![Characterization of Potential Drug Targeting Folate Transporter Proteins from Eukaryotic Pathogens [Version 2; Peer Review: 2 Approved with Reservations]](http://data.docslib.org/img/34befdc336e9d24632610f99553080cf-1.webp)
F1000Research 2017, 6:36 Last updated: 28 JUL 2021
RESEARCH ARTICLE
Characterization of potential drug targeting folate transporter proteins from Eukaryotic Pathogens [version 2; peer review: 2 approved with reservations]
Previously titled: Genome-wide characterization of folate transporter proteins of eukaryotic pathogens
Mofolusho O. Falade 1, Benson Otarigho 1,2
1Cellular Parasitology Programme, Cell Biology and Genetics Unit, Department of Zoology, University of Ibadan, Ibadan, Nigeria 2Department of Biological Science, Edo University, Iyamho, Nigeria
v2 First published: 12 Jan 2017, 6:36 Open Peer Review https://doi.org/10.12688/f1000research.10561.1 Latest published: 13 Jul 2017, 6:36 https://doi.org/10.12688/f1000research.10561.2 Reviewer Status
Invited Reviewers Abstract Background: Medically important pathogens are responsible for the 1 2 death of millions every year. For many of these pathogens, there are limited options for therapy and resistance to commonly used drugs is version 2 fast emerging. The availability of genome sequences of many (revision) report report eukaryotic microbes is providing critical biological information for 13 Jul 2017 understanding parasite biology and identifying new drug and vaccine targets. version 1 Methods: We developed automated search strategies in the 12 Jan 2017 report report Eukaryotic Pathogen Database Resources (EuPathDB) to construct a protein list and retrieve protein sequences of folate transporters encoded in the genomes of 200 eukaryotic microbes. The folate 1. Raphael D. Isokpehi, Bethune-Cookman transporters were categorized according to features including University , Daytona Beach, USA mitochondrial localization, number of transmembrane helix, and protein sequence relatedness. 2. Gajinder Singh , International Centre for Results: We identified 234 folate transporter proteins associated with Genetic Engineering and 63 eukaryotic microbes including 48 protozoa, 13 fungi the others being algae and bacteria. Phylogenetic analysis placed 219 proteins Biotechnology (ICGEB), New Delhi, India into a major clade and 15 proteins into a minor clade. All the folate Any reports and responses or comments on the transporter sequences from the malaria parasite, Plasmodium, belonged to the major clade. The identified folate transporters include article can be found at the end of the article. folate-binding protein YgfZ, folate/pteridine transporter, folate/biopterin transporter, reduced folate carrier family protein and folate/methotrexate transporter FT1. About 60% of the identified proteins are reported for the first time. Phylogeny computation shows the similarity of the proteins identified. Conclusion: These findings offer new possibilities for potential drug development targeting folate-salvage proteins in eukaryotic pathogens.
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Keywords Folate transporter, Eukaryotic pathogens, Drug discovery, Putative homologues
This article is included in the Neglected Tropical Diseases collection.
Corresponding author: Mofolusho O. Falade ([email protected]) Author roles: Falade MO: Conceptualization, Data Curation, Formal Analysis, Methodology, Writing – Original Draft Preparation, Writing – Review & Editing; Otarigho B: Conceptualization, Data Curation, Formal Analysis, Methodology, Project Administration, Supervision, Writing – Original Draft Preparation, Writing – Review & Editing Competing interests: No competing interests were disclosed. Grant information: B.O. was supported by a TWAS-CNPq fellowship (FP number: 3240274297) The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Copyright: © 2017 Falade MO and Otarigho B. 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 work is properly cited. Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication). How to cite this article: Falade MO and Otarigho B. Characterization of potential drug targeting folate transporter proteins from Eukaryotic Pathogens [version 2; peer review: 2 approved with reservations] F1000Research 2017, 6:36 https://doi.org/10.12688/f1000research.10561.2 First published: 12 Jan 2017, 6:36 https://doi.org/10.12688/f1000research.10561.1
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Antifolate drugs target this pathway and are the most important REVISE D Amendments from Version 1 and successful antimicrobial chemotherapies targeting a range of We have improved the manuscript following the suggestions bacterial and eukaryotic pathogens. While most parasitic proto- of the reviewers. We have addressed their major and minor zoa can synthesize folates from simple precursors, such as GTP, concerns. p-aminobenzoic acid (pABA) and glutamate, higher animals and 20 • Title has been modified from the first version humans cannot . Additionally, a few of these parasites can also 21 • Abstract has also been corrected as suggested salvage folate as nutrient from their host . These folate compounds • Work flow diagram has been removed and improved detail of are important for synthesis of DNA, RNA and membrane lipids workflow stated and are transported via receptor-mediated or/and carrier-mediated • Spelling errors and abbreviations have been corrected transmembrane proteins; folate transporters20–22. Importantly, • Errors in the Methodology section have been identified and antifolate chemotherapies that target the biosynthesis and corrected processing of folate cofactors have been effective in the • Key results including, percentages have been added to the chemotherapy of bacterial and protozoan parasites21. More result section importantly, the folate pathway has also been confirmed as being • The Protein names/sequence verification step resulted in the essential in some eukaryotic pathogens such as Plasmodium, 234 proteins trypanosomes and Leishmania19. • The integrated results from the search strategies in the databases provided the input for the phylogenetic analyses • Table 1 is now Dataset 2 and content of tables and dataset In addition to the folate biosynthesis pathway, proteins that have been modified mediate transport of useful nutrients such as folic acid have been • All figures have been modified as suggested identified as important chemotherapeutic drug targets18,19,23. Hence, • Discussion and conclusions have been modified and the folate pathway, metabolites and transporters continue to be suggested reference added to discussion extensively studied for identification of new enzymes includ- • Newick format phylogenic tree has been submitted in ing transporters, which may serve as new drug targets22. Recent Supplementary Datasets estimates have ascribed eight different membrane transporters to See referee reports eukaryotes24.
Proteins that mediate transportation of folates have been well Introduction studied in a few parasites such as Plasmodium falciparum, A heterogeneous diversity of eukaryotic pathogens is responsi- Trypanosoma brucei, Leishmania donovani and Leishmania ble for the most economically important diseases of humans and major25,26. These studies have provided information on mode of animals1,2. As a result of underdevelopment, a lack of social infra- action of drugs25,27,28 in addition to studies describing mechanisms structure and insufficient funding of public health facilities, most of parasite drug resistance25–32. However, folate transport proteins of these pathogens are endemic to resource-poor countries in sub- remain unidentified and uncharacterized in many other eukaryo- Saharan Africa, South-East Asia and South America, where they tic pathogens. This is despite the sequencing of the genomes of are responsible for high morbidity and mortality1–3. Of these, par- most eukaryotic microbes, which has produced a vast wealth of asitic protozoa form a major group, with the apicomplexans and data that could aid in identification of druggable pathogen-specific kinetoplastid parasites represented by important members, which proteins33–39. It is therefore imperative to search and identify cause diseases such as malaria, cryptosporidiosis, toxoplasmosis, from these parasite genomes additional proteins such as folate babesiosis, leishmaniasis, Human African trypanosomiasis and transporters that may serve as novel drug targets40,41. south American trypanosomiasis or Chagas’ disease causing most of the morbidity and mortality4,5. Other important diseases Therefore, in an attempt to identify and characterize targets for caused by protozoans include giardiasis, amoebic dysentery6,7 and novel therapeutics, we report herein an extensive search of folate trichomoniasis8. A vicious cycle of poverty and disease exists for transporters from pathogen genomes. In addition, we investigated most of these parasites with a high infection and death rate in the evolutionary relationship of these transporters in a bid to affected populations9–11. The appreciable burden of disease caused determine similarities and differences that make them attractive by these parasites has been aggravated by the lack of a licensed drug targets. The knowledge provided may assist in the design of vaccine for most of them12. Furthermore, current drugs of choice new antifolates for protozoan parasites. for treatment for many of the parasites have significant side effects, with the added emergence of drug resistant strains13–15. Despite the Methods urgent demand for new therapies for control, few drugs have been We extracted protein sequences of approximately 200-pathogens developed to combat these parasites16. A major limitation to the that mediate transportation or salvage of folates from Eukaryo- development of new drugs is the paucity of new drug targets. There tic Pathogen Genome Database Resources (http://eupathdb.org/ is therefore a need for discovery of novel and alternate potential eupathdb/), and from the literature using a key-word search. We chemotherapeutic targets that can help in drug development efforts also searched the 200-pathogen genome sequences archived at for disease control16–18. A possible approach to selective antimi- the Eukaryotic Pathogen Genome Database Resources (http:// crobial chemotherapy has been to exploit the inhibition of unique eupathdb.org/eupathdb/). The search was for all proteins that medi- targets, vital to the pathogen and absent in mammals17,18. ate transportation or folate salvage alone or folate salvage and related compounds (such as pteridine, biopterin and methotrex- A metabolic pathway that has been exploited considerably for ate) together. This database gives public access to most sequenced the development of drugs is the folate biosynthetic pathway19. emerging/re-emerging infectious pathogen genomes42. We utilized
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the word “folate” for search on the gene text and “folic acid” was the highest number of folate transporters are Phytophthora para- used to confirm the hits. Hit results containing proteins annotated sitica INRA-310, P. infestans T30–4 and Leptomonas pyrrhocoris as folate-binding protein YgfZ, folate/pteridine transporter, folate/ H10 with 20, 16 and 16 proteins, respectively. While Aspergillus biopterin transporter, reduced folate carrier family protein, folate/ clavatus NRRL 1, A. flavus NRRL3357, A. macrogynus ATCC methotrexate transporter FT1, Folate transporters alone and other 38327, Crithidia fasciculata strain Cf-Cl and others have one folate related proteins were retrieved. The complete list of proteins folate transporter protein each (Dataset 244). The different proteins extracted from EuPathdb is presented in Dataset 143. The folate identified to be involved in folate salvage or related molecules transporters were classified based on type of transporter, number were folate-binding protein YgfZ, folate/pteridine transporter, of transmembrane helix (TMH) and localization (either cell or folate/biopterin transporter, reduced folate carrier family protein, mitochondrial membrane) of transporter. Gene sequences were folate/methotrexate transporter FT1 and folate transporters having obtained in FASTA format for transporter proteins using the a 4%, 11%, 56%, 1%, 3% and 21% identity, respectively. Proteins sequence download tool on EuPathDB (http://eupathdb.org/ that did not belong to these groups were classified as others (4%) eupathdb/). (Figure 1A). A good number of the proteins identified had -pre dicted transmembrane helixes with a few having none (Figure 1B). To ensure that most of the retrieved proteins had not been pre- Furthermore, a number of the transporters possess signal peptides viously studied, we performed a literature search on PubMed (Dataset 143), which may be required for targeting to cellular loca- (http://www.ncbi.nlm.nih.gov/pubmed/?term=) and Google Scholar tions. Deciphering the sequence of the targeting signal may indicate (https://scholar.google.com) using the query “folate transporter + its product destination. Parasite name”. The protein sequence information (Dataset 143) obtained from literature search was used for a BLAST search on Our literature search for parasite folate transporters on PubMed EupathDB (http://eupathdb.org/eupathdb/), UniprotDB (http://www. and Google Scholar indicated 60% (38 out 63) of the proteins uniprot.org) and GeneDB (http://www.genedb.org/Homepage). were identified for the first time as presented in Dataset 244, The protein information are included in Dataset 143 and summa- while 40% have been previously investigated. Besides, the Leish- rised in Dataset 244, which are marked as either identified in other mania folate transporters we came across were not found on the literature or in this research work. Sequence data were edited on EupathDB resource. We thus performed a BLAST search of textEdit mac version and uploaded to Molecular Evolutionary Kinetoplastida on EupathDB, the returned hits were folate/ Genetics Analysis (MEGA) platform version 7.0 obtained from biopterin transporter for L. infantum. The only Plasmodium http://www.megasoftware.net45. The 234 sequences were aligned species with results for proteins that salvage folate was P. falci- using muscle tools with large alignment (Max iterations = 2) selected parum. Our study, however, describes for the first time the presence while other settings were left at defaults. Evolutionary history was of these transporters in other Plasmodium species. There were inferred using the Neighbor-Joining method46. The percentage of no transporter proteins deposited in EupathDB for P. malariae replicate trees in which the associated taxa clustered together in and P. ovale. However, folate transporters I and II were retrieved the bootstrap test (500 replicates) was also analysed47. The tree was from our search of GeneDB for P. malariae and P. ovale curtisi, drawn to scale, with branch lengths in the same units as those of respectively. the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the number of dif- Our analysis of folate transporters indicate the presence in Plas- ferences method48. While uniform rate and complete deletion was modium species of two proteoforms; folate transporter I and II selected for substitution rates and data subset, respectively. Other (Dataset 143). All Leishmania species identified possess folate/biopt- parameters were at default settings. All positions containing gaps erin transporters and not folate transporters. Trypanosome species and missing data were eliminated. The newick format of the tree have both folate/pteridine and folate transporters; T. cruzi Dm28c, was exported and opened on FigTree 1.4.2 platform downloaded T. cruzi Sylvio X10/1 and T. cruzi CL Brener Esmeraldo-like, from http://tree.bio.ed.ac.uk/software/figtree/49. The final tree was T. cruzi CL Brener Non-Esmeraldo-like and T. cruzi marinkellei constructed using radial tree layout. Additional analysis consisted strain B7 all have folate/pteridine transporter while T. brucei of sub-phylogenies based on the transporter type. Since folate- TREU927, T. brucei Lister strain 427, T. brucei gambiense DAL972, binding protein YgfZ, folate/pteridine transporter, folate/biopterin T. congolense IL3000 possess folate transporters. Eukaryotic para- transporter, putative, reduced folate carrier family protein, folate/ sites like Eimeria acervulina Houghton, E. brunetti Houghton, methotrexate transporter FT1, putative folate transporters alone and E. maxima Weybridge, E. necatrix Houghton, E. praecox Houghton, others have 10, 25, 132, 2, 7, 49 and 9. So we decided to reconstruct E. tenella strain Houghton and Neospora caninum Liverpool all the phylogeny based folate transporter, folate-biopterin transporter boast folate/methotrexate transporter FT1. The folate-binding pro- after considering the identification number, the species diversity in tein YgfZ was found in the fungus, Allomyces macrogynus ATCC each category. 38327, the protist C. fasciculata strain Cf-Cl, C. immitis RS, the feline protozoon, Hammondia hammondi strain H.H.34, Sarco- Results cystis neurona SN3, S. punctatus DAOM BR117, T. gondii GT1, A methodological search for folate transporters in all eukaryo- T. gondii ME49, T. gondii VEG and T. brucei TREU927. Parasites tic microbe genomes examined under EuPathDB with validation such as Microsporidium daphniae UGP3 and the amoeba Naegle- via GenBank, GeneDB and UniProt contained a total of 234 pro- ria fowleri ATCC 30863 possess the reduced folate carrier family teins (detail features of proteins are presented in Dataset 143). We protein. We observed that 7% of the identified proteins are local- identified these transporters in 28 pathogen species (containing ized on the mitochondrial membrane of some pathogens such as 63 strains) cutting across 12 phyla (Dataset 244). The parasites with the fungi Aspergillus clavatus NRRL 1, A. flavus NRRL3357,
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C. immitis RS, the yeast Cryptococcus neoformans var. grubii Dataset 1. Complete list of proteins extracted from Eupthadb and H99, Fusarium graminearum PH-1, A. capsulatus G186AR, Lepto- literature search, including their properties monas pyrrhocoris H10, the food fungus Neosartorya fischeri NRRL 43 181, Phytophthora parasitica INRA-310 and P. ultimum DAOM http://dx.doi.org/10.5256/f1000research.10561.d167325 BR144. The remaining proteins are localized on the plasma These data are available in a .xlsx file membrane (Dataset 143).
