al., 2009) and transcription factors TRF4 and SNAP50 (Thomas et al., 2099) are frequently associated, with a few of these chromatin modifications also detected at internal TSS (small bent arrow) (Siegel et al., 2009; Wright et al., 2010). The polycistronic RNAs (pre-mRNAs) are individualized in monocistronic mRNAs after the addition of a capped splice leader RNA through a trans-splicing reaction coupled to polyadenylation. These processing reactions are guided by polypyrimidine tracts (PolyPy) that are present in every intergenic region. Mature mRNAs are exported to the cytoplasm where their stability and translation efficiencies are largely dependent on cis-acting elements present in their untranslated region (UTR) (Araujo et al., 2011). Transcriptomic analyses also showed that polycistronic pre-mRNAs can suffer alternative RNA processing that may result in changes in the initiator AUG, thereby altering protein translation (A), targeting and/or function (B). Alternative splicing and polyadenylation can also result in the inclusion/exclusion of regulatory elements present in the 5′ UTRs (C) or 3′ UTRs (D), thereby altering gene expression (Kolev et al., 2010; Nilsson et al., 2010; Siegel et al., 2010).

Figure 4

Retention and loss of RNAi genes in trypanosomatids. (A) Schematic representation of the mechanism of RNA interference (RNAi) present in some trypanosomes. Double-stranded RNA (dsRNA) derived endogenously or by transfection procedures is processed by an RNase III enzyme known as DICER into small interfering dsRNA molecules (siRNA) 19–23 nt long. siRNAs associate with Argonaute (AGO), the catalytic core of the RISC (RNA-induced silencing complex), with one siRNA strand being released and the guide siRNA strand then mediating the degradation or inhibiting the translation of the target mRNA. Genes encoding components of the RNAi machinery occur only in T. brucei and in a sub-group of Leishmania. (B) Phylogenetic analysis of trypanosomatid species based on the predicted protein sequences of the housekeeping gene GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and ubiquitin. The neighbor-joining tree was generated using sequences found in the parasite genome databases (www.genedb.org) and MEGA4 software. The absence or presence of genes encoding components of active RNAi machinery (as identified by Lye et al., 2010) is indicated on the right.