Reduction of Toxoplasma gondii development due to inhibition of parasite antioxidant enzymes by ...

46 Pages·2015·3.36 MB·English

AAC Accepted Manuscript Posted Online 21 September 2015
Antimicrob. Agents Chemother. doi:10.1128/AAC.00057-15
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
1 Reduction of Toxoplasma gondii development due to inhibition of parasite
2 antioxidant enzymes by a dinuclear iron(III) compound
3 PORTES, J.A.1,2; SOUZA, T.G.1; DOS SANTOS, T.A.T.1,5; DA SILVA, L.L.R. 5;
4 RIBEIRO, T.P.7; PEREIRA, M.D.7; HORN JR., A.6; FERNANDES, C.6;
5 DAMATTA, R.A.5; DE SOUZA, W.2,3,4; SEABRA, S.H.1* D
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6 1Laboratório de Tecnologia em Cultura de Células, Centro Universitário w
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7 Estadual da Zona Oeste (UEZO), Rio de Janeiro, RJ, Brazil. a
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8 2Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica d
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9 Carlos Chagas Filho, Universidade Federal do Rio de Janeiro (UFRJ), RJ, m

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10 Brazil. tt
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11 3Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagem /a
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12 (INBEB) e Centro Nacional de Biologia Estrutural e Bioimagem (CENABIO). a
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13 4Instituto Nacional de Metrologia, Qualidade e Tecnologia (Inmetro). .
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14 5Laboratório de Biologia Celular e Tecidual, Centro de Biociências e /
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15 Biotecnologia, Universidade Estadual do Norte Fluminense Darcy Ribeiro N
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16 (UENF), Campos dos Goytacazes, RJ, Brazil. e
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17 6Laboratório de Ciências Químicas, Centro de Ciência e Tecnologia, be
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18 Universidade Estadual do Norte Fluminense Darcy Ribeiro (UENF), Campos 3
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19 dos Goytacazes, RJ, Brazil. 0
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20 7Laboratório de Citotoxicidade e Genotoxicidade, Instituto de Química, b
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21 Departamento de Bioquímica, Universidade Federal do Rio de Janeiro (UFRJ), u
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22 Rio de Janeiro, RJ, Brazil. t
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24 *Corresponding author: Sergio Henrique Seabra, Phone: [phone]; Fax:
25 55 21 2332-7535; E-mail: [email protected]
26
27 Abstract
28 Toxoplasma gondii, which is the agent of toxoplasmosis, is an obligate
29 intracellular protozoan that can infect a wide range of vertebrate cells. Here, we
30 describe the cytotoxic effects of the dinuclear iron compound
31 [Fe(HPClNOL)(SO4)]2-µ-oxo, where HPClNOL is the ligand 1-(bis-pyridin-2- D
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32 ylmethyl-amino)-3-chloropropan-2-ol, on T. gondii infecting LLC-MK2 host cells.
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33 This compound was not toxic to LLC-MK2 cells at concentrations up to 200 μM a
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34 but was very active against the parasite, with an IC50 of 3.6 μM after 48 h of f
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35 treatment. Cyst formation was observed after treatment as indicated by the

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36 appearance of a cyst wall, Dolichos biflorus lectin staining, and scanning and tp
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37 transmission electron microscopy characteristics. Ultraestructural changes were a
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38 also seen in T. gondii, including membrane blebs and clefts in the cytoplasm as
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39 with inclusions similar to amylopectin granules, which are typically found in .o
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40 bradyzoites. An analysis of cell death pathways in the parasite revealed that a o
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41 combination of apoptosis and autophagy was caused by the compound. N
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42 Fluorescence assays demonstrated that the redox environment in the LLC-MK2 e
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43 cells becomes oxidant in the presence of the iron compound. Furthermore, a e
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44 reduction of superoxide dismutase and catalase activities in the treated 3
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45 parasites and the presence of reactive oxygen species within the 01
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46 parasitophorous vacuoles were observed, indicating an impaired protozoan b
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47 response against these radicals. These findings suggest that this compound u
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48 disturbs the redox equilibrium of T. gondii, inducing cystogenesis and parasite t
49 death.
50 Keywords: Toxoplasma gondii; iron(III) compound; oxidative stress; apoptosis;
51 autophagy.
52 1. Introduction
53 Toxoplasma gondii is an intracellular parasitic protozoan and the agent of
54 toxoplasmosis, with a worldwide distribution in warm-blooded animals, including
55 humans (1). The following forms of T. gondii can infect hosts: tachyzoites, which
56 are present during the acute phase of toxoplasmosis; bradyzoites, which are D
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57 typically found inside tissue cysts in the brain and skeletal muscles during the
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58 chronic phase of the infection; and sporozoites, which are present inside a
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59 oocysts that are produced during the sexual cycle that occurs in the intestine of
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60 felines, which are the definitive hosts (2). As the host adaptive immune m

