The endosymbiont genomes.

Table 2 summarizes the genome analyses of both symbionts. The genome of the A. deanei endosymbiont contains 821,813 bp, with almost 31% G+C content and 787 CDSs. Of these, 640 (81.3%) were characterized as known CDSs, 94 (11.9%) as hypothetical, and 53 (6.7%) as rRNA or tRNA. The average CDS length is 987 bp, and coding regions account for 88% of the genome, indicating that the genome is highly compact. There are three copies of each rRNA and 44 tRNAs, suggesting a functional translation metabolism. The endosymbiont of S. culicis has a genome composed of 820,037 bps and 769 CDSs, 637 (83.5%) coding for known proteins, 78 (9.5%) annotated as hypothetical proteins, and 54 (6.0%) as rRNA or tRNA. The G+C content (32.6%) is similar to but slightly higher than that of the A. deanei endosymbiont (30.96%). A. deanei and S. culicis endosymbiont genomes are composed of 88 and 87% of CDSs with few regions formed by non-coding sequences.

A direct comparison between the two endosymbionts indicated that they share 507 genes that meet the criteria for inclusion in a cluster as described in the Materials and Methods. This represents approximately 70% of the annotated genes in both genomes, indicating a certain degree of genetic similarity. Figure 2A shows the full alignment of the A. deanei and S. culicis symbionts. This alignment indicates the occurrence of an inversion involving approximately one half of the genomes. However, this inversion would be validated by experimental work. The observed differences agree with phylogenetic analyses suggesting the classification of these symbionts as different species, Candidatus Kinetoplastibacterium crithidii and Candidatus Kinetoplastibacterium blastocrithidii [2], [23].

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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.

https://doi.org/10.1371/journal.pone.0060209.g002

The origins of symbionts in trypanosomatids.

Previous phylogenetic studies based on sequencing of the small-subunit ribosomal DNA suggested that symbionts of trypanosomatids descended from a common ancestor, a β-proteobacteria of the Bordetella genus [2], [22], [23]. Comparisons of the endosymbiont genomes with the KEGG database revealed eight organisms that share high numbers of similar CDSs: Bordetella petrii, A. xylosoxidans, Bordetella avium, Bordetella parapertussis, Pusillimonas, Bordetella bronchiseptica and Taylorella equigenitalis. All these species are phylogenetically related to β-proteobacteria belonging to the Alcaligenaceae family. The genus Taylorella consists of two species, T. equigenitalis and T. asinigenitalis, which are microaerophilic, slow-growing gram-negative bacteria belonging to the family Alcaligenaceae [26], [27]. T. equigenitalis is an intracellular facultative pathogen in horses that causes contagious equine metritis (CEM), a sexually transmitted infection [28].

Based on these facts, clustering analysis was performed to compare these genomes and establish the genetic similarity among them. The clustering analysis compared the genomes of A. deanei and S. culicis endosymbionts, T. equigenitalis MCE9, T. asinigenitalis MCE3, B. petrii DSM 12804, A. xylosoxidans A8 and Wolbachia pipiens (WMel). For the A. deanei endosymbiont, the highest numbers of shared clusters are observed for A. xylosoxidans (490 clusters) and B. petrii (483 clusters), followed by T. asinigenitalis (376 clusters) and T. equigenitalis (375 clusters). However, considering the genome length, T. equigenitalis and T. asinigenitalis had the greater proportion of genes in clusters (24.1 and 24.67% of the annotated genes, respectively). The values for A. xylosoxidans and B. petrii are 7.59 and 9.61%, respectively. Note that the A. xylosoxidans plasmids pA81 and pA82 are not included in these comparisons. The S. culicis endosymbiont shares a high number of clusters (74%) with other genomes; considering 714 annotated genes (rRNA and tRNA genes were not taken into account), 544 (76.19%) were similar to genes of the other microorganisms. The highest number of clusters is shared between A. xylosoxidans (501 clusters) and B. petrii (495 clusters), followed by T. asinigenitalis (390) and T. equigenitalis (388 clusters). Using W. pipiens (wMel), an endosymbiont of Drosophila melanogaster, as an out-group, we found 70 clusters for A. deanei and 73 clusters for S. culicis. Wolbachia also shares a lower number of clusters with T. asinigenitalis (79) and T. equigenitalis (81).

T. equigenitalis MCE9 and T. asinigenitalis MCE3 contain 1,695,860 and 1,638,559 bps, respectively. Therefore, the A. deanei and S. culicis symbiont genomes are reduced when compared to Taylorella, which also have reduced genomes when compared to Bordetella or Achromobacter [26], [27]. Alignments indicate the existence of similar sequences between the Taylorella and the kinetoplastid symbionts (Figure 2B and C), corroborating the results obtained in the clustering analyses. Much less similarity is observed between A. deanei and W. pipientis wMel, as well as between W. pipientis and T. asinigenitalis using the same alignment parameters (Figure 2D and E). Both Taylorella genomes are AT-rich (37.4 and 38.3% for T. equigenitalis and T. asinigenitalis, respectively), a characteristic also shared with both symbionts. Therefore, it is possible that the process of adaptation to intracellular life involved substantial base-composition modification, as most symbiotic bacteria are AT-rich [29], [30].