Approximately 15% (34/234) of the folate transporters identi- Dataset 2. Eukaryotic microbes from which folate transporters fied possess signal peptides with the trypanosomes with the most were identified signal peptides. Deductions can be made of the probable destination http://dx.doi.org/10.5256/f1000research.10561.d16732644 within the cell of any transporter by its signal peptide sequence; These data are available in a .xlsx file. thus, further work may seek to decipher the sequence of the targeting signal to determine its localization. The proteins identified all have transmembrane helixes with the exception of the Discussion alveolate Chromera velia CCMP2878, apicomplexan P. berghei Folate transporters are important proteins involved in the salvage ANKA, S. neurona SN3, the kinetoplastid T. brucei TREU927, of folate, cofactors and related molecules in eukaryotic pathogens T. grayi ANR4 and protist Vitrella brassicaformis CCMP3155 important for metabolism and survival in their respective hosts21. with Gene ID’s Cvel_17766, PBANKA_0713700, SN3_01500005, We identified proteins that could mediate the salvage of folates into Tb927.8.6480, Tgr.2739.1000 and Vbra_15327, respectively cells and/or mitochondria from eukaryotic microbe genomes in (Dataset 143). EuPathDB. Many of these proteins are involved in folate The phylogenetic tree (Figure 2) shows the evolutionary position, biosynthesis or transport and are present in most of the eukaryo- history and relationship of all the folate transporters identified in tic microbe genomes we queried. In this study, 234 genes encod- this work. The type of transporter or species/strain was used for ing homologues of folate salvaging proteins were identified in constructing phylogenic trees, with the 234 proteins identified the genome of 64 strains, representing 28 species of eukaryotic forming two clades, a major and minor. The major clade lacked a microbes. Some of the pathogens among the microbes queried sub-clade, while the minor clade possessed a sub-clade. All proteins include P. falciparum 3D7 and IT, P. knowlesi H, P. berghei ANKA, identified were distributed between the two major clades; except P. chabaudi chabaudi, T. brucei Lister 427, T. brucei TREU927, for folate/methotrexate transporter and mitochondrial folate trans- T. brucei gambiense DAL972, Encephalitozoon cuniculi GB-M1. porter, with the latter present on the major clade and the former The pathogens range from bacteria through to fungi, intracellular on the minor clade exclusively. All the species are represented on parasites such as Plasmodium and leishmania species, to extra- both clades, however, V. brassicaformis CCMP3155, Plasmodium cellular parasites such as trypanosome species. This suggests a species, A. clavatus NRRL, A. flavus NRRL3357, A. macrogynus widespread presence of the proteins cutting across a range of ATCC 38327, C. fasciculata strain Cf-Cl, C. immitis RS, C. immitis pathogens that infect humans and animals. RS, C. muris RN66, C. neoformans var. grubii H99, C. neoformans var. grubii H99, Leishmania species, N. bombycis CQ1, N. caninum About 40% of the proteins we identified have been previously Liverpool, F. graminearum PH-1 and H. hammondi strain H.H.34 identified and characterized in parasites such as Plasmodium are exclusively on the major clade. There are some parasites that falciparum22,30, Trypanosome species26, Leishmania species and were identified once, as shown in Dataset 143; these are mostly in Toxoplasma gondii50. It has been estimated that over half of the the large clade. Some of these pathogens include P. ultimum DAOM drugs currently on the market target integral membrane proteins BR144, which has mitochondrial folate transporter/carrier proteins ion channel blockers like verapamil, and serotonin transporter similar to Homo sapiens, E. cuniculi GB-M1, which has proteins inhibitors feature prominently in the WHO model list of essential similar to folate transporter, and S. punctatus DAOM BR117, which medicines51,52. Unfortunately, a great number of these transport- has folate-binding protein YgfZ. These were the only proteins of ers have not been adequately explored as drug targets53. Folate the aforementioned species identified in this work. However, transporters therefore represent attractive drug targets for M. daphniae UGP3, which had reduced folate carrier domain treatment of infectious diseases. Thus their identification from containing protein, was the only parasite that was found in the other eukaryotic pathogens could open a window for novel small clade. Improving on our phylogenetic analysis, we performed chemotherapeutics for disease control54,55. a sub-phylogenetic reconstruction (Figure 3–Figure 5) based on the substrate type of the transport proteins. After phylogenetic In Plasmodium two folate transporters have been identified, namely analysis each sub-phylogeny show a clear characterization PfFT1 and PfT2. These transporters have been shown to mediate except for folate-biopterin transporters (Figure 4), which fell in the salvage of folate derivatives and precursors in P. falciparum a different clade save for Leptomonas species and C. velia. The and proposed blocking of their salvage activities may improve newick formats of the phylogenetic trees in Figure 2–Figure 5 are the antimalarial efficacy of several classes of antimalarial drugs. presented as Supplementary Dataset 1–Supplementary Dataset 4. In our work we identified folate transporters for other plasmodial
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Figure 2. Phylogenetic tree showing the relatedness of all the proteins identified to transport folate in different pathogen species. Tree was inferred using the Neighbor-Joining method with bootstrap test at 1000 replicates.
Page 7 of 24 5FW45*#@*@5SZQBOPTPNB@FWBOTJ@TUSBJO@45*#@@*@GPMBUF@USBOTQPSUFS@QVUBUJWF@QVUBUJWF
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Page 8of24 Figure 3. Phylogenetic tree showing the relatedness of folate transporters alone in different pathogen species. Tree was inferred using the Neighbor-Joining method with bootstrap test at 1000 replicates. F1000Research 2017, 6:36 Last updated: 28 JUL 2021
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/$-*7@]/FPTQPSB@DBOJOVN@-JWFSQPPM]QVUBUJWF@GPMBUFNFUIPUSFYBUF@USBOTQPSUFS F1000Research 2017,6:36Lastupdated:28JUL2021
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Figure 5. Phylogenetic tree showing the relatedness of folate-binding protein YgfZ alone in different pathogen species. Tree was inferred using the Neighbor-Joining method with bootstrap test at 1000 replicates. F1000Research 2017, 6:36 Last updated: 28 JUL 2021
species, which, like P. falciparum, may also be chemotherapeu- phylogenetically distinct clades in the eukaryotic pathogens exam- tic targets. Transport of folate in higher eukaryotes is made pos- ined. The clustering of these proteins suggests that these transport sible by a high affinity folate-biopterin transporters FBT or BT1 proteins have highly conserved regions often required for basic family22,30. In the trypanosomes and related kinetoplastids, a cellular function or stability60,71–87. Thus, antifolate chemothera- member of these transporters, the folate biopterin transporter pic drugs that are effective against one pathogen might have some (FBT) family of proteins was identified in Leishmania28. It is effect on others. However, the converse may be the case for the thought that MFS proteins are related to the FBT. These proteins free-living non-parasitic photosynthetic algae, Chromera velia have been characterized in a few protozoa and cyanobacteria56. and Vitrella brassicaformis, protists related to apicomplexans88,89. Results from our study describing the presence of these trans- These groups of algae live freely in their environment, which porters across several phyla corroborate results from other works unlike apicomplexans that depend on a host animal to survive88. establishing the conservation of folate transport function among This adaptation may explain the difference in the clustering of FBT family proteins from plants and protists22,56. their transporters after phylogenetic analysis, which separated on the minor clade from other apicomplexans that separated on the Malaria parasites encode transporters belonging to the organo anion major clade. This suggests a high level of evolutionary divergence transporter (OAT) folate-biopterin transporter (FBT), glycoside- between folate transporters in both the apicomplexans and these pentoside-hexuronide: cation symporter (GPH), families, which algae based on life-style adaptations. are closely related to the major facilitator super-family of mem- brane proteins57. The inhibition of these transporters by blockers of Conclusion organic anion transporters such as probenecid has been implicated In summary, we have retrieved information on 234 folate trans- in sensitization of Plasmodium resistant parasites to antifolates58,59. porter proteins from Eukaryotic Pathogen Database (EuPathDB) Thus, in Plasmodium chemotherapy, identification of folate trans- resources. The folate transporter proteins were categorized into porters could lead to screening for compounds that interfere with potential drug targeting features including mitochondrial locali- folate transport and salvage for antimalarial chemotherapy22,30. zation, number of transmembrane helix, and protein sequence We identified several types of folate transporters that have been relatedness. The identification of folate salvage proteins in diverse described and functionally characterized in Leishmania with some eukaryotes extend the evolutionary diversity of these proteins implicated in the import of the antifolate methotrexate60,61. Thus and suggests they might offer new possibilities for potential far, only protozoan transporters in Plasmodium, Leishmania, and drug development targeting folate-salvaging routes in eukaryotic Trypanosoma brucei have been characterized and these are known pathogens. to mediate the uptake of the vitamins folate and/or biopterin22,62,63. Thus in parasites species of medical importance folate transporter Data availability proteins may provide new targets for therapy. Dataset 1: Complete list of proteins extracted from Eupthadb and literature search, including their properties. These data We also identified folate salvaging proteins from fungi such as are available in a .xlsx file. Doi, 10.5256/f1000research.10561. Coccidioides immitis and A. clavatus, fungi found in soil64–66, d16732543 vegetable64 and waters in tropical and subtropical areas67. These fungi are known to occasionally become pathogenic and act as Dataset 2: Eukaryotic microbes from which folate transport- opportunistic pathogens for animals and man66. Coccidioidomyco- ers were identified. These data are available in a .xlsx file. Doi, sis caused by C. immitis in association with AIDS has been known 10.5256/f1000research.10561.d16732644 to be a fatal disease68. Treatment of acute and chronic infections with antifungals such as amphotericin B have not been adequate, hence folate transporters may present new targets in these group of pathogens. Identification was also made on pathogens such as Author contributions C. fasciculata that parasitize several species of insects including M.O.F. and B.O. Conceptualized and Designed the study. M.O.F. mosquitoes and has been widely used to test new therapeutic strat- and B.O. structured methodology. B.O. performed analysis. egies against parasitic infections69. As a model organism, folate M.O.F and B.O. wrote manuscript. transporters identified in C. fasciculata may be useful in research for developing new drugs in medically important Kinetoplasts as Competing interests has been shown for other targets in this protozoan parasite70. No competing interests were disclosed.
