1. Summary

2. Background

Table 1. Diversity of the presence and cellular location of PFOR in protists.

Figure 3. Pathogen specific bio-activation of nitro-based prodrugs.

Figure 3. Pathogen specific bio-activation of nitro-based prodrugs.

Table 2. Proportion of bacterial-like genes estimated from whole genome sequencing of some protozoan parasites.

2.1. Pyruvate-ferredoxin oxidoreductase

The ability to metabolize pyruvate through the process of oxidative decarboxylation remains a fundamental biochemical reaction in all living organisms (Inui et al. 1999). The pyruvate dehydrogenase multi-enzyme complex (PDC), an enzyme present in the mitochondria of almost all aerobic organisms, catalyzes this reaction (Hucho 1975). However, in most anaerobic and microaerophilic organisms, the enzyme PFOR, which is absent in higher eukaryotes, is responsible for catalyzing this reaction (Chabrière et al. 1999, Charon et al. 1999). PFOR is an ancient, oxygen sensitive, iron-sulfur protein that belongs to the 2-oxo acid ferredoxin oxidoreductase (OFOR) superfamily. It is found in diverse cellular locations in some anaerobic organisms (Emelyanov et al. 2011). Archaea, most anaerobic bacteria, and some anaerobic eukaryotes possess this enzyme (Blamey et al. 1993). The fermentative decarboxylation of pyruvate to acetyl-CoA and carbon dioxide (CO₂) by PFOR generates electrons that are transferred to either ferredoxin or flavodoxin (Marczak et al. 1983, Moreno et al. 1985). Due to the reversibility of this reaction, whereby pyruvate will be regenerated, PFOR is also called pyruvate synthase.

According to previous studies, the innate function of PFOR is to protect anaerobic organisms against oxidative stress by catalyzing the transfer of electrons that eventually reduce and inactivate free oxygen radicals during unfavorable oxidative conditions (Raj, et al. 2014). In an alternative pathway, the reduced ferredoxin derived from the decarboxylation of pyruvate can be re-oxidized by ferredoxin: NAD oxidoreductase (FNOR). This involves the transfer of electrons from ferredoxin to NAD(P) (Ctrnacta et al. 2006). The resulting NAD(P)H then transfers the electrons to oxygen radicals by another reaction catalyzed by NAD(P)H oxidase to form water molecules (Mastronicola et al. 2016). In this way, PFOR contributes its redox function together with other enzymes as an antioxidant detoxification system.

Figure 4. Metabolic fate of pyruvate in mitochondriate and some amitochondriate organisms.

Figure 4. Metabolic fate of pyruvate in mitochondriate and some amitochondriate organisms.

During the fundamental metabolic pathway of glycolysis, glucose is converted to pyruvate along a series of phosphorylation and dehydrogenation reactions. The fate of pyruvate is mainly dependent on the presence of mitochondrion. In mitochondriate organisms, pyruvate is further dehydrogenated to acetyl CoA by the pyruvate dehydrogenase enzyme complex (PDC). Acetyl CoA enters the tricarboxylic acid cycle as the primary substrate for oxidative reactions that result in the production of CO₂ and ATP molecules for intermediary metabolism. In some amitochondriate organisms, however, pyruvate undergoes fermentative decarboxylation to produce Acetyl CoA in a reversible redox reaction catalyzed by the pyruvate-ferredoxin oxidoreductase (PFOR).

Figure 5. PFOR-mediated anaerobic metabolism of pyruvate.

Figure 5. PFOR-mediated anaerobic metabolism of pyruvate.

Adapted and modified from: UniProtKB™ (www.uniprot.org/uniprot/P94692-D5IGG6)

In the fermentative decarboxylation of pyruvate, PFOR catalyzes the reduction of two molecules of ferredoxin using Co-enzyme A as a co-factor to produce acetyl-CoA, carbon dioxide and hydrogen ions. The reduced ferredoxin can be utilized as an electron donor for further redox reactions.

In the presence of nitro-based prodrugs, this same redox machinery is used as a drug target to make pathogens vulnerable to nitro-radical-induced cytotoxic damage. In this instance, the resulting reduced ferredoxin serves as an electron donor by transferring electrons to nitro-prodrugs. These then become pharmacologically activated and subsequently transformed into nitroso and hydroxylamine derivatives that cause cytotoxic damage (Horner et al. 1999, Ragsdale 2003).

Figure 6. PFOR-mediated re-oxidation of ferredoxin and bio-activation of nitro-based prodrugs.

Figure 6. PFOR-mediated re-oxidation of ferredoxin and bio-activation of nitro-based prodrugs.

In the presence of sufficient concentrations of the inactive prodrug, reduced ferredoxin donates electrons to the prodrug instead of acetyl-Co A by the catalytic activity of PFOR. In this reverse redox reaction, ferredoxin is re-oxidized and the prodrug is activated to its cytotoxic radical intermediate.

Figure 7. A summary of the PFOR-mediated fermentative decarboxylation of pyruvate and reductive activation of nitro-based prodrugs.

Figure 7. A summary of the PFOR-mediated fermentative decarboxylation of pyruvate and reductive activation of nitro-based prodrugs.

This figure depicts the reversible redox reaction catalyzed by PFOR in the presence of very high concentrations of the inactive prodrug. In the forward reaction, PFOR catalyzes the decarboxylation of pyruvate to acetyl-Co A using oxidized ferredoxin as electron acceptor. The resulting reduced ferredoxin then donates electrons to the inactive prodrug to reductively bio-activate it. This cycle continues until the concentration of the radical intermediates of the prodrug reaches therapeutic levels within the pathogen to disrupt nucleic acid and protein synthesis.

2.1.1. Activation of metronidazole by the PFOR of G. duodenalis and E. histolytica

Metronidazole is a synthetic nitro-imidazole derivative effective against most obligate anaerobic bacteria and protozoan parasites such as G. duodenalis, E. histolytica, T. vaginalis (Upcroft et al. 2001, Pal et al. 2009). It exerts its antimicrobial effect by interfering with nucleic acid synthesis resulting in DNA damage in the target pathogens (Lamp et al. 1999). Electrons from the initial redox reaction catalyzed by PFOR are transferred to ferredoxin, which has a sufficiently low redox potential to reductively activate metronidazole to its toxic radical metabolites. Through a series of electron transfer reactions, different radical intermediates are formed that act primarily as alkylating agents that disrupt DNA synthesis and growth of invading pathogens (Dingsdag et al. 2018). The different radical metabolites of metronidazole have been illustrated in Figure 8 below. Strains of G. duodenalis and E. histolytica with decreased or absent expression of pfor have been shown to exhibit increased metronidazole resistance (Dan et al. 2000). However, some recent studies suggest the involvement of alternative enzymes that also play central roles in the activation of, and resistance to, metronidazole (Leitsch et al. 2011, Ansell et al. 2017, Müller et al. 2019, Lopes-Oliveira et al. 2020). For example, T. vaginalis has been shown in some studies to display a complete resistance to metronidazole only after both PFOR and NAD-dependent malic enzyme are inactivated in the hydrogenosome (Rasoloson et al. 2002, Hrdý, Cammack et al. 2005).

The initial step in the bio-activation of metronidozole involves the transfer of one electron from ferredoxin to metronidazole to form its radical anion. In the presence of molecular oxygen the radical anion is re-oxidized to the parent compound in a futile cycle, hence, the overall bio-activation reaction is stalled at this stage. In anaerobic conditions, the radical anion recieves a further electron to form the 5-nitroso intermediate. In a subsequent reduction reaction involving the transfer of two electrons, the 5-nitroso intermediate is converted to a 5-(N–hydroxy)-amino intermediate, which then undergoes a spotaneous ring fision to produce hydroxethyl oxamic acid and acetamide. These fission products are the proposed end products of the bioactivation process that contribute immensely to the toxicity of metronidazole in invading pathogens.

2.1.2. Interaction between nitazoxanide and the PFOR of C. parvum

Nitazoxanide is a nitrothiazolyl-salicylamide derivative which exhibits broad-spectrum antimicrobial activity against a variety of pathogens including anaerobic and microaerophilic bacteria, protozoa, helminths, and viruses (Shakya et al. 2018, Bharti et al. 2021). Recent preclinical studies have demonstrated potential antineoplastic activity of nitazoxanide that can be further explored in the field of oncology (Pal et al. 2020, Abd El-Fadeal et al. 2021, Kiehl et al. 2021). The antiviral activity of nitazoxanide against the SARS-CoV-2 infection is currently being explored (Meneses et al. 2020, Lokhande et al. 2021, Rocco et al. 2021). Interestingly, nitazoxanide interacts with PFOR in a completely different manner than metronidazole. Some in-vitro studies postulate an interference of the redox function of PFOR by nitazoxanide (Hoffman et al. 2007, Scior et al. 2015). It deactivates the oxidative decarboxylation of pyruvate along with the electron transfer machinery that produces reduced ferredoxin in the presence of the coenzyme thiamine pyrophosphate (TPP) (Hoffman et al. 2007). This is the generally accepted mechanism by which it inactivates the metabolism, growth and survival of C. parvum (Gargala 2008). A similar mechanism of action is postulated for the interaction between the PFOR of some anaerobic bacteria and nitazoxanide (Warren et al. 2012, Kennedy et al. 2016).

4. Aim

5. Objectives

• To analyze the phylogenetic distribution of redox enzymes of selected protozoan parasites using proteins encoded by housekeeping genes as controls.
• To use comparative bioinformatics approaches to test the hypothesis that these redox enzymes were most likely acquired through horizontal gene transfer from bacterial origin.
• To use proteome-wide analysis and gene expression analysis to identify other genes that were putatively acquired through horizontal gene transfer.

6. Expected Outcomes

7. Methodology

7.1. Source of protein sequences

Protein sequences for the PFOR of the selected protozoan parasites with their respective gene and accession numbers were retrieved from the UniProt™ protein database. (https://www.uniprot.org/uniprot/). Three control proteins of each parasite: glyceraldehayde-3-phosphate dehydrogenase (GAPDH) (EC 1.2.1.12), tubulin alpha chain (TUBa), and DNA-directed RNA polymerase II subunit beta I (RPB1) (EC 2.7.7.6) were retrieved using the same procedure. These proteins were selected because they are encoded by housekeeping genes and are well conserved in protozoa.

Table 3. Gene and accession numbers of the redox enzymes.

Table 3. Gene and accession numbers of the redox enzymes.

Parasite	Enzyme	Gene number	Accession number	Redox drug
G. duodenalis	PFOR	Q24982	AAA74894.1	Metronidazole
E. histolytica	PFOR	N9TJX2	ENY63830.1	Metronidazole
C. parvum	PFOR	A1DRF1	ABK91849.1	Nitazoxanide

Table showing the gene and accession numbers of the redox enzymes with the corresponding pro-drug substrates of selected protozoan parasites that were obtained from the UniProt™ Protein Database

Table 4. Gene and accession numbers of the control proteins.

Table 4. Gene and accession numbers of the control proteins.

Parasite	Control protein1	Gene/Accession number	Control protein 2	Gene/Accession number	Control protein 3	Gene/Accession number
G. duodenalis	GAPDH	P53429 AAB18421.1	TUB	A8BPC0EDO78046.1	RPB1	Q8MUU2 AAM77743.1
E. histolytica	GAPDH	C4LVR9BAN37492.1	TUB	P31017AAA57315.1	RPB1	Q6IUR3 AAT40981.1
C. parvum	GAPDH	Q7YYQ9BAJ77164.1	TUB	Q9UAC3AAD20239.1	RPB1	A0A7G2HJ78 CAD98371.1

Table showing the gene and accession numbers of the control proteins of selected protozoan parasites that were obtained from the UniProt™ protein database.

7.3. Inclusion-Exclusion approach of BLAST search

The total number of distinct species that possess the control proteins and the corresponding redox enzymes were derived from the taxonomy reports of the BLAST search results. In the BLAST pyramid approach, the first BLAST search was performed by excluding the protein sequence of the protozoan species of interest whilst including the sequences of all other species belonging to its genus. In the next query, the genus was excluded whilst including the next available taxonomic level. This was followed by subsequent inclusion-exclusion queries in increasing order along the taxonomic hierarchy until the eukaryotic domain. In a final step, all eukaryotes were excluded whilst including all prokaryotes. The proportion of organisms in each taxon that had hits for the redox enzymes was represented as percentage deviation. Since the total number of distinct species in each taxon was not available in the NCBI database, the percentage deviation was determined by comparing the total number of species that had hits for the redox enzymes to the total number of organisms that had hits for GAPDH.

Table 5. Illustration of BLAST search pyramid approach.

Table 5. Illustration of BLAST search pyramid approach.

	Eukaryota	Procaryotae
	Alveolata	Eukaryota
	Apicomplexa	Alveolata
	Conoidasida	Apicomplexa
	Eucoccidiorida	Conoidasida
	Cryptosporidiidae	Eucoccidiorida
	Cryptosporidium	Cryptosporidiidae
	Cryptosporidium parvum	Cryptosporidium
	EXCLUDE	INCLUDE

This table illustrates how the BLAST searches are run using C. parvum protein sequences as query. In the first step, a BLAST search is done with hits excluding the species C. parvum while including all other species of the genus Cryptosporidium. In the next step, all species of the genus Cryptosporidium are excluded whilst including organisms of the next taxonomical hierarchy. This is done sequentially until all eukaryotic organisms are excluded to filter hits for prokaryotes only.

8. Results

8.1. BLAST Pyramid

The three selected control proteins are encoded by housekeeping genes in eukaryotic organisms. As expected, the BLAST search using the corresponding proteins from each of the five protozoan species as the query showed positive hits with a steady increase in the total number of distinct organisms in each level along the taxonomic hierarchy from species to domain. The total number of hits in the eukaryotic domain compared to the prokaryotic domain showed a further increase for GAPDH but not for TUBa and RPB1. Since the total number of prokaryotic organisms is larger than that of eukaryotic organisms, it can be concluded that GAPDH is more conserved in both domains and represents a better control protein compared to TUBa and RPB1. This was to be expected, as bacteria do not possess microtubules and have different types of nucleic acid polymerases. Therefore, GAPDH was used for further comparative analysis against PFOR.

Figure 9. The distribution of control proteins along the taxonomic hierarchy using query proteins of C. parvum shows that GAPDH is relatively more conserved in both eukaryotes and prokaryotes. .

Figure 9. The distribution of control proteins along the taxonomic hierarchy using query proteins of C. parvum shows that GAPDH is relatively more conserved in both eukaryotes and prokaryotes. .

8.2. Percentage deviation of PFOR from GAPDH

At the genus level of Giardia, Entamoeba and Cryptosporidium, there was no deviation observed in the total number of hits for PFOR compared to GAPDH in the NBCI database. From these findings, it can be extrapolated that all species in the genus Giardia, Entamoeba and Cryptosporidium possess PFOR. Above the genus level, the total number of organisms that had hits for PFOR compared to GAPDH was reduced.

Figure 10. Graph showing no significant deviation in the total number of hits for PFOR compared to GAPDH at the genus level of Giardia. Above the genus level to the eukaryotic domain, there was a gradual increase in the percentage deviation. Approximately 50% of all prokaryotes that express GAPDH showed no hits for PFOR.

Figure 10. Graph showing no significant deviation in the total number of hits for PFOR compared to GAPDH at the genus level of Giardia. Above the genus level to the eukaryotic domain, there was a gradual increase in the percentage deviation. Approximately 50% of all prokaryotes that express GAPDH showed no hits for PFOR.

Figure 11. This graph shows an equal expression of GAPDH and PFOR in all the species of the genus Entamoeba. There is an approximately 72% deviation at the level of Amoebozoa, 50% in the eukaryotic domain and 64% in the prokaryotic domain. .

Figure 11. This graph shows an equal expression of GAPDH and PFOR in all the species of the genus Entamoeba. There is an approximately 72% deviation at the level of Amoebozoa, 50% in the eukaryotic domain and 64% in the prokaryotic domain. .

Figure 12. This graph also shows equal expression of GAPDH and PFOR in all the species of the genus Cryptosporidium. In the taxa above the genus level, relatively fewer organisms had positive hits for PFOR compared to GAPDH.

Figure 12. This graph also shows equal expression of GAPDH and PFOR in all the species of the genus Cryptosporidium. In the taxa above the genus level, relatively fewer organisms had positive hits for PFOR compared to GAPDH.

8.3. Phylogenetic trees

The multiple alignments of 43 GAPDH and 33 PFOR protein sequences of representative eukaryotic and prokaryotic species were used to generate the corresponding phylogenies as shown in fig. 16-18. The percentages of replicate phylogenies in which the species were clustered in the bootstrap test of 1000 replicates are shown next to the branches. The trees are drawn to scale, with branch lengths in the same units as those of the evolutionary distances used for the inference. The evolutionary distances were computed using the Poisson correction method and are in the units of the number of amino acid substitutions per site.

Figure 13. The phylogeny of the selected eukaryotic and prokaryotic species based on the protein sequences of GAPDH shows a pattern of relatedness that is in consonance with the expected phylogeny of eukaryotes. The topology was inferred using the Neighbor-Joining method. The optimal unrooted tree with the sum of branch length = 4.58236819 is shown from a total of 659 positions in the final dataset.

