Alloferon regulates the growth and movement of Trichomonas vaginalis by altering hydrogenosomes

doi:10.21203/rs.3.rs-4345816/v1

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Alloferon regulates the growth and movement of Trichomonas vaginalis by altering hydrogenosomes

https://doi.org/10.21203/rs.3.rs-4345816/v1

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Background

Trichomoniasis infected with Trichomonas vaginalis (T. vaginalis), can cause mild symptoms like itching and burning but can also lead to more serious adverse outcomes. While typically treated with metronidazole, this medication can face resistance from T. vaginalis and some individuals may experience side effects. Hence, the research on effective therapeutic methods is essential to improve traditional therapy for trichomoniasis.

Method

To investigate the potential of alloferon as a therapeutic agent for parasitic infection, we measured the activity of hydrogenosomes in T. vaginalis using flow cytometric analysis and observed the morphology of hydrogenosomes through a transmission electron microscopy. In addition, the cell cycle of T. vaginalis was assessed using cell cycle analysis. To the synergetic effect of alloferon and metronidazole, the movement of T. vaginalis was observed through a microscopy and video recording.

Result

T. vaginalis treated with alloferon reduced the activity of its energy-producing organelles, hydrogenosomes and changed structure of hydrogenosome. In addition, alloferon induced cell cycle arrest in the S phase of T. vaginalis, thereby leading to decreased proliferation. While metronidazole alone at its minimum lethal concentration was ineffective, combining it with alloferon, significantly suppressed motility and proliferation in T. vaginalis.

Conclusion

Alloferon induces decreased growth and movement of T. vaginalis by altering the morphology and size of hydrogenosomes. Our findings suggest that alloferon could be a synergistic agent in combination therapy with metronidazole for trichomoniasis.

Trichomonas vaginalis

Alloferon

Hydrogenosome

Metronidazole

Trichomoniasis is a prevalent sexually transmitted infection (STI) caused by the parasite Trichomonas vaginalis [1, 2]. Globally, estimated 2016 global prevalence rates were 5.3% in women and 0.6% in men, with an estimated 156 million new cases of T. vaginalis infection worldwide in 2020 [3, 4, 5]. Infection with T. vaginalis has been associated with multiple adverse outcomes in women, including premature birth, low birth weight in newborns, infertility, delayed pregnancy, and increased risk of contracting human immunodeficiency virus (HIV), the virus that causes acquired immunodeficiency syndrome (ADIS) [6, 7, 8, 9, 10, 11]. In men, approximately 75% of T. vaginalis infections were asymptomatic. However, recent studies have revealed that the parasite may be associated with aggressive prostatic cancer in man and cervical cancer in women infected with T. vaginalis [12, 13, 14]. Metronidazole is commonly used to treat trichomoniasis caused by T. vaginalis. While typically effective, therapeutic use of metronidazole can have potentially severe side effects including neuropathy, cerebellar dysfunction, visual impairment, and encephalopathy [15, 16, 17]. With these factors, there is a need to develop a new therapeutic agent to trichomoniasis more efficiently with fewer side effects.

Alloferon is a small peptide first extracted from the blood of Calliphora vicina larvae following bacterial challenge. Alloferon was isolated to assess their antiviral, anti-inflammatory, and antitumoral properties [18, 19, 20, 21, 22, 23, 24]. However, the potential antiparasitic of alloferon remains unexplored. Consequently, we investigated the antiparasitic effect of alloferon and its potential synergistic action with metronidazole.

T. vaginalis contains organelles similar to mitochondria called hydrogenosomes. However, their metabolic pathway is distinct from those found in mitochondria. Hydrogenosomes have not been found in multicellular animals or plants and, are only present in parasites [25, 26, 27]. These organelles oxidize pyruvate or malate under anaerobic conditions and generate ATP via oxygen-sensitive enzymes, such as pyruvate:ferredoxin oxidoreductase and Fe–Fe hydrogenase, leading to produce molecular hydrogen [28, 29]. Several studies have shown targeting hydrogenosomes to disrupt their size and morphology, highlighting their potential as therapeutic strategy [30, 31, 32, 33]. Thus, in this study, we show that hydrogenosome dysfunction using alloferon leads to a decrease in proliferation. Our findings demonstrate that alloferon could be a novel therapeutic agent for the treatment of trichomoniasis.

Cell culture

We used T. vaginalis strain T106 was kindly provided by Prof. Jae Sook Ryu (Hanyang University). The strain was cultured in screw-capped glass tubes containing complex trypticase–yeast extract–maltose (TYM) medium. This medium consisted of trypticase peptone, yeast extract, maltose monohydrate, L-ascorbic acid, L-cysteine, phosphates (i.e., monopotassium, and dipotassium phosphates), horse serum, and antibiotics (penicillin 100 U/ml, 100 µg/ml of streptomycin; Welgene, Kyungsan, Korea) (Table 1). T. vaginalis was incubated at 37℃ in a 5% CO₂ chamber with loosely capped tubes for ventilation. Viability of T. vaginalis was assessed using trypan blue staining.

