Abstract
Toxoplasma gondii, the etiological agent of toxoplasmosis, infects nucleated cells and then resides and multiplies within a parasitophorous vacuole. For this purpose, the parasite secretes many virulence factors for the purpose of invading and subverting the host microbicidal defenses in order to facilitate its survival in the intracellular milieu. Essential metals are structural components of proteins and enzymes or cofactors of enzymatic reactions responsible for these parasitic survival mechanisms. However, an excess of non-essential or essential metals can lead to parasite death. Thus, infected host cells were incubated with 20 μM ZnCl2 in conjunction with 3 μM CdCl2 or HgCl2 for 12 h in order to investigate cellular events and organelle damage related to intracellular parasite death and elimination. In the presence of these metals, the tachyzoites undergo lipid uptake and transport impairment, functional and structural mitochondrial disorders, DNA condensation, and acidification of the parasitophorous vacuole, thus leading to parasite death. Additional research has suggested that lysosome-vacuole fusion was involved in parasite elimination since acid phosphatases were found inside the parasitophorous vacuole, and vacuoles containing parasites were also positive for autophagy. In conclusion, low concentrations of CdCl2, HgCl2, and ZnCl2 can cause damage to Toxoplasma gondii organelles, leading to loss of viability, organelle death, and elimination without causing toxic effects to host cells.
![](http://media.springernature.com/m312/springer-static/image/art%3A10.1007%2Fs00436-018-5806-x/MediaObjects/436_2018_5806_Fig1_HTML.gif)
![](http://media.springernature.com/m312/springer-static/image/art%3A10.1007%2Fs00436-018-5806-x/MediaObjects/436_2018_5806_Fig2_HTML.gif)
![](http://media.springernature.com/m312/springer-static/image/art%3A10.1007%2Fs00436-018-5806-x/MediaObjects/436_2018_5806_Fig3_HTML.gif)
![](http://media.springernature.com/m312/springer-static/image/art%3A10.1007%2Fs00436-018-5806-x/MediaObjects/436_2018_5806_Fig4_HTML.gif)
![](http://media.springernature.com/m312/springer-static/image/art%3A10.1007%2Fs00436-018-5806-x/MediaObjects/436_2018_5806_Fig5_HTML.gif)
![](http://media.springernature.com/m312/springer-static/image/art%3A10.1007%2Fs00436-018-5806-x/MediaObjects/436_2018_5806_Fig6_HTML.gif)
![](http://media.springernature.com/m312/springer-static/image/art%3A10.1007%2Fs00436-018-5806-x/MediaObjects/436_2018_5806_Fig7_HTML.gif)
![](http://media.springernature.com/m312/springer-static/image/art%3A10.1007%2Fs00436-018-5806-x/MediaObjects/436_2018_5806_Fig8_HTML.gif)
Similar content being viewed by others
References
Andreini C, Banci L, Bertini I, Rosato A (2006a) Counting the zinc-proteins encoded in the human genome. J Proteome Res 5:196–201
Andreini C, Banci L, Bertini I, Rosato A (2006b) Zinc through the three domains of life. J Proteome Res 5:3173–3178
Andreini C, Bertini I, Cavallaro G, Hollyday GL, Thornton JM (2008) Metal ions in biological catalysis: from enzyme databases to general principles. J Biol Inorg Chem 13:1205–1218
Babula P, Masarik M, Adam V, Eckschlager T, Stiborova M, Trnkova L, Skutkova H, Provaznik I, Hubalek J, Kizek R (2012) Mammalian metallothioneins: properties and functions. Metallomics 4:739–750
Belyaeva EA, Dymkowska D, Wieckowski MR, Wojtczak L (2006) Reactive oxygen species produced by the mitochondrial respiratory chain are involved in Cd 2+-induced injury of rat ascites hepatoma AS-30D cells. Biochim Biophys Acta 1757:1568–1574
Beraldo H, Gambino D (2004) The wide pharmacological versatility of semicarbazones, thiosemicarbazones and their metal complexes. Mini Rev Med Chem 4:31–39
Bernardi P (1999) Mitochondrial transport of cations: channels, exchangers, and permeability transition. Physiol Rev 79:1127–1155
Biederbick A, Kern HF, Elsasser HP (1995) Monodansylcadaverine (MDC) is a specific in vivo marker for autophagic vacuoles. Eur J Cell Biol 66:3–14
Bisanz C, Bastien O, Grando D, Jouet J, Maréchal E, Cesbron-Delauw MF (2006) Toxoplasma gondii acyl-lipid metabolism: de novo synthesis from apicoplast-generated fatty acids versus scavenging of host cell precursors. Biochem J 394:197–205
Bray TM, Bettger WJ (1990) The physiological role of zinc as an antioxidant. Free Radic Biol Med 8:281–291
Carruthers V, Giddings OK, Sibley LD (1999) Secretion of micronemal proteins is associated with Toxoplasma invasion of host cells. Cell Microbiol 1:225–235
Carvalho CS, Melo EJT (2006) Acidification of the parasitophorous vacuole containing Toxoplasma gondii in the presence of hydroxyurea. An Acad Bras Cienc 78:475–484
Carvalho LP, Beiral HJV, de Melo EJT (2017) The relationship among mitochondrial dysfunction and antibiotic activities on Toxoplasma gondii in vitro. W J Pharmac Sci 9:225–233
Carvalho LP, Melo EJT (2016) Non-essential and essential metal effects on intracellular Toxoplasma gondii. Eur J Pharm Sci 3:23–32
Carvalho LP, Melo EJT (2017) Life and death of Trypanosoma cruzi in presence of metals. Biometals 30:955–974
Cesbron-Delauw M-F, Gendrin C, Travier L, Ruffiot P, Mercier C (2008) Apicomplexa in mammalian cells: trafficking to the parasitophorous vacuole. Traffic 9:657–664
Chazotte B (2011) Labelling mitochondria with MitoTracker dyes. Cold Spring Har Protoc (8):990–1002
Clough B, Frickel EM (2017) The Toxoplasma parasitophorous vacuole: an evolving host-parasite frontier. Trends Parasitol 33:473–488
Coppens I, Dunn JD, Romano JD, Pypaert M, Zhang H, Boorthroyd JC, Joiner KA (2006) Toxoplasma gondii sequesters lysosomes from mammalian hosts in the vacuolar space. Cell 125:261–274
Demoro B, Caruso F, Rossi M, Benítez D, Gonzalez M, Cerecetto H, Parajón-Costa H, Castiglioni J, Galizzi M, Docampo R, Otero L, Gambino D (2010) Risedronate metal complexes potentially active against Chagas disease. J Inorg Biochem 104:1252–1258
Deretic V, Levine B (2009) Autophagy, immunity, and microbial adaptations. Cell Host Microbe 5:527–549
Eide DJ (2006) Zinc transporters and the cellular trafficking of zinc. Biochim Biophys Acta 1763:711–722
Elmore S (2007) Apoptosis: a review of programmed cell death. Toxicol Pathol 35:495–516
Fernández M, Varela J, Correia I, Birriel E, Castiglioni J, Moreno V, Pessoa JC, Cerecetto H, González M, Gambino D (2013) A new series of heteroleptic oxidovanadium(IV) compounds with phenanthroline-derived co-ligands: selective Trypanosoma cruzi growth inhibitors. Dalton Trans 42:11900–11911
Fontaine E, Bernardi P (1999) Progress on the mitochondrial permeability transition pore: regulation by complex I and ubiquinone analogs. J Bioenerg Biomembr 31:1988–1989
Grubman A, White AR, Liddell JR (2014) Mitochondrial metals as a potential therapeutic target in neurodegeneration. Br J Pharmacol 171:2159–2173
Hajj HE, Lebrun M, Fourmaux MN, Vial H, Dubremetz JF (2006) Characterization, biosynthesis and fate of ROP7, a ROP2 related rhoptry protein of Toxoplasma gondii. Mol Biochem Parasitol 146:98–100
Hakansson S, Charron AJ, Sibley LD (2001) Toxoplasma evacuoles: a two-step process of secretion and fusion forms the parasitophorous vacuole. EMBO J 20:3132–3144
Johnson LV, Walsh ML, Chen LB, Buchanan JM (1980) Localization of mitochondria in living cells with rhodamine 123. Proc Natl Acad Sc USA. Cell Biol 77:990–994
Kielian MC, Cohn ZA (1980) Phagosome-lysosome fusion. Characterization of intracellular membrane fusion in mouse macrophages. J Cell Biol 85:754–765
Klaassen CD, Liu J, Choudhuri S (1999) Metallothionein: an intracellular protein to protect against cadmium toxicity. Annu Rev Pharmacol Toxicol 39:267–294
Levine B (2005) Eating oneself and uninvited guests: autophagy-related pathways in cellular defense. Cell 120:159–162
Lipsky NG, Pagano RE (1985) Intracellular translocation of fluorescent sphingolipids in cultured fibroblasts: endogenously synthesized sphingomyelin and glucocerebroside analogues pass through the Golgi apparatus an route to the plasma membrane. J Cell Biol 100:27–34
Liu K, Liu P-C, Liu R, Wu X (2015) Dual AO/EB staining to detect apoptosis in osteosarcoma cells compared with flow cytometry. Med Sci Monit Basic Res 21:15–20
Marchetti C (2013) Role of calcium channels in heavy metal toxicity. ISRN Toxicol 2013:184360
Martinez-Finley EJ, Chakraborty S, Fretham SJB, Aschner M (2012) Cellular transport and homeostasis of essential and nonessential metals. Metallomics 4:593–605
Melo EJT, Attias M, Souza W (2000) The single mitochondrion of tachyzoites of Toxoplasma gondii. J Struct Biol 130:27–33
Melo EJT, Carvalho TU, Souza W (1992) Penetration of Toxoplasma gondii into host cells induces changes in the distribution of the mitochondria and the endoplasmic reticulum. Cell Struct Funct 17:311–317
Melo EJT, Souza W (1996) Pathway of C6-NBD-ceramide on the host cell infected with Toxoplasma gondii. Cell Struct Funct 21:47–52
Morales ME, Derbes RS, Ade CM, Ortego JC, Stark J, Deininger PL, Roy-Engel AM (2016) Heavy metal exposure influences double strand break DNA repair outcomes. PLoS One 11:0151367
Morey JR, Mcdevitt CA, Kehl-Fie TE (2015) Host-imposed manganese starvation of invading pathogens: two routes to the same destination. Biometals 28:509–519
Morisaki JH, Heuser JE, Sibley LD (1995) Invasion of Toxoplasma gondii occurs by active penetration of the host cell. J Cell Sci 108:2457–2464
Nam H-W (2009) GRA proteins of Toxoplasma gondii: maintenance of host-parasite interactions across the parasitophorous vacuolar membrane. Korean J Parasitol 47:29–37
Pagano RE, Martin OC, Hang HC, Haugland RP (1991) A novel fluorescent ceramide analogue for studying membrane traffic in animal cells: accumulation at the Golgi apparatus results in altered spectral properties of the sphingolipid precursor. J Cell Biol 113:1267–1279
Parkin G (2004) Synthetic analogues relevant to the structure and function of zinc enzymes. Chem Rev 104:699–767
Pierrel F, Cobine PA, Winge DR (2007) Metal ion availability in mitochondria. Biometals 20:675–682
Reyes-Caballero H, Campanello GC, Giedroc DP (2011) Metalloregulatory proteins: metal selectivity and allosteric switching. Biophys Chem 156:103–114
Rines AK, Ardehali H (2013) Transition metals and mitochondrial metabolism in the heart. J Mol Cell Cardiol 55:50–57
Romano JD, Sonda S, Bergbower E, Smith ME, Coppens I (2013) Toxoplasma gondii salvages sphingolipids from the host Golgi through the rerouting of selected Rab vesicles to the parasitophorous vacuole. Mol Biol Cell 24:1975–1995
Roohani N, Hurrell R, Kelishadi R, Schulin R (2013) Zinc and its importance for human health: an integrative review. J Res Med Sci 18:144–157
Schmid D, Münz C (2007) Innate and adaptive immunity through autophagy. Immunity 27:11–21
Schwab JC, Beckers CJ, Joiner KA (1994) The parasitophorous vacuole membrane surrounding intracellular Toxoplasma gondii functions as a molecular sieve. Proc Natl Acad Sci U S A 91:509–513
Sears ME (2013) Chelation: harnessing and enhancing heavy metal detoxification - a review. Sci W J 2013:219840
Soares AMS, Carvalho LP, Melo EJT, Costa HP, Vasconcelos IM, Oliveira JT (2015) A protein extract and a cysteine protease inhibitor enriched fraction from Jatropha curcas seed cake have in vitro anti-Toxoplasma gondii activity. Exp Parasitol 153:111–117
Szewczyk A, Wojtczak L (2002) Mitochondria as a pharmacological target. Pharmacol Rev 54:101–127
Tomavo S (2014) Evolutionary repurposing of endosomal systems for apical organelle biogenesis in Toxoplasma gondii. Int J Parasitol 44:133–138
Tsujikawa K, Imai T, Kakutami M, Kayamori Y, Mimura T, Otaki N, Kimura M, Fukuyama R, Shimizu N (1991) Localization of metallothionein in nuclei of growing primary cultured adult rat hepatocytes. FEBS Lett 283:239–242
Vašák M (2005) Advances in metallothionein structure and functions. J Trace Elem Med Biol 19:13–17
Weinberg ED (1966) Roles of metallic ions in host-parasite interactions differential metallic ion growth requirements of virulent and avirulent bacterial strains. Bacteriol Rev 30:136–115
Yapici NB, Bi Y, Li P, Chen X, Yan X, Mandalapu SR, Faucett M, Jockusch S, Ju J, Gibson KM, Paean WJ, Bi L (2015) Highly stable and sensitive fluorescent probes (LysoProbes) for lysosomal labeling and tracking. Sci Rep 5:8576
Acknowledgements
The authors would like to thank FAPERJ (Fundação de Amparo à Pesquisa do Rio de Janeiro) and CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
de Carvalho, L.P., de Melo, E.J.T. Further aspects of Toxoplasma gondii elimination in the presence of metals. Parasitol Res 117, 1245–1256 (2018). https://doi.org/10.1007/s00436-018-5806-x
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00436-018-5806-x