We noticed that P. parasitica INRA-310 and L. pyrrhocoris H10 Grant information had the highest number of folate transporters identified. Their util- B.O. was supported by a TWAS-CNPq fellowship (FP number: ity as model fungal (P. parasitica) and monoxenous kinetoplast 3240274297). may provide models instrumental for developing new antifolates for fungal and protozoan diseases. The relatedness of these pro- The funders had no role in study design, data collection and analysis, teins across the different pathogens show that there are two major decision to publish, or preparation of the manuscript.
Page 11 of 24 F1000Research 2017, 6:36 Last updated: 28 JUL 2021
Supplementary material Supplementary Dataset 1: Newick format for the phylogenetic tree showing the relatedness of all the proteins identified to transport folate in different pathogen species. Tree was inferred using the Neighbor-Joining method with bootstrap test at 1000 replicates. Click here to access the data.
Supplementary Dataset 2: Newick format for the phylogenetic tree showing the relatedness of folate transporters alone in different pathogen species. Tree was inferred using the Neighbor-Joining method with bootstrap test at 1000 replicates. Click here to access the data.
Supplementary Dataset 3: Newick format for the phylogenetic tree showing the relatedness of folate/biopterin transporter alone in different pathogen species. Tree was inferred using the Neighbor-Joining method with bootstrap test at 1000 replicates. Click here to access the data.
Supplementary Dataset 4: Newick format for the phylogenetic tree showing the relatedness of folate-binding protein YgfZ alone in different pathogen species. Tree was inferred using the Neighbor-Joining method with bootstrap test at 1000 replicates. Click here to access the data.
References
1. Fadiel A, Isokpehi RD, Stambouli N, et al.: Protozoan parasite aquaporins. Expert 1551–62. Rev Proteomic. 2009; 6(2): 199–211. PubMed Abstract | Publisher Full Text PubMed Abstract | Publisher Full Text 15. Castillo E, Dea-Ayuela MA, Bolás-Fernández F, et al.: The kinetoplastid 2. Prole DL, Taylor CW: Identification and analysis of putative homologues of chemotherapy revisited: current drugs, recent advances and future mechanosensitive channels in pathogenic protozoa. PLoS One. 2013; 8(6): perspectives. Curr Med Chem. 2010; 17(33): 4027–51. e66068. PubMed Abstract | Publisher Full Text PubMed Abstract | Publisher Full Text | Free Full Text 16. Dias DA, Urban S, Roessner U: A historical overview of natural products in drug 3. Wiser M: Protozoa and human disease. Garland Science. 2010. discovery. Metabolites. 2012; 2(2): 303–36. Reference Source PubMed Abstract | Publisher Full Text | Free Full Text 4. Turrens JF: Oxidative stress and antioxidant defenses: a target for the 17. Croft SL: The current status of antiparasite chemotherapy. Parasitology. 1997; treatment of diseases caused by parasitic protozoa. Mol Aspects Med. 2004; 114(Suppl): S3–15. 25(1–2): 211–20. PubMed Abstract PubMed Abstract | Publisher Full Text 18. Pink R, Hudson A, Mouriès MA, et al.: Opportunities and challenges in 5. Shiadeh MN, Niyyati M, Fallahi S, et al.: Human parasitic protozoan infection to antiparasitic drug discovery. Nat Rev Drug Discov. 2005; 4(9): 727–40. infertility: a systematic review. Parasitol Res. 2016; 115(2): 469–77. PubMed Abstract | Publisher Full Text PubMed Abstract | Publisher Full Text 19. Paiardini A, Fiascarelli A, Rinaldo S, et al.: Screening and in vitro testing of 6. Khanum H, Kadir R, Arju T, et al.: Detection of Entamoeba histolytica, Giardia antifolate inhibitors of human cytosolic serine hydroxymethyltransferase. lamblia and Cryptospodium sp. Infection among diarrheal patients. Bangladesh ChemMedChem. 2015; 10(3): 490–7. J Zoology. 2015; 43(1): 1–7. PubMed Abstract | Publisher Full Text | Free Full Text Publisher Full Text 20. Nzila A, Ward SA, Marsh K, et al.: Comparative folate metabolism in humans 7. Herman ML, Surawicz CM: Intestinal Parasites. In: Textbook of Pediatric and malaria parasites (part I): pointers for malaria treatment from cancer Gastroenterology, Hepatology and Nutrition. Springer International Publishing. 2016; chemotherapy. Trends Parasitol. 2005; 21(6): 292–8. 185–193. PubMed Abstract | Publisher Full Text | Free Full Text Publisher Full Text 21. Nzila A, Ward SA, Marsh K, et al.: Comparative folate metabolism in humans 8. Ton Nu PA, Nguyen VQ, Cao NT, et al.: Prevalence of Trichomonas vaginalis and malaria parasites (part II): activities as yet untargeted or specific to infection in symptomatic and asymptomatic women in Central Vietnam. J Infect Plasmodium. Trends Parasitol. 2005; 21(7): 334–9. Dev Ctries. 2015; 9(06): 655–60. PubMed Abstract | Publisher Full Text | Free Full Text PubMed Abstract | Publisher Full Text 22. Salcedo-Sora JE, Ward SA: The folate metabolic network of Falciparum malaria. 9. Garcia LS, Bruckner DA: Diagnostic medical parasitology. Washington, DC. Mol Biochem Parasitol. 2013; 188(1): 51–62. 2001; 131–5. PubMed Abstract | Publisher Full Text 10. Macpherson CN: Human behaviour and the epidemiology of parasitic 23. Costi MP, Ferrari S: Update on antifolate drugs targets. Curr Drug Targets. 2001; zoonoses. Int J Parasitol. 2005; 35(11–12): 1319–31. 2(2): 135–66. PubMed Abstract | Publisher Full Text PubMed Abstract | Publisher Full Text 11. Andrews KT, Fisher G, Skinner-Adams TS: Drug repurposing and human 24. Zhao R, Diop-Bove N, Visentin M, et al.: Mechanisms of membrane transport of parasitic protozoan diseases. Int J Parasitol Drugs Drug Resis. 2014; 4(2): folates into cells and across epithelia. Annu Rev Nutr. 2011; 31: 177–201. 95–111. PubMed Abstract | Publisher Full Text | Free Full Text PubMed Abstract | Publisher Full Text | Free Full Text 25. Müller IB, Hyde JE: Antimalarial drugs: modes of action and mechanisms of 12. Ouattara A, Laurens MB: Vaccines against malaria. Clin Infect Dis. 2015; 60(6): parasite resistance. Future Microbiol. 2010; 5(12): 1857–73. 930–6. PubMed Abstract | Publisher Full Text PubMed Abstract | Publisher Full Text | Free Full Text 26. Emmer BT, Nakayasu ES, Souther C, et al.: Global analysis of protein 13. Monzote L, Siddiq A: Drug development to protozoan diseases. Open Med Chem palmitoylation in African trypanosomes. Eukaryot Cell. 2011; 10(3): 455–63. J. 2011; 5(1): 1–3. PubMed Abstract | Publisher Full Text | Free Full Text PubMed Abstract | Publisher Full Text | Free Full Text 27. Müller IB, Hyde JE: Folate metabolism in human malaria parasites--75 years on. 14. Petersen I, Eastman R, Lanzer M: Drug-resistant malaria: molecular Mol Biochem Parasitol. 2013; 188(1): 63–77. mechanisms and implications for public health. FEBS Lett. 2011; 585(11): PubMed Abstract | Publisher Full Text
Page 12 of 24 F1000Research 2017, 6:36 Last updated: 28 JUL 2021
28. Vickers TJ, Beverley SM: Folate metabolic pathways in Leishmania. Essays 2016; 7: 10519. Biochem. 2011; 51: 63–80. PubMed Abstract | Publisher Full Text | Free Full Text PubMed Abstract | Publisher Full Text | Free Full Text 53. Mansour TE: Chemotherapeutic targets in parasites: contemporary strategies. 29. Borst P, Ouellette M: New mechanisms of drug resistance in parasitic protozoa. Cambridge University Press; 2002. Annu Rev Microbiol. 1995; 49(1): 427–60. Reference Source PubMed Abstract | Publisher Full Text 54. Alam A, Goyal M, Iqbal MS, et al.: Novel antimalarial drug targets: hope for new 30. Wang P, Brobey RK, Horii T, et al.: Utilization of exogenous folate in the human antimalarial drugs. Expert Rev Clin Pharmacol. 2009; 2(5): 469–89. malaria parasite Plasmodium falciparum and its critical role in antifolate drug PubMed Abstract | Publisher Full Text synergy. Mol Microbiol. 1999; 32(6): 1254–62. 55. Dean P, Major P, Nakjang S, et al.: Transport proteins of parasitic protists and PubMed Abstract | Publisher Full Text their role in nutrient salvage. Front Plant Sci. 2014; 5: 153. 31. Ouellette M, Légaré D, Papadopoulou B: Multidrug resistance and ABC PubMed Abstract | Publisher Full Text | Free Full Text transporters in parasitic protozoa. J Mol Microbiol Biotechnol. 2001; 3(2): 201–6. 56. Saier MH Jr, Beatty JT, Goffeau A, et al.: The major facilitator superfamily. J Mol PubMed Abstract Microbiol Biotechnol. 1999; 1(2): 257–79. 32. Stein WD, Sanchez CP, Lanzer M: Virulence and drug resistance in malaria PubMed Abstract parasites. Trends Parasitol. 2009; 25(10): 441–3. 57. Sowunmi A, Fehintola FA, Adedeji AA, et al.: Open randomized study of PubMed Abstract | Publisher Full Text pyrimethamine-sulphadoxine vs. pyrimethamine-sulphadoxine plus 33. Agüero F, Al-Lazikani B, Aslett M, et al.: Genomic-scale prioritization of drug probenecid for the treatment of uncomplicated Plasmodium falciparum malaria targets: the TDR Targets database. Nat Rev Drug Discov. 2008; 7(11): 900–7. in children. Trop Med Int Health. 2004; 9(5): 606–14. PubMed Abstract | Publisher Full Text | Free Full Text PubMed Abstract | Publisher Full Text 34. Franzén O, Jerlström-Hultqvist J, Castro E, et al.: Draft genome sequencing of 58. Nzila A, Mberu E, Bray P, et al.: Chemosensitization of Plasmodium falciparum giardia intestinalis assemblage B isolate GS: is human giardiasis caused by by probenecid in vitro. Antimicrob Agents Chemother. 2003; 47(7): 2108–12. two different species? PLoS Pathog. 2009; 5(8): e1000560. PubMed Abstract | Publisher Full Text | Free Full Text PubMed Abstract | Publisher Full Text | Free Full Text 59. Richard D, Kündig C, Ouellette M: A new type of high affinity folic acid 35. Butt AM, Nasrullah I, Tahir S, et al.: Comparative genomics analysis of transporter in the protozoan parasite Leishmania and deletion of its gene in Mycobacterium ulcerans for the identification of putative essential genes and methotrexate-resistant cells. J Biol Chem. 2002; 277(33): 29460–7. therapeutic candidates. PLoS One. 2012; 7(8): e43080. PubMed Abstract | Publisher Full Text PubMed Abstract | Publisher Full Text | Free Full Text 60. Richard D, Leprohon P, Drummelsmith J, et al.: Growth phase regulation of the 36. Tiffin N, Adie E, Turner F,et al.: Computational disease gene identification: a main folate transporter of Leishmania infantum and its role in methotrexate concert of methods prioritizes type 2 diabetes and obesity candidate genes. resistance. J Biol Chem. 2004; 279(52): 54494–501. Nucleic Acids Res. 2006; 34(10): 3067–81. PubMed Abstract | Publisher Full Text PubMed Abstract | Publisher Full Text | Free Full Text 61. Kündig C, Haimeur A, Légaré D, et al.: Increased transport of pteridines 37. Götz S, García-Gómez JM, Terol J, et al.: High-throughput functional annotation compensates for mutations in the high affinity folate transporter and and data mining with the Blast2GO suite. Nucleic Acids Res. 2008; 36(10): contributes to methotrexate resistance in the protozoan parasite Leishmania 3420–35. tarentolae. EMBO J. 1999; 18(9): 2342–51. PubMed Abstract | Publisher Full Text | Free Full Text PubMed Abstract | Publisher Full Text | Free Full Text 38. Nikolskaya T, Bugrim A, Nikolsky Y, et al.: Methods for identification of novel 62. Gottesdiener KM: A new VSG expression site-associated gene (ESAG) in the protein drug targets and biomarkers utilizing functional networks. United promoter region of Trypanosoma brucei encodes a protein with 10 potential States patent US 8000949 B2; 2011. transmembrane domains. Mol Biochem Parasitol. 1994; 63(1): 143–51. Reference Source PubMed Abstract | Publisher Full Text 39. Panwar B, Menon R, Eksi R, et al.: Genome-wide Functional Annotation of 63. Stewart RA, Meyer KF: Isolation of Coccidioides immitis (Stiles) from the soil. Human Protein-coding Splice Variants Using Multiple Instance Learning. Exp Biol Med. 1932; 29(8): 937–8. J Proteome Res. 2016; 15(6): 1747–53. Publisher Full Text PubMed Abstract | Publisher Full Text 64. Greene DR, Koenig G, Fisher MC, et al.: Soil isolation and molecular 40. Antony AC: Folate receptors. Annu Rev Nutr. 1996; 16(1): 501–21. identification of Coccidioides immitis. Mycologia. 2000; 92(3): 406–10. PubMed Abstract | Publisher Full Text Publisher Full Text 41. Girardin F: Membrane transporter proteins: a challenge for CNS drug 65. Litvintseva AP, Marsden-Haug N, Hurst S, et al.: Valley fever: finding new places development. Dialogues Clin Neurosci. 2006; 8(3): 311–21. for an old disease: Coccidioides immitis found in Washington State soil PubMed Abstract | Free Full Text associated with recent human infection. Clin Infect Dis. 2015; 60(1): e1–3. 42. Aurrecoechea C, Heiges M, Wang H, et al.: ApiDB: integrated resources for PubMed Abstract | Publisher Full Text | Free Full Text the apicomplexan bioinformatics resource center. Nucleic Acids Res. 2007; 66. Hajji M, Kanoun S, Nasri M, et al.: Purification and characterization of an 35(Database issue): D427–30. alkaline serine-protease produced by a new isolated Aspergillus clavatus ES1. PubMed Abstract | Publisher Full Text | Free Full Text Process Biochem. 2007; 42(5): 791–7. 43. Falade MO, Otarigho B: Dataset 1 in: Characterization of potential drug Publisher Full Text targeting folate transporter proteins from Eukaryotic Pathoge. F1000Research. 67. Rodríguez-Cerdeira C, Arenas R, Moreno-Coutiño G, et al.: Systemic fungal 2017. infections in patients with human inmunodeficiency virus. Actas Dermosifiliogr. Data Source 2014; 105(1): 5–17. 44. Falade MO, Otarigho B: Dataset 2 in: Characterization of potential drug PubMed Abstract | Publisher Full Text targeting folate transporter proteins from Eukaryotic Pathogens. 68. Awadelkariem FM, Hunter KJ, Kirby GC, et al.: Crithidia fasciculata as feeder F1000Research. 2017. cells for malaria parasites. Exp Parasitol. 1995; 80(1): 98–106. Data Source PubMed Abstract | Publisher Full Text 45. Saitou N, Nei M: The neighbor-joining method: a new method for 69. Krungkrai J, Cerami A, Henderson GB: Pyrimidine biosynthesis in parasitic reconstructing phylogenetic trees. Mol Biol Evol. 1987; 4(4): 406–25. protozoa: purification of a monofunctional dihydroorotase from Plasmodium PubMed Abstract | Publisher Full Text berghei and Crithidia fasciculata. Biochemistry. 1990; 29(26): 6270–5. 46. Sanderson MJ: Confidence limits on phylogenies: the bootstrap revisited. PubMed Abstract | Publisher Full Text Cladistics. 1989; 5(2): 113–29. 70. Eyal E, Najmanovich R, Mcconkey BJ, et al.: Importance of solvent accessibility Publisher Full Text and contact surfaces in modeling side-chain conformations in proteins. J Comput Chem. 2004; 25(5): 712–24. 47. Nei M, Kumar S: Molecular evolution and phylogenetics. Oxford university press; PubMed Abstract Publisher Full Text 2000. | Reference Source 71. Fedorova ND, Khaldi N, Joardar VS, et al.: Genomic islands in the pathogenic filamentous fungus Aspergillus fumigatus. PLoS Genet. 2008; 4(4): e1000046. 48. Rambaut A: FigTree 1.4. 2 software. Institute of Evolutionary Biology, Univ. PubMed Abstract Publisher Full Text Free Full Text Edinburgh. | | 72. Nierman WC, Yu J, Fedorova-Abrams ND, et al.: Genome sequence of 49. Massimine KM, Doan LT, Atreya CA, et al.: Toxoplasma gondii is capable Aspergillus flavus NRRL 3357, a strain that causes aflatoxin contamination of of exogenous folate transport. A likely expansion of the BT1 family of food and feed. Genome Announc. 2015; 3(2): e00168–15. transmembrane proteins. Mol Biochem Parasitol. 2005; 144(1): 44–54. PubMed Abstract Publisher Full Text Free Full Text PubMed Abstract Publisher Full Text | | | 73. Katinka MD, Duprat S, Cornillot E, et al.: Genome sequence and gene 50. Swaan PW: Membrane Transport Proteins and Drug Transport. Burger’s compaction of the eukaryote parasite Encephalitozoon cuniculi. Nature. 2001; Medicinal Chemistry and Drug Discovery. 2003. 414(6862): 450–3. Publisher Full Text PubMed Abstract | Publisher Full Text 51. World Health Organization: WHO model list of essential medicines. 2015; 1–55. 74. Rogers MB, Hilley JD, Dickens NJ, et al.: Chromosome and gene copy number Reference Source variation allow major structural change between species and strains of 52. Kenthirapalan S, Waters AP, Matuschewski K, et al.: Functional profiles of orphan Leishmania. Genome Res. 2011; 21(12): 2129–42. membrane transporters in the life cycle of the malaria parasite. Nat Commun. PubMed Abstract | Publisher Full Text | Free Full Text
Page 13 of 24 F1000Research 2017, 6:36 Last updated: 28 JUL 2021
75. Selman M, Sak B, Kváč M, et al.: Extremely reduced levels of heterozygosity in 82. El-Sayed NM, Myler PJ, Blandin G, et al.: Comparative genomics of the vertebrate pathogen Encephalitozoon cuniculi. Eukaryot Cell. 2013; 12(4): trypanosomatid parasitic protozoa. Science. 2005; 309(5733): 404–9. 496–502. PubMed Abstract | Publisher Full Text PubMed Abstract | Publisher Full Text | Free Full Text 83. El-Sayed NM, Myler PJ, Bartholomeu DC, et al.: The genome sequence of 76. Kaur K, Coons T, Emmett K, et al.: Methotrexate-resistant Leishmania donovani Trypanosoma cruzi, etiologic agent of Chagas disease. Science. 2005; genetically deficient in the folate-methotrexate transporter. J Biol Chem. 1988; 309(5733): 409–15. 263(15): 7020–8. PubMed Abstract | Publisher Full Text PubMed Abstract 84. Massimine KM, Doan LT, Atreya CA, et al.: Toxoplasma gondii is capable 77. Beck JT, Ullman B: Affinity labeling of the folate-methotrexate transporter from of exogenous folate transport. A likely expansion of the BT1 family of Leishmania donovani. Biochemistry. 1989; 28(17): 6931–7. transmembrane proteins. Mol Biochem Parasitol. 2005; 144(1): 44–54. PubMed Abstract | Publisher Full Text PubMed Abstract | Publisher Full Text 78. Ouameur AA, Girard I, Légaré D, et al.: Functional analysis and complex gene 85. Kelly S, Ivens A, Manna PT, et al.: A draft genome for the African crocodilian rearrangements of the folate/biopterin transporter (FBT) gene family in the trypanosome Trypanosoma grayi. Sci Data. 2014; 1: 140024. protozoan parasite Leishmania. Mol Biochem Parasitol. 2008; 162(2): 155–64. PubMed Abstract | Publisher Full Text | Free Full Text PubMed Abstract | Publisher Full Text 86. Stoco PH, Wagner G, Talavera-Lopez C, et al.: Genome of the avirulent human- 79. Ubeda JM, Légaré D, Raymond F, et al.: Modulation of gene expression in drug infective trypanosome--Trypanosoma rangeli. PLoS Negl Trop Dis. 2014; 8(9): resistant Leishmania is associated with gene amplification, gene deletion and e3176. chromosome aneuploidy. Genome Biol. 2008; 9(7): R115. PubMed Abstract | Publisher Full Text | Free Full Text PubMed Abstract | Publisher Full Text | Free Full Text 87. Kumar S, Stecher G, Tamura K: MEGA7: Molecular Evolutionary Genetics 80. Flegontov P, Butenko A, Firsov S, et al.: Genome of Leptomonas pyrrhocoris: a Analysis Version 7.0 for Bigger Datasets. Mol Biol Evol. 2016; 33(7): 1870–4. high-quality reference for monoxenous trypanosomatids and new insights into PubMed Abstract | Publisher Full Text evolution of Leishmania. Sci Rep. 2016; 6: 23704. 88. Janouškovec J, Horák A, Barott KL, et al.: Environmental distribution of coral- PubMed Abstract | Publisher Full Text | Free Full Text associated relatives of apicomplexan parasites. ISME J. 2013; 7(2): 444–7. 81. Kraeva N, Butenko A, Hlaváčová J, et al.: Leptomonas seymouri: Adaptations PubMed Abstract | Publisher Full Text | Free Full Text to the Dixenous Life Cycle Analyzed by Genome Sequencing, Transcriptome 89. Woo YH, Ansari H, Otto TD, et al.: Chromerid genomes reveal the evolutionary Profiling and Co-infection with Leishmania donovani. PLoS Pathog. 2015; 11(8): path from photosynthetic algae to obligate intracellular parasites. eLife. 2015; e1005127. 4: e06974. PubMed Abstract | Publisher Full Text | Free Full Text PubMed Abstract | Publisher Full Text | Free Full Text
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Open Peer Review
Current Peer Review Status:
Version 2
Reviewer Report 04 September 2017 https://doi.org/10.5256/f1000research.12807.r24210
© 2017 Isokpehi R. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Raphael D. Isokpehi College of Science, Engineering and Mathematics (CSEM) , Bethune-Cookman University , Daytona Beach, FL, USA
General Comments 1. Several revisions are noted especially the datasets and the phylogenetic trees. However, the narrative contain sections that needs to be improved for accuracy. 2. Correct abbreviations. For example “EuPathdb” should be “EuPathDB” Specific Comments. Suggested revisions are provided.
INTRODUCTION 1. The statement of objectives of the research needs to be clear. “Therefore, in an attempt to identify and characterize targets for novel therapeutics, we report herein an extensive search of folate transporters from pathogen genomes. In addition, we investigated the evolutionary relationship of these transporters in a bid to determine similarities and differences that make them attractive drug targets. The knowledge provided may assist in the design of new antifolates for protozoan parasites.”
Suggested Revision: “The objectives of the research reported in this article were to (1) characterize folate transporters encoded in genomes of eukaryotic pathogens; and (2) determine evolutionary relationship of folate transporters in genomes of eukaryotic genomes. These research objectives will help advance the development of effective anti-folate drugs to reduce the morbidity and mortality associated with eukaryotic pathogens.”
METHODS 1. Divide the methods section according to the two objectives. 2. approximately 200-pathogens
Suggested revision: delete “-“. The purpose of the dash is not justified. 3. The folate transporters were classified based on type of transporter, number of
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transmembrane helix (TMH) and localization (either cell or mitochondrial membrane) of transporter. Gene sequences were obtained in FASTA format for transporter proteins using the sequence download tool on EuPathDB (http://eupathdb.org/eupathdb/).
Suggested revision: “The folate transporters were characterized according to type of transporter, number of transmembrane helix (TMH) and localization (either cell or mitochondrial membrane) of transporter. Gene sequences were obtained in FASTA format for transporter proteins using the sequence download tool on EuPathDB (http://eupathdb.org/eupathdb/).”
Dataset 1 1. Dataset 1.Complete list of proteins extracted from Eupthadb and literature search, including their properties. Correct the spelling of EuPathDB.
Dataset 2 1. Actinobacillus is included in Dataset 2. Correct to Ajellomyces since the Strain/Species field is A. capsulatus G186AR. Provide the correct taxonomic lineage (http://www.uniprot.org/taxonomy/5037). Update the abstract, phylogenetic tree and discussion since no bacterial folate transport was characterized in the research. The protein sequence mislabeled clusters with Allomyces. 2. Provide the purpose of the color coding. 3. Label the eukaryotic microbe group according to the higher level classification of all living organisms1 e.g. fungi, protozoa and chromista. 4. To facilitate secondary analysis of Dataset 2, move legend to another sheet and remove blank rows and blank columns.
RESULTS 1. The results section should have subsection headings to help with readability and understanding of the article. 2. Replace “Methodological search” with “Systematic search”. 3. Replace “we came across” with “we observed”. 4. “A good number of the proteins identified had predicted transmembrane helixes with a few having none (Figure 1B).” Specify a quantity or percentage for the “good number”. 5. “Furthermore, a number of the transporters possess signal peptides (Dataset 1), which may be required for targeting to cellular locations. Deciphering the sequence of the targeting signal may indicate its product destination.” How many transporters possess the signal peptide sequence? 6. “We identified these transporters in 28 pathogen species (containing 63 strains) cutting across 12 phyla”
Suggested revision: “We obtained information on folate transporters encoded in genomes of 63 eukaryotic microbes consisting of 26 pathogenic genera and two genera of photosynthetic algae closely related to the apicomplexan pathogens. The genera are Ajellomyces, Allomyces, Aspergillus, Chromera, Coccidioides, Crithidia, Cryptococcus, Cryptosporidium, Eimeria,
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Encephalitozoon, Gibberella, Hammondia, Leishmania, Leptomonas, Mitosporidium, Naegleria, Neosartorya, Neospora, Nosema, Phytophthora, Plasmodium, Pythium, Sarcocystis, Spizellomyces, Theileria, Toxoplasma, Trypanosoma, and Vitrella.
DISCUSSION 1. Replace “were identified in the genome of 64 strains” with “were identified in the genomes of 63 strains”.
References 1. Ruggiero MA, Gordon DP, Orrell TM, Bailly N, et al.: A higher level classification of all living organisms.PLoS One. 2015; 10 (4): e0119248 PubMed Abstract | Publisher Full Text
Competing Interests: No competing interests were disclosed.
I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
Reviewer Report 26 July 2017 https://doi.org/10.5256/f1000research.12807.r24209
© 2017 Singh G. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Gajinder Singh Molecular Medicine Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), New Delhi, Delhi, India
I am disappointed to see that most of the issues identified in the original manuscript remain unaddressed including grammatical mistakes. e.g. 1. The current literature on folate transporters as drug targets has not been discussed, including information on their essentiality.
2. Using keyword and BLAST based searches will miss and misidentify folate transporters, thus more sensitive HMM profile based methods should be employed or please provide a justification for not doing the same.
3. Many sentences identified as unclear in the previous report remain unchanged.
I would request authors to provide a point by point response to my previous comments. I would be very happy to reconsider their response.
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Competing Interests: No competing interests were disclosed.
I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
Version 1
Reviewer Report 06 March 2017 https://doi.org/10.5256/f1000research.11380.r20419
© 2017 Singh G. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Gajinder Singh Molecular Medicine Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), New Delhi, Delhi, India
Below are my major concerns: 1. The abstract does not provides an adequate summary of the article.
The authors have claimed much higher scope of their work than actually reported. While their work is restricted to folate transporters, they have claimed to work on whole folate pathway. In the Abstract: "We applied a combination of bioinformatics methods to examine the genomes of pathogens in the EupathDB for genes encoding homologues of proteins that mediate folate salvage in a bid to identify and assign putative functions." "These findings offer new possibilities for potential drugConclusion: development targeting folate- salvage proteins."
2. No proper justification for the work is provided.
a) While folate pathway is well established as drug target, the authors have only identified folate transporters. So please give examples of drugs (with names) which are known to target folate transporters in any organism. If no sufficient information is provided, the usefulness of the work is severely reduced.
b) How many of folate transporters are essential in species where essentiality data is available such as P. berghei and T. gondii?
3. The methodology to identify transporters is not comprehensive.
Since authors have used key-word searches to identify folate transporters, they are likely to miss many transporters not labelled as such. An appropriate methodology will incorporate
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profile (such as HMM) based searches. Thus the number of transporters identified by authors is most likely to be an underestimate.
4. The methodology is not clear.
Authors write that "we utilized the word “folate” for search on the gene text and “folic acid” was used to confirm the hits", then how only transporters were retrieved? The Figure 1 is confusing. Where is BLAST used here?
5. The manuscript is written very poorly with so many scientific and grammatical mistakes that it is very difficult for the reader to follow the manuscript. Below are some examples.
a) "The mitochondrion is the predicted location of the majority of the proteins, with 15% possessing signal peptides." - how can mitochondria be majority location if only 15% have signal peptides and even less with mitochondrial signal peptide? Shouldn't the majority then be cytoplasmic?
b) "We identified 234 proteins to be involve in folate transport".
c) "Since folate-binding protein YgfZ, folate/ pteridine transporter, folate/biopterin transporter, putative, reduced folate carrier family protein, folate/methotrexate transporter FT1, putative folate transporters alone and others have 10, 25, 132, 2, 7, 49 and 9." What are these numbers?
d) "So we decided to reconstruct the phylogeny based folate transporter, folate-biopterin transporter after considering the identification number, the species diversity in each category."
e) "The different proteins identified to be involved in folate salvage or related molecules were folatebinding protein YgfZ, folate/pteridine transporter, folate/biopterin transporter, reduced folate carrier family protein, folate/methotrexate transporter FT1 and folate transporters having a 4%, 11%, 56%, 1%, 3% and 21% identity, respectively." What does this statement mean?
f) Does Table 1 really need to be 4 page long?
g) "The only Plasmodium species with results for proteins that salvage folate was P. falciparum"
h) "However, folate transporters I and II were retrieved from our search of GeneDB for P. malariae and P. ovale curtisi, respectively." What are these transporter classes?
i) "Some of these pathogens include P. ultimum DAOM BR144, which has mitochondrial folate transporter/carrier proteins similar to Homo sapiens, E. cuniculi GB-M1, which has proteins similar to folate transporter, and S. punctatus DAOM BR117, which has folate- binding protein YgfZ."
j) "After phylogenetic analysis each sub-phylogeny show a clear characterization except for
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folate-biopterin transporters"
h) "In this study, 234 genes encoding homologues of folate salvaging proteins were identified in the genome of 64 strains, representing 28 species of eukaryotic pathogens. Some of the pathogens include P. falciparum 3D7 and IT, P. knowlesi H, P. berghei ANKA, P. chabaudi chabaudi, T. brucei Lister 427, T. brucei TREU927, T. brucei gambiense DAL972, Encephalitozoon cuniculi GB-M1. The pathogens range from bacteria through to fungi, intracellular parasites such as Plasmodium and leishmania species, to extracellular parasites such as trypanosome species" Which bacteria was included in the study?
i) "It has been estimated that over half of the drugs currently on the market target integral membrane proteins of which membrane transporters are a part, but unfortunately, these transporters have not been adequately explored as drug targets. Folate transporters therefore represent attractive drug targets for treatment of infectious diseases." Please tell us how many drugs are available in the market which target folate transporters, which is a more relavant statistic with respect to this study.
j) "In the trypanosomes and related kinetoplastids, a member of these transporters, the folate biopterin transporter (FBT) family of proteins was identified in Leishmania."
k) "It is thought that MFS proteins are related to the FBT." What is MFS?
l) "Results from our study describing the presence of these transporters across several phyla corroborate results other researches, establishing the conservation of folate transport function among FBT family proteins from species from plants and protists".
m) "The clustering of these proteins suggests that these transport proteins have highly conserved regions often required for basic cellular function or stability". The clustering does not suggest that these transport proteins have highly conserved regions often required for basic cellular function or stability.
n) " We also performed phylogenetic comparisons of identified proteins. .".
Competing Interests: No competing interests were disclosed.
I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
Reviewer Report 27 February 2017 https://doi.org/10.5256/f1000research.11380.r20535
© 2017 Isokpehi R. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Raphael D. Isokpehi College of Science, Engineering and Mathematics (CSEM) , Bethune-Cookman University , Daytona Beach, FL, USA
Summary of Referee’s Report The manuscript presents a strong justification for research on folate transporter proteins as drug targets for diseases caused by eukaryotic pathogens including the malaria parasite. The manuscript reports a data curation effort that involves the use of the Eukaryotic Pathogens Database (EuPathDB) Resource. Several novel results to guide future research are included such as (i) a list of 234 folate transporter proteins from 63 eukaryotic microbes including eukaryotic pathogens; and (ii) phylogenetic trees of relatedness of the protein sequences. The authors observed the clustering of the protein sequences that indicate the possibility that antifolate drugs could be effective for multiple eukaryotic pathogens.
Major concerns are: (i) The need for a clearer description of the workflow for the construction of the protein list. (ii) There is inadequate support for the statement that “60% of the proteins were identified for the first time”. (iii) Confusion between retrieval and identification of protein sequences. The workflow diagram indicates retrieval of sequences but narrative text describes identification in multiple sections. The potential drug targeting categorization of the retrieved protein sequences is a key contribution of the research study.
Some minor concerns include (i) typographic errors such as spelling of abbreviations (e.g. EuPathDB, PubMed and UniProt); (ii) classification of A. (Ajellomyces) capsulatus G186AR as a bacteria; and (iii) need to quantify the statistics associated with observations (Examples: in abstract “Many of the genomes”; in discussion: “A few of the proteins” ).
Title and Abstract: Is the title appropriate for the content of the article? The manuscript title is “Genome-wide characterization of folate transporter proteins of eukaryotic pathogens”. “Genome-wide characterization” does not effectively describe the accomplishments of the research reported. The manuscript in the conclusion section (Page 15) states “we have identified and classified 234 proteins…”. The workflow (Figure 1) provides categorization of proteins by features such as cellular location, presence of signal peptide and number of transmembrane helices. Figure 2 has the title “Categorization of proteins identified”. A suggested revised title is “Categorization of potential drug targeting folate transporter proteins from eukaryotic pathogens”. The “potential drug targeting” is obtained from the conclusion section.
Does the abstract represent a suitable summary of the work? There are sentences in the abstract that should be revised to accurately represent a suitable summary of the research performed. Comments/suggestions are provided below. 1. Quantify the observations described as many. For example “genome sequences of many”: How many?
2. “eukaryotic protozoa” > “eukaryotic microbes”. The term “eukaryotic microbes” encompasses pathogens and non-pathogens (e.g. Chromera velia and Vitrella brassicaformis1 ).
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3. “important data” > “critical biological information”
4. “pathway are important for” > “pathways are necessary for”
5. “Methods: We applied a combination of bioinformatics methods to examine the genomes of pathogens in the EupathDB for genes encoding homologues of proteins that mediate folate salvage in a bid to identify and assign putative functions.” > “Methods: We developed automated search strategies in the Eukaryotic Pathogen Database Resources (EuPathDB) to construct a protein list and retrieve protein sequences of folate transporters encoded in the genomes of 200 eukaryotic microbes. The folate transporters were categorized according to features including mitochondrial localization, number of transmembrane helix, and protein sequence relatedness.
6. Provide key result(s) of the protein list retrieval and phylogenetic comparison of the retrieved proteins. For example, We constructed a list of 234 folate transporter proteins associated with 63 eukaryotic microbes including ??? algae, ??? fungi and ??? protozoa. Seven percent of the proteins were predicted to localize on the mitochondrial membrane. Phylogenetic tree revealed major (??? proteins) and minor (??? Proteins) clades. All the folate transporter sequences from the malaria parasite, Plasmodium, belonged to the major clade.
7. “The mitochondrion is the predicted location of the majority of the proteins”. This statement is not supported by Figure 2D, where 7% of the protein sequences are labelled as Mitochondrial folate transporters.
Article content: Have the design, methods and analysis of the results from the study been explained and are they appropriate for the topic being studied?
Design and Methods: 1. Figure 1 presents a conceptual hierarchical methodology. The rectangle labelled “Protein names/ sequences verification” has arrows to PubMed, UniProt, Membranetransporters.org, NCBI, GeneDB, Google Scholar and Phylogenetic analyses. It appears that the integrated results from the search strategies in the databases provided the input for the phylogenetic analyses. Please clarify.
2. Which step of the workflow resulted in the list of 234 proteins?
3. How many proteins were retrieved from the initial search using EuPathDB?
4. Protein Features Retrieved rectangle: Was the retrieval of protein features performed on only the 234 proteins?
5. There is adequate explanation of the methods for phylogenetic analyses. Please provide the Newick format phylogenetic tree as a supplementary dataset.
Analysis of the Results: 1. Table 1 is a major curation effort presented in the manuscript.
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a. The title of Table 1 should be updated to “Eukaryotic microbes from which folate transporters were identified”. The list includes non-pathogens. b. The content of the table (especially the Kingdom entries) should be checked for accuracy. The Kingdom column could be updated to Eukaryotic Microbe Group with entries as algae, protozoa or fungi. c. The column entries for A. capsulatus G186AR (mislabelled as bacteria) should be updated as the organism name is for a fungus (genus Ajellomyces). This update will also affect the Phylogenetic Tree (Figure 3). The node labelled Actinobacillus clusters with Ajellomyces macrogynus. d. An updated Table 1 should be presented as Dataset 2 in a spreadsheet file. This would enable secondary data analysis by other researchers. e. A new Table 1 could consist of columns for Eukaryotic Microbe Group, Genera of Eukaryotic Microbe, List of Species/Strain and Number of Folate Transport Proteins. This will provide reader with an overview of how the 234 proteins is distributed into the genera of the eukaryotic microbes. f. References listed for confirmation searches. Page 8, Paragraph 1, Sentence 1: “Our literature search for parasite folate transporters on PubMed and Google Scholar indicated 60% (38 out 63) of the proteins were identified for the first time as presented in Table 1. Comment: Among the references included in Table 1, only eight references (22, 73 to 77 and 82) on the basis of the article title provide experimental assessments of the folate transporters. Reference 85 is a reference for MEGA7 software. In the sentence “proteins” should be eukaryotic microbes. The proportion of eukaryotic microbes whose folate transporter(s) have been previously investigated with functional assays should be revised.
2. Figure 2. Categorization of proteins identified. Authors should consider representing Figure 2A and 2B as bar graphs. Figures 2C, 2D and 2E have only two categories that can be described in the Results section.
3. Discussion a. “genomes of 64 strains”. Table 1 has 63 eukaryotic microbes. b. Discuss Chromera velia and Vitrella brassicaformis as organismal systems for investigating folate transporter function. See Woo et al.1
4. Conclusion Consider revising “In summary, we have identified and classified 234 proteins…” to “In summary, we have retrieved information on 234 folate transporter proteins from the Eukaryotic Pathogen Database (EuPathDB) resources. The folate transporter proteins were categorized into potential drug targeting features including mitochondrial localization, number of transmembrane helix, and protein sequence relatedness."
Data (if applicable): Has enough information been provided to be able to replicate the experiment? Are the data in a usable format/structure and have all the data been provided? 1. Table 1 needs to be revised and converted to a Dataset.
2. Please provide the Newick format phylogenetic tree as a supplementary dataset.
3. The Gene Identifiers [Gene ID] in Dataset 1 can be used to retrieve the protein sequences
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from EuPathDB.
References 1. Woo YH, Ansari H, Otto TD, Klinger CM, et al.: Chromerid genomes reveal the evolutionary path from photosynthetic algae to obligate intracellular parasites.Elife. 2015; 4: e06974 PubMed Abstract | Publisher Full Text
Competing Interests: No competing interests were disclosed.
I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
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