Figure 13. The phylogeny of the selected eukaryotic and prokaryotic species based on the protein sequences of GAPDH shows a pattern of relatedness that is in consonance with the expected phylogeny of eukaryotes. The topology was inferred using the Neighbor-Joining method. The optimal unrooted tree with the sum of branch length = 4.58236819 is shown from a total of 659 positions in the final dataset.

Figure 14. The phylogeny based on the protein sequences of PFOR was inferred using the maximum likelihood method. The unrooted tree with the highest log likelihood (-48017.90) is shown from a total of 1331 positions in the final dataset. The PFOR of Entamoeba invadens shows a monophyletic relationship with Giardia and that of Euglena gracilis is monophyletic with Cryptosporidium. This phylogenetic incongruence is however not entirely conclusive of a bacterial origin of pfor, since no protozoa is monophyletic with the selected bacterial species.

Figure 14. The phylogeny based on the protein sequences of PFOR was inferred using the maximum likelihood method. The unrooted tree with the highest log likelihood (-48017.90) is shown from a total of 1331 positions in the final dataset. The PFOR of Entamoeba invadens shows a monophyletic relationship with Giardia and that of Euglena gracilis is monophyletic with Cryptosporidium. This phylogenetic incongruence is however not entirely conclusive of a bacterial origin of pfor, since no protozoa is monophyletic with the selected bacterial species.

8.4. Heat maps, density plots and cladograms

All scores from the proteome screening against the HMM profile libraries were normalized to 1 as the highest score obtained for each HMM, and a minimum cut-off significance value of 0.25 was used to represent a positive hit. As expected, there was a positive hit for the control proteins in almost all the selected eukaryotic species, which indicates a high degree of sensitivity of the screening program. All selected prokaryotic species recorded positive hits for GAPDH but, as expected, had relatively low hits for RPB1 and virtually no hit for TUBa. In the density plots, it was observed that GAPDH was relatively very well conserved in all eukaryotic taxa as well as the prokaryotic domain. Even though TUBa and RPB1 were well distributed in the eukaryotic taxa, there was a poor representation in the prokaryotic domain. The results from the proteome analysis approach correlated considerably well with the results from the BLAST pyramid approach. As expected, all animal, plant and fungal species showed no positive hits for PFOR. These results represent very good negative controls and hence, a high degree of specificity of the screening program.

All selected species in the stramenopile group had no hits for PFOR with the exception of B. hominis. All species in the alveolate group had no hits for PFOR except C. parvum that showed a significant hit. The only two rhizarian species with available proteome sequences in the ensembl genomes database had no hit for PFOR. All the selected protozoa in the phylum Metamonada recorded hits for PFOR. All species in the euglenozoa group had no hit for PFOR. Although Entamoeba and Dictyostelium are closely related amoebozoans, the two species in the genus Entamoeba showed positive hits for PFOR whilst the species in the genus Dictyostelium had no significant hit. All the species in the genus Cryptosporidium recorded significant hits for PFOR, but the closely related species in the genus Eimeria and Cyclospora cayetanensis had no significant hits. A similar pattern was depicted in all the species of the genus Entamoeba versus those of Dictyostelium, Cavenderia, Tiegemostilium, and Planoprotostelium. For the metamonada group, there was unfortunately no further species for the expanded cladogram. About 41% of the selected bacterial species showed significant hits for PFOR.

Table 6. Heat map of normalized scores from the whole proteome analysis of the selected protozoan species.

Table 6. Heat map of normalized scores from the whole proteome analysis of the selected protozoan species.

Table 7. Heat map of normalized scores from the whole proteome analysis the selected animal, plant and bacterial species.

Table 7. Heat map of normalized scores from the whole proteome analysis the selected animal, plant and bacterial species.

Figure 15. Density plots showing the distribution of control proteins compared to that of NTR and PFOR in the major eukaryotic taxa and the prokaryotic domain. .

Figure 15. Density plots showing the distribution of control proteins compared to that of NTR and PFOR in the major eukaryotic taxa and the prokaryotic domain. .

• Cladogram of selected straminopile, alveolate and rhizaria species (SAR) showing the distribution of PFOR and control proteins

Figure 16. In the SAR clade, it can be observed that, B. hominis is the only straminopile that has positive hit for PFOR. Furthermore, only Cryptosporidium shows a positive hit for PFOR in the alveolate clade. It is important to note that even the closely related Eimeria and Cyclospora have no hits for PFOR.

Figure 16. In the SAR clade, it can be observed that, B. hominis is the only straminopile that has positive hit for PFOR. Furthermore, only Cryptosporidium shows a positive hit for PFOR in the alveolate clade. It is important to note that even the closely related Eimeria and Cyclospora have no hits for PFOR.

• Cladogram of selected protozoan species of the phyla Discoba, Amoebozoa and Metamonada showing the distribution of NTR, PFOR and control proteins

Figure 17. This cladogram shows exclusively no expression of PFOR in all the selected protozoa in the discoba clade. For the phylum Amoebozoa, PFOR is positive in the genus Entamoeba but not Dictyostelium, despite their close relatedness. All available organisms with proteomes in the metamonad clade show positive hits for PFOR. .

Figure 17. This cladogram shows exclusively no expression of PFOR in all the selected protozoa in the discoba clade. For the phylum Amoebozoa, PFOR is positive in the genus Entamoeba but not Dictyostelium, despite their close relatedness. All available organisms with proteomes in the metamonad clade show positive hits for PFOR. .

• Cladogram of selected opithokont species showing the distribution of NTR, PFOR and control proteins

Figure 18. As expected, animals and fungi do not express PFOR, hence there was no significant hit recorded in all species. The controls proteins, however, are positive for all selected species.

Figure 18. As expected, animals and fungi do not express PFOR, hence there was no significant hit recorded in all species. The controls proteins, however, are positive for all selected species.

• Cladogram of selected plant and archaeplastida species showing the distribution of NTR, PFOR and control proteins

Figure 19. All plant species had no hits for PFOR. For the selected archaeplastida species, Chlamydomonas and Chlorella show hits for PFOR.

Figure 19. All plant species had no hits for PFOR. For the selected archaeplastida species, Chlamydomonas and Chlorella show hits for PFOR.

• Expanded cladogram of the genera Cryptosporidium and Entamoeba

Figure 20. An expanded coverage of all organisms with available whole proteome sequences from the ensemblgenomes database shows a differential expression of PFOR in the genus Cryptosporidium against the closely related genera Eimeria and Cyclospora, and the genus Entamoeba against the closely related genera Dictyostelium, Cavenderia, Tiegemostilium and Planoprotostelium. .

Figure 20. An expanded coverage of all organisms with available whole proteome sequences from the ensemblgenomes database shows a differential expression of PFOR in the genus Cryptosporidium against the closely related genera Eimeria and Cyclospora, and the genus Entamoeba against the closely related genera Dictyostelium, Cavenderia, Tiegemostilium and Planoprotostelium. .

8.5. Proteome BLAST and gene enrichment analysis

8.5.1. Results for E. histolytica

The predicted proteome of E. histolytica from the ensembl genomes database had 7,455 proteins. The BLAST search showed that 44.4% of these proteins had no hits in the proteomes of D. discoideum and D. vulgaris. 11.9% had hits in both D. discoideum and D. vulgaris. 42.7% of the proteins in E. histolytica showed hits in the proteome of D. discoideum whilst only 1% had hits in D. vulgaris. Out of this 1%, PFOR had the highest score of 1,329. The scores of all proteins were increased by one and the logarithmic values were plotted with Entamoeba versus Desulfovibrio on the vertical axis and Entamoeba versus Dictyostelium on the horizontal axis as shown in Figure 27.

Figure 21. Illustration of the total number of proteins in Entamoeba that have significant hits in Dictyostelium and Desulfovibrio. Seventy-four proteins occur exclusively in Entamoeba and Desulfovibrio. These proteins are potential candidates of horizontal gene transfer.

Figure 21. Illustration of the total number of proteins in Entamoeba that have significant hits in Dictyostelium and Desulfovibrio. Seventy-four proteins occur exclusively in Entamoeba and Desulfovibrio. These proteins are potential candidates of horizontal gene transfer.

Figure 22. Every spot represents one protein of E. histolytica. Proteins with hits in only D. vulgaris are located on the vertical axis and those with hits in only D. discoideum are on the horizontal axis. E. histolytica proteins with no hits in either organism are located at the origin whilst those with hits in both D. discoideum and D. vulgaris are located in the middle of the graph. PFOR is marked with an arrow. Log is to base 10.

Figure 22. Every spot represents one protein of E. histolytica. Proteins with hits in only D. vulgaris are located on the vertical axis and those with hits in only D. discoideum are on the horizontal axis. E. histolytica proteins with no hits in either organism are located at the origin whilst those with hits in both D. discoideum and D. vulgaris are located in the middle of the graph. PFOR is marked with an arrow. Log is to base 10.

