title: Nitric oxide maintains cell survival of Trichomonas vaginalis upon iron depletion.
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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Kweishan, Taoyuan, Taiwan. m9701203@stmail.cgu.edu.tw.|Molecular Regulation and Bioinformatics Laboratory, Department of Parasitology, College of Medicine, Chang Gung University, Kweishan, Taoyuan, Taiwan. m9701203@stmail.cgu.edu.tw.
authors.forename: Wei-Hung
authors.initials: WH
authors.lastname: Cheng
authors.affiliation: Molecular Regulation and Bioinformatics Laboratory, Department of Parasitology, College of Medicine, Chang Gung University, Kweishan, Taoyuan, Taiwan. d9801201@mail.cgu.edu.tw.|Molecular Medicine Research Center, Chang Gung University, Kweishan, Taoyuan, Taiwan. d9801201@mail.cgu.edu.tw.
authors.forename: Kuo-Yang
authors.initials: KY
authors.lastname: Huang
authors.affiliation: Molecular Medicine Research Center, Chang Gung University, Kweishan, Taoyuan, Taiwan. pjhuang@mail.cgu.edu.tw.|Bioinformatics Center, Chang Gung University, Kweishan, Taoyuan, Taiwan. pjhuang@mail.cgu.edu.tw.
authors.forename: Po-Jung
authors.initials: PJ
authors.lastname: Huang
authors.affiliation: Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Kweishan, Taoyuan, Taiwan. johsuan_@hotmail.com.|Molecular Regulation and Bioinformatics Laboratory, Department of Parasitology, College of Medicine, Chang Gung University, Kweishan, Taoyuan, Taiwan. johsuan_@hotmail.com.
authors.forename: Jo-Hsuan
authors.initials: JH
authors.lastname: Hsu
authors.affiliation: Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Kweishan, Taoyuan, Taiwan. d9701204@stmail.cgu.edu.tw.|Molecular Regulation and Bioinformatics Laboratory, Department of Parasitology, College of Medicine, Chang Gung University, Kweishan, Taoyuan, Taiwan. d9701204@stmail.cgu.edu.tw.
authors.forename: Yi-Kai
authors.initials: YK
authors.lastname: Fang
authors.affiliation: Molecular Infectious Disease Research Center, Chang Gung Memorial Hospital, Kweishan, Taoyuan, Taiwan. chchiu@adm.cgmh.org.tw.
authors.forename: Cheng-Hsun
authors.initials: CH
authors.lastname: Chiu
authors.affiliation: Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Kweishan, Taoyuan, Taiwan. petang@mail.cgu.edu.tw.|Molecular Regulation and Bioinformatics Laboratory, Department of Parasitology, College of Medicine, Chang Gung University, Kweishan, Taoyuan, Taiwan. petang@mail.cgu.edu.tw.|Bioinformatics Center, Chang Gung University, Kweishan, Taoyuan, Taiwan. petang@mail.cgu.edu.tw.|Molecular Infectious Disease Research Center, Chang Gung Memorial Hospital, Kweishan, Taoyuan, Taiwan. petang@mail.cgu.edu.tw.
authors.forename: Petrus
authors.initials: P
authors.lastname: Tang
mesh_terms: D000222:Adaptation, Physiological; D000818:Animals; D001483:Base Sequence; D001562:Benzimidazoles; D002232:Carbocyanines; D002470:Cell Survival / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D000090463:Iron Deficiencies; D007976:Leupeptins / Q000494:pharmacology; D008954:Models, Biological; D009569:Nitric Oxide / Q000494:pharmacology*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors*; D015800:Protozoan Proteins / Q000037:antagonists & inhibitors; D017382:Reactive Oxygen Species / Q000494:pharmacology; D017423:Sequence Analysis, RNA; D059467:Transcriptome; D014245:Trichomonas Infections / Q000469:parasitology*; D014246:Trichomonas vaginalis / Q000187:drug effects* / Q000235:genetics / Q000502:physiology; D019323:omega-N-Methylarginine / Q000494:pharmacology
chemical_list: D001562:Benzimidazoles; D002232:Carbocyanines; D004791:Enzyme Inhibitors; D007976:Leupeptins; D015800:Protozoan Proteins; D017382:Reactive Oxygen Species; C068624:5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine; D019323:omega-N-Methylarginine; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde