Predicting the proteins of Angomonas deanei, Strigomonas culicis and their respective endosymbionts reveals new aspects of the trypanosomatidae family

Abstract

Endosymbiont-bearing trypanosomatids have been considered excellent models for the study of cell evolution because the host protozoan co-evolves with an intracellular bacterium in a mutualistic relationship. Such protozoa inhabit a single invertebrate host during their entire life cycle and exhibit special characteristics that group them in a particular phylogenetic cluster of the Trypanosomatidae family, thus classified as monoxenics. In an effort to better understand such symbiotic association, we used DNA pyrosequencing and a reference-guided assembly to generate reads that predicted 16,960 and 12,162 open reading frames (ORFs) in two symbiont-bearing trypanosomatids, Angomonas deanei (previously named as Crithidia deanei) and Strigomonas culicis (first known as Blastocrithidia culicis), respectively. Identification of each ORF was based primarily on TriTrypDB using tblastn, and each ORF was confirmed by employing getorf from EMBOSS and Newbler 2.6 when necessary. The monoxenic organisms revealed conserved housekeeping functions when compared to other trypanosomatids, especially compared with Leishmania major. However, major differences were found in ORFs corresponding to the cytoskeleton, the kinetoplast, and the paraflagellar structure. The monoxenic organisms also contain a large number of genes for cytosolic calpain-like and surface gp63 metalloproteases and a reduced number of compartmentalized cysteine proteases in comparison to other TriTryp organisms, reflecting adaptations to the presence of the symbiont. The assembled bacterial endosymbiont sequences exhibit a high A+T content with a total of 787 and 769 ORFs for the Angomonas deanei and Strigomonas culicis endosymbionts, respectively, and indicate that these organisms hold a common ancestor related to the Alcaligenaceae family. Importantly, both symbionts contain enzymes that complement essential host cell biosynthetic pathways, such as those for amino acid, lipid and purine/pyrimidine metabolism. These findings increase our understanding of the intricate symbiotic relationship between the bacterium and the trypanosomatid host and provide clues to better understand eukaryotic cell evolution.

Figure 1. Venn diagram illustrating the distribution of MCL protein clusters.

The diagram shows the cluster distribution comparing endosymbiont-bearing trypanosomatids (group A), Leishmania sp. (group B) and Trypanosoma sp. (group C). Protein clusters with less clear phylogenetic distributions are identified as others.

Figure 2. Genome alignments.

The figure shows the alignment of the A. deanei endosymbiont (Endo-A. deanei) and the S. culicis endosymbiont (Endo-S. culicis) (A); between Endo-A. deanei and T. asinigenitalis (B), T. equigenitalis (C), or Wolbachia (D); and between Wolbachia and T. asinigenitalis (E). Alignments were performed with the ACT program based on tblastx analyses. Red (direct similarity) and blue lines (indirect similarity) connect similar regions with at least 700 bp and a score cutoff of 700. The numbers on the right indicate the size of the entire sequence for each organism.

Figure 3. Phylogenetic of histones of A. deanei, S. culicis, and other trypanosomatids.

Histone protein (panel A) and nucleotide (panel B) sequences were generated by MUSCLE tool using 10 iterations in the Geneious package . Trees were constructed using the Geneious Tree Builder, by employing Jukes-Cantor genetic distance model with a neighbor-joining method and no out-groups. The consensus trees were generated from 100 bootstrap replicates of all detected histone genes, as shown below. Scale bars are indicated for each consensus tree. The trees in panel A are based in a collection of sequences of all trypanosomatids. The nucleotide sequences used for dihydrofolate reductase-thymidylate synthase are: T. cruzi, XM_810234; T. brucei, XM_841078; T. vivax, HE573023; L. mexicana, FR799559; L. major, XM_001680805; L. infantum, XM_001680805; and C. fasciculata, M22852.

Figure 4. Microsatellite content in the genomes of A. deanei, S. culicis, and their endosymbionts.

Panel (A) shows the percentage of repetitive nucleotides for each repeat length. The total numbers of nucleotides are derived from microsatellite sequences divided by the total number of assembled nucleotides. Panel (B) shows the microsatellite density. The values indicate the number of microsatellite loci divided by the genome length×100.

Figure 5. Oxidative stress-related genes in the genomes of A. deanei, S. culicis and L. major.

The figure shows the number of ORFs for the indicated enzymes for each species.

Figure 6. Schematic representation of the cell division machinery found in the endosymbionts.

Panel (A) indicates the basic model derived from a gram-negative bacterium with the localization of each component (shown on the right). Panel (B) represents the components found in the endosymbiont of A. deanei, and Panel (C) shows the steps in the assembly of the Z-ring. The missing components of the A. deanei endosymbiont are drawn in red.

Figure 7. Main metabolic exchanges between host and endosymbionts.

Schematic representation of the amino acids, vitamins, and cofactors exchanged between A. deanei and S. culicis and their respective symbionts. Dotted lines indicate pathways that have or might have contributions from both partners, whereas metabolites inside one of the circles, representing the symbiont or host, indicate that one partner holds candidate genes coding for enzymes of the whole biosynthetic pathway. *Candidate genes were only found for the symbiont of S. culicis and not for the symbiont of A. deanei. BCAA (branched-chain amino acids) are leucine, isoleucine and valine.

Figure 8. Purine production, acquisition, and utilization in A. deanei and S. culicis.

The figure illustrates the production, acquisition and utilization of purines in the host trypanosomes considering the presence of endosymbiont enzymes. This model suggests that the trypanosomatid acquires purines from the symbiont, which synthesizes them de novo. Some ecto-localized proteins, such as apyrase (APY) and adenosine deaminase (ADA), could be responsible for the generation of extracellular nucleosides, nucleobases, and purines. Nucleobases and purines could be acquired by the parasite through membrane transporters (T) or diffusion and could be incorporated into DNA, RNA, and kDNA molecules after “purine salvage pathway” processing. Abbreviations: NTP (nucleoside tri-phosphate), NDP (nucleoside di-phosphate), NMP (nucleoside mono- phosphate), N (nucleobase), ADO (adenosine), INO (inosine).