8.5.2. Results for C. parvum

Of the 3,805 predicted proteins in the proteome of C. parvum, the BLAST search showed that 44.4% had no hits in the proteomes of E. tenella and D. vulgaris. 13% of the proteins had hits in E. tenella and D. vulgaris. 41.7% of the proteins in C. parvum showed hits in the proteome of E. tenella whilst 0.9% had hits in D. vulgaris. Out of this 0.9%, PFOR had the highest score of 1,202. The scores of all proteins were increased by one and the logarithmic values were plotted with Entamoeba versus Desulfovibrio on the vertical axis and Entamoeba versus Dictyostelium on the horizontal axis as shown in Figure 29.

Figure 23. Illustration of the total number of proteins in Cryptosporidium that have significant hits in Eimeria and Desulfovibrio. Thirty-five proteins occur exclusively in Cryptosporidium and Desulfovibrio. These proteins are potential candidates of horizontal gene transfer.

Figure 23. Illustration of the total number of proteins in Cryptosporidium that have significant hits in Eimeria and Desulfovibrio. Thirty-five proteins occur exclusively in Cryptosporidium and Desulfovibrio. These proteins are potential candidates of horizontal gene transfer.

Figure 24. Every spot represents one protein of C. parvum. Proteins with hits in only D. vulgaris are located on the vertical axis and those with hits in only E. tenella are on the horizontal axis. C. parvum proteins with no hits in either organism are located at the origin whilst those with hits in both E. tenella and D. vulgaris are located in the middle of the graph. PFOR is marked with an arrow. Log is to base 10.

Figure 24. Every spot represents one protein of C. parvum. Proteins with hits in only D. vulgaris are located on the vertical axis and those with hits in only E. tenella are on the horizontal axis. C. parvum proteins with no hits in either organism are located at the origin whilst those with hits in both E. tenella and D. vulgaris are located in the middle of the graph. PFOR is marked with an arrow. Log is to base 10.

8.5.3. Gene enrichment analysis

Due to the unavailability of a reference genome for C. parvum at the GO website (http://geneontology.org/), the gene enrichment analysis was limited to E. histolytica. The gene IDs of proteins that had significantly high similarity based on the proteome BLAST scores for E. histolytica against D. vulgaris were analyzed.

Biological process was used as annotation data category to analyze the gene list of 40 query proteins against the reference genome of E. histolytica with 7,959 genes. Nine of the 40 query genes were unmapped, hence the remaining 31 genes were used for the gene enrichment analysis. 19 genes from the reference list and 3 genes from the query list mapped to the pyruvate metabolic pathway. Based on the reference list, 0.239% of genes were expected to map to the pyruvate metabolic pathway. Since the query list contained 31 genes, the number of genes expected to map to this pathway was 0.074.

The Fold-Enrichment of the genes observed in the query list to the expected outcome was 40.54, which indicates that the pyruvate metabolic pathway is overrepresented in the query list. The raw p-value for the statistical analysis as determined by Fisher’s exact test and the False Discovery Rate as calculated by the Benjamini-Hochberg procedure were 7.75^-0.5 and 3.35^-0.2 respectively. These findings suggest that the number of genes from the query list that were mapped to the pyruvate metabolic pathway did not occur by chance, as determined by comparison to the reference genome of E. histolytica. This small p-value also indicates that the result is non-random and potentially interesting, and worth looking at in closer detail.

The other genes that were also significantly enriched were mapped to the carbohydrate metabolic process and pathways involved in the generation of precursor metabolites and energy. The list of all the genes that were significantly enriched is shown in Table 8.

Table 8. Results of the gene enrichment analysis from the gene ontology website.

Table 8. Results of the gene enrichment analysis from the gene ontology website.

Mapped IDs of enriched genes	Expressed proteins
XP_657019	Pyruvate: ferredoxin oxidoreductase
XP_651627	Type A flavoprotein
XP_657332	Pyruvate phosphate dikinase
XP_651155	Alkyl sulfatase
XP_653620	Phosphotransferase
XP_654182	Phosphoglycerate mutase
XP_656980	Branched-chain amino acid aminotransferase
XP_655990	acetate kinase
XP_656909	Polysaccharide deacetylase
XP_651609	Ribose 5-phosphate isomerase
XP_656356	Carbohydrate degrading enzyme

The table above shows only results for the statistically significant gene enrichment data with p and FDR values < 0.05. The second and third columns contain the number of genes in the reference list and the query list that map to the corresponding metabolic processes. The fourth column contains the expected number of genes that map to the corresponding metabolic processes based on the reference list. The GO biological processes with the highest enrichment of 40.54 was obtained from genes that mapped to the pyruvate metabolic pathway. A plus sign indicates over-representation and a negative sign indicates under-representation. The table below shows the list of all genes that were significantly enriched.

Figure 25. Ancestor chart for the pathways related to the pyruvate metabolic process, GO: 0006090. Source: (http://geneontology.org/).

Figure 25. Ancestor chart for the pathways related to the pyruvate metabolic process, GO: 0006090. Source: (http://geneontology.org/).

Table 9. List of all GO terms that are direct descendants of the pyruvate metabolic process.

Table 9. List of all GO terms that are direct descendants of the pyruvate metabolic process.

9. Discussion

10. Conclusion

11. Final remarks

• Since metronidazole is an established, well tolerated drug for treating infections caused by PFOR-possessing pathogens, it can be considered as a potential drug candidate for the treatment of infections caused by Cryptosporidium, Spironucleus and Blastocystis.
• In this project, there was a considerable selection bias from using non-redundant protein sequences as the sole database for the BLAST searches and a relatively skewed coverage of pathogenic protozoa against free-living protozoa. Moreover, unavailability of the total number of distinct organisms in each taxon and possible bacterial contamination with PFOR protein sequences from the gastrointestinal tract were challenging factors.
• The unavailability of whole proteome sequences of other closely related species in the phylum Metamonada in the ensemblgenomes database posed a significant limitation in performing further analyses for the distribution of PFOR.
• The unavailability of reference genome for C. parvum in the Gene Ontology database was a major limiting factor in performing comparative studies with the results of the gene enrichment analysis from the exclusively present proteins in E. histolytica and D. vulgaris.

