Abstract
Members of the Naegleria genus are free-living amoebae, and the only pathogenic specie described to date for humans is N. fowleri. However, as the complete genome of this specie has not been reported, non-pathogenic N. gruberi is employed to describe molecular pathways in N. fowleri. Regardless, certain mechanisms, such as autophagy, have not yet been characterized in N. gruberi. Autophagy is involved in different cellular processes in some protozoa, including the recycling of unnecessary organelles, development, and cell differentiation. In this work, we characterized autophagy in N. gruberi using the specific inducer rapamycin. The formation of autophagy vacuoles in treated trophozoites was observed by ultrastructural analysis, and real time quantitative PCR demonstrated overexpression of the atg8 gene. In addition, we detected an increase in the vacuolar acidification of treated amoebae using the LysoTracker. Finally, confocal microscopy was utilized to identify Atg8 protein signal in the cytoplasm of N. gruberi trophozoites induced with rapamycin and even in trophozoites induced to encyst. In conclusion, N. gruberi possesses an Atg8 protein homolog that is overexpressed during the autophagic mechanism induced by rapamycin and also during encystation of this free-living amoeba.
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Acknowledgments
We are grateful to Angelica Silva-Olivares, Maria Luisa Bazán-Tejeda, and Gabriel Maldonado-Shibayama for their valuable technical assistance. This work was supported by grant number 237523 of the Mexican Council for Science and Technology (CONACyT).
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The animal management protocol was approved by the institutional committee (CICUAL, protocol number 0152–15). Our institution fulfills all the technical specifications for the production, care, and use of laboratory animals, which is certified by official national law NOM-062-ZOO-1999. The rabbit was euthanized by an overdose of sodium pentobarbital at the end of the experiments and handled according to the guidelines of the AVMA Guidelines for the Euthanasia of Animals (2013 Edition).
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Fig. S1
Rapamycin does not affect the viability and morphology of N. gruberi trophozoites. a Trypan blue exclusion assays were performed using N. gruberi trophozoites incubated with 5 μM rapamycin in free-serum medium for different times. Viability was unaffected under all conditions evaluated. The percentage of viability was determined by counting dead cells using light microscopy (N=3). b Light microscopy of rapamycin-treated cells showed the typical amoebic morphology at 48 h compared with the controls. Bars 20 μm. (GIF 171 kb)
Fig. S2
Intracellular acidification during autophagy in N. gruberi trophozoites induced with rapamycin. Trophozoites were treated with rapamycin for different times and compared with untreated amoebae. a Acidic cytoplasmic structures were detected with LysoTracker (Red), and nuclei were stained with DAPI (Blue); samples were analyzed by confocal microscopy. b The red signal was quantified, and the results are represented in the graphic as the fluorescence intensity for each treatment time. An increment in the acidification was observed in rapamycin-treated trophozoites at 24 and 48 h, where the signal was statistically significant. Bars 20 μm. **p ≤ 0.003 vs. Control group. (GIF 427 kb)
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Cárdenas-Zúñiga, R., Sánchez-Monroy, V., Bermúdez-Cruz, R.M. et al. Ubiquitin-like Atg8 protein is expressed during autophagy and the encystation process in Naegleria gruberi . Parasitol Res 116, 303–312 (2017). https://doi.org/10.1007/s00436-016-5293-x
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DOI: https://doi.org/10.1007/s00436-016-5293-x