The degree of similarity and even identity of the endosymbionts with Taylorella genomes and even with genomes of other species such as Bordetella and Achromobacter reinforce the origin of both endosymbionts from an ancestor of the Alcaligenaceae group. Both endosymbionts are similar to T. equigenitalis, T. asinigenitalis, B. petrii, and A. xylosoxidans and to other species of this family to different degrees. In absolute numbers, B. petrii and A. xylosoxidans have the highest numbers of clusters in common with the symbionts. However, considering the genome length, Taylorella species have the highest proportions of clusters in common with the A. deanei and S. culicis endosymbionts. A phylogenomic analysis using 235 orthologs was performed in order to establish the evolutionary history among A. xylosoxidans A8, B. petrii DSM 12804, T. asinigenitalis MCE3, T. equigenitalis MCE9, Ca. K. blastocrithidii and Ca. K. crithidii. The results indicated that symbionts present in both trypanosomatid species are closely related to the Alcaligenaceae family (Figure S1). Pseudomonas aeruginosa PA7 was the Gammaproteobacteria used as outgroup. These data corroborate the results from Alves et al. 2011 [11].

Although the genome lengths of both trypanosomatid bacteria are slightly larger than those of Buchnera sp. [31], they are several fold larger than those of symbiotic bacteria, which have extremely reduced genomes [32]. Analysis of the B. pertussis and B. parapertussis genomes revealed a process of gene loss during host adaptation [33], [34]. This process was proposed to be associated with mobile DNA elements such as Insertion Sequences (IS) and the presence of pseudo genes [33], [34]. However, the mechanism(s) involved in the length reduction observed for the genomes of the two symbionts studied here needs further investigation. Our data enable future studies examining the relationship between endosymbiosis in trypanosomatids and the origin of organelles in eukaryotic cells.

The microtubule cytoskeleton and flagellum of the host trypanosomatids.

The cytoskeleton is composed of structures such as the microtubular subpelicular corset, the axoneme, the basal body, and the paraflagellar rod [35]. Thus, the cytoskeleton controls several characteristics of trypanosomatids such as their shape, the positions of structures, the flagellar beating and the host colonization. The presence of the symbiont has been related to unique characteristics of the host trypanosomatid.

Six members of the tubulin superfamily (α, β, δ, γ, ε and ζ) are present in A. deanei and S. culicis. Accordingly, δ and ε-tubulins are present in organisms that possess basal bodies and flagella [36]. γ-tubulin is localized in the basal body of A. deanei [14] as in other trypanosomatids [35]. Additionally, in common with other trypanosomatids, five centrins were identified in A. deanei and S. culicis. Furthermore, symbiont-containing trypanosomatids contain ε-tubulin, as in algae genomes, which can be related to the replication and inheritance of the centriole and basal bodies [37], [38]. Interestingly, the absence of microtubules that form the subpelicular corset in areas where the mitochondrion touches the plasma membrane is unique to symbiont-containing trypanosomatids [15]. However, we cannot explain this atypical microtubule distribution based on database searches. Moreover, no classical eukaryotic microtubule associated proteins (MAPs) or intermediate filament homologues were identified in symbiont-bearing or other trypanosomatids, except for TOG/MOR1 and Asp.

Actin and other protein homologues that play roles in the binding and nucleation of actin filaments are present in A. deanei and S. culicis. However, the ARP 2/3 complex, which is involved in the nucleation of actin, is absent in symbiont-bearing species. As actin seems to be necessary for endocytosis in trypanosomatids [39], the absence of some proteins involved in actin nucleation may be related to the low rates of endocytosis of these protozoa (unpublished data). Indeed, both symbiont-bearing trypanosomatids have low nutritional requirements, as the symbiotic bacterium completes essential metabolic routes of the host cell [3].

Trypanosomatids are the only organisms from the orders Euglenida and Kinetoplastida that have a paraflagellar rod. This structure is continuously associated with axoneme and it contains two major proteins designated PFR1 and PFR2 [35]. Importantly, only PFR1 was identified in A. deanei and S. culicis. Perhaps we missed PFR2 since these PFR proteins are highly repetitive and their assemblies are difficult. Nevertheless, these species have a reduced paraflagellar rod located at the proximal area of the flagellum [15], [16], although the same pattern of flagellar beating described for other trypanosomatids is observed for A. deanei [40]. The paraflagellar rod components (PFC) 4, PFC 10, PFC 16, and PFC 18 were detected in the A. deanei database, whereas in S. culicis PFC 11 was also identified. Other minor components of the paraflagellar rod could not be detected. Accordingly, RNA interference (RNAi) knockdown of PFCs such as PFC3 does not impair the flagellar movement of T. brucei [41], differently from PFC4 and PFC6 depletion [42].

Several other minor flagellar proteins detected in these and other trypanosomatids are absent in A. deanei and S. culicis, especially the flagellar membrane proteins and those involved in intraflagellar transport (kinesins). Symbiont-containing species had adenylate kinase B (ADKB) but not ADKA, in contrast to other trypanosomatids, which express both. These proteins are involved in the maintenance of ATP supply to the distal portion of the flagellum [43], [44].

