Figures Abstract Some non-pathogenic trypanosomatids maintain a mutualistic relationship with a betaproteobacterium of the Alcaligenaceae family. Intensive nutritional exchanges have been reported between the two partners, indicating that these protozoa are excellent biological models to study metabolic co-evolution.
We previously sequenced and herein investigate the entire genomes of five trypanosomatids which harbor a symbiotic bacterium SHTs for Symbiont-Haboring Trypanosomatids and the respective bacteria TPEs for Trypanosomatid Proteobacterial Endosymbiont , as well as two trypanosomatids without symbionts RTs for Regular Trypanosomatids , for the presence of genes of the classical pathways for vitamin biosynthesis.
Our data show that genes for the biosynthetic pathways of thiamine, biotin, and nicotinic acid are absent from all trypanosomatid genomes. This is in agreement with the absolute growth requirement for these vitamins in all protozoa of the family. Also absent from the genomes of RTs are the genes for the synthesis of pantothenic acid, folic acid, riboflavin, and vitamin B6.
This is also in agreement with the available data showing that RTs are auxotrophic for these essential vitamins. On the other hand, SHTs are autotrophic for such vitamins. Indeed, all the genes of the corresponding biosynthetic pathways were identified, most of them in the symbiont genomes, while a few genes, mostly of eukaryotic origin, were found in the host genomes. The only exceptions to the latter are: Their presence in trypanosomatids may result from lateral gene transfer.
Taken together, our results reinforce the idea that the low nutritional requirement of SHTs is associated with the presence of the symbiotic bacterium, which contains most genes for vitamin production. June 26, ; Accepted: September 25, ; Published: November 19, Copyright: This is an open-access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
The research leading to these results was funded by: Teixeira and Erney P. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
The authors have declared that no competing interests exist. Introduction All flagellates of the Trypanosomatidae family Euglenozoa, Kinetoplastida are parasites, with hosts among plants, vertebrates human included and invertebrates mainly insects. The non-pathogenic, insect-exclusive parasites contain the largest number of trypanosomatid species, whose most common habitat is the digestive tube of dipterans and hemipterans. Cultures of insect trypanosomatids, also referred to as monoxenics, were first obtained in the s.
However, most designated species of these protozoa have not been cultivated and are only known from morphological descriptions recorded in drawings published since the end of the nineteenth century . The modest number of available cultures of insect trypanosomatids is in part due to the difficulties inherent to growing these organisms in artificial media.
This is related to the fastidiousness of insect trypanosomatids, which require nutritionally very rich and complex media in order to grow  — . The first defined medium for an insect trypanosomatid was published in  , as an attempt to cultivate Crithidia fasciculata, a species isolated from mosquitoes.
The identity of the flagellate, however, cannot be taken at face value because some confusion prevailed at the time up until today as concerns the authenticity of the strains and species of insect trypanosomatids. In most cases, the cultivation of insect trypanosomatids requires all essential amino acids, vitamins of the B-complex, para-aminobenzoate pABA , inositol, and choline, in addition to purines, glucose, and salts  — . Earlier, Newton  ,  had described the much simpler nutritional requirements of Strigomonas oncopelti, which in addition to the B vitamins needed only methionine, adenine, glucose, and salts for its growth.
Later, it was shown that S. At the current time, six symbiont-bearing trypanosomatids have been identified, which belong to the genera Angomonas and Strigomonas  and harbor a single bacterium per cell . Such symbiotic bacteria are usually referred to as TPEs Trypanosomatid Proteobacterial Endosymbiont , they are vertically transmitted, after a synchronized division with other host cell structures .
Trypanosomatids harboring symbionts are called SHTs, in contrast to regular trypanosomatids RTs which do not contain symbionts. From early on, it was suspected that the symbiont was responsible for the enhanced nutritional capabilities of the SHTs, a fact supported by the loss of these capabilities in strains cured of the symbiont aposymbiotic strains by chloramphenicol treatment  — . Further nutritional studies have shown that, indeed, the requirements of the SHTs are minimal compared to those of RTs  , .
Such nutritional studies suggested that SHTs require neither hemin nor the amino acids that are essential for the growth of RTs. A recent investigation revealed that SHTs have the complete set of genes that code for enzymes of the heme pathway . A similar search on all available metabolic pathways also showed that SHTs have all the gene sequences for the enzymes involved in the synthesis of most essential amino acids . Genomic analyses further revealed that the genes for heme, as well as for the synthesis of essential amino acids, are unequally distributed between the host and the endosymbiont genomes, with most of them being located in the bacterium  — .
As concerns the need for vitamins by RTs, very little is known mainly because their growth media are very complex, making it difficult to define their specific nutritional requirements. Despite this, various papers addressed indirect aspects of vitamin metabolism  ,  — . The development of a defined medium for RTs from insects had initially established that seven vitamins are essential to sustain protozoan growth in culture medium: Studies on the nutritional requirements of insect trypanosomatids did not progress in a satisfactory way, but interestingly demonstrated that SHTs of the genus Angomonas are nutritionally much less exigent than RTs .