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61 response weakens, parasite tissue cysts rupture and release bradyzoites tt
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62 through an unknown mechanism. These recrudescent infections permit parasite a
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63 conversion to the rapidly-dividing tachyzoite stage and cause significant a
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64 morbidity, including Toxoplasma encephalitis (3,4). .
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65 Transmission in humans occurs via the ingestion of food or water /
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66 contaminated with oocysts shed by cats, via the ingestion of undercooked or N
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67 raw meat containing tissue cysts, or congenitally, particularly when the mother e
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68 acquires the infection for the first time during pregnancy (5). In e
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69 immunocompetent organisms, T. gondii infection is rarely severe and is often 3
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70 asymptomatic. In contrast, in immunocompromised individuals, the most 0
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71 common condition associated with this infection is encephalitis, which causes b
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72 headache, disorientation, lethargy, hemiparesis, altered reflexes and u
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73 convulsions (6). Pneumonia and myocarditis may also occur in these t
74 individuals. In children infected in utero, the parasite invades the brain and
75 retina, resulting in potentially severe consequences, including reduced visual
76 acuity, mental retardation, intracranial calcifications and hydrocephalus (6).
77 Associations have recently been made between parasite infection and
78 neurological disorders, such as schizophrenia (7).
79 Currently, the most effective therapy for toxoplasmosis is the
80 administration of anti-folate compounds, such as the combination of
81 pyrimethamine and sulfadiazine. Despite the efficacy of this therapy, these D
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82 drugs are often associated with many side effects, which are primarily observed
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83 in AIDS patients and include bone marrow suppression and hematological a
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84 toxicity, which occur in association with pyrimethamine, and/or hypersensitivity
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85 and allergic skin reactions, which are associated with sulfadiazine (8, 9, 10). m

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86 In light of the medical relevance of toxoplasmosis, the development of tt
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87 new therapies for this parasitic disease is essential. However, the main a
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88 challenge in this field is the development of compounds that are capable of a
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89 reaching the protozoan inside the host cell at concentrations that are toxic to the .
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90 parasite but safe for the host. /
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91 Some reports in the literature demonstrate that coordination compounds N
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92 may be an interesting alternative for antiparasite therapy. For example, e
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93 compounds containing copper or cobalt ions bound to the HmtpO ligand, where e
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94 HmtpO is [5-methyl-1,2,4-triazol [1,5-a] pyrimidin-7 (4H)-one], strongly affect the 3
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95 energy metabolism of Leishmania infantum and L. braziliensis cells, disrupting 0
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96 the membrane structure of organelles and inducing cell death (11). These b
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97 compounds were also active in vitro against the trypomastigote and amastigote u
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98 forms of Trypanosoma cruzi at concentrations similar to those of drugs that are t
99 commonly used in clinical therapy, such as benznidazol; however, these
100 compounds were associated with reduced toxicity to host cells and an improved
101 selectivity index. Furthermore, in vivo tests demonstrated that these compounds
102 promoted a significantly lower parasite burden than benznidazol treatment (12).
103 Horn Jr. et al. (13) reported that HPClNOL [1-(bis-pyridin-2-ylmethyl-
104 amino)-3-chloropropan-2-ol] is a promising ligand for the development of
105 metallopharmaceuticals because the associated copper and iron complexes D
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106 exhibit interesting biological activities. The associated copper complexes
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107 [Cu(HPClNOL)Cl]+ exhibited nuclease activity and were cytotoxic to leukemia a
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108 cancer cells (14). Iron complexes with the same ligand were also biologically
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109 tested, and the mononuclear compound [Fe(HPClNOL)(Cl)2] protected m

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110 Saccharomyces cerevisiae cells against oxidative stress, mimicking superoxide tt
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111 dismutase and catalase (15). This same compound and its dinuclear a
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112 counterparts [Fe(HPClNOL)(SO4)]2-µ-oxo and [Fe(HPClNOL)Cl]2-µ-oxo as
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113 accelerated DNA hydrolysis approximately 108-fold compared to the .
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114 spontaneous DNA cleavage rate, revealing an impressive nuclease activity. /
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115 However, the activities of these compounds against cancer cells were modest N
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116 and associated with a very low toxicity for normal human peripheral blood e
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117 mononuclear cells (16). This lack of toxicity for normal cells prompted us to e
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118 evaluate the activity of these compounds in antiparasitic therapies because the 3
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119 main challenge of these therapies is preserving host cell viability. 0
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120 Thus, we report the evaluation of the anti-Toxoplasma activity of the b
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121 compound [Fe(HPClNOL)(SO4)]2-µ-oxo (Figure 1), which significantly reduced u
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122 the level of parasite infection in the host cell. Furthermore, the associated iron t
123 complex induces the production of reactive oxygen species in the cell and
124 promotes a dramatic reduction in the activity of the parasite antioxidant
125 enzymes superoxide dismutase (SOD) and catalase (CAT), indicating that the
126 mode of action of this compound involves the impairment of this protective
127 system.
128
129 2. Materials and methods
130 D
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131 2.1. Parasites
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132 The tachyzoites used in this study were from the virulent RH strain of T. a
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133 gondii and were maintained via intraperitoneal infections in Swiss mice. After 48
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134 h of infection, the parasites were collected via a peritoneal wash with m

