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Nitric oxide maintains cell survival of Trichomonas vaginalis upon iron depletion.

Cheng WH, Huang KY, Huang PJ, Hsu JH, Fang YK, Chiu CH, Tang P - Parasit Vectors (2015)

Bottom Line: The free radical signaling molecules reactive oxygen species (ROS) and reactive nitrogen species (RNS) have been proven to participate in iron deficiency in eukaryotes.We found that the inhibition of proteasome activity shortened the survival of iron-deficient cells compared with untreated iron-deficient cells.Surprisingly, the addition of arginine restored both NO level and the survival of proteasome-inhibited cells, suggesting that proteasome-derived NO is crucial for cell survival under iron-limited conditions.

View Article: PubMed Central - PubMed

Affiliation: Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Kweishan, Taoyuan, Taiwan. m9701203@stmail.cgu.edu.tw.

ABSTRACT

Background: Iron plays a pivotal role in the pathogenesis of Trichomonas vaginalis, the causative agent of highly prevalent human trichomoniasis. T. vaginalis resides in the vaginal region, where the iron concentration is constantly changing. Hence, T. vaginalis must adapt to variations in iron availability to establish and maintain an infection. The free radical signaling molecules reactive oxygen species (ROS) and reactive nitrogen species (RNS) have been proven to participate in iron deficiency in eukaryotes. However, little is known about the roles of these molecules in iron-deficient T. vaginalis.

Methods: T. vaginalis cultured in iron-rich and -deficient conditions were collected for all experiments in this study. Next generation RNA sequencing was conducted to investigate the impact of iron on transcriptome of T. vaginalis. The cell viabilities were monitored after the trophozoites treated with the inhibitors of nitric oxide (NO) synthase (L-NG-monomethyl arginine, L-NMMA) and proteasome (MG132). Hydrogenosomal membrane potential was measured using JC-1 staining.

Results: We demonstrated that NO rather than ROS accumulates in iron-deficient T. vaginalis. The level of NO was blocked by MG132 and L-NMMA, indicating that NO production is through a proteasome and arginine dependent pathway. We found that the inhibition of proteasome activity shortened the survival of iron-deficient cells compared with untreated iron-deficient cells. Surprisingly, the addition of arginine restored both NO level and the survival of proteasome-inhibited cells, suggesting that proteasome-derived NO is crucial for cell survival under iron-limited conditions. Additionally, NO maintains the hydrogenosomal membrane potential, a determinant for cell survival, emphasizing the cytoprotective effect of NO on iron-deficient T. vaginalis. Collectively, we determined that NO produced by the proteasome prolonged the survival of iron-deficient T. vaginalis via maintenance of the hydrogenosomal functions.

Conclusion: The findings in this study provide a novel role of NO in adaptation to iron-deficient stress in T. vaginalis and shed light on a potential therapeutic strategy for trichomoniasis.

No MeSH data available.


Related in: MedlinePlus

NO maintains the hydrogenosomal membrane potential of iron-deficient T. vaginalis.a The histograms indicate the red signal of the cells treated with DIP (180 μM), DIP-MG132 (10 μM), and DIP-MG132-arginine (5 mM). Red fluorescence represents a hydrogenosome with high membrane potential. The red fluorescence intensities of negative control group (CCCP treated cells, 50 μM) and experimental groups were detected by using flow cytometry after JC-1 staining. b Quantification data of the red fluorescence intensities (geometric mean) in panel a are shown. All tested groups were collected 6 h after drug treatments, and the data are presented as the mean ± SD of three independent experiments. *(p < 0.05) and **(p < 0.01), based on the differences between the indicated groups
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Fig5: NO maintains the hydrogenosomal membrane potential of iron-deficient T. vaginalis.a The histograms indicate the red signal of the cells treated with DIP (180 μM), DIP-MG132 (10 μM), and DIP-MG132-arginine (5 mM). Red fluorescence represents a hydrogenosome with high membrane potential. The red fluorescence intensities of negative control group (CCCP treated cells, 50 μM) and experimental groups were detected by using flow cytometry after JC-1 staining. b Quantification data of the red fluorescence intensities (geometric mean) in panel a are shown. All tested groups were collected 6 h after drug treatments, and the data are presented as the mean ± SD of three independent experiments. *(p < 0.05) and **(p < 0.01), based on the differences between the indicated groups