Taken together, the differences in the composition and function of the cytoskeleton in symbiont-containing trypanosomatids seem to represent adaptations to incorporate the endosymbiont. Further exploration of these differences could enable a better understanding of how endosymbiosis was established.

The kinetoplast.

The kinetoplast is an enlarged portion of the single mitochondrion that contains the mitochondrial DNA, which exhibits an unusual arrangement of catenated circles that form a network. The kinetoplast shape and the kDNA topology vary according to species and developmental stage. Endosymbiont-containing trypanosomatids show differences in the morphology and topology of the kDNA network when compared to other species of the same family. Both species present a loose kDNA arrangement, but in A. deanei, the kinetoplast has a trapezoid-like shape with a characteristic transversal electron-dense band, whereas in S. culicis the disk shape structure is wider at the center in relation to the extremities [2], [17].

Differences in kDNA arrangement are related to low molecular weight basic proteins such as kinetoplast-associated protein (KAP), taking part in the organization and segregation of the kDNA network [45], [46]. Our data indicate that KAP4 and KAP3 homologues are present in A. deanei, while KAP4, KAP2 homologues, and ScKAP-like protein are found in S. culicis (Table S1). In addition, a conserved nine amino acid domain in the N-terminal region, most likely a mitochondrial import signal [47], [48], is found in AdKAP4 and ScKAP4 (amino acid positions 10 to 16) (Figure S2). Furthermore, ScKAP2 has a conserved domain called the High Mobility Group (HMG), indicating that this protein may be involved in protein-protein interactions. These KAPs might be related to the typical kDNA condensation of symbiont-bearing trypanosomatids.

Housekeeping genes.

Histones, which are responsible for structuring the chromatin, are highly conserved proteins that appeared in the eukaryotic branch of evolution. Although well conserved, Trypanosomatidae histones display differences in the N and C-terminal sequences, sites of post-translational modifications, when compared to other eukaryotes. Phylogenetic analysis revealed that histones and their variants in both A. deanei and S. culicis are clustered in a separate branch, between the Trypanosoma and Leishmania species (Figure 3A). Similar phylogenetic distribution is seen for the dihydrofolate reductase-thymidylate synthase when we performed the analysis using nucleotide sequences (Figure 3B). Nevertheless, the symbiont-bearing species show conservation in the sites of post-translation when compared to other trypanosomes as shown in supplementary Figure S3. In A. deanei and S. culicis the proteins related to the chromatin assembly are also maintained, including histones and histone-modifying enzymes as shown in Tables S2–S7 and Figure S4 of the supporting information. For a more detailed analysis about housekeeping genes of A. deanei and S. culicis see Text S1.

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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 [120]. 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.

https://doi.org/10.1371/journal.pone.0060209.g003

DNA replication, repair, transcription, translation and signal transduction in A. deanei and S. culicis functions can be respectively attributed at least to 914 ORFs and 643 ORFs (Table 3). Most of the genes are exclusive to the protozoan and are absent in the endosymbiont (Table 4), thus indicating that these processes are exclusive to the host organism as shown in the supplementary Tables S8–S13, typically containing a conserved spliced-leader RNA as found in other trypanosomes (see Figure S5 for more information). A total of 133 and 130 proteins with similar functions are detectable in the endosymbionts of both species, with up to 95% amino acid identity to proteins of Bordetella sp. and A. xylosoxidans.

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Table 3. Numbers of ORFs identified in A. deanei and S. culicis and their symbionts, according to the mechanisms of DNA replication and repair, signal transduction, transcription and translation.

https://doi.org/10.1371/journal.pone.0060209.t003

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Table 4. Summary of the origin of ORFs found in A. deanei and S. culicis.

https://doi.org/10.1371/journal.pone.0060209.t004

Similar DNA repair proteins are present in both eukaryote and prokaryote predicted sequences. These findings demonstrate that the endosymbionts conserved essential housekeeping proteins despite their genome reduction. Some differences were found in mismatch repair (MMR) between symbiont-bearing trypanosomatid genomes. As microsatellite instability is considered the molecular fingerprint of the MMR system, we compared the abundance of tandem repeats in the genomes of A. deanei and S. culicis and their respective endosymbionts. We noticed that the genomes of S. culicis and its endosymbiont are more repetitive than the genomes of A. deanei and its endosymbiont (Figure 4A). However, the higher repetitive content of the genomes of S. culicis and its endosymbiont is not only due to the higher number of microsatellite loci (Figure 4B) but also to the expansion of the size of the microsatellite sequences. These data suggest that microsatellites of S. culicis and its endosymbiont evolved faster than those of A. deanei and its endosymbiont. Interestingly, we identified some missing components of the MMR machinery in S. culicis that are present in A. deanei, such as exonuclease I (Exo I), a 5′-3′ exonuclease that is implicated in the excision step of the DNA mismatch repair pathway (Table S9). Several studies have correlated the silencing of the ExoI protein and/or mutations of the ExoI gene and microsatellite instability with development of lymphomas and colorectal cancer [49], [50], [51]. Therefore, we speculate that deficiencies in the MMR machinery in S. culicis may be related to the high proportion of microsatellites in its genome. The association between microsatellite instability and MMR deficiency has already been described for T. cruzi strains [52], [53]. The same variability pattern is observed for each symbiont, despite the fact that the MMR machinery seems to be complete in both symbiotic bacteria (Table S10). It is tempting to speculate that this finding may indicate that the parasite and its endosymbiont are exposed to the same environment and therefore may be subjected to similar selective pressures imposed by an external oxidative condition.

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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.

https://doi.org/10.1371/journal.pone.0060209.g004

A. deanei and S. culicis have 607 and 421 putative kinase-encoding genes, respectively (Table 5). Thirty one of the A. deanei kinases were classified in the AGC family, 31 as atypical, 49 as CAMK, 15 as CK1, 108 as CMGC, 64 as STE, 1 as TKL, 81 as others, and 227 that could not be classified in any of these families. No typical tyrosine kinases (TK) are present in A. deanei or S. culicis, as in other trypanosomes, although tyrosine residues are subjected to phosphorylation [54], [55]. Several phosphatases have also been described in trypanosomes, pointing toward their regulatory role in the development of these organisms. The T. brucei PTP (TbPTP1) is associated with the cytoskeleton and has been reported to be intrinsically involved in this parasite’s cycle [56]. Similar sequences are found in the A. deanei genome, including PTP1, which is not found in the S. culicis database. Additionally, a large number of other PTPs appear in both genomes, including ectophosphatases (Table S14).

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Table 5. Kinase families identified in trypanosomatids.

https://doi.org/10.1371/journal.pone.0060209.t005

Two major signal transduction pathways are described in trypanosomatids: one is the cyclic AMP-dependent route and the other is the mitogen-activated protein kinase pathway [57]. The major components of these pathways, including phosphatidylinositol signaling, mTOR and MAPK signaling pathways are identified in A. deanei and S. culicis. These pathways may regulate cellular activities such as gene expression, mitosis, differentiation, and cell survival/apoptosis (Table 6).

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Table 6. Representative ORFs involved in the signal transduction pathways in A. deanei and S. culicis.

https://doi.org/10.1371/journal.pone.0060209.t006

Most genes encoding heat shock proteins are present in symbiont-bearing species, as was previously described in other trypanosomatids (Table S15). Genes for redox molecules and antioxidant enzymes, which are part of the oxidative stress response, are also present in the A. deanei and S. culicis genomes. Both contain slightly more copies of ascorbate peroxidase, methionine sulfoxide reductase, glucose-6-phosphate dehydrogenase, and trypanothione reductase genes than L. major. In particular, several genes related to the oxidative stress response are present in higher copy numbers in symbiont-bearing trypanosomatids than in L. major (Figure 5).

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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.

https://doi.org/10.1371/journal.pone.0060209.g005

A. deanei sequences codify enzymes involved in RNAi, a mechanism described in various organisms that promotes the specific degradation of mRNA. RNAi is initiated by the recognition of double-stranded RNA through the action of endoribonucleases known as Dicer and Slicer, members of the Argonaut (Ago) protein family (RNase H-type) [58]. The cleavage of double-stranded RNA results in a complex that specifically cleaves mRNA molecules that are homologous to the double-stranded sequence. A. deanei contains the gene coding Dicer-like protein II (AGDE14022) and Ago1 (AGDE11548), homologous to enzymes in T. brucei and Leishmania braziliensis (Ngo et al., 1998; Lye et al., 2010). In addition, A. deanei contains the RNA interference factor (RIF) 4 (AGDE09645) with an exonuclease domain of the DnaQ superfamily, as described in T. brucei. A fragmented RIF5 sequence was also found in the sequence AGDE15656. These proteins were shown to interact with Ago1 as was recently demonstrated in T. brucei [59], suggesting that RNAi might be active in A. deanei. None of these sequences were found in the S. culicis database.

Cell cycle control in host trypanosomes.

In eukaryotes, DNA replication is coordinated with cell division by a cyclin-CDK complex that triggers DNA duplication during the S phase of the cell cycle. Multiple copies of the CRK gene (cdc2-related protein kinase) are found in A. deanei and four genes coding for two different CRKs are present in S. culicis. Both proteins exhibit structural features of the kinase subunits that make up the CDK complex, as they contain the cyclin-binding PSTAIRE motif, an ATP-binding domain and a catalytic domain. These motifs and domains are not the same in different CRKs (Figure S6), strongly suggesting that these CRKs might control different stages of the cell cycle. A. deanei contains four genes coding for cyclins. Three of these genes are homologues to mitotic cyclin from S. cerevisiae and T. brucei. However, none of them contain the typical destruction domain present in T. brucei mitotic cyclin [60]. The fourth codes for a S. cerevisiae Clb5 homolog, an S-phase cyclin. These data indicate that more than one CRK and more than one cyclin would be involved in the cell cycle control of symbiont-containing trypanosomatids, suggesting that tight regulation must occur to guarantee the precise maintenance of only one symbiont per cell [14].

Cell cycle control in the endosymbionts.

