|Year : 2021 | Volume
| Issue : 1 | Page : 1-8
Identification of autophagy-related protein 3 in the ancient protist Trichomonas vaginalis
Chang-Huei Tsao1, Hsin-An Lin2, Hsin-Chung Lin3, Ruei-Min Chen3, Chien-Fu F Chen4, Yu-Chun Lin5, Kuo-Yang Huang6
1 Department of Medical research, Tri-Service General Hospital; Department of Microbiology and Immunology, National Defense Medical Center, Taipei, Taiwan
2 Division of Infection, Department of Medicine, Tri-Service General Hospital, SongShan Branch, Taipei, Taiwan
3 Division of Clinical Pathology, Department of Pathology, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan
4 Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan
5 Department of Pathology, Tri-Service General Hospital; Graduate Institute of Pathology and Parasitology, National Defense Medical Center, Taipei, Taiwan
6 Graduate Institute of Pathology and Parasitology, National Defense Medical Center, Taipei, Taiwan
|Date of Submission||05-Feb-2020|
|Date of Decision||01-Jun-2020|
|Date of Acceptance||10-Jul-2020|
|Date of Web Publication||02-Sep-2020|
Dr. Kuo-Yang Huang
Graduate Institute of Pathology and Parasitology, National Defense Medical Center, No.161, Sec. 6, Minquan E. Road, Neihu Dist, Taipei City 114
Source of Support: None, Conflict of Interest: None
Background: Autophagy has been suggested to be involved in the pathogenesis of protists. While the molecular mechanisms of autophagy are mainly studied in model organisms, functional characterization of autophagy-related (Atg) proteins is poorly understood in deep-branching protists. Trichomoniasis is the most common nonviral sexually transmitted infection caused by Trichomonas vaginalis. Bioinformatics analysis of the T. vaginalis genome reveals that the parasite possesses the genes encoding proteins of the Atg8 conjugation system. Herein, we sought to characterize whether the T. vaginalis Atg3 ortholog (TVAG_447140), a putative component of the TvAtg8 conjugation system, regulates autophagy in this parasite. Methods: The recombinant protein of T. vaginalis Atg3 ortholog (TvAtg3) (rTvAtg3) and the polyclonal antibody against rTvAtg3 were generated. The expression and localization was monitored upon autophagy induction by glucose restriction (GR) compared with glucose-rich cultivation. The role of TvAtg3 in autophagy was clarified using small interfering RNA targeting TvAtg3 gene. Results: Phylogenic analysis of Atg3 proteins from different organisms showed that T. vaginalis was not in a close evolutionary relationship with any other protozoan. The expression of TvAtg3 was upregulated in the late-stationary phase of GR culture, implying its involvement in autophagy. Immunofluorescence analysis revealed a much higher TvAtg3 fluorescent intensity located on the round and/or linear structures close to the nucleus. Silencing Tvatg3 expression suppressed GR-induced TvAtg8 expression and autophagic vacuoles formation. Conclusions: These findings suggest the potential role of TvAtg3 in T. vaginalis autophagy and enhance our understanding of the autophagy regulatory network in the deep-branching eukaryotes.
Keywords: Trichomonas vaginalis, autophagy, autophagy-related protein 3
|How to cite this article:|
Tsao CH, Lin HA, Lin HC, Chen RM, Chen CFF, Lin YC, Huang KY. Identification of autophagy-related protein 3 in the ancient protist Trichomonas vaginalis. J Med Sci 2021;41:1-8
|How to cite this URL:|
Tsao CH, Lin HA, Lin HC, Chen RM, Chen CFF, Lin YC, Huang KY. Identification of autophagy-related protein 3 in the ancient protist Trichomonas vaginalis. J Med Sci [serial online] 2021 [cited 2021 Feb 28];41:1-8. Available from: https://www.jmedscindmc.com/text.asp?2021/41/1/1/294292
| Introduction|| |
Autophagy is one of two major bulk protein degradation systems and is widely conserved in eukaryotes. Increasing evidence suggests that autophagy has many physiological functions, including development and differentiation, immunity, and regulation of lifespan. Starvation-induced autophagy is commonly considered as a cell survival mechanism in response to nutrient deprivation., The core step of autophagic process is the formation of autophagosomes, which is mediated by the autophagy-related protein 8 (Atg8) and Atg5/Atg12 conjugation systems. Upon autophagy induction, the cytosolic form of Atg8 is cleaved by the protease Atg4, conjugated to Atg7 and Atg3, and finally formed an amide bond with phosphatidylethanolamine, the lipidated form of Atg8 on the autophagosomal membrane. Hence, Atg8 is widely used as a reliable marker of autophagosomes during autophagosome formation.
It has been shown that autophagy may contribute to the pathogenesis of several protists, For example, autophagy correlates with the development of Trypanosoma cruzi and plays a role in differentiation in Leishmania major, Acanthamoeba castellamii, and Entamoeba invadens. It remains controversial whether autophagy is a survival response to death stimuli or an alternative form of programmed cell death., Autophagy has been proposed as a cell death mechanism in Blastocystis hominis, Trypanosoma brucei, Toxoplasma gondii, and Plasmodium berghei. In contrast, other studies demonstrated that the components of the Atg8 conjugation system in Plasmodium falciparum and T. gondii are essential for survival., Recently, Atg8 has been proved to be involved in endosomal and phagosomal acidification in Entamoeba histolytica. It is noteworthy that only a limited number of Atg orthologs were identified in several protists, drawing our attention to the noncanonical autophagic machinery of these ancient eukaryotes.,
Trichomonas vaginalis is a unicellular protozoan parasite that causes human trichomoniasis and annually infects approximately 276 million people worldwide. T. vaginalis colonizes the urogenital tract of humans and leads to serious consequences for women's health, including vaginitis, preterm delivery, infertility, low birth weight infants, and susceptibility to cervical cancer. The complications of trichomoniasis coupled with the enhanced risk factor for HIV transmission and lethal prostate cancer. A genome survey of T. vaginalis reveals that the parasite possesses the genes encoding proteins of the Atg8 conjugation system but lacks those encoding Atg12-Atg5,,, suggesting that the Atg8 conjugation system may be the minimal machinery for autophagosome formation in trichomonads. While the identification and functional characterization of most Atg genes are widely studied in a number of model organisms, the autophagic machinery and its biological significance remain poorly understood in trichomonads.
Glucose restriction (GR) has been shown to induce an autophagy-like response in T. vaginalis, providing a great platform for the exploration of autophagy in this parasite. In the present study, we aim to clarify whether the T. vaginalis Atg3 ortholog (TvAtg3) may participate in the process of autophagy. We have examined the expression and localization of TvAtg3 upon autophagy induction by GR compared with glucose-rich condition. Furthermore, the role of TvAtg3 in modulation of autophagy was validated. These results not only proved that TvAtg3 is a key player in autophagosome formation, but also paved the way for future investigations on the regulatory network of autophagy in trichomonads.
| Methods|| |
Parasites and culture conditions
T. vaginalis isolate (ATCC30236) was grown in YIS medium, pH 5.8, supplemented with 10% heat-inactivated horse serum and 1% glucose (high-glucose medium, HGM) at 37°C. For glucose-restricted (GR) cultivation, trophozoites were maintained in the same medium without glucose supplement at 37°C as previously described.
Sequences and phylogenetic analysis
Atg3 protein sequences of different organisms were downloaded from the NCBI protein database (http://www.ncbi.nlm.nih.gov/protein). The TvAtg3 was identified as previously described and its protein sequence was retrieved from TrichDB. To obtain the sequence identity of Atg3 orthologs from various organisms, the proteins sequences were aligned using ClustalW multiple alignment tool in the local BioEdit sequence alignment editor. A phylogenetic tree was constructed by the T-REX web server using the neighbor joining method, with the reliability testing of each branch by 100 bootstrap replications.
Autophagic vacuoles and lysosomes detection
AVs and lysosomes were monitored as previously described. Briefly, 1 × 10 trophozoites were co-stained with Cyto-ID™ (1 mL of 1X Assay Buffer containing 2 μl of Cyto-ID™ Green Detection Reagent, 100 μl/sample) (Enzol) and 1 μM Lysotracker Red DND-99 (Invitrogen) for 30 min at 37°C. Ten μl of the cell suspension was applied to POC-R chamber (Zeiss). Fluorescent images were obtained using confocal microscopy (Zeiss LSM510). Standard GFP and RFP filter sets were used for imaging the autophagic and lysosomal signals, respectively. Images were analyzed with LSM 510 software.
Production of the polyclonal antibody against rTvAtg3
The recombinant TvAtg3 (TVAG_447140) protein was generated by pTrcHis and pTrcHis2 TOPO® TA expression Kit (Invitrogen) according to the manufacturer's instructions. Briefly, the full-length TvAtg3 coding sequence (786 bp) was amplified and cloned into the pTrcHis vector to produce the recombinant plasmid construct, followed by sequencing with pTrcHis forward and reverse primers (Tri-I Biotect). The construct was then introduced into Escherichia More Details coli (TOP10) for the expression of TvAtg3 recombinant protein. After induction with 1 mM isopropyl-β-D-thiogalactopyranoside, the expression of histidine (HIS)-tagged recombinant protein was analyzed by 15% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and Western blotting using mouse anti-His antibody (GeneTex). The recombinant protein was purified by His-bound resin (Novagen) column chromatography. Rabbit polyclonal antiserum against the recombinant protein was commercially produced (AbKing), followed by purification using NHS Mag Sepharose (GE healthcare) conjugated with His-tagged recombinant proteins.
Western blot analysis
Trophozoites (1 × 10 cells) were harvested and lysed in 100 μl SDS sample buffer (125 mM Tris-HCl, 1% SDS, 20% glycerol, 0.05% bromophenol blue, and 5% 2-mercaptoethanol), followed by heating at 95°C for 5 min. Whole cell lysates were separated on 15% SDS-PAGE and the gels were transferred to nitrocellulose membrane (GE Healthcare) using a semi-dry transfer unit (Bio-Rad) under 15 V for 1 h. The membranes were blocked with 5% skim milk in TTBS buffer (Tris-buffered saline containing 0.1% Tween 20) for 1 h. The membranes were incubated with the primary antibodies in blocking buffer at 4°C overnight. The following primary antibodies were used: mouse anti-His (1: 2000 dilution); rabbit anti-TvAtg3 (1: 2000 dilution); and rabbit anti-GAPDH (Abcam) (1: 2000 dilution) antibodies. After being washed with TTBS for three times, the bound antibody was detected by anti-mouse or anti-rabbit IgG secondary antibodies (GeneTex) (1:5000 dilution) in blocking buffer for 1 h. Stable peroxide solution and enhanced solution (1:1) (Thermo Scientific) were added on the membranes. The signals of protein bands were visualized and quantified using Biospectrum Imaging System (UVP) and Image J, respectively. The signal of GAPDH was used as a loading control for Western blotting.
Indirect immunofluorescence assay
Trophozoites were fixed onto microscopic slides with 4% formaldehyde in phosphate-buffered saline (PBS) for 20 min at room temperature (RT). Cells were washed with PBS and permeabilized with 0.1% Triton X-100/PBS for 10 min at RT and then blocked with 3% bovine serum albumin (BSA)/PBS for 1 h at RT. Cells were incubated with anti-TvAtg3 antibody (1:500 dilution) in 3% BSA-0.1% Triton X-100/PBS for 1 h at RT. After being washed with PBS for three times, cells were incubated with Alexa fluor 488 or 594 anti-rabbit IgG antibody (1:500 dilution) (Invitrogen) for 1 h at RT. Cells were washed and stained with 4',6-diamidino-2-phenylindole to label the nuclei (0.1 μg/ml, Sigma) for 15 min. Slides were mounted with the mounting medium (0.17 M KHCO3, 50% glycerol) and examined under a confocal laser scanning microscopy (Zeiss LSM510).
Small interfering RNA transfection
Log phase trophozoites (1 × 10 cells) grown in GR were harvested and then transfected with 30 nM of negative control small interfering RNA (siRNA) (Biotools) or siRNAs targeting TvAtg3 gene using Lipofectamine™ RNAiMax (Invitrogen). The sequences of siRNA targeting TvAtg3 were as follows:
si-TvAtg3#1, 5'-GCUGCAGGCGA UUGUUUAATT-3'(sense) and 5'UUAAACAAUCGCCUGCAGCTT-3' (anti- sense);
si-TvAtg3#2, 5'-CCCACUUACAUUAGAACAATT-3' (sense) and 5'-UUGUUCUAAUGUAAGUGGGTT-3' (anti- sense);
si-TvAtg3#3, 5'-GCAACAAAGAACCUUUCUATT-3' (sense) and 5'UAGAAAGGUUCUUUGU UGCTT-3' (anti-sense). After incubation for 4 h at 37 °C, the trophozoites (2 × 10 cells) containing siRNA complex were inoculated into 10 ml of fresh GR medium and cultured for 24 h. Whole cell lysates were extracted from each transfected group 24 h posttransfection and the knockdown efficiency of siRNAs was evaluated by Western blotting.
Quantitative data were expressed as mean ± standard error of the mean of three independent experiments unless otherwise indicated. Student's t-test (two-tailed) was used to evaluate the significant differences between groups. P < 0.05 was considered statistically significant.
| Results|| |
In silico analysis of the Atg3 ortholog in Trichomonas vaginalis
We previously identified the ortholog of Atg3 in the T. vaginalis genome (TVAG_447140), which contained the conserved functional domains for Atg3 [Figure 1]a. Amino acid sequences of Atg3 of different organisms were aligned using Clustal W to indicate regions of similarity [Figure 1]b. A previous study reported that Atg3 directly interacts with Atg8 through the WEDL sequence in Saccharomyces cerevisiae. However, the Atg3 orthologs in T. vaginalis and other protozoans do not possess the WEDL sequence, suggesting that there is a unique mechanism mediating the interaction between Atg3 and Atg8 in protists. We further analyzed the amino acid sequence identity of TvAtg3 to their corresponding proteins in yeast, mammals, and other protozoan parasites [Figure 1]c. TvAtg3 showed less than 30% sequence identity to those of the model organisms and other protists. Additionally, a phylogenetic tree constructed by a set of Atg3 proteins demonstrated that T. vaginalis was not in a close evolutionary relationship with any other protozoan [Figure 1]d. These results suggest that T. vaginalis may possess distinct autophagic machinery from other protists and experimental characterization of TvAtg3 is required to unveil its possible autophagy-related function or additional functions beyond autophagy.
|Figure 1: Sequence identities and phylogenic analysis of TvAtg3 to their orthologs in other organisms. (a) Functional domain analysis of TvAtg3. (b) The sequence identities of TvAtg3 to their orthologs were analyzed using the BioEdit sequence alignment editor. (c) The sequence identities of Atg3 were presented as a heat map. (d) Phylogenetic trees were constructed by the T-REX web server using the neighbor joining method. Tg = Toxoplasma gondii; Pf = Plasmodium falciparum; Lm = Leishmania major; Eh = Entamoeba histolytica; Ei = Entamoeba invadens; Tb = Trypanosoma brucei; Tc = Trypanosoma cruzi; Sc = Saccharomyces cerevisiae; Ce = Caenorhabditis elegans; Dm = Drosophila melanogaster; Mm = Mus musculus; Hs = Homo sapiens|
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The expression and localization of Trichomonas vaginalis Atg3 ortholog under high-glucose medium and glucose-restricted conditions
Atg3 is generally considered essential for Atg8 conjugation to the autophagosomal membrane. To investigate whether TvAtg3 participates in the autophagic process, the TvAtg3 coding sequence was cloned into an expression vector and the recombinant protein (rTvAtg3) was generated via a prokaryotic expression system [Figure 2]a and [Figure 2]b. The raised TvAtg3 polyclonal antibody was tested to recognize rTvAtg3, exhibiting great specificity of the interaction [Figure 2]c. The expression levels of TvAtg3 were examined under GR compared with HGM, which represent autophagy-inducing and non-inducing conditions, respectively. TvAtg3 was upregulated (1.26 fold) in the late-stationary phase of GR culture (GR-48 h) [Figure 2]d. In addition, GR-cultured cells treated with the autophagy inhibitor wortmannin (Wort) suppressed the expression of TvAtg3 in the early-stationary phase (GR-24 h) [Figure 2]e. These results suggest the involvement of TvAtg3 in GR-induced autophagy.