List of abbreviations

References

1. Abd El-Fadeal, N. M., M. S. Nafie, K. E.-K. M, A. El-Mistekawy, H. M. F. Mohammad, A. M. Elbahaie, A. A. Hashish, S. Y. Alomar, S. Y. Aloyouni, M. El-Dosoky, K. M. Morsy and S. A. Zaitone (2021). "Antitumor Activity of Nitazoxanide against Colon Cancers: Molecular Docking and Experimental Studies Based on Wnt/β-Catenin Signaling Inhibition." Int J Mol Sci 22(10). [CrossRef]
2. Ackerley, D. F., C. F. Gonzalez, M. Keyhan, R. Blake, 2nd and A. Matin (2004). "Mechanism of chromate reduction by the Escherichia coli protein, NfsA, and the role of different chromate reductases in minimizing oxidative stress during chromate reduction." Environ Microbiol 6(8): 851-860. [CrossRef]
3. Adl, S. M., A. G. Simpson, C. E. Lane, J. Lukeš, D. Bass, S. S. Bowser, M. W. Brown, F. Burki, M. Dunthorn, V. Hampl, A. Heiss, M. Hoppenrath, E. Lara, L. Le Gall, D. H. Lynn, H. McManus, E. A. Mitchell, S. E. Mozley-Stanridge, L. W. Parfrey, J. Pawlowski, S. Rueckert, L. Shadwick, C. L. Schoch, A. Smirnov and F. W. Spiegel (2012). "The revised classification of eukaryotes." J Eukaryot Microbiol 59(5): 429-493. [CrossRef]
4. Alsmark, U. C., T. Sicheritz-Ponten, P. G. Foster, R. P. Hirt and T. M. Embley (2009). "Horizontal gene transfer in eukaryotic parasites: a case study of Entamoeba histolytica and Trichomonas vaginalis." Methods Mol Biol 532: 489-500. [CrossRef]
5. Altschul, S. F., T. L. Madden, A. A. Schäffer, J. Zhang, Z. Zhang, W. Miller and D. J. Lipman (1997). "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs." Nucleic Acids Res 25(17): 3389-3402. [CrossRef]
6. Altschul, S. F., J. C. Wootton, E. M. Gertz, R. Agarwala, A. Morgulis, A. A. Schäffer and Y. K. Yu (2005). "Protein database searches using compositionally adjusted substitution matrices." Febs j 272(20): 5101-5109. [CrossRef]
7. Andersson, J. O. (2011). "Evolution of patchily distributed proteins shared between eukaryotes and prokaryotes: Dictyostelium as a case study." J Mol Microbiol Biotechnol 20(2): 83-95. [CrossRef]
8. Ansell, B. R., L. Baker, S. J. Emery, M. J. McConville, S. G. Svärd, R. B. Gasser and A. R. Jex (2017). "Transcriptomics Indicates Active and Passive Metronidazole Resistance Mechanisms in Three Seminal Giardia Lines." Front Microbiol 8: 398. [CrossRef]
9. Benchimol, M. (2009). "Hydrogenosomes under microscopy." Tissue Cell 41(3): 151-168. [CrossRef]
10. Benchimol, M., J. C. Almeida and W. de Souza (1996). "Further studies on the organization of the hydrogenosome in Tritrichomonas foetus." Tissue Cell 28(3): 287-299. [CrossRef]
11. Benchimol, M., I. de Andrade Rosa, R. da Silva Fontes and A. J. Burla Dias (2008). "Trichomonas adhere and phagocytose sperm cells: adhesion seems to be a prominent stage during interaction." Parasitol Res 102(4): 597-604. [CrossRef]
12. Bendesky, A., D. Menéndez and P. Ostrosky-Wegman (2002). "Is metronidazole carcinogenic?" Mutat Res 511(2): 133-144. [CrossRef]
13. Bharti, C., S. Sharma, N. Goswami, H. Sharma, S. A. Rabbani and S. Kumar (2021). "Role of nitazoxanide as a repurposed drug in the treatment and management of various diseases." Drugs Today (Barc) 57(7): 455-473. [CrossRef]
14. Blamey, J. M. and M. W. Adams (1993). "Purification and characterization of pyruvate ferredoxin oxidoreductase from the hyperthermophilic archaeon Pyrococcus furiosus." Biochim Biophys Acta 1161(1): 19-27. [CrossRef]
15. Bontell, I. L., N. Hall, K. E. Ashelford, J. P. Dubey, J. P. Boyle, J. Lindh and J. E. Smith (2009). "Whole genome sequencing of a natural recombinant Toxoplasma gondii strain reveals chromosome sorting and local allelic variants." Genome Biol 10(5): R53. [CrossRef]
16. Boxma, B., R. M. de Graaf, G. W. van der Staay, T. A. van Alen, G. Ricard, T. Gabaldón, A. H. van Hoek, S. Y. Moon-van der Staay, W. J. Koopman, J. J. van Hellemond, A. G. Tielens, T. Friedrich, M. Veenhuis, M. A. Huynen and J. H. Hackstein (2005). "An anaerobic mitochondrion that produces hydrogen." Nature 434(7029): 74-79. [CrossRef]
17. Bradic, M., S. D. Warring, G. E. Tooley, P. Scheid, W. E. Secor, K. M. Land, P. J. Huang, T. W. Chen, C. C. Lee, P. Tang, S. A. Sullivan and J. M. Carlton (2017). "Genetic Indicators of Drug Resistance in the Highly Repetitive Genome of Trichomonas vaginalis." Genome Biol Evol 9(6): 1658-1672. [CrossRef]
18. Carlton, J. M., R. P. Hirt, J. C. Silva, A. L. Delcher, M. Schatz, Q. Zhao, J. R. Wortman, S. L. Bidwell, U. C. Alsmark, S. Besteiro, T. Sicheritz-Ponten, C. J. Noel, J. B. Dacks, P. G. Foster, C. Simillion, Y. Van de Peer, D. Miranda-Saavedra, G. J. Barton, G. D. Westrop, S. Müller, D. Dessi, P. L. Fiori, Q. Ren, I. Paulsen, H. Zhang, F. D. Bastida-Corcuera, A. Simoes-Barbosa, M. T. Brown, R. D. Hayes, M. Mukherjee, C. Y. Okumura, R. Schneider, A. J. Smith, S. Vanacova, M. Villalvazo, B. J. Haas, M. Pertea, T. V. Feldblyum, T. R. Utterback, C. L. Shu, K. Osoegawa, P. J. de Jong, I. Hrdy, L. Horvathova, Z. Zubacova, P. Dolezal, S. B. Malik, J. M. Logsdon, Jr., K. Henze, A. Gupta, C. C. Wang, R. L. Dunne, J. A. Upcroft, P. Upcroft, O. White, S. L. Salzberg, P. Tang, C. H. Chiu, Y. S. Lee, T. M. Embley, G. H. Coombs, J. C. Mottram, J. Tachezy, C. M. Fraser-Liggett and P. J. Johnson (2007). "Draft genome sequence of the sexually transmitted pathogen Trichomonas vaginalis." Science 315(5809): 207-212. [CrossRef]
19. Chabrière, E., M. H. Charon, A. Volbeda, L. Pieulle, E. C. Hatchikian and J. C. Fontecilla-Camps (1999). "Crystal structures of the key anaerobic enzyme pyruvate:ferredoxin oxidoreductase, free and in complex with pyruvate." Nat Struct Biol 6(2): 182-190. [CrossRef]
20. Chapwanya, A., A. Y. Usman and P. C. Irons (2016). "Comparative aspects of immunity and vaccination in human and bovine trichomoniasis: a review." Trop Anim Health Prod 48(1): 1-7. [CrossRef]
21. Charon, M. H., A. Volbeda, E. Chabriere, L. Pieulle and J. C. Fontecilla-Camps (1999). "Structure and electron transfer mechanism of pyruvate:ferredoxin oxidoreductase." Curr Opin Struct Biol 9(6): 663-669. [CrossRef]
22. Chung, M. C., P. L. Bosquesi and J. L. dos Santos (2011). "A prodrug approach to improve the physico-chemical properties and decrease the genotoxicity of nitro compounds." Curr Pharm Des 17(32): 3515-3526. [CrossRef]
23. Ctrnacta, V., J. G. Ault, F. Stejskal and J. S. Keithly (2006). "Localization of pyruvate:NADP+ oxidoreductase in sporozoites of Cryptosporidium parvum." J Eukaryot Microbiol 53(4): 225-231. [CrossRef]
24. Dan, M., A. L. Wang and C. C. Wang (2000). "Inhibition of pyruvate-ferredoxin oxidoreductase gene expression in Giardia lamblia by a virus-mediated hammerhead ribozyme." Mol Microbiol 36(2): 447-456. [CrossRef]
25. Daubin, V. and G. J. Szöllősi (2016). "Horizontal Gene Transfer and the History of Life." Cold Spring Harb Perspect Biol 8(4): a018036. [CrossRef]
26. Deng, Y., H. Xu, Y. Su, S. Liu, L. Xu, Z. Guo, J. Wu, C. Cheng and J. Feng (2019). "Horizontal gene transfer contributes to virulence and antibiotic resistance of Vibrio harveyi 345 based on complete genome sequence analysis." BMC Genomics 20(1): 761. [CrossRef]