Thus, while the autotrophy of SHTs for most of the B vitamins was evidenced, nothing was known about pathways for the synthesis of other vitamins. Furthermore, any direct evidence of the symbiont contribution to the vitamin synthetic capabilities of the host trypanosomatid was missing.
The acquisition of metabolic capabilities through a mutualistic symbiosis with bacteria is widespread among eukaryotes. The sap-feeding insects are well studied examples of this  — . The great majority of these associations enables the synthesis of the essential amino acids not available in the poor diet of the insect hosts.
In some cases, the bacterial symbionts are able to produce vitamins of the B complex and cofactors. Such is the case of the endosymbiont, Wigglesworthia glossinidia, of the tsetse fly and also Candidatus Baumannia cicadellinicola, an endosymbiont of the sharpshooter  , . The latter is in a dual bacterial symbiosis, where one partner Ca. Sulcia muelleri supplies amino acids to the host whereas the other Ca. This makes the sharpshooter less nutritionally exigent .
In recent studies, we reported on the sequencing of the entire genomes of five species of TPEs  and we also annotated the proteins of two SHT species and their respective symbionts . Moreover, we sequenced to a draft-level the genomes of the five host species as well as of two RTs .
In this paper, we analyze these genomes for the presence of genes involved in the synthesis of vitamins. The participation of both host and symbiont in the production of vitamins is presented and discussed in association with previous data on the nutritional requirements of RTs and SHTs.
In order to get a broader view, we compared our findings with other trypanosomatids and bacteria from the Alcaligenaceae family based on KEGG . Materials and Methods 2. Analyzed organisms and their genome sequences The genomes of the following SHTs and of the respective symbionts were examined: Their corresponding symbionts are referred to as: The endosymbiont genomes were finished to a closed circle as previously described .
The genomes of two RTs were also analyzed: The identified segments of DNA were then extracted from the genomes and manually curated for completion and proper location of start and stop codons by using the GBrowse genome browser .
For each enzyme characterized in this work, corresponding putative orthologous genes from all domains of life were collected from the public databases by BLAST search E-value cutoff of 1e, maximum of 10, matches accepted against the full NCBI NR protein database, collecting sequences from taxonomic groups as widespread as possible and keeping one from each species or genus, if the tree was too large for subsequent phylogenetic analysis.
All endosymbiont genes analyzed here have been previously sequenced  ; gene identifiers are available in Table S3. For comparison, we used in our analyses the genome annotations of trypanosomatids Trypanosoma brucei, T. However, care should be taken since these data may lack manual curation. Phylogenetic analyses All analyses were performed at the protein sequence level.
Each alignment was submitted to bootstrap analysis with pseudo-replicates. Trees were initially drawn and formatted using TreeGraph2  and Dendroscope  , with subsequent cosmetic adjustments performed with the Inkscape vector image editor http: CodonW  was used to perform correspondence analyses of codon usage and to calculate codon adaptation index scores for the candidate HGT genes using an endosymbiont gene as a negative control.
The genomes of two RTs, C. Indeed, the enormous diversity present in the Trypanosomatidae family is sometimes not fully appreciated, leading to apparent conflicts in the interpretation of metabolic data, as happened with the early studies on the nutrition of Crithidia species. Data on the nutritional requirements of C. Many years elapsed until it was realized that these organisms were quite distinct phylogenetically, and in fact belonged to different genera .
It became clear that S. According to our present data, these extra nutritional capabilities largely result from the contribution of the endosymbiont to the metabolism of their trypanosomatid hosts as will be discussed here when analyzing vitamin biosynthesis in SHTs. Autotrophy of SHTs for the synthesis of riboflavin, pantothenic acid, vitamin B6 and folic acid 3.
Riboflavin is essential for the growth of RTs, as well as for the aposymbiotic strains of SHTs  ,  ,  , but not for the symbiont-carrying strains of SHTs, which are autotrophic for this vitamin  ,  , . Riboflavin is synthesized from guanosine 5'-triphosphate GTP and ribulose 5'-phosphate Figure 1 , and is the precursor for the essential flavin cofactors of redox reactions: On the other hand, TPEs have all the genes responsible for such synthesis, except for a poorly characterized step in the pathway, probably involving a phosphoric monoester hydrolase Figure 1 , IV-V.
However, it is uncertain which enzyme is responsible for this dephosphorylation process although it was suggested that a phosphatase of low substrate specificity might be involved  , .
Bacteria from the Alcaligenaceae family have all the enzymes for the synthesis of riboflavin as is the case for TPEs, missing only the uncharacterized one Figure S1. Since SHTs do not require riboflavin, it can be assumed that the dephosphorylation reaction is catalyzed by any of a cohort of phosphatases of broad substrate range.