The genome sequence of Leishmania (Leishmania) amazonensis: functional annotation and extended analysis of gene models

Abstract

We present the sequencing and annotation of the Leishmania (Leishmania) amazonensis genome, an etiological agent of human cutaneous leishmaniasis in the Amazon region of Brazil. L. (L.) amazonensis shares features with Leishmania (L.) mexicana but also exhibits unique characteristics regarding geographical distribution and clinical manifestations of cutaneous lesions (e.g. borderline disseminated cutaneous leishmaniasis). Predicted genes were scored for orthologous gene families and conserved domains in comparison with other human pathogenic Leishmania spp. Carboxypeptidase, aminotransferase, and 3'-nucleotidase genes and ATPase, thioredoxin, and chaperone-related domains were represented more abundantly in L. (L.) amazonensis and L. (L.) mexicana species. Phylogenetic analysis revealed that these two species share groups of amastin surface proteins unique to the genus that could be related to specific features of disease outcomes and host cell interactions. Additionally, we describe a hypothetical hybrid interactome of potentially secreted L. (L.) amazonensis proteins and host proteins under the assumption that parasite factors mimic their mammalian counterparts. The model predicts an interaction between an L. (L.) amazonensis heat-shock protein and mammalian Toll-like receptor 9, which is implicated in important immune responses such as cytokine and nitric oxide production. The analysis presented here represents valuable information for future studies of leishmaniasis pathogenicity and treatment.

Keywords: Leishmania amazonensis; amastin; genome; heat-shock protein; interactome.

Figure 1.

Overview of the L. (L.) mexicana complex. (A) Classification of the Leishmania genus, subgenus and species complex (adapted from the WHO reports and Bates, 2007). Leishmania (L.) amazonensis and L. (L.) mexicana belong to the L. (L.) mexicana complex, subgenus Leishmania, and are causative agents of New World cutaneous leishmaniasis in which diffuse or disseminated lesions are hallmarks. The genomes of the species marked in red were employed in the present comparative analyses [*L. (S.) tarentolae was employed only in the amastin phylogenetic study]. (B) Large parasitophorous vacuoles (PVs) of L. (L.) amazonensis. Phase contrast microscopy image (left) of a bone marrow-derived macrophage containing a spacious PV (asterisk) lined with rounded amastigotes. Bar = 10 μm. Field-emission scanning electron micrograph (right) of an amastigote-hosting macrophage. The fractured sample indicated that amastigote forms (in red) were contained in a spacious PV. Bar = 5 μm.

Figure 2.

Bioinformatics analysis workflow used in the present study. Sequenced reads from the L. (L.) amazonensis genome were assembled into 2627 scaffolds and 8100 genes were predicted using comparative and ab initio prediction tools. The functional analysis of these predicted genes included: (i) AutoFACT functional annotation, which revealed that 45% of the predicted genes were unclassified or with unassigned function; (ii) screening for orthologous families of genes among Leishmania spp. (OrthoMCL); and (iii) screening for information about conserved protein domains deposited in CDD and PFAM databases. Expanded or exclusive orthologous proteins, or those conserved domains detected in the L. (L.) amazonensis genome were selected for interactome analysis with mammalian host proteins. This selection involved screening for possibly secreted proteins (using SecretomeP and TargetP) that also were orthologous to immune function-related proteins in humans and mice.

Figure 3.

Diagrammatic representation of (A) species-specific orthologous gene families (OrthoMCL analysis) and (B) conserved domains (CDD–PFAM analysis). A core of 6784 orthologous families and 1881 domains was conserved in all studied Leishmania species [L. (L.) amazonensis, L. (L.) mexicana, L. (L.) major, L. (L.) infantum, and L. (V.) braziliensis]. We detected 8 orthologous families and 20 conserved domains that were exclusive to L. (L.) mexicana complex. A complete list of orthologous families and conserved domains is presented in Supplementary data, Table S4 and S5, respectively.

Figure 4.

Bayesian consensus phylogeny of amastin surface proteins. The phylogram is represented by a consensus of 214 amastin sequences. The root was inferred using midpoint rooting. WAG was used as the substitution matrix for the protein alignment. Posterior probabilities exceeding 0.5 are shown in the branches. The tree topology suggests early branching of similar amastins shared by different species (blue). These branches were classified as Leishmania pre-speciation amastins, composed by α, β, and γ subfamily clades. We highlighted the terminal taxa (late branching or apomorphic) of species-specific δ-amastin clades of L. (L.) major (yellow), L. (V.) braziliensis (green), and L. (L.) infantum (purple). Complex-specific clades of L. (L.) amazonensis and L. (L.) mexicana amastin surface proteins are in red. The scale of the generated tree (see 0.4 bar) represents the number of substitutions per sequence position. The classification of amastin clades in subfamilies α, β, γ, and δ was based on the amastin phylogeny performed by Jackson et al. (2010).

Figure 5.

Interactomes of potentially secreted L. (L.) amazonensis [A30200 (A) and A45910 (B)] and mammalian immune cell proteins. The secreted parasite gene products are represented by red nodes in the interactome. The expression statuses of these parasite proteins during the amastigote stage were inferred using blastp with the proteomic database of L. (L.) mexicana amastigotes. The secreted components of L. (L.) amazonensis amastigotes share 28% identity and 94% coverage (A30200, A) and 69% identity and 90% coverage (A45910, B) with the mammalian HYOU1 and HSPA5 proteins, respectively. Both secreted components could directly interact with TLR9. We propose that orthologs of mammalian HYOU1 and HSPA5 are secreted by L. (L.) amazonensis amastigotes, interfering with host cell functions such as signaling and the production of NO and ILs. Arrows represent direct interactions and dashed arrows represent indirect interactions. The interactome was built using Ingenuity software, considering only proteins expressed in human and mouse immune cells and considering experimentally identified protein–protein interactions.