27. Denoeud, F., M. Roussel, B. Noel, I. Wawrzyniak, C. Da Silva, M. Diogon, E. Viscogliosi, C. Brochier-Armanet, A. Couloux, J. Poulain, B. Segurens, V. Anthouard, C. Texier, N. Blot, P. Poirier, G. C. Ng, K. S. Tan, F. Artiguenave, O. Jaillon, J. M. Aury, F. Delbac, P. Wincker, C. P. Vivarès and H. El Alaoui (2011). "Genome sequence of the stramenopile Blastocystis, a human anaerobic parasite." Genome Biol 12(3): R29. [CrossRef]
28. Dingsdag, S. A. and N. Hunter (2018). "Metronidazole: an update on metabolism, structure-cytotoxicity and resistance mechanisms." J Antimicrob Chemother 73(2): 265-279. [CrossRef]
29. Docampo, R., S. N. Moreno and R. P. Mason (1987). "Free radical intermediates in the reaction of pyruvate:ferredoxin oxidoreductase in Tritrichomonas foetus hydrogenosomes." J Biol Chem 262(26): 12417-12420. [CrossRef]
30. Doolittle, W. F. (1998). "You are what you eat: a gene transfer ratchet could account for bacterial genes in eukaryotic nuclear genomes." Trends Genet 14(8): 307-311. [CrossRef]
31. Dubini, A., F. Mus, M. Seibert, A. R. Grossman and M. C. Posewitz (2009). "Flexibility in anaerobic metabolism as revealed in a mutant of Chlamydomonas reinhardtii lacking hydrogenase activity." J Biol Chem 284(11): 7201-7213. [CrossRef]
32. Dyall, S. D., W. Yan, M. G. Delgadillo-Correa, A. Lunceford, J. A. Loo, C. F. Clarke and P. J. Johnson (2004). "Non-mitochondrial complex I proteins in a hydrogenosomal oxidoreductase complex." Nature 431(7012): 1103-1107. [CrossRef]
33. Embley, T. M., M. van der Giezen, D. S. Horner, P. L. Dyal and P. Foster (2003). "Mitochondria and hydrogenosomes are two forms of the same fundamental organelle." Philos Trans R Soc Lond B Biol Sci 358(1429): 191-201; discussion 201-192. [CrossRef]
34. Eme, L., E. Gentekaki, B. Curtis, J. M. Archibald and A. J. Roger (2017). "Lateral Gene Transfer in the Adaptation of the Anaerobic Parasite Blastocystis to the Gut." Curr Biol 27(6): 807-820. [CrossRef]
35. Emelyanov, V. V. and A. V. Goldberg (2011). "Fermentation enzymes of Giardia intestinalis, pyruvate:ferredoxin oxidoreductase and hydrogenase, do not localize to its mitosomes." Microbiology (Reading) 157(Pt 6): 1602-1611. [CrossRef]
36. Felsenstein, J. (1985). "Confidence limits on phylogenies: An approach using the bootstrap." Evolution 39(4): 783-791. [CrossRef]
37. Field, J., B. Rosenthal and J. Samuelson (2000). "Early lateral transfer of genes encoding malic enzyme, acetyl-CoA synthetase and alcohol dehydrogenases from anaerobic prokaryotes to Entamoeba histolytica." Mol Microbiol 38(3): 446-455. [CrossRef]
38. Fleck, L. E., E. J. North, R. E. Lee, L. R. Mulcahy, G. Casadei and K. Lewis (2014). "A screen for and validation of prodrug antimicrobials." Antimicrob Agents Chemother 58(3): 1410-1419. [CrossRef]
39. Freeman, C. D., N. E. Klutman and K. C. Lamp (1997). "Metronidazole. A therapeutic review and update." Drugs 54(5): 679-708. [CrossRef]
40. Fritz-Laylin, L. K., S. E. Prochnik, M. L. Ginger, J. B. Dacks, M. L. Carpenter, M. C. Field, A. Kuo, A. Paredez, J. Chapman, J. Pham, S. Shu, R. Neupane, M. Cipriano, J. Mancuso, H. Tu, A. Salamov, E. Lindquist, H. Shapiro, S. Lucas, I. V. Grigoriev, W. Z. Cande, C. Fulton, D. S. Rokhsar and S. C. Dawson (2010). "The genome of Naegleria gruberi illuminates early eukaryotic versatility." Cell 140(5): 631-642. [CrossRef]
41. Gardner, M. J., N. Hall, E. Fung, O. White, M. Berriman, R. W. Hyman, J. M. Carlton, A. Pain, K. E. Nelson, S. Bowman, I. T. Paulsen, K. James, J. A. Eisen, K. Rutherford, S. L. Salzberg, A. Craig, S. Kyes, M. S. Chan, V. Nene, S. J. Shallom, B. Suh, J. Peterson, S. Angiuoli, M. Pertea, J. Allen, J. Selengut, D. Haft, M. W. Mather, A. B. Vaidya, D. M. Martin, A. H. Fairlamb, M. J. Fraunholz, D. S. Roos, S. A. Ralph, G. I. McFadden, L. M. Cummings, G. M. Subramanian, C. Mungall, J. C. Venter, D. J. Carucci, S. L. Hoffman, C. Newbold, R. W. Davis, C. M. Fraser and B. Barrell (2002). "Genome sequence of the human malaria parasite Plasmodium falciparum." Nature 419(6906): 498-511. [CrossRef]
42. Gargala, G. (2008). "Drug treatment and novel drug target against Cryptosporidium." Parasite 15(3): 275-281. [CrossRef]
43. Gomaa, F., D. R. Utter, C. Powers, D. J. Beaudoin, V. P. Edgcomb, H. L. Filipsson, C. M. Hansel, S. D. Wankel, Y. Zhang and J. M. Bernhard (2021). "Multiple integrated metabolic strategies allow foraminiferan protists to thrive in anoxic marine sediments." Sci Adv 7(22). [CrossRef]
44. Hackstein, J. H., A. Akhmanova, B. Boxma, H. R. Harhangi and F. G. Voncken (1999). "Hydrogenosomes: eukaryotic adaptations to anaerobic environments." Trends Microbiol 7(11): 441-447. [CrossRef]
45. Hedges, S. B., H. Chen, S. Kumar, D. Y. Wang, A. S. Thompson and H. Watanabe (2001). "A genomic timescale for the origin of eukaryotes." BMC Evol Biol 1: 4. [CrossRef]
46. Higuera, A., X. Villamizar, G. Herrera, J. C. Giraldo, A. L. Vasquez, P. Urbano, O. Villalobos, C. Tovar and J. D. Ramírez (2020). "Molecular detection and genotyping of intestinal protozoa from different biogeographical regions of Colombia." PeerJ 8: e8554. [CrossRef]
47. Hoffman, P. S., G. Sisson, M. A. Croxen, K. Welch, W. D. Harman, N. Cremades and M. G. Morash (2007). "Antiparasitic drug nitazoxanide inhibits the pyruvate oxidoreductases of Helicobacter pylori, selected anaerobic bacteria and parasites, and Campylobacter jejuni." Antimicrob Agents Chemother 51(3): 868-876. [CrossRef]
48. Horner, D. S., R. P. Hirt and T. M. Embley (1999). "A single eubacterial origin of eukaryotic pyruvate: ferredoxin oxidoreductase genes: implications for the evolution of anaerobic eukaryotes." Mol Biol Evol 16(9): 1280-1291. [CrossRef]
49. Hrdý, I., R. Cammack, P. Stopka, J. Kulda and J. Tachezy (2005). "Alternative pathway of metronidazole activation in Trichomonas vaginalis hydrogenosomes." Antimicrob Agents Chemother 49(12): 5033-5036. [CrossRef]
50. Hrdy, I., R. P. Hirt, P. Dolezal, L. Bardonová, P. G. Foster, J. Tachezy and T. M. Embley (2004). "Trichomonas hydrogenosomes contain the NADH dehydrogenase module of mitochondrial complex I." Nature 432(7017): 618-622. [CrossRef]
51. Hrdý, I. and M. Müller (1995). "Primary structure and eubacterial relationships of the pyruvate:ferredoxin oxidoreductase of the amitochondriate eukaryote Trichomonas vaginalis." J Mol Evol 41(3): 388-396. [CrossRef]
52. Huang, J., N. Mullapudi, T. Sicheritz-Ponten and J. C. Kissinger (2004). "A first glimpse into the pattern and scale of gene transfer in Apicomplexa." Int J Parasitol 34(3): 265-274. [CrossRef]
53. Hucho, F. (1975). "The pyruvate dehydrogenase multienzyme complex." Angew Chem Int Ed Engl 14(9): 591-601. [CrossRef]
54. Hug, L. A., A. Stechmann and A. J. Roger (2010). "Phylogenetic distributions and histories of proteins involved in anaerobic pyruvate metabolism in eukaryotes." Mol Biol Evol 27(2): 311-324. [CrossRef]
55. Husnik, F. and J. P. McCutcheon (2018). "Functional horizontal gene transfer from bacteria to eukaryotes." Nat Rev Microbiol 16(2): 67-79. [CrossRef]
56. Inui, H. and Y. Nakano (1999). "[Vitamin B1]." Nihon Rinsho 57(10): 2187-2192.
57. Johnson, P. J., C. J. Lahti and P. J. Bradley (1993). "Biogenesis of the hydrogenosome in the anaerobic protist Trichomonas vaginalis." J Parasitol 79(5): 664-670. [CrossRef]
58. Juhas, M. (2015). "Horizontal gene transfer in human pathogens." Crit Rev Microbiol 41(1): 101-108. [CrossRef]
59. Keeling, P. J. (2009). "Functional and ecological impacts of horizontal gene transfer in eukaryotes." Curr Opin Genet Dev 19(6): 613-619. [CrossRef]
60. Keeling, P. J. and J. D. Palmer (2008). "Horizontal gene transfer in eukaryotic evolution." Nat Rev Genet 9(8): 605-618. [CrossRef]
61. Kennedy, A. J., A. M. Bruce, C. Gineste, T. E. Ballard, I. N. Olekhnovich, T. L. Macdonald and P. S. Hoffman (2016). "Synthesis and Antimicrobial Evaluation of Amixicile-Based Inhibitors of the Pyruvate-Ferredoxin Oxidoreductases of Anaerobic Bacteria and Epsilonproteobacteria." Antimicrob Agents Chemother 60(7): 3980-3987. [CrossRef]
62. Kiehl, I. G. A., E. Riccetto, A. C. C. Salustiano, M. V. Ossick, K. L. Ferrari, H. B. Assalin, O. Ikari and L. O. Reis (2021). "Boosting bladder cancer treatment by intravesical nitazoxanide and bacillus calmette-guérin association." World J Urol 39(4): 1187-1194. [CrossRef]
63. Kulda, J. (1999). "Trichomonads, hydrogenosomes and drug resistance." Int J Parasitol 29(2): 199-212. [CrossRef]
64. Kumar, S., G. Stecher, M. Li, C. Knyaz and K. Tamura (2018). "MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms." Mol Biol Evol 35(6): 1547-1549. [CrossRef]
65. Kusdian, G. and S. B. Gould (2014). "The biology of Trichomonas vaginalis in the light of urogenital tract infection." Mol Biochem Parasitol 198(2): 92-99. [CrossRef]
66. Lacroix, B. and V. Citovsky (2016). "Transfer of DNA from Bacteria to Eukaryotes." mBio 7(4). [CrossRef]
67. Lacroix, B. and V. Citovsky (2018). "Beyond Agrobacterium-Mediated Transformation: Horizontal Gene Transfer from Bacteria to Eukaryotes." Curr Top Microbiol Immunol 418: 443-462. [CrossRef]
68. Lamp, K. C., C. D. Freeman, N. E. Klutman and M. K. Lacy (1999). "Pharmacokinetics and pharmacodynamics of the nitroimidazole antimicrobials." Clin Pharmacokinet 36(5): 353-373. [CrossRef]
69. Leitsch, D., A. G. Burgess, L. A. Dunn, K. G. Krauer, K. Tan, M. Duchêne, P. Upcroft, L. Eckmann and J. A. Upcroft (2011). "Pyruvate:ferredoxin oxidoreductase and thioredoxin reductase are involved in 5-nitroimidazole activation while flavin metabolism is linked to 5-nitroimidazole resistance in Giardia lamblia." J Antimicrob Chemother 66(8): 1756-1765. [CrossRef]
70. Lewis, W. H., A. E. Lind, K. M. Sendra, H. Onsbring, T. A. Williams, G. F. Esteban, R. P. Hirt, T. J. G. Ettema and T. M. Embley (2020). "Convergent Evolution of Hydrogenosomes from Mitochondria by Gene Transfer and Loss." Mol Biol Evol 37(2): 524-539. [CrossRef]
71. Lindmark, D. G. and M. Müller (1973). "Hydrogenosome, a cytoplasmic organelle of the anaerobic flagellate Tritrichomonas foetus, and its role in pyruvate metabolism." J Biol Chem 248(22): 7724-7728. [CrossRef]
72. Loftus, B., I. Anderson, R. Davies, U. C. Alsmark, J. Samuelson, P. Amedeo, P. Roncaglia, M. Berriman, R. P. Hirt, B. J. Mann, T. Nozaki, B. Suh, M. Pop, M. Duchene, J. Ackers, E. Tannich, M. Leippe, M. Hofer, I. Bruchhaus, U. Willhoeft, A. Bhattacharya, T. Chillingworth, C. Churcher, Z. Hance, B. Harris, D. Harris, K. Jagels, S. Moule, K. Mungall, D. Ormond, R. Squares, S. Whitehead, M. A. Quail, E. Rabbinowitsch, H. Norbertczak, C. Price, Z. Wang, N. Guillén, C. Gilchrist, S. E. Stroup, S. Bhattacharya, A. Lohia, P. G. Foster, T. Sicheritz-Ponten, C. Weber, U. Singh, C. Mukherjee, N. M. El-Sayed, W. A. Petri, Jr., C. G. Clark, T. M. Embley, B. Barrell, C. M. Fraser and N. Hall (2005). "The genome of the protist parasite Entamoeba histolytica." Nature 433(7028): 865-868. [CrossRef]
73. Lokhande, A. S. and P. V. Devarajan (2021). "A review on possible mechanistic insights of Nitazoxanide for repurposing in COVID-19." Eur J Pharmacol 891: 173748. [CrossRef]
74. Lopes-Oliveira, L. A. P., M. Fantinatti and A. M. Da-Cruz (2020). "In vitro-induction of metronidazole-resistant Giardia duodenalis is not associated with nucleotide alterations in the genes involved in pro-drug activation." Mem Inst Oswaldo Cruz 115: e200303. [CrossRef]
75. Makiuchi, T. and T. Nozaki (2014). "Highly divergent mitochondrion-related organelles in anaerobic parasitic protozoa." Biochimie 100: 3-17. [CrossRef]
76. Marczak, R., T. E. Gorrell and M. Müller (1983). "Hydrogenosomal ferredoxin of the anaerobic protozoon, Tritrichomonas foetus." J Biol Chem 258(20): 12427-12433. [CrossRef]
77. Martinez, V. and E. Caumes (2001). "[Metronidazole]." Ann Dermatol Venereol 128(8-9): 903-909.
78. Mastronicola, D., M. Falabella, E. Forte, F. Testa, P. Sarti and A. Giuffrè (2016). "Antioxidant defence systems in the protozoan pathogen Giardia intestinalis." Mol Biochem Parasitol 206(1-2): 56-66. [CrossRef]
79. Meneses Calderón, J., M. D. R. Figueroa Flores, L. Paniagua Coria, J. C. Briones Garduño, J. Meneses Figueroa, M. J. Vargas Contretas, L. De la Cruz Ávila, S. Díaz Meza, R. Ramírez Chacón, S. Padmanabhan and H. Mendieta Zerón (2020). "Nitazoxanide against COVID-19 in three explorative scenarios." J Infect Dev Ctries 14(9): 982-986. [CrossRef]
80. Moreno, S. N. and R. Docampo (1985). "Mechanism of toxicity of nitro compounds used in the chemotherapy of trichomoniasis." Environ Health Perspect 64: 199-208. [CrossRef]
81. Müller, J., S. Braga, M. Heller and N. Müller (2019). "Resistance formation to nitro drugs in Giardia lamblia: No common markers identified by comparative proteomics." Int J Parasitol Drugs Drug Resist 9: 112-119. [CrossRef]
82. nergy metabolism of anaerobic protists-a special case in eukaryotic evolution." Evolutionary relationships among protozoa.
83. Müller, M. (1993). "The hydrogenosome." J Gen Microbiol 139(12): 2879-2889. [CrossRef]
84. Narikawa, S. (1986). "Distribution of metronidazole susceptibility factors in obligate anaerobes." J Antimicrob Chemother 18(5): 565-574. [CrossRef]
85. Nepali, K., H. Y. Lee and J. P. Liou (2019). "Nitro-Group-Containing Drugs." J Med Chem 62(6): 2851-2893. [CrossRef]
86. Nixon, J. E., A. Wang, J. Field, H. G. Morrison, A. G. McArthur, M. L. Sogin, B. J. Loftus and J. Samuelson (2002). "Evidence for lateral transfer of genes encoding ferredoxins, nitroreductases, NADH oxidase, and alcohol dehydrogenase 3 from anaerobic prokaryotes to Giardia lamblia and Entamoeba histolytica." Eukaryot Cell 1(2): 181-190. [CrossRef]
87. Noth, J., D. Krawietz, A. Hemschemeier and T. Happe (2013). "Pyruvate:ferredoxin oxidoreductase is coupled to light-independent hydrogen production in Chlamydomonas reinhardtii." J Biol Chem 288(6): 4368-4377. [CrossRef]
88. Ocaña, K. A. and A. M. Dávila (2011). "Phylogenomics-based reconstruction of protozoan species tree." Evol Bioinform Online 7: 107-121. [CrossRef]
89. Pal, A. K., M. Nandave and G. Kaithwas (2020). "Chemoprophylactic activity of nitazoxanide in experimental model of mammary gland carcinoma in rats." 3 Biotech 10(8): 338. [CrossRef]
90. Pal, D., S. Banerjee, J. Cui, A. Schwartz, S. K. Ghosh and J. Samuelson (2009). "Giardia, Entamoeba, and Trichomonas enzymes activate metronidazole (nitroreductases) and inactivate metronidazole (nitroimidazole reductases)." Antimicrob Agents Chemother 53(2): 458-464. [CrossRef]
91. E. Boucher and I. B. Lambert (2002). "Regulation of the nfsA Gene in Escherichia coli by SoxS." J Bacteriol 184(1): 51-58. [CrossRef]
92. Peterson, F. J., R. P. Mason, J. Hovsepian and J. L. Holtzman (1979). "Oxygen-sensitive and -insensitive nitroreduction by Escherichia coli and rat hepatic microsomes." J Biol Chem 254(10): 4009-4014. [CrossRef]
93. Rada, P., P. Doležal, P. L. Jedelský, D. Bursac, A. J. Perry, M. Šedinová, K. Smíšková, M. Novotný, N. C. Beltrán, I. Hrdý, T. Lithgow and J. Tachezy (2011). "The core components of organelle biogenesis and membrane transport in the hydrogenosomes of Trichomonas vaginalis." PLoS One 6(9): e24428. [CrossRef]
94. Ragsdale, S. W. (2003). "Pyruvate ferredoxin oxidoreductase and its radical intermediate." Chem Rev 103(6): 2333-2346. [CrossRef]
95. Raj, D., E. Ghosh, A. K. Mukherjee, T. Nozaki and S. Ganguly (2014). "Differential gene expression in Giardia lamblia under oxidative stress: significance in eukaryotic evolution." Gene 535(2): 131-139. [CrossRef]
96. Rasoloson, D., S. Vanacova, E. Tomkova, J. Razga, I. Hrdy, J. Tachezy and J. Kulda (2002). "Mechanisms of in vitro development of resistance to metronidazole in Trichomonas vaginalis." Microbiology (Reading) 148(Pt 8): 2467-2477. [CrossRef]
97. Rau, J. and A. Stolz (2003). "Oxygen-insensitive nitroreductases NfsA and NfsB of Escherichia coli function under anaerobic conditions as lawsone-dependent Azo reductases." Appl Environ Microbiol 69(6): 3448-3455. [CrossRef]
98. Ricard, G., N. R. McEwan, B. E. Dutilh, J. P. Jouany, D. Macheboeuf, M. Mitsumori, F. M. McIntosh, T. Michalowski, T. Nagamine, N. Nelson, C. J. Newbold, E. Nsabimana, A. Takenaka, N. A. Thomas, K. Ushida, J. H. Hackstein and M. A. Huynen (2006). "Horizontal gene transfer from Bacteria to rumen Ciliates indicates adaptation to their anaerobic, carbohydrates-rich environment." BMC Genomics 7: 22. [CrossRef]
99. Rocco, P. R. M., P. L. Silva, F. F. Cruz, M. A. C. Melo-Junior, P. Tierno, M. A. Moura, L. F. G. De Oliveira, C. C. Lima, E. A. Dos Santos, W. F. Junior, A. Fernandes, K. G. Franchini, E. Magri, N. F. de Moraes, J. M. J. Gonçalves, M. N. Carbonieri, I. S. Dos Santos, N. F. Paes, P. V. M. Maciel, R. P. Rocha, A. F. de Carvalho, P. A. Alves, J. L. Proença-Módena, A. T. Cordeiro, D. B. B. Trivella, R. E. Marques, R. R. Luiz, P. Pelosi and E. S. J. R. Lapa (2021). "Early use of nitazoxanide in mild COVID-19 disease: randomised, placebo-controlled trial." Eur Respir J 58(1). [CrossRef]
100. Rodríguez, M. A., R. M. García-Pérez, L. Mendoza, T. Sánchez, N. Guillen and E. Orozco (1998). "The pyruvate:ferredoxin oxidoreductase enzyme is located in the plasma membrane and in a cytoplasmic structure in Entamoeba." Microb Pathog 25(1): 1-10. [CrossRef]
101. Roger, A. J., C. G. Clark and W. F. Doolittle (1996). "A possible mitochondrial gene in the early-branching amitochondriate protist Trichomonas vaginalis." Proc Natl Acad Sci U S A 93(25): 14618-14622. [CrossRef]
102. Roldán, M. D., E. Pérez-Reinado, F. Castillo and C. Moreno-Vivián (2008). "Reduction of polynitroaromatic compounds: the bacterial nitroreductases." FEMS Microbiol Rev 32(3): 474-500. [CrossRef]
103. Rosenthal, B., Z. Mai, D. Caplivski, S. Ghosh, H. de la Vega, T. Graf and J. Samuelson (1997). "Evidence for the bacterial origin of genes encoding fermentation enzymes of the amitochondriate protozoan parasite Entamoeba histolytica." J Bacteriol 179(11): 3736-3745. [CrossRef]
104. Rotte, C., F. Stejskal, G. Zhu, J. S. Keithly and W. Martin (2001). "Pyruvate : NADP+ oxidoreductase from the mitochondrion of Euglena gracilis and from the apicomplexan Cryptosporidium parvum: a biochemical relic linking pyruvate metabolism in mitochondriate and amitochondriate protists." Mol Biol Evol 18(5): 710-720. [CrossRef]
105. Saitou, N. and M. Nei (1987). "The neighbor-joining method: a new method for reconstructing phylogenetic trees." Mol Biol Evol 4(4): 406-425.
106. Schönknecht, G., W. H. Chen, C. M. Ternes, G. G. Barbier, R. P. Shrestha, M. Stanke, A. Bräutigam, B. J. Baker, J. F. Banfield, R. M. Garavito, K. Carr, C. Wilkerson, S. A. Rensing, D. Gagneul, N. E. Dickenson, C. Oesterhelt, M. J. Lercher and A. P. Weber (2013). "Gene transfer from bacteria and archaea facilitated evolution of an extremophilic eukaryote." Science 339(6124): 1207-1210. [CrossRef]
107. Scior, T., J. Lozano-Aponte, S. Ajmani, E. Hernández-Montero, F. Chávez-Silva, E. Hernández-Núñez, R. Moo-Puc, A. Fraguela-Collar and G. Navarrete-Vázquez (2015). "Antiprotozoal Nitazoxanide Derivatives: Synthesis, Bioassays and QSAR Study Combined with Docking for Mechanistic Insight." Curr Comput Aided Drug Des 11(1): 21-31. [CrossRef]
108. Shakya, A., H. R. Bhat and S. K. Ghosh (2018). "Update on Nitazoxanide: A Multifunctional Chemotherapeutic Agent." Curr Drug Discov Technol 15(3): 201-213. [CrossRef]
109. Sibbald, S. J., L. Eme, J. M. Archibald and A. J. Roger (2020). "Lateral Gene Transfer Mechanisms and Pan-genomes in Eukaryotes." Trends Parasitol 36(11): 927-941. [CrossRef]
110. Sisson, G., A. Goodwin, A. Raudonikiene, N. J. Hughes, A. K. Mukhopadhyay, D. E. Berg and P. S. Hoffman (2002). "Enzymes associated with reductive activation and action of nitazoxanide, nitrofurans, and metronidazole in Helicobacter pylori." Antimicrob Agents Chemother 46(7): 2116-2123. [CrossRef]
111. Soucy, S. M., J. Huang and J. P. Gogarten (2015). "Horizontal gene transfer: building the web of life." Nat Rev Genet 16(8): 472-482. [CrossRef]
112. Upcroft, P. and J. A. Upcroft (2001). "Drug targets and mechanisms of resistance in the anaerobic protozoa." Clin Microbiol Rev 14(1): 150-164. [CrossRef]
113. Van Etten, J. and D. Bhattacharya (2020). "Horizontal Gene Transfer in Eukaryotes: Not if, but How Much?" Trends Genet 36(12): 915-925. [CrossRef]
114. van Lis, R., C. Baffert, Y. Couté, W. Nitschke and A. Atteia (2013). "Chlamydomonas reinhardtii chloroplasts contain a homodimeric pyruvate:ferredoxin oxidoreductase that functions with FDX1." Plant Physiol 161(1): 57-71. [CrossRef]
115. Warren, C. A., E. van Opstal, T. E. Ballard, A. Kennedy, X. Wang, M. Riggins, I. Olekhnovich, M. Warthan, G. L. Kolling, R. L. Guerrant, T. L. Macdonald and P. S. Hoffman (2012). "Amixicile, a novel inhibitor of pyruvate: ferredoxin oxidoreductase, shows efficacy against Clostridium difficile in a mouse infection model." Antimicrob Agents Chemother 56(8): 4103-4111. [CrossRef]
116. Wawrzyniak, I., M. Roussel, M. Diogon, A. Couloux, C. Texier, K. S. Tan, C. P. Vivarès, F. Delbac, P. Wincker and H. El Alaoui (2008). "Complete circular DNA in the mitochondria-like organelles of Blastocystis hominis." Int J Parasitol 38(12): 1377-1382. [CrossRef]
117. Wyres, K. L., R. R. Wick, L. M. Judd, R. Froumine, A. Tokolyi, C. L. Gorrie, M. M. C. Lam, S. Duchêne, A. Jenney and K. E. Holt (2019). "Distinct evolutionary dynamics of horizontal gene transfer in drug resistant and virulent clones of Klebsiella pneumoniae." PLoS Genet 15(4): e1008114. [CrossRef]
118. Xiong, J., G. Wang, J. Cheng, M. Tian, X. Pan, A. Warren, C. Jiang, D. Yuan and W. Miao (2015). "Genome of the facultative scuticociliatosis pathogen Pseudocohnilembus persalinus provides insight into its virulence through horizontal gene transfer." Sci Rep 5: 15470. [CrossRef]