|Figure 2: The expression of TvAtg3 in HG and glucose-restricted cultivation. (a) The coding sequence of TvAtg3 was cloned into pTrcHis vector, which was introduced into E. coli for protein expression. The expression of recombinant protein was induced by isopropyl-β-D-thiogalactopyranoside for 1, 2, and 3 h. The His-tagged recombinant protein was separated by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analyzed by Western blotting using anti-His antibody. (b) The recombinant TvAtg3 protein was purified by His-bound resin column chromatography and analyzed by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Lane Ct, control recombinant protein; Lane S, soluble fraction; Lane FT, flow-through fraction; Lane W, wash fraction. The numbers represent each fraction collected from elution. (c) The raised TvAtg3 polyclonal antibody (α-TvAtg3) was used to recognize the recombinant protein (rTvAtg3) for determination of the specificity. (d) The protein expression of TvAtg3 in high-glucose medium and glucose-restricted cultivation. Cell lysates collected from the high-glucose medium and glucose-restricted cultures with different incubation time were fractionated by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the expression of TvAtg3 was analyzed by Western blotting analysis using anti-TvAtg3 antibody. (e) The protein expression of TvAtg3 in glucose-restricted-cultured cells treated with Wort. Trophozoites cultured under glucose-restricted (24 and 48 h) were treated with 100 μM Wort and the expression of TvAtg3 was determined compared with that of the DMSO-treated control (Ct). The expression of GAPDH was used as an internal for Western blotting. Quantitation of TvAtg3 expression was presented as a fold change to GAPDH|
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The localization of Trichomonas vaginalis Atg3 ortholog under high-glucose medium and glucose-restricted conditions
Immunofluorescence analysis revealed a much higher TvAtg3 fluorescent intensity expressed on the round and/or linear structures close to the nucleus of the GR-and HGM-cultured cells [Figure 3]a, the position resembling the Golgi apparatus of the parasite. Consistent with the Western blot analysis, there was a significant decrease in TvAtg3 fluorescent intensity in GR-cultured cells treated with Wort [Figure 3]b. In silico analysis by Euk-mPLoc 2.0 (http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/) suggests that TvAtg3 may localize in the cytoplasm; however, the precise localization of TvAtg3 needs further confirmation.
|Figure 3: Localization of TvAtg3 in high-glucose medium and glucose-restricted cultivation. (a) Subcellular localization of TvAtg3 in high-glucose medium-and glucose-restricted-cultured cells. The distribution of TvAtg3 in high-glucose medium and glucose-restricted-cultivated cells (24 h) was detected using anti-TvAtg3 antibody followed by confocal microscopy analysis. Scale bar = 5 μm. NC: the negative control probed only with a secondary antibody. (b) Localization of TvAtg3 after autophagy inhibition by Wort. Glucose-restricted-cultivated cells (24 h) were treated with Wort (50 and 100 μM) for 3 h and the distribution of TvAtg3 was monitored compared with that of the DMSO-treated control (Ctrl)|
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Trichomonas vaginalis Atg3 ortholog is required for autophagy induced by glucose restriction
To further ascertain the role of TvAtg3 in autophagosome formation, GR-cultured cells were transfected with siRNAs targeting TvAtg3 and the autophagic status was determined compared with that of the control cells transfected with a non-targeting siRNA. Among the three siRNAs tested, 24 h post transfection with si-TvAtg3 #3 showed a significant reduction in TvAtg3 protein expression [Figure 4]a. Moreover, knockdown of TvAtg3 expression suppressed the accumulation of TvAtg8 [Figure 4]a and formation of AVs (the AVs per cell for the control group and cells transfected with si-TvAtg3 #3 decreased from 4.85 ± 0.33 to 2.55 ± 0.32, P < 0.001) in GR-cultured cells [Figure 4]b, supporting that TvAtg3 is essential for autophagosome formation.