Bacterial cell division is a highly regulated event that mainly depends on two structures, the peptidoglycan layer and the Z ring. The first step in the segregation of the bacterium is the formation of a polymerized Z ring at the middle of the cell. This structure acts as a platform for the recruitment of other essential proteins named Filament Temperature Sensitive (Fts), which are mainly involved in the formation and stabilization of the Z ring [61], [62] and in establishing the peptidoglycan septum formation site in most bacteria [63] (Figure 6A).

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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.

https://doi.org/10.1371/journal.pone.0060209.g006

Two fts sequences were identified in A. deanei and S. culicis symbionts based on Bordetella genes (Table 7). One of them is FtsZ, which requires integral membrane proteins such as Zip A and FtsA for anchoring. However, these sequences are absent in the symbionts. FtsZ should also interact with FtsE, which is absent in both symbionts. This protein is homologous to the ATP-binding cassette of ABC transporters and co-localizes with the division septum [64]. The lack of these proteins could be related to the absence of a classical Z ring in these symbionts. The other sequence is FtsK that docks FtsQ, FtsB and FtsL, which are related to the formation of the peptidoglycan layer in E. coli and B. subtilis [65], [66], [67], but these proteins are absent in symbionts, as in most bacteria that exhibit reduced peptidoglycan production [64]. RodA, a homologous integral membrane protein involved in bacterial cell growth, is detected in the endosymbionts. RodA could replace FtsW, which is absent in both symbionts. FtsW is essential for the localization of FtsI (PBP3) in the Z ring [68], which is absent in the symbiotic bacteria.

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Table 7. Members of the Fts family and PBPs that are present in endosymbionts of A. deanei and S. culicis.

https://doi.org/10.1371/journal.pone.0060209.t007

Endosymbionts have only one bifunctional synthase (PBP1A), while E. coli has PBP1A, PBP1B, and PBP1C. Cells require at least one of these synthases for viability. The peptidoglycan layer is functional in trypanosomatid symbionts, as shown by treatment with β-lactam antibiotics affecting the division of the bacterium, generating filamentous structures and culminating in cell lysis. PBP1 and PBP2 have also been detected at the symbiont envelope [6]. PBP1B interacts with the two essential division proteins, FtsN and PBP3/FtsI, which are absent in the symbiont. PBP1B can also interact with PBP2 that is identified in both symbiont databases (see Table 7).

A sequence encoding a minor PBP described in E. coli was also identified in the symbionts. This protein is known as a putative PBP precursor (PBP5/dacC). This PBP is involved in the regulation of the peptidoglycan structure, along with 3 other minor PBPs described in E. coli, but these are absent from the symbiont (Table 7). On the other hand, all the enzymes involved in the synthesis of activated nucleotide precursors for the assembly of the peptidoglycan layer are present in the symbiont genome, except for Braun’s lipoprotein (Lpp), which forms the lipid-anchored disaccharide-pentapeptide monomer subunit [69]. In E. coli strains, mutations in Lpp genes result in a significant reduction of the permeability barrier, although small effects on the maintenance of the cell growth and metabolism were observed in these cells [70], [71].

Taken together, we consider that gene loss in the dcw cluster [72] (represented in Figure 6) explains the lack of the FtsZ ring in the endosymbiont during its division process [73]. Moreover, the symbiont envelope contains a reduced peptidoglycan layer and lacks a septum during its division process, which can be related to the facilitation of metabolic exchanges, as well as to the control of division by the host protozoan [6]. These losses could be understood since the host trypanosomatid is controlling the number of symbiotic bacteria per cell. This phenomenon has been described for obligatory intracellular bacteria that co-evolve in eukaryotic cells, as well as for the organelles of prokaryotic origin, the chloroplast and the mitochondrion [74], [75].

Oxidative phosphorylation.

FoF1-ATP synthase and the entire mitochondrial electron transport chain are present in A. deanei and S. culicis, although some subunits are missing (Table 8). These species have a rotenone-insensitive NADH:ubiquinone oxidoredutase in complex I, as do other trypanosomatids [77]. Ten complex II (succinate:ubiquinone reductase) subunits of the twelve identified in T. cruzi [78] are also present in both trypanosomatids. Many subunits from complex III, composed of cytochrome c reductase, are found in A. deanei and S. culicis. In addition, these protozoa contain genes for cytochrome c, as previously suggested by biochemical studies in other symbiont-containing trypanosomatids [3], [79].

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Table 8. Respiratory chain complexes identified in the predicted proteome of A. deanei, S. culicis and their respective endosymbionts.

https://doi.org/10.1371/journal.pone.0060209.t008

Both symbionts contain sequences with hits for all subunits of complex I, NADH:ubiquinone oxidoredutase, similar to E. coli (Table 8). Complexes II and III, including cytochrome c, and complex IV (cytochrome c oxidase, succinate:ubiquinone reductase and cytochrome c reductase, respectively) are not found in either symbiont. However, we detected the presence of cytochrome d as found in Allochromatium vinosum, and also a cytochrome d oxidase with a sequence close to that of B. parapertussis. All portions of the FoF1-ATP synthase were identified in symbionts, although not every subunit of each portion was found.

Lipid metabolism.

The sphingophospholipid (SPL) content in A. deanei and its symbiont has been previously described, with phosphatidylcholine (PC) representing the major SPL in the host, whereas cardiolipin predominates in the symbiotic bacterium [5], [80]. The synthetic pathway of phosphatidylglycerol from glycerol phosphate is present in both host trypanosomatids (Table S16). The biosynthetic pathways of PC and PE from CDP-choline and CDP-ethanolamine (Kennedy pathways), that synthesize PC and PE respectively, are incomplete in A. deanei and S. culicis. Nevertheless, the methylation pathway (Greenberg pathway), which converts PE in PC, seems to be absent in both trypanosomatids, even though one enzyme sequence was identified in A. deanei.

The symbiont of A. deanei exhibits two routes for phosphatidylethanolamine (PE) synthesis, starting from CDP-diacylglycerol and producing phosphatidylserine as an intermediate (Table S17). Interestingly, this last step of the pathway is not found in the S. culicis endosymbiont. Importantly, both symbionts lack genes that encode proteins of PC biosynthetic pathways, reinforcing the idea that this phospholipid is mainly obtained from the host protozoa [5]. Remarkably, phoshpatidylglycerophosphatase A, which produces the intermediate phosphatidylglycerol necessary for cardiolipin biosynthesis, was not found in either protozoa but is present in both symbionts. As cardiolipin is present in the inner membranes of host mitochondria, the symbionts may complete cardiolipin biosynthesis.

Pathways for sphingolipid production, including the synthesis of ceramide from sphingosine-1P, are present in A. deanei, while S. culicis lacks enzymes of this pathway (Table S16). Both host trypanosomatids have glycerol kinase and 3-glycerophosphate acyltransferase, enzymes for the synthesis of 1,2-diacyl-sn-glycerol and triacylglycerol from D-glycerate. In endosymbionts, glycerolipid metabolism seems to be reduced to two enzymes: 3-glycerophosphate acyltransferase and 1-acylglycerol-3-phosphate O-acyltransferase (Table S17), suggesting metabolic complementation between partners.

Furthermore, both hosts contain enzymes of the biosynthesis pathway for ergosterol production from zymosterol, as well as the pathway of sterol biosynthesis that produces lanosterol from farnesyl-PP. These pathways are only complete in A. deanei. The symbionts do not have enzymes for sterol biosynthesis, in accordance with our previous biochemical analysis [80].

Metabolism of amino acids, vitamins, cofactors and hemin.

Symbiosis in trypanosomatids is characterized by intensive metabolic exchanges, reducing the nutritional requirements of these trypanosomatids when compared to species without the symbiotic bacterium, or to aposymbiotic strains. Several biochemical studies have been carried out analyzing the biosynthetic pathways involved in this intricate relationship as recently reviewed [76], and our genomic data corroborate these findings. A schematic description of the potential metabolic interactions concerning the metabolism of amino acids, vitamins, cofactors, and hemin is provided in Figure 7.

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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.

https://doi.org/10.1371/journal.pone.0060209.g007

Both symbiotic bacteria have genes potentially encoding for all necessary enzymes for lysine, phenylalanine, tryptophan and tyrosine synthesis, in agreement with previous experimental data [40]. Tyrosine is required in the growth medium of A. deanei [81], but it is not essential for S. oncolpelti or S. culicis [41], [82], [83]. Here, in the symbiotic bacteria, we found enzymes involved in tyrosine synthesis, as well as indications that phenylalanine and tyrosine can be interconverted. In fact, protozoan growth is very slow in absence of phenylalanine and tryptophan [81], which may indicate that larger amounts of these amino acids are required for rapid cell proliferation.

Our data indicate that branched-chain amino acid (BCAA) synthesis mainly occurs in the symbionts except for the last step, with the branched-chain amino acid aminotransferase found in the host protozoan.

Among the pathways that (might) involve contributions from both partners, two have previously been characterized in detail, the urea cycle and heme synthesis. The urea cycle is complete in both symbiont-harboring trypanosomatids. Symbiotic bacteria contribute with ornithine carbamoyltransferase, which converts ornithine to citrulline, and with ornithine acetyltransferase, which transforms acetylornithine in ornithine. Conversely, aposymbiotic strains and symbiont-free Crithidia species need exogenous arginine or citrulline for cell proliferation [8] [68]. Our genomic data corroborate these studies.

Contrary to symbiont-free trypanosomatids, A. deanei and S. culicis do not require any source of heme for growth because the bacterium contains the required enzymes to produce heme precursors that complete the heme synthesis pathway in the host cell [7], [9], [10], [11], [84]. Our results support the idea that heme biosynthesis is mainly accomplished by the endosymbiont, with the last three steps of this pathway performed by the host trypanosomatid, and in most cases also by the bacterium as described in [11]. Furthermore, this metabolic route may represent the result of extensive gene loss and multiple lateral gene transfer events in trypanosomatids [11].

According to our genomic analyses, the symbiotic bacteria also perform the synthesis of histidine, folate, riboflavin, and coenzyme A, but one step is missing in the middle of each pathway, making them candidates for metabolic interchange with the host. In the case of folate and coenzyme A biosynthesis, one candidate gene was found in the host trypanosomatid. Moreover, none of these four metabolites are required in the growth medium of A. deanei and S. culicis [85], suggesting that these pathways are fully functional.

Candidate genes for the ubiquinone biosynthetic pathway were found in S. culicis but none for A. deanei endosymbionts. For the route with chorismate as precursor, only the first out of nine steps is missing in the S. culicis endosymbiont; moreover a candidate gene for that step is found in S. culicis genome. Only a few steps of these pathways are absent in A. deanei and S. culicis host organisms. In L. major, the ubiquinone ring synthesis has been described as having either acetate (via chorismate as in prokaryotes) or aromatic amino acids (as in mammalian cells) as precursors [45].

Methionine is considered essential for the growth of A. deanei, S. culicis and S. oncopelti [41], [81], [82]. We were not able to identify one enzyme among the four involved in the synthesis of methionine from either pyruvate or serine via cysteine in the genomes of A. deanei and S. culicis. No candidate to complement this pathway was found in the symbiotic bacteria.

Purine and pyrimidine metabolism for nucleotide production.

Trypanosomatids are not able to synthesize the purine ring de novo [86], [87], [88]. We observed that endosymbiont-bearing trypanosomatids contain sequences encoding ectonucleotidases from the E-NTPDase family and the adenosine deaminase family (Table S18), which are required for the hydrolysis and deamination of extracellular nucleotides [89], [90]. Interestingly, sequences encoding 5′-nucleotidases are not found in either symbiont-bearing trypanosomatid. The absence of this enzyme can be related to the presence of the endosymbiont, which can supply adenosine to the host cell, as we found all genes involved in the de novo pathway in the symbionts, indicating that they are able to complement the purine requirements of the host (Figure 8). However, we cannot discard the possibility that adenosine is transported to the intracellular medium by carriers of monophosphate nucleoside or by the presence of other enzymes that have the same function as 5′-nucleotidase. On the other hand, the lack of 5′-nucleotidase in A. deanei and S. culicis can be related to the fact that such protozoa are only insect parasites. According to this idea, several studies have shown the importance of ectonucleotidases in the establishment of infection by some trypanosomatid species [91]. The high activity of ectonucleotidases with concomitant production of adenosine, a known immune system inhibitor, lead to high susceptibility to Leishmania infection because adenosine can induce anti-inflammatory effects on the host [92], [93].

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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).

https://doi.org/10.1371/journal.pone.0060209.g008

Nucleoside transporters can take up nucleosides and nucleobases generated by ectonucleotidase activity. Genes encoding nucleoside transporters are present in both trypanosomatid genomes (Table S19), enabling cells to obtain exogenous purines from the medium. Furthermore, A. deanei and S. culicis contain intracellular enzymes that can convert purines to nucleotides, such as adenine phosphoribosyltransferase, hypoxanthine-guanine phosphoribosyltransferase, adenylate kinase, AMP deaminase, inosine monophosphate dehydrogenase and GMP synthetase. These data indicate that these organisms can interconvert intracellular purines into nucleotides. In contrast, both endosymbionts lack all the genes encoding enzymes related to purine salvage. Nevertheless, the symbiotic bacteria have genes encoding all the enzymes expected to participate in the de novo synthesis of purine nucleotides as previously proposed [94], [95]. One interesting possibility is that the symbiotic bacterium is able to supply the host trypanosomatid with purines. According to this idea, the endosymbiont participates in the de novo purine nucleotide pathway of A. deanei, as the aposymbiotic strain is unable to utilize glycine for the synthesis of purine nucleotides, only for pyrimidine nucleotide production [87].

Protozoa are generally, but not universally considered to be capable of synthesizing pyrimidines from glutamine and aspartic acid, which are used as precursors. Our results indicate that both symbiont-bearing trypanosomatids carry out de novo pyrimidine synthesis (Table S19). Interestingly, in silico analyses also revealed the presence of all the genes for de novo pyrimidine synthesis in both symbiont genomes, but not for the pyrimidine salvage pathway. A previous report indicated that A. deanei was able to synthesize purine and pyrimidine nucleotides from glycine (“de novo” pathway) and purine nucleotides from adenine and guanine (“salvage” pathway). Adenine would be incorporated into both adenine and guanine nucleotides, whereas guanine was only incorporated into guanine nucleotides, suggesting a metabolic block at the level of GMP reductase [87].

Deoxyribonucleotides are derived from the corresponding ribonucleotides by reactions in which the 2′-carbon atom of the D-ribose portion of the ribonucleotide is directly reduced to form the 2′-deoxy derivative. This reaction requires a pair of hydrogen atoms that are donated by NADPH via the intermediate-carrying protein thioredoxin. The disulfide thioredoxin is reduced by NADPH in a reaction catalyzed by thioredoxin reductase, providing the reducing equivalents for the ribonucleotide reductase, as observed for the endosymbionts that could provide 2′-deoxy derivatives. In folate metabolism, the formation of thymine nucleotides requires methylation of dUMP to produce dTMP, a reaction catalyzed by thymidilate kinase, which is present in A. deanei, S. culicis, and their respective endosymbionts. Figure 8 summarizes the purine and pyrimidine metabolisms in A. deanei and S. culicis considering the metabolic complementarity between the protozoan and the endosymbiont.