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135 phosphate-buffered saline (PBS), pH 7.2, and then centrifuged at 1,000 g for 10 tt
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136 min. The pellet was washed twice with PBS and RPMI-1640 medium. The a
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137 parasites were used within 30 - 40 min of their removal from the peritoneal a
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138 cavity. All animal studies were reviewed and approved by the ethics committee .
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139 of animal use of the Biophysics Institute Carlos Chagas Filho (code: IBCCF99). /
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140 N
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141 2.2. LLC-MK2 cells e
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142 LLC-MK2 kidney epithelial cells (Rhesus monkey, Macaca mulata) were e
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143 grown in 25 cm2 culture flasks (SPL Life Sciences) containing RPMI 1640 3
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144 medium supplemented with 10% fetal bovine serum (FBS) at 37˚C in a 5% CO2 01
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145 atmosphere. Infection by the parasite was conducted in subconfluent cultures in b
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146 flasks or over coverslips in 24-well tissue culture plates (SPL Life Sciences). u
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147 t
148 2.3. Compound
149 The iron(III) compound [Fe(HPClNOL)(SO4)]2-µ-oxo was obtained as
150 previously described (17). For in vitro studies, this compound was dissolved in
151 RPMI 1640 medium and stored at -20˚C.
152 2.4. Cell viability assays
153 The possible toxic effects of the compound on the host cell were D
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154 evaluated based on the reduction of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-
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155 diphenyltetrazolium bromide] and the release of lactate dehydrogenase (LDH). a
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156 For these assays, 1 x 105 cells were seeded per well in 96-well plates and d
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157 cultured with RPMI 1640 medium supplemented with 5% FBS. After 24 h, the m

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158 cells were washed and directly subjected to compound treatment at tt
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159 concentrations derived from serial dilutions in RPMI 1640 medium a
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160 supplemented with 5% FBS; the dilutions included concentrations from 200 to a
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161 1.69 μmol L-1. As a negative control, the cells were cultured in RPMI 1640 .
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162 medium supplemented with 5% FBS without the addition of the compound. As a /
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163 positive control, the cells were directly subjected to 10% Triton X-100 and N
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164 cultured as previously described. e
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165 After 24 h of treatment, the culture supernatant was removed and used e
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166 for LDH measurement (see below) and 15 μl of MTT (5 mg/ml) in RPMI 1640 3
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167 medium solution was added to each well for 4 h. The formazan crystals were 0
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168 subsequently solubilized by the addition of 100 μl of pure DMSO. The plate was b
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169 centrifuged at 400 g for 7 min, and 100 μL of the supernatant was collected, u
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170 transferred to a new 96-well plate and read in a Versamax microplate reader t
171 (Molecular Devices) at 570 nm using the 6.0 SoftMax Pro® software. LDH
172 concentrations (18, 19) were measured in the culture supernatants of the LLC-
173 MK2 cells using the Doles® kit with 50 μl of the cultured supernatant, according
174 to the manufacturer’s protocol. The data were plotted using the Graphpad Prism
175 6.0® software. The presented data are representative of three independent
176 experiments.
177
178 2.5. Anti-proliferative assays D
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179 Approximately 2x105 LLC-MK2 cells were seeded per well over w
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180 coverslips in a 24-well plate 1 day before the assay. The cells were infected a
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181 with parasites in RPMI 1640 medium using a 5:1 parasite:host cell ratio based
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182 on the host cell count on the day of the infection. Tachyzoites were allowed to m

