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Inhibition of the mitochondrial respiratory chain in gills of Rhamdia quelen experimentally infected by Pseudomonas aeruginosa: Interplay with reactive oxygen species
Microbial Pathogenesis 107 (2017) 349e353
Contents lists available at ScienceDirect
Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
Inhibition of the mitochondrial respiratory chain in gills of Rhamdia quelen experimentally infected by Pseudomonas aeruginosa: Interplay with reactive oxygen species Matheus D. Baldissera a, *, Carine F. Souza b, Mateus Grings c, Belisa S. Parmeggiani c, Guilhian Leipnitz d, Karen L.S. Moreira e, Maria Izabel U.M. da Rocha e, Marcelo L. da Veiga e, Roberto C.V. Santos a, Lenita M. Stefani f, Bernardo Baldisserotto b, ** a
Department of Microbiology and Parasitology, Universidade Federal de Santa Maria (UFSM), Santa Maria, RS, Brazil Department of Physiology and Pharmacology, Universidade Federal de Santa Maria (UFSM), Santa Maria, RS, Brazil c ~o em Ci^ sicas da Saúde, Universidade Federal do Rio Grande do Sul s Graduaça gicas: Bioquímica, Instituto de Ci^ Programa de Po encias Biolo encias Ba (UFRGS), Porto Alegre, RS, Brazil d ~o em Ci^ sicas da Saúde, Universidade s Graduaça gicas: Bioquímica, Departamento de Bioquímica, Instituto de Ci^ Programa de Po encias Biolo encias Ba Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil e Department of Morphology, Universidade Federal de Santa Maria (UFSM), Santa Maria, RS, Brazil f , SC, Brazil Graduate School of Animal Science, Universidade do Estado de Santa Catarina (UDESC), Chapeco b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 31 March 2017 Received in revised form 10 April 2017 Accepted 11 April 2017 Available online 13 April 2017
It has long been recognized that there are several infectious diseases linked to the impairment of enzymatic complexes of the mitochondrial respiratory chain, with consequent production of reactive oxygen species (ROS), that contribute to disease pathogenesis. In this study, we determined whether the inhibition on mitochondrial respiratory chain might be considered a pathway involved in the production of ROS in gills of Rhamdia quelen experimentally infected by P. aeruginosa. The animals were divided into two groups with six fish each: uninfected (the negative control group) and infected (the positive control group). On day 7 post-infection (PI), animals were euthanized and the gills were collected to assess the activities of complexes I-III, II and IV of the respiratory chain, as well as ROS levels. The activities of complexes I-III, II and IV of the respiratory chain in gills decreased, while the ROS levels increased in infected compared to uninfected animals. Moreover, a significant negative correlation was found between enzymatic activity of the complexes I-III and IV related to ROS levels in P. aeruginosa infected animals, corroborating to our hypothesis that inhibition on complexes of respiratory chain leads to ROS formation. Also, microscopic severe gill damage and destruction of primary and secondary lamellae were observed in infected animals, with the presence of hyperplasia, leukocytic infiltration and telangiectasia. In summary, we have demonstrated, for the first time, that experimental infection by P. aeruginosa inhibits the activities of mitochondrial complexes of respiratory chain and, consequently, impairs the cellular energy homeostasis. Moreover, the inhibition on mitochondrial complexes I-III and IV are linked to the ROS production, contributing to disease pathogenesis. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Branchial tissue Cytochrome c oxidoreductase DCIP-oxidoreductase Cytochrome c oxidase Fish pathogen
1. Introduction Pseudomonas aeruginosa is a Gram-negative opportunist
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] [email protected] (B. Baldisserotto). http://dx.doi.org/10.1016/j.micpath.2017.04.017 0882-4010/© 2017 Elsevier Ltd. All rights reserved.
(M.D.
Baldissera),
pathogen widely distributed in aquaculture farms, responsible for considerable economic losses for fish producers [1]. This bacterium leads to the development of the so called red skin disease in fish, which occurs during a stressful condition, such as inappropriate handling or during transportation [2], affecting mainly the Nile tilapia (Oreochromis niloticus), Mozambique tilapia (O. mossambicus) [1] and the silver catfish (Rhamdia quelen) [3]. This disease is characterized by petechial hemorrhages, darkness of the skin, abdominal ascitis, ulcerative syndrome, rotten fish gills,
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septicemia and behavioral alterations [4,5], with severe histopathological lesions of the primary and secondary lamellae of gills [1]. Recently, a study conducted by Baldissera et al. [5] demonstrated that infections caused by P. aeruginosa leads to oxidative stress due to the excessive production of reactive oxygen species (ROS) as an attempt to control the disease, but the effects of ROS on mitochondrial synthesis of adenosine triphosphate (ATP) during this bacterial infection remains unknown. Mitochondria are vital intracellular organelles that accommodate a large amount of four different oxidative phosphorylation complexes (complexes I-IV) in their inner or cristae membranes, that are involved in biological and physiological energy for intracellular metabolic pathways [6]. These complexes accept and transfer electrons through oxidation-reduction reactions and translocation of protons across the inner membrane. Also, the proton efflux generated in the respiratory chain drives the formation of ATP from adenosine diphosphate (ADP), and inorganic phosphate through the enzyme ATP synthase, known as complex V [7]. Recently, studies have demonstrated that redox reactions are linked to dysfunctions on the electron transport chain due to the formation of ROS and superoxide anions, that are considered products of metabolic redox reactions [6,8]. Also, mitochondria are considered the primary sites of intracellular ROS formation, and depending on the formation rates, ROS may contribute to signaling events and can mediate mitochondrial dysfunction in pathologies through oxidative modifications of mitochondrial components [6]. During mitochondrial ATP synthesis, the main sites of oxidant formation in mitochondria include mitochondrial electron transport chain complexes I and III, as well as the transfer of electrons that generates superoxide radical. Moreover, mitochondrial targets of oxidant species include the enzyme succinate dehydrogenase (SDH), and the complexes I and IV of the respiratory chain. Mitochondrial involvement in diseases has been linked to its central role on ROS production and to the harmful effects of ROS on this organelle, as observed during other pathological conditions, such as parasitic infections [9]. According to Haegler et al. [10], impaired mitochondrial respiratory chain function results in an oxidative phosphorylation disorder and decreased ATP synthesis associated to cell death. In this sense, a recent study conducted by Garrabou et al. [11] demonstrated that sepsis is associated with mitochondrial dysfunction and impaired oxygen consumption, and consequently, inhibition of the mitochondrial complexes I, III and IV, that contributes to disease evolution. Based on these evidences, our hypothesis is that the impairment of mitochondrial complexes of the respiratