Energy production pathways in T. vaginalis

Trichomonas vaginalis obtains most of its energy through carbohydrate metabolism, which involves breaking down glucose to produce lactate, acetate, molecular hydrogen and adenosine triphosphate (ATP). Malate can also serve as an energy source. Figures 2a and b depict the structure (Honigberg and King, Reference Honigberg and King1964; Honigberg and Brugerolle, Reference Honigberg, Brugerolle and Honigberg1990; Bouchemal et al., Reference Bouchemal, Bories and Loiseau2017) and energy production pathways of T. vaginalis. One pathway begins in the cytoplasm where glucose undergoes glycolysis to form pyruvate, which is then passively transported into hydrogenosomes (Fig. 2a). Inside the hydrogenosome (Fig. 2b), the enzyme pyruvate:ferredoxin oxidoreductase (PFOR) facilitates oxidation (loss of electrons) of pyruvate by ferredoxin (Fdx) to produce acetyl-CoA. The reduced Fdx is oxidized by the enzyme hydrogenase (HYD), which uses the electrons gained from Fdx to produce molecular hydrogen (H₂). Figure 2b depicts a second energy production pathway in the hydrogenosomes that uses malate (Dolezal et al., Reference Dolezal, Vanacova, Tachezy and Hrdy2004; Hrdy et al., Reference Hrdy, Cammack, Stopka, Kulda and Tachezy2005). This pathway utilizes malate dehydrogenase (MDH) and nicotinamide adenine dinucleotide:ferredoxin oxidoreductase (NADH:FOR). Malate is decarboxylated by MDH to yield pyruvate and CO₂, transferring electrons to NAD⁺ whereas pyruvate enters the first pathway to produce acetyl-CoA and ATP. Similar to PFOR, NADH:FOR facilitates the transfer of electrons to Fdx, allowing the reaction to proceed to H₂ production. ATP and acetate production in the hydrogenosomes occurs through a substrate-level phosphorylation catalysed by two enzymes: acetate:succinate CoA-transferase (ASCT) and succinyl-CoA synthetase (SCS), called the ASCT/SCS cycle (Fig. 2b; pathway 3).

Fig. 2. MTZ activation through the energy production pathway in T. vaginalis. (a) Overview of T. vaginalis and the MTZ drug activation pathway. (1) MTZ enters the cell by passive diffusion and is subsequently metabolized in the hydrogenosome. (2) Pyruvate is produced from glucose through glycolysis in the cytosol. (3) Inside the hydrogenosome, PFOR facilitates oxidative decarboxylation of pyruvate by reducing and transferring electrons to Fdx. Fdx then reduces the nitro group of MTZ, which creates a cytotoxic nitro radical anion. (4) Activated MTZ then interacts with T. vaginalis DNA causing damage and death of the organism. (b) Detailed energy production and MTZ drug activation pathway in the hydrogenosome. Solid line: Major energy production pathway: Pyruvate is the major intermediate product produced from glucose through the glycolytic pathway (pathway 1) as well as through an alternative malate-dependent pathway (pathway 2). PFOR then transfers electrons from pyruvate to the electron acceptor Fdx, producing acetyl-CoA, which is through ASCT/SCS cycle reduced to acetate, producing ATP (pathway 3). The reduced Fdx is used to produce H₂ by the HYD enzyme. Dashed line: Drug activation pathway – Once MTZ enters the hydrogenosome, it competes with HYD for the electron carrying Fdx. MTZ is reduced to a nitro radical anion that interacts with T. vaginalis DNA causing damage and death of the organism. PFOR, pyruvate:ferredoxin oxidoreductase; Fdx, ferredoxin; HYD, hydrogenase; H₂, hydrogen; CO₂, carbon dioxide; MTZ, metronidazole; MDH, malate dehydrogenase; NADPH, nicotinamide adenine dinucleotide phosphate; NADH:FOR, nicotinamide adenine dinucleotide:ferredoxin oxidoreductase; PEPCK, phosphoenolpyruvate carboxykinase; GTP/GDP, guanosine triphosphate/guanosine diphosphate; PDC, pyruvate decarboxylase; LDH, lactate dehydrogenase; ADH, alcohol dehydrogenase; ASCT, acetate:succinate CoA-transferase; SCS, succinyl CoA synthetase; OX, oxidized; RED, reduced.

Antioxidant defense pathway in T. vaginalis

The antioxidant defense pathway takes place in the cytosol of T. vaginalis. Figure 3a illustrates this pathway under normal conditions, i.e. in the absence of a 5-nitroimidazole. The reactive oxygen species (ROS), superoxide (O₂⁻), is reduced to molecular oxygen (O₂) and hydrogen peroxide (H₂O₂) by the enzyme superoxide dismutase (SOD). The O₂ is then reduced by either nicotinamide adenine dinucleotide (NADH) oxidase to H₂O or by flavin reductase 1 (FR1) to H₂O₂. The H₂O₂ produced by these processes must be further reduced to avoid its cytotoxic side-effects. This is accomplished indirectly by the activity of the flavin enzyme thioredoxin reductase (TrxR) and its accompanying protein and enzyme thioredoxin (Trx) and thioredoxin peroxidase (TrxP), respectively. TrxR facilitates the reduction of Trx by nicotinamide adenine dinucleotide phosphate (NADPH). Reduced Trx subsequently activates enzymes involved in other metabolic pathways. Importantly, Trx activates TrxP, which then reduces H₂O₂ to H₂O.

Fig. 3. Aerobic resistance to MTZ arises from deficient O₂ scavenging pathways. (a) Normal T. vaginalis antioxidant defense pathways – (1) ROS, such as O₂⁻, can be harmful to trichomonads, and multiple pathways can control and ‘neutralize’ ROS. SOD reduces O₂⁻ to molecular O₂ while also producing water and H₂O₂. (2) Intracellular O₂ can be reduced to H₂O₂ by FR1 or by (3) NADH oxidase into H₂O. The cytotoxic H₂O₂ produced by these pathways further metabolized by the trichomonad. (4) H₂O₂ can be indirectly reduced to H₂O, through the activation of the flavin-containing enzyme TrxR by NADPH. Electrons from NADPH are transferred from TrxR to Trx, which activates thioredoxin-dependent peroxidases such as TrxP to reduce H₂O₂ to H₂O. (b) Aerobic resistance pathway – (1) TrxR, like Fdx and NTR, can reduce MTZ. Once reduced, MTZ can form covalent adducts with TrxR and Trx. These adducts inhibit the activities of the enzymes, leading to increased levels of cytotoxic H₂O₂. (2) FR1 and NADH oxidase activities are significantly decreased or absent in MTZ resistant strains. (3) Intracellular O₂ levels increase in the absence of normally functioning O₂-scavenging pathways, resulting in the inactivation of MTZ from its nitro radical anion in a process known as futile cycling. ROS, reactive oxygen species; O₂⁻, superoxide; SOD, superoxide dismutase; O₂, oxygen; H₂O₂, hydrogen peroxide; FR1, flavin reductase 1; NADH, nicotinamide adenine dinucleotide; TrxR, thioredoxin reductase; NTR, nitroreductase; NADPH, nicotinamide adenine dinucleotide phosphate; Trx, thioredoxin; TrxP, thioredoxin peroxidase; MTZ, metronidazole; OX, oxidized; RED, reduced.

Drug activation of 5-nitroimidazoles in T. vaginalis

MTZ or other 5-nitroimidazoles used for Trichomonas treatment enter the parasites in an inactive form through passive diffusion (Muller, Reference Muller1986). 5-Nitroimidazoles need to be reduced in order to actively interfere with T. vaginalis processes (Fig. 2a). MTZ is activated in the hydrogenosomes by the energy-production pathway through competition with HYD for the electrons of Fdx (Fig. 2b) (Lloyd and Kristensen, Reference Lloyd and Kristensen1985, Lloyd et al., Reference Lloyd, Yarlett and Yarlett1986). Both Fdx and nitroreductase (NTR) can reduce MTZ to its active nitro radical anion form (Yarlett et al., Reference Yarlett, Gorrell, Marczak and Muller1985; Yarlett et al., Reference Yarlett, Yarlett and Lloyd1986a, Reference Yarlett, Yarlett and Lloyd1986b; Pal et al., Reference Pal, Banerjee, Cui, Schwartz, Ghosh and Samuelson2009). In this active form, MTZ interferes with, and disrupts, DNA synthesis and repair, leading to cell death (Declerck and De Ranter, Reference Declerck and De Ranter1986).

TrxR, such as Fdx and NTR, can activate MTZ (Leitsch et al., Reference Leitsch, Kolarich, Binder, Stadlmann, Altmann and Duchene2009). This provides an alternative theory to the mechanism of action and target metabolic mechanisms that 5-nitroimidazoles affect. Upon activation, MTZ can form a ‘covalent adduct’ with TrxR and other enzymes and proteins involved in the antioxidant defense pathway. The inactivation of TrxR prevents Trx activation which in turn blocks TrxP from reducing H₂O₂, which is cytotoxic. Left unchecked, this will lead to death of the organism. Thus, antioxidant defenses (i.e. oxygen scavenging mechanisms) could be the main mechanism of action of MTZ instead of DNA disruption.