In this way, both symbiont-containing protozoa express a unique complement of nutritionally indispensable salvage and interconversion enzymes that enable the acquisition of purines from the medium. The intracellular purines can be acquired through the medium by the action of ectonucleotidases and nucleoside transporters.

Surface proteins and protease gene families.

One remarkable characteristic of trypanosomatid genomes is the large expansion of gene families encoding surface proteins [107]. Experimental data indicated that these genes encode surface proteins involved in interactions with the hosts. We selected eight gene families encoding surface proteins present in T. cruzi, T. brucei and Leishmania spp. to search for homologous sequences in the genomes of the two symbiont-bearing trypanosomatids. Because the draft assemblies of these genomes are still fragmented, we also used a read-based analysis to search for sequences with homology to these multigene families. It is well known that misassemblies frequently occur for tandemly repeated genes, as most repetitive copies collapse into only one or two copies. A total of 3,624,411 reads (corresponding to 1,595 Mb of sequences) from the A. deanei genome and 2,666,239 reads (corresponding to 924 Mb) from the S. culicis genome were used in this comparison. In A. deanei and S. culicis, we identified gene families encoding amastins, gp63, and cysteine peptidases (Table S21). As expected, we could not identify sequences homologous to mucin-like glycoproteins typical of T. cruzi [108], variant surface glycoprotein (VSG) characteristic of African trypanosomes, or trans-sialidases present in the genomes of all Trypanosoma species.

Calpain-like cysteine peptidases constitute the largest gene family identified in the A. deanei (85 members) and S. culicis (62 members) genomes, and they are also abundant in trypanosomatids [46]. The presence of the N-terminal fatty acid acylation motif was found in some members of calpain-like cysteine peptidases, indicating that some of these peptidases are associated with membranes, as has also been shown for other members of the family [109], [110]. The relatively large amount of calpain-like peptidases may be related to the presence of the endosymbiont, which would require a more complex regulation of the cell cycle and intracellular organelle distribution [14], as cytosolic calpains were found to regulate cytoskeletal remodeling, signal transduction, and cell differentiation [46].

A second large gene family in the A. deanei and S. culicis genomes encoding surface proteins with proteolytic activity is gp63. In our genomic analyses, we identified 37 and 9 genes containing sequences homologous to the gp63 of Leishmania and Trypanosoma spp. in the genomes of A. deanei and S. culicis, respectively. Proteins belonging to this group of zinc metalloproteases, also known as major surface protease (MSP) or leishmanolysin, have been characterized in various species of Leishmania and Trypanosoma [111]. Extensive studies on the role of this family in Leishmania indicate that they are involved in several aspects of host-parasite interaction including resistance to complement-mediated lysis, cell attachment, entry, and survival in macrophages [112]. Gene deletion studies in T. brucei indicated that the TbMSP of bloodstream trypanosomes acts in concert with phospholipase C to remove the variant surface protein from the membrane, required for parasite differentiation into the procyclic insect form [113]. Gp63-like molecules have been observed on the cell surface of symbiont-harboring trypanosomatids [114]. Importantly, the symbiont containing A. deanei displays a higher amount (2-fold) of leishmanolysin-like molecules at the surface compared to the aposymbiotic strain, which are unable to colonize insects [4]. As anti-gp63 antibodies decrease protozoan-insect interactions [21], our results reinforce the idea that the presence of such interactions caused the expansion of this gene family in endosymbiont-bearing organisms.

In contrast, only two copies of lysosomal cathepsin-like cysteine peptidases were identified in the A. deanei (AGDE05983 and AGDE10254) and S. culicis genomes (STCU01417 and STCU06430). The two A. deanei sequences encode identical cathepsin-B-like proteins, whereas the two S. culicis genes encode proteases of the cathepsin-L-like group. This class of cysteine peptidase is represented by cruzain or cruzipain, major lysosomal proteinases of T. cruzi expressed by parasites found in insect and vertebrate hosts, and encoded by a large gene family [115], [116]. In T. cruzi, these enzymes have important roles in various aspects of the host/parasite relationship and in intracellular digestion as a nutrient source [115]. Conversely, the low copy number of this class of lysosomal peptidase in symbiont-containing trypanosomatids seems to be related to their low nutritional requirements.

Amastins constitute a third large gene family in the A. deanei and S. culicis genomes that encodes surface proteins. Initially described in T. cruzi [117], amastin genes have also been identified in various Leishmania species [118], in A. deanei and in another related insect parasite, Leptomonas seymouri [119]. In Leishmania, amastins constitute the largest gene family with gene expression that is regulated during the parasite life cycle. As amastin has no sequence similarity to any other known protein, its function remains unknown. In this work, we identified 31 genes with sequences belonging to all four sub-families of amastins in the genome of A. deanei and 14 copies of amastin genes in S. culicis. Similar to Leishmania, members of all four amastin subfamilies were identified in symbiont-containing species (see Figure S7).