Table 1

Content of the TYM medium
Materials	Amount
Trypticase peptone	10.0 g
Yeast extract	5.0 g
Maltose monohydrate	2.5 g
L-Ascorbic acid	0.5 g
L-Cysteine	0.5 g
KH₂PO₄	0.5 g
K₂HPO₄	0.5 g
Horse serum	50 mL
Penicillin-Streptomycin	4 mL
Distilled water	Total 500 mL

Flow Cytometric Analysis

T. vaginalis were treated with 2 or 4 µg/ml alloferon and 30 µM deferoxamine (positive controls; Sigma, St. Louis, MO, USA) for 4 days. T. vaginalis were harvested and washed with PBS. Following treatment, cells were harvested, washed with PBS, resuspended in PBS, and seeded at 5 x 10⁵ cells/tube in FACS tubes. Hydrogenosomal membrane potential was measured by incubating T. vaginalis with DiOC₆ (40 nM in PBS; Sigma) for 15 min at 37℃ in FACS tube. To detect reactive oxygen species (ROS), T. vaginalis were incubated with DCFH-DA (10 µM in PBS; Sigma) for 15 min at room temperature. At least 5,000 cells per sample were gated and analyzed for fluorescent intensity using a FACSCaliburTM cytometer (BD Biosciences, San Jose, CA, USA). Data analysis was performed with FlowJo software (BD Biosciences, Franklin Lakes, NJ, USA).

Cell Cycle Analysis

T. vaginalis was treated with 2 or 4 µg/ml alloferon (with untreated parasites) for 4 days in TYM medium. Subsequently, cultured cells were harvested, washed with PBS, resuspended in PBS, and seeded at 1 x 10⁶ cell/tube in FACS tubes. After dropwise fixation in 70% ethanol overnight at -20℃, cells were washed with PBS and FACS buffer (PBS with 0.5% BSA and 0.1% sodium azide). Cell were stained with in 0.5 ml propidium iodide (PI)/RNase staining buffer (BD Pharmingen, San Diego, CA, USA) for 15 min at room temperature and analyzed by flow cytometry. Data were analyzed using FlowJo software (BD Biosciences) after acquisition with a FACSCaliburTM cytometer (BD Biosciences).

Transmission electron microscopy (TEM)

T. vaginalis were treated with or without 2 or 4 µg/ml alloferon and 30 µM deferoxamine for 4 days in TYM medium. Following collection and PBS washes, cells were fixed with 2.5% glutaraldehyde at 4℃, embedded, sectioned, stained with uranyl acetate and lead citrate, and visualized using a JEM-1400 transmission electron microscope (JEOL USA, Peabody, MA, USA).

Video Microscopy

To measure the minimum lethal concentration (MLC) of metronidazole against T. vaginalis, 4 x 10⁴ cells were seeded per well in a 96-well culture plate (150 µl/well). Cells were then treated with 100 µl of varying metronidazole concentrations (0.12–250 µg/ml; Sigma) and incubated at 37℃ overnight. Following treatment, cell viability was assessed using trypan blue staining, and their movement was observed at 200x magnification under a microscopy (EVOS M5000; Invitrogen, Carlsbad, CA, USA). To further confirm the effect of alloferon and its combination with metronidazole on motility, cells were seeded in 96-well plates and treated with 2 or 4 µg/ml alloferon or cotreatment of alloferon metronidazole (1.9 µg/ml), for 24 h. T. vaginalis motility was evaluated microscopically at 200X magnification, followed by a video recording of their movements.

Statistical Analysis

Data were presented as mean ± SD. For comparisons between groups, an unpaired two-tailed t-test was used. For comparisons of three or more groups, one-way analysis of variance (ANOVA) followed by Tukey multiple comparisons test after performed. Statistical significance was set at p < 0.05. Analyses were performed using GraphPad Prism 8.0.2 (GraphPad Software, La Jolla, CA, USA).

Alloferon disrupts the hydrogenosomal function by decreasing membrane potential in T. vaginalis.