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183 interact with the host cells for 1 h, the cell monolayer was washed twice with tt
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184 PBS to remove non-adhered parasites, and the iron(III) compound was added a
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185 at different concentrations (2.5-25 µM) in RPMI 1640 medium supplemented a
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186 with FBS. After 48 h or 6 days of treatment, the cells were fixed with fresh 4% .
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187 formaldehyde in PBS, stained with Giemsa and observed by light microscopy. /
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188 The samples subjected to the 6-day treatment received new culture medium N
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189 containing the compound every 2 days. This procedure was repeated for all of e
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190 the 6-day assays described in this paper. The proliferation index was calculated e
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191 by multiplying the mean number of internalized T. gondii per cell by the 3
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192 percentage of infected cells on two different coverslips per experiment (20). The 0
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193 data were plotted using the Graphpad Prism 6.0® software. The presented b
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194 results represent the mean ± standard deviation of at least three independent u
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195 experiments, and asterisks indicate statistically significant differences at P < t
196 0.05. For the calculations of the IC50 (concentration for 50% parasite growth
197 inhibition), the percentage of growth inhibition was plotted as a function of the
198 drug concentration by fitting the values to non-linear curve analysis. The
199 regression analyses were performed using Sigma Plot 8.0 software (Systat
200 Software Inc., Chicago, IL, USA).
201
202 2.6. Electron microscopic analysis
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203 To observe the ultrastructure of intracellular parasites using transmission o
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204 electron microscopy, cells in culture flasks were fixed for 1 h in a solution lo
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205 containing 2.5% glutaraldehyde and 4% recently prepared formaldehyde in 0.1 de
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206 mol L-1 sodium cacodylate buffer, pH 7.4, after 48 h or 6 days of treatment with fr
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207 10 µmol L-1 of the compound. The cells were scraped from the flasks with a
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208 rubber policeman, washed with sodium cacodylate buffer and post-fixed for 1 h :
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209 in the dark with a solution containing 1% osmium tetroxide in 0.1 M sodium a
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210 cacodylate buffer. The cells were subsequently washed in the same buffer, s
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211 dehydrated in acetone, and embedded in Epon. Ultrathin sections were stained r
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212 with uranyl acetate and lead citrate and observed under a Zeiss 900 o
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213 transmission electron microscope. o
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214 For scanning electron microscopy, cells over coverslips were fixed and m
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215 post-fixed after 48 h or 6 days of treatment with 10 µmol L-1 or 25 µmol L-1 of the r

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216 compound, as described above, and dehydrated in a graded acetone series. ,
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217 The cells were critical point-dried and mounted on stubs, and the upper portion 1
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218 of the cells was scraped off with an adhesive tape, revealing the internal y

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219 organization of the parasitophorous vacuole (21). The samples were then e
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220 coated with gold (20–30 nm) and observed using a Jeol JSM 6490LV scanning
221 electron microscope.
222
223 2.7. Immunofluorescence and cell death assays
224 LLC-MK2 cells over coverslips were infected with tachyzoites, treated
225 with 10 µmol L-1 of the compound or left untreated for 48 h or 6 days, washed
226 with PBS and fixed with 4% freshly prepared formaldehyde in PHEM buffer (60
227 mmol L-1 Pipes, 20 mmol L-1 Hepes, 10 mmol L-1 EGTA, 5 mmol L-1 MgCl2, and
228 70 mmol L-1 KCl, pH 7.2). After fixation, the cells were washed, permeabilized D
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229 with 2% Triton X-100 in PHEM buffer for 10 min, incubated with 100 mmol L-1 w
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230 NH4Cl for 30 min and then incubated with PHEM buffer containing 3% bovine ad
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231 serum albumin (PHEM-BSA) for 30 min at room temperature. The cells were
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232 incubated for 1 h in the presence of Dolichos biflorus lectin conjugated with m

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233 fluorescein isothiocyanate (DBA-FITC) (10 µg ml-1) (Sigma–Aldrich Co., St. tt
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234 Louis, MO, USA) or with the LC3B rabbit polyclonal antibody (1:1000 dilution). a
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235 After labeling with LC3B, the cells were incubated with goat anti-rabbit Alexa- a
s
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236 546 (1:100 dilution) (Molecular Probes). After labelling, the cells were washed .
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237 with PHEM and the coverslips with cells were mounted in Prolong Gold with or /
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238 without 4',6-diamidino-2-phenylindole (DAPI). The percentage of cells with N
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239 vacuoles positive for LC3B was quantified by a direct count of the total number e
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240 positive cells among 100 infected cells on two different coverslips per e
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241 experiment. DNA fragmentation was also assayed after infection and treatment. 3
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242 Infected cells were treated with 10 µmol L-1 of the compound or left untreated 0
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243 for 6 days, washed and fixed as described above. After fixation, the cells were b
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244 washed, permeabilized with cold 70% ethanol for 10 min and processed as u
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245 recommended by the manufacturer of the Click-iT TUNEL Alexa Fluor 488 t
246 Imaging Assay kit (Molecular Probes). After labeling, the coverslips were
247 prepared as described above and observed using a Zeiss LSM-710 confocal
248 laser-scanning microscope.