Aerobic resistance

Aerobic resistance arises due to deficiencies associated with oxygen-scavenging mechanisms of the antioxidant-defense pathway (Lloyd and Pedersen, Reference Lloyd and Pedersen1985; Yarlett et al., Reference Yarlett, Yarlett and Lloyd1986a, Reference Yarlett, Yarlett and Lloyd1986b; Ellis et al., Reference Ellis, Yarlett, Cole, Humphreys and Lloyd1994). Decreased or absent FR1, TrxR and possibly NADH oxidase and SOD activity leads to increased intracellular oxygen levels (Fig. 3b) (Muller and Gorrell, Reference Muller and Gorrell1983; Rasoloson et al., Reference Rasoloson, Tomkova, Cammack, Kulda and Tachezy2001; Leitsch et al., Reference Leitsch, Kolarich and Duchene2010). Downregulated expression of FR1 in response to toxic TrxR-MTZ adducts limits the production of H₂O₂ and increases intracellular O₂ leading to inactivation (oxidation) of MTZ (Leitsch et al., Reference Leitsch, Drinic, Kolarich and Duchene2012; Leitsch et al., Reference Leitsch, Janssen, Kolarich, Johnson and Duchene2014). Although increased O₂ levels and MTZ inactivate cytosolic metabolic pathways, the hydrogenosomal metabolic pathways are still functional in aerobically resistant T. vaginalis strains. This is characterized by increased rates of glucose consumption and increased production of acetate and CO₂ (Ellis et al., Reference Ellis, Cole and Lloyd1992).

Anaerobic resistance

Trichomonas vaginalis isolates that exhibit anaerobic resistance tend to have much higher in vitro MLC values compared to aerobically-resistant isolates. Anaerobic resistance has mainly been observed in vitro and induced under laboratory conditions rather than arising clinically. It is characterized by the disruption of enzymes that participate in the energy production pathway (Land et al., Reference Land, Clemens and Johnson2001; Land et al., Reference Land, Delgadillo and Johnson2002). Decreased levels of hydrogenosomal enzymes have been associated with smaller hydrogenosomes in laboratory-induced resistant strains compared to MTZ-sensitive strains (Wright et al., Reference Wright, Webb, O'Donoghue, Upcroft and Upcroft2010). However, loss of PFOR activity alone does not lead to resistance as Fdx functions in the malate-dependent pathway through NADH:FOR and MDH (Fig. 2b) (Rasoloson et al., Reference Rasoloson, Vanacova, Tomkova, Razga, Hrdy, Tachezy and Kulda2002). Thus, full resistance only occurs when both PFOR and NADH:FOR activities are lost. Another alternative energy pathway proposed in resistant T. vaginalis strains bypasses PFOR and the need to pass on electrons to Fdx. It involves the enzyme keto acid oxidoreductase (KOR), which, like PFOR, can reduce pyruvate and other metabolites to meet the energy demands of T. vaginalis (Brown et al., Reference Brown, Upcroft, Dodd, Chen and Upcroft1999).

The role of genetic diversity and differential gene expression in 5-nitroimidazole resistance

Many techniques have been used to characterize and analyse the genotypic, phenotypic and genetic relatedness of T. vaginalis strains from different geographical regions. These techniques involve analysing random-amplified polymorphic DNA patterns, restriction fragment length polymorphisms, multi-locus sequence typing and internal transcribed spacer (ITS) region sequences (Vanacova et al., Reference Vanacova, Tachezy, Kulda and Flegr1997; Snipes et al., Reference Snipes, Gamard, Narcisi, Beard, Lehmann and Secor2000; Stiles et al., Reference Stiles, Shah, Xue, Meade, Lushbaugh, Cleary and Finley2000; Hampl et al., Reference Hampl, Vanacova, Kulda and Flegr2001; Abdel-Magied et al., Reference Abdel-Magied, El-Kholya, Abou El-Khair, Abdelmegeed, Hamoudaa, Mohamed and El-Tantawy2017). Genotypic and phenotypic analyses demonstrate the genetic relatedness of MTZ-resistant T. vaginalis strains and suggest T. vaginalis can be divided into two distinct populations (Cornelius et al., Reference Cornelius, Mena, Lushbaugh and Meade2010; Rabiee et al., Reference Rabiee, Bazmani, Matini and Fallah2012). Type 1 populations are the more ancestral lineage whereas type 2 populations appear to have diverged more recently (Conrad et al., Reference Conrad, Gorman, Schillinger, Fiori, Arroyo, Malla, Dubey, Gonzalez, Blank, Secor and Carlton2012), with type 2 having higher mean MLCs for MTZ (Bradic et al., Reference Bradic, Secor, Conrad and Carlton2012; Conrad et al., Reference Conrad, Gorman, Schillinger, Fiori, Arroyo, Malla, Dubey, Gonzalez, Blank, Secor and Carlton2012). Some genes involved in 5-nitroimidazole resistance possess mutations and are differentially expressed in resistant strains (Mead et al., Reference Mead, Fernadez, Romagnoli and Secor2006; Bradic et al., Reference Bradic, Warring, Tooley, Scheid, Secor, Land, Huang, Chen, Lee, Tang, Sullivan and Carlton2017).

Fdx gene mutations

Some MTZ-resistant T. vaginalis isolates have decreased fdx gene transcription (Quon et al., Reference Quon, d'Oliveira and Johnson1992). Two different point mutations have been identified at nucleotide positions −178 and −239 upstream of the transcription start site. The −239 mutation is more relevant and is characterized by an A→T transversion (adenine to thymidine). One study observed a 28-bp region surrounding the −239 mutation that binds a 23-kDa DNA-binding protein thought important for transcription of the fdx gene (Quon et al., Reference Quon, d'Oliveira and Johnson1992). MTZ-resistant isolates with this mutation have a lower binding affinity for DNA-binding protein that could lead to reduced transcription. This −239 A→T mutation has been observed more recently (Heidari et al., Reference Heidari, Bandehpour, Seyyed-Tabaei, Valadkhani, Haghighi, Abadi and Kazemi2013). An additional study analysed the amino-acid sequence of Fdx and determined regions coding for amino-acids 43–48 are more susceptible to mutation (Wiwanitkit, Reference Wiwanitkit2008).

NTR 4/6 gene mutation

NTR is one of the enzymes responsible for the reduction of 5-nitroimidazoles (Pal et al., Reference Pal, Banerjee, Cui, Schwartz, Ghosh and Samuelson2009). A recent study found single-nucleotide polymorphisms (SNPs) in two NTR genes (ntr4 and ntr6) (Paulish-Miller et al., Reference Paulish-Miller, Augostini, Schuyler, Smith, Mordechai, Adelson, Gygax, Secor and Hilbert2014). These SNPs were nonsense mutations (introduction of a stop codon) associated with MTZ resistance. Isolates with either mutation or both were more likely to belong to the type 2 population, which is associated with greater MTZ resistance (Paulish-Miller et al., Reference Paulish-Miller, Augostini, Schuyler, Smith, Mordechai, Adelson, Gygax, Secor and Hilbert2014).

ITS1 point mutation

The ITS regions that flank the 5.8S subunit of the ribosomal DNA gene of T. vaginalis are not as highly conserved as the ribosomal coding sequences they flank, leading to a greater frequency of genetic polymorphisms between organisms of the same species. A point mutation at the 66th nucleotide of the ITS region consisting of a cysteine being replaced with a thymidine (C66T) is associated with higher levels of MTZ resistance (Snipes et al., Reference Snipes, Gamard, Narcisi, Beard, Lehmann and Secor2000; Xiao et al., Reference Xiao, Xie, Fang, Gao, Zhu, Song, Zhong and Lun2006; Kazemi et al., Reference Kazemi, Hooshyar, Zareikar, Bandehpour, Arbabi, Talari, Alizadeh and Kazemi2010).

Iron metabolism affects gene expression in T. vaginalis

Iron plays an important role in the metabolic processes (as a cofactor in many enzymes) and transcriptional regulation of genes for key enzymes in T. vaginalis (Argaez-Correa et al., Reference Argaez-Correa, Alvarez-Sanchez, Arana-Argaez, Ramirez-Camacho, Novelo-Castilla, Coral-Martinez and Torres-Romero2019). The restriction of iron in T. vaginalis leads to the down-regulation of genes important for energy metabolism and drug activation like PFOR and Fdx (Horvathova et al., Reference Horvathova, Safarikova, Basler, Hrdy, Campo, Shin, Huang, Huang, Lin, Tang and Tachezy2012). Decreased expression leads to development of 5-nitroimidazole resistance. Iron is a key structural and metabolic component of PFOR and Fdx; decreasing iron stores available to T. vaginalis isolates decrease their ability to synthesize these critically important enzymes. Supplemental addition of iron with MTZ treatment decreases MLCs under both aerobic and anaerobic conditions in MTZ-sensitive strains (Elwakil et al., Reference Elwakil, Tawfik, Alam-Eldin and Nassar2017).

Expression of surface carbohydrate genes

Trichomonas vaginalis membranes contain glycoproteins and glycolipids that act as cell receptors and are involved in host–parasite interactions. The plasma membranes of MTZ-resistant and MTZ-sensitive T. vaginalis strains differ in their surface carbohydrate structures (Dias Filho et al., Reference Dias Filho, Andrade, de Souza, Esteves and Angluster1992). Trichomonas vaginalis also has a P-glycoprotein gene (Tvpgp1) that codes for an ATP binding cassette (ABC) transmembrane protein (Johnson et al., Reference Johnson, Schuck and Delgadillo1994). ABC proteins are involved in the transport of drugs out of cells. Tvpgp1 is expressed 2–20-fold higher in MTZ-resistant T. vaginalis strains compared to sensitive strains (Johnson et al., Reference Johnson, Schuck and Delgadillo1994).

Extracellular ATP and ADP level modulation by T. vaginalis

Extracellular ATP is involved in host–parasite interactions such as signal transduction, cytoadherence, immune response and inflammation. The enzymes that T. vaginalis employs for the hydrolysis of extracellular ATP, adenosine diphosphate (ADP) and adenosine monophosphate, are NTPDase1 and ecto-5′-nucleotidase; these enzymes can be incorporated into the plasma membrane of T. vaginalis and are involved in signal transduction and cytoadhesion. Prolonged exposure/treatment with MTZ or TDZ results in the inhibition of these enzymes in MTZ-sensitive strains (Tasca et al., Reference Tasca, Borges, Bonan, De Carli, Battastini and Sarkis2003). Thus, modulation of extracellular ATP and ADP levels may be a defense strategy of T. vaginalis in response to unfavourable environmental conditions (i.e. drug pressure) (Tasca et al., Reference Tasca, Borges, Bonan, De Carli, Battastini and Sarkis2003).

Trichomonas vaginalis virus (TVV)

TVV is a 4.5 kbp dsRNA virus that infects some T. vaginalis isolates (Wang and Wang, Reference Wang and Wang1985). Four strains of TVV have been described since its discovery in the mid-1980s with TVV1 being the most common strain, followed by TVV2 and TVV3, with TVV4 being least common (Graves et al., Reference Graves, Ghosh, Kissinger and Muzny2019). TVV2 and TVV3 are associated with expression of the surface immunogen P270. Some studies found no clear associations of TVV presence on MTZ resistance (Wang and Wang, Reference Wang and Wang1985; Flegr et al., Reference Flegr, Cerkasov, Kulda, Tachezy and Stokrov1987), whereas another study found increased MTZ susceptibility in TVV-infected T. vaginalis (Malla et al., Reference Malla, Kaul, Sehgal and Gupta2011); additional research is needed to clarify the effect of TVV infection on 5-nitroimidazole resistance in T. vaginalis.

Mycoplasma hominis

Mycoplasma hominis is a bacterial pathogen of the lower genital tract that naturally infects T. vaginalis (van Belkum et al., Reference van Belkum, van der Schee, van der Meijden, Verbrugh and Sluiters2001). Some studies have found that M. hominis infection of T. vaginalis was associated with increased MTZ resistance (Xiao et al., Reference Xiao, Xie, Fang, Gao, Zhu, Song, Zhong and Lun2006; Furnkranz et al., Reference Furnkranz, Henrich and Walochnik2018). However, other studies indicate M. hominis infection has no clinical significance (Butler et al., Reference Butler, Augostini and Secor2010; Fraga et al., Reference Fraga, Rodriguez, Fernandez, Mondeja, Sariego, Fernandez-Calienes and Rojas2012; da Luz Becker et al., Reference da Luz Becker, dos Santos, Frasson, de Vargas Rigo, Macedo and Tasca2015). Mycoplasma hominis can downregulate expression of Fdx, FR1 and PFOR genes involved in MTZ resistance mechanisms (Furnkranz et al., Reference Furnkranz, Henrich and Walochnik2018); however, similar to TVV, how M. hominis affects T. vaginalis susceptibility to MTZ needs further investigation.