|Figure 4: TvAtg3 is required for autophagy. (a) Knockdown of TvAtg3 expression by specific siRNAs. Glucose-restricted-cultured cells were transfected with siRNAs (si-TvAtg3 #1, #2 and #3) targeting TvAtg3 or with a non-targeting siRNA as a negative control (Ct). The expression levels of TvAtg3 and TvAtg8 were analyzed and quantified at 24 h post transfection. (b) The effect of TvAtg3 knockdown on the formation of autophagic vacuoles. TvAtg3-knockdown cells (si-TvAtg3 #3) were costained with the autophagic green fluorescent dye and Lysotracker Red, and the formation of autophagic vacuoles and lysosomes was detected compared with that in the control group (si-Ctrl). Each dot represents one cell. Quantitation of autophagic vacuoles in TvAtg3-knockdown cells compared with that in the control group. The average number of autophagic vacuoles was determined by counting the autophagic green fluorescent signals in cells in different microscopic fields (30 cells, 10 cells/group). **P < 0.01|
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| Discussion|| |
Multiple membrane sources may be implicated in autophagosome formation, including endoplasmic reticulum (ER), mitochondria, and Golgi. It has been shown that mammalian ER is interconnected with the phagophores, the small cup-shaped membrane precursor formed upon autophagy induction. In addition, lipid delivery from mitochondria to newly forming autophagosomes in starved cells was observed, suggesting that mitochondria supply membranes for autophagosome formation. Another study in S. cerevisiae reported that two post-Golgi Sec proteins may redirect Golgi-derived membrane to the phagophore assembly site for autophagosome formation upon starvation. It is not clear what the membrane sources and key proteins are for autophagosome formation in protists during autophagy. The unique localization of TvAtg3 provided a clue for further investigations into the detailed mechanism of membrane sources for autophagosome formation.
The role (s) of Atg3 in protists has been studied in Acanthamoeba castellanii, T. gondii and T. brucei. A. castellanii Atg3 has been shown to mediate Atg8 lipidation and regulate cyst formation. In addition to the function participating in TgAtg8 conjugation to autophagosomes, TgAtg3 is critical for maintaining mitochondrial integrity and intracellular development of the tachyzoites. A recent study also reported that Atg3 conjugation to Atg12 regulates mitochondrial homeostasis and cell death in mammalian cells, suggesting the pleiotropic roles for Atg3 beyond autophagy. In procyclic T. brucei, depletion of TbAtg3 delays cell death under starvation, suggesting the involvement of TbAtg3 in cell death. The dual roles of Atg3 in determining the cell fates of these parasites reveal that autophagy can serve as a pro-survival or pro-death mechanism in different protists. Further characterization of TvAtg3-associated complex will not only establish parasite-specific autophagy networks but unravel novel role (s) of TvAtg3 in trichomonads.
| Conclusions|| |
Collectively, this is the first report to functionally characterize the component of the Atg8 conjugation system in trichomonads. We verified that TvAtg3 is involved in autophagosome formation induced by GR. The localization of TvAtg3 highlights the distinct regulatory network for autophagosome formation. It remains to be determined whether TvAtg3 interacts with TvAtg8 or other TvAtg proteins to mediate non-selective or selective autophagy in T. vaginalis.
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Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]