Mentions: Previous studies have proven that NO activates cGMP signaling to modulate mitochondrial biogenesis and activity in mammals [34]. Mitochondrial membrane potential is believed to be a determinant of metabolic activity and health status of a cell [35]. We utilized a cell-permeable dye JC-1 to monitor the hydrogenosomal membrane potential of T. vaginalis, which reflects the hydrogenosomal functions [17, 26]. Using JC-1 staining, we monitored the fluctuations in the red fluorescence-containing cells, which possessed hydrogenosomes with high membrane potential, in different iron concentrations. We used the cells treated with CCCP, a disruptor of mitochondrial membrane potential, as a negative control. As shown in Fig. 5a and b, the fluorescence intensities were reduced in the CCCP-treated cells, indicating that the measurement is suitable for analysis of hydrogenosomal membrane potential. We found that the red fluorescence intensity in MG132 treated iron-deficient cells was decreased significantly (p < 0.01) compared with untreated cells (Fig. 5b). Interestingly, the MG132-mediated reduction in red signal can be recovered by the addition of arginine (Fig. 5b), suggesting that NO maintains the hydrogenosomal membrane potential in iron-deficient T. vaginalis. These observations indicate that the hydrogenosomal membrane potential of MG132-treated cells can be reversed after co-treatment with arginine, confirming that NO functions in hydrogenosomal membrane potential maintenance. These data demonstrate that NO serves as a “keeper” for maintaining the functions of the hydrogenosome, which is positively correlated with prolonged survival in iron-deficient T. vaginalis.Fig. 5


Nitric oxide maintains cell survival of Trichomonas vaginalis upon iron depletion.

Cheng WH, Huang KY, Huang PJ, Hsu JH, Fang YK, Chiu CH, Tang P - Parasit Vectors (2015)

NO maintains the hydrogenosomal membrane potential of iron-deficient T. vaginalis.a The histograms indicate the red signal of the cells treated with DIP (180 μM), DIP-MG132 (10 μM), and DIP-MG132-arginine (5 mM). Red fluorescence represents a hydrogenosome with high membrane potential. The red fluorescence intensities of negative control group (CCCP treated cells, 50 μM) and experimental groups were detected by using flow cytometry after JC-1 staining. b Quantification data of the red fluorescence intensities (geometric mean) in panel a are shown. All tested groups were collected 6 h after drug treatments, and the data are presented as the mean ± SD of three independent experiments. *(p < 0.05) and **(p < 0.01), based on the differences between the indicated groups
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4513698&req=5

Fig5: NO maintains the hydrogenosomal membrane potential of iron-deficient T. vaginalis.a The histograms indicate the red signal of the cells treated with DIP (180 μM), DIP-MG132 (10 μM), and DIP-MG132-arginine (5 mM). Red fluorescence represents a hydrogenosome with high membrane potential. The red fluorescence intensities of negative control group (CCCP treated cells, 50 μM) and experimental groups were detected by using flow cytometry after JC-1 staining. b Quantification data of the red fluorescence intensities (geometric mean) in panel a are shown. All tested groups were collected 6 h after drug treatments, and the data are presented as the mean ± SD of three independent experiments. *(p < 0.05) and **(p < 0.01), based on the differences between the indicated groups
Mentions: Previous studies have proven that NO activates cGMP signaling to modulate mitochondrial biogenesis and activity in mammals [34]. Mitochondrial membrane potential is believed to be a determinant of metabolic activity and health status of a cell [35]. We utilized a cell-permeable dye JC-1 to monitor the hydrogenosomal membrane potential of T. vaginalis, which reflects the hydrogenosomal functions [17, 26]. Using JC-1 staining, we monitored the fluctuations in the red fluorescence-containing cells, which possessed hydrogenosomes with high membrane potential, in different iron concentrations. We used the cells treated with CCCP, a disruptor of mitochondrial membrane potential, as a negative control. As shown in Fig. 5a and b, the fluorescence intensities were reduced in the CCCP-treated cells, indicating that the measurement is suitable for analysis of hydrogenosomal membrane potential. We found that the red fluorescence intensity in MG132 treated iron-deficient cells was decreased significantly (p < 0.01) compared with untreated cells (Fig. 5b). Interestingly, the MG132-mediated reduction in red signal can be recovered by the addition of arginine (Fig. 5b), suggesting that NO maintains the hydrogenosomal membrane potential in iron-deficient T. vaginalis. These observations indicate that the hydrogenosomal membrane potential of MG132-treated cells can be reversed after co-treatment with arginine, confirming that NO functions in hydrogenosomal membrane potential maintenance. These data demonstrate that NO serves as a “keeper” for maintaining the functions of the hydrogenosome, which is positively correlated with prolonged survival in iron-deficient T. vaginalis.Fig. 5

Bottom Line: The free radical signaling molecules reactive oxygen species (ROS) and reactive nitrogen species (RNS) have been proven to participate in iron deficiency in eukaryotes.We found that the inhibition of proteasome activity shortened the survival of iron-deficient cells compared with untreated iron-deficient cells.Surprisingly, the addition of arginine restored both NO level and the survival of proteasome-inhibited cells, suggesting that proteasome-derived NO is crucial for cell survival under iron-limited conditions.

View Article: PubMed Central - PubMed

Affiliation: Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Kweishan, Taoyuan, Taiwan. m9701203@stmail.cgu.edu.tw.

ABSTRACT

Background: Iron plays a pivotal role in the pathogenesis of Trichomonas vaginalis, the causative agent of highly prevalent human trichomoniasis. T. vaginalis resides in the vaginal region, where the iron concentration is constantly changing. Hence, T. vaginalis must adapt to variations in iron availability to establish and maintain an infection. The free radical signaling molecules reactive oxygen species (ROS) and reactive nitrogen species (RNS) have been proven to participate in iron deficiency in eukaryotes. However, little is known about the roles of these molecules in iron-deficient T. vaginalis.

Methods: T. vaginalis cultured in iron-rich and -deficient conditions were collected for all experiments in this study. Next generation RNA sequencing was conducted to investigate the impact of iron on transcriptome of T. vaginalis. The cell viabilities were monitored after the trophozoites treated with the inhibitors of nitric oxide (NO) synthase (L-NG-monomethyl arginine, L-NMMA) and proteasome (MG132). Hydrogenosomal membrane potential was measured using JC-1 staining.

Results: We demonstrated that NO rather than ROS accumulates in iron-deficient T. vaginalis. The level of NO was blocked by MG132 and L-NMMA, indicating that NO production is through a proteasome and arginine dependent pathway. We found that the inhibition of proteasome activity shortened the survival of iron-deficient cells compared with untreated iron-deficient cells. Surprisingly, the addition of arginine restored both NO level and the survival of proteasome-inhibited cells, suggesting that proteasome-derived NO is crucial for cell survival under iron-limited conditions. Additionally, NO maintains the hydrogenosomal membrane potential, a determinant for cell survival, emphasizing the cytoprotective effect of NO on iron-deficient T. vaginalis. Collectively, we determined that NO produced by the proteasome prolonged the survival of iron-deficient T. vaginalis via maintenance of the hydrogenosomal functions.

Conclusion: The findings in this study provide a novel role of NO in adaptation to iron-deficient stress in T. vaginalis and shed light on a potential therapeutic strategy for trichomoniasis.

No MeSH data available.


Related in: MedlinePlus