Based on finding that alloferon regulates antioxidant protein [34], we investigated its potential effect on hydrogenosomal membrane potential and ROS level in T. vaginalis. The parasite was treated with 2 or 4 µg/ml alloferon in TMY medium for 4 days. The chosen alloferon concentration has been demonstrated to be highly effective in various cell types [20]. Deferoxamine (DFO), an iron chelator, is known to regulate hydrogenosomal membrane potential, morphology, and size in T. vaginalis [35, 36, 37]. We investigated the effects of alloferon treatment on these parameters both at short-term (early response) and long-term (considering T. vaginalis doubling time) intervals [38]. Despite variation in treatment duration and alloferon concentration, hydrogenosomal activity consistently decreased. Compared with the untreated parasites, 2 and 4 µg/ml alloferon treatment for one-day inhibited hydrogenosomal membrane potential by 11.4 ± 5.9%, and 21.0 ± 4.6%, respectively, whereas 2 and 4 µg/ml alloferon treatment for 4 days inhibited hydrogenosomal membrane potential by 18.7 ± 3.7%, and 24.2 ± 4.4%, respectively (Fig. 1A and B). Notably, alloferon treatment did not alter ROS levels (Fig. 1C and D). These results suggest that alloferon may reduce hydrogenosomal activity through mechanisms independent of ROS generation.

Alloferon induces alterations in the morphology of hydrogenosomes.

We examined the morphology of hydrogenosome in alloferon-treated T. vaginalis. Typically, these organelles are spherical or slightly elongated. However, exposure to drug like metronidazole, colchicine, and cytochalasin disrupts their morphology, leading to the formation of bizarre and enlarged structures [31, 32]. Interestingly, alloferon induced morphological changes in T. vaginalis hydrogenosome, characterized by the presence of enlarged organelles emergence of internal membranes. Additionally, the hydrogenosomal matrix appeared altered and tended to merge with each other. These morphological changes were observed alike in DFO-treated hydrogenosomes (Fig. 2). Therefore, our findings suggest that alloferon treatment induced structural alterations in the hydrogenosomes of T. vaginalis.

Alloferon inhibits parasite growth and induces cell cycle arrest.

Several studies have reported that hydrogenosome associated with the cell cycle. We investigated the cell cycle and measured cell proliferation following treatment of alloferon in T. vaginalis. Treatment with alloferon suppressed the growth of T. vaginalis, as evidenced by a decrease un cell number over time. Moreover, a high (4 µg/ml) alloferon exerted a more growth-inhibitory effect than a low dose (2 µg/ml) alloferon. The cell number, compared with the untreated parasites, decreased by 14.0 ± 1.1%, and 20.2 ± 5.7% in 2 and 4 µg/ml alloferon-treated cells, respectively (Fig. 3A). Alloferon-treated T. vaginalis exhibited cell cycle arrest in the S phase and decreased cell number in the G2 phase. The untreated parasites and alloferon-treated T. vaginalis (2 and 4 µg/ml) displayed S phase percentage of 6.5 ± 0.2%, 12.3 ± 0.8%, and 14.6 ± 1.4%, respectively, whereas percentage of cells in G2 phase showed 65.9 ± 2.1%, 55.4 ± 0.6%, and 47.2 ± 0.8%, respectively (Fig. 3B and C). These findings suggest that alloferon inhibits T. vaginalis proliferation by regulating cell cycle progression, with a specific effect on S and G2 phase. No significant changes were observed in the G0/G1 or Sub G1 phases.

Combined alloferon and metronidazole therapy shows a synergistic therapeutic effect against T. vaginalis.

To investigate the effect of combined alloferon and metronidazole therapy on T. vaginalis, we determined the MLC of metronidazole after a one-day exposure and evaluated its movement. The antiparasitic effect was observed at a minimum lethal concentration, 1.95 µg/ml metronidazole (Fig. 4A). T. vaginalis was treated with the combination of alloferon and metronidazole exhibited significantly reduced movement and proliferation compared to untreated parasites or those treating individual drug treatments (Fig. 4B). Therefore, our findings support the potential of combined alloferon and metronidazole therapy as an effective antiparasitic strategy against T. vaginalis.

Alloferon regulates antioxidant protein, leading to their downregulation and subsequent intracellular ROS generation [34]. Given alloferon ability to regulate redox-related proteins, we investigated the potential of alloferon as an antiparasitic agent. Many parasites are known to be susceptible to oxidative stress, making the generation of ROS or inhibition of endogenous antioxidant enzymes promising approaches for developing novel antiparasitic drugs [39]. While this study found no significant increase in ROS production by alloferon in T. vaginalis, it suggests a different mechanism of antiparasitic action: induction of hydrogenosomal dysfunction (Fig. 1A and B). These organelles, similar in size to mitochondria, act as redox organelles to generate energy for the cell. Targeting hydrogenosome metabolism, previously identified as a key antiparasitic strategy against T. vaginalis, may explain effect of alloferon despite the lack of ROS response [27, 40]. T. vaginalis relies on oxygen-sensitive enzymes like private:ferredoxin oxidoreductase (PFOR) and ferredoxin, along with malic enzyme, to generate ATP using substrates like pyruvate and malate. The malic enzyme converts malate to pyruvate through oxidative decarboxylation. Subsequently, PFOR oxidizes pyruvate, leading to ATP synthesis and the release of electrons transferred to ferredoxin [26, 28, 29]. Therefore, we investigated potential change in key hydrogenosomal enzymes. However, alloferon treatment did not alter the levels of these enzymes, suggesting an alternative mechanism for disruption of hydrogenosome function (Additional file1: Figure S1).

Hydrogenosome, key energy-producing organelles in T. vaginalis, are important drug targets. Metronidazole, the primary treatment for trichomoniasis, disrupts by inducing abnormal size and crista-like invaginations of the inner membrane [31, 40]. Treatment with alloferon triggered significant morphological changes in T. vaginalis hydrogenosomes, which were also characterized by the presence of enlarged structures, internal membrane, and abnormal shapes. Interestingly, a previous study reported duplicate structures are frequently observed during the S/G2 phase. As cells approach the premitotic phase, hydrogenosomes undergo morphological changes, including the formation of septal structures through internal membrane invagination and the emergence of new shapes [41]. Our analysis revealed that alloferon treatment altered the cell cycle of T. vaginalis, inducing an increase in the S phase and arrest in the premitotic G2 phase, suggesting potential associations between alloferon induced alterations and cell cycle progression (Fig. 3). This finding mirrored the reported effect of metronidazole, known to prolong the S phase due to DNA damage-induced repair mechanisms [42]. Therefore, our results confirm that alloferon exerts an inhibitory effect on T. vaginalis proliferation by regulating its cell cycle progression.

Currently used for treating T. vaginalis infection, metronidazole (a nitromidazole drug) was first introduced in 1959 and remains the standard treatment [43, 44]. However, problems regarding side effects and emerging resistance necessitate the development of alternative treatment options [45, 46, 47]. Combination therapy, using multiple drugs synergistically, offers potential solutions for improved efficacy, reduced side effects, and resistance prevention [48, 49]. Hence, we investigated the synergy effect of combination treatment with alloferon and metronidazole against T. vaginalis. While treatment with metronidazole alone (MLC) did not affect parasite movement, combining it with alloferon led to a significant decrease in motility (Fig. 4). According to previous reports, concentration of metronidazole reaches 10.62–24.88 µg/ml in serum after a 1,000 mg/day dose [50]. Therefore, our study suggests that the used metronidazole concentration in this study was sufficient to reduce side effects and resistance concerns while offering enhanced antiparasitic activity when combined with alloferon. This finding implies the potential of alloferon as valuable supplement to metronidazole based trichomoniasis therapy.

Taken together, our study demonstrates that alloferon exerts antiparasitic against T. vaginalis through multiple mechanisms, including (1) decreasing hydrogenosomal membrane potential, (2) altering hydrogenosome morphology, and (3) inducing cell cycle arrest in the S phase. Importantly, combining alloferon with metronidazole synergistically enhances its antiparasitic activity compared to metronidazole alone. These findings suggest the potential of alloferon as a valuable therapeutic agent for trichomoniasis.

STI

Sexually transmitted infection

T. vaginalis

Trichomonas vaginalis

HIV

Human immunodeficiency virus

ADIS

Acquired immunodeficiency syndrome

TYM

Trypticase–yeast extract–maltose

TEM

Transmission electron microscopy

MLC

Minimum lethal concentration

ANOVA

One-way analysis of variance

DFO

Deferoxamine

PFOR

Private:ferredoxin oxidoreductase.

Ethics approval and consent to participate:

Not applicable.

Consent for publication:

Not applicable.

Competing interests:

The authors declare that they have no competing interests.

Funding:

This work was supported by the National Research Foundation of Korea (No.: 2020R1C1C1009334).

Author Contribution

Conceptualization, J.S.K.; methodology, H.J., Y.K. and J.S.K; formal analysis, H.J. and Y.K.; investigation, H.J., S.S., T.A., H.A. and S.J.; resources, J.S.K.; data curation, H.J., Y.K. and J.S.K.; writing—original draft preparation, H.J. and Y.K.; visualization, H.J.; project administration, J.S.K.; funding acquisition, Y.K.

Acknowledgement

We thank all members of the Institute of Allergy and Clinical Immunology and N therapeutics for their scientific discussions and support.

Availability of data and materials:

The original data presented in the study are included in the article /supplementary material.

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No competing interests reported.

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Alloferon regulates the growth and movement of Trichomonas vaginalis by altering hydrogenosomes

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Abstract

Figures

Background

Methods

Cell culture

Flow Cytometric Analysis

Cell Cycle Analysis

Transmission electron microscopy (TEM)

Video Microscopy

Statistical Analysis

Results

Discussion

Conclusion

Abbreviations

Declarations

Ethics approval and consent to participate:

Funding:

Author Contribution

Acknowledgement

Availability of data and materials:

References

Additional Declarations

Supplementary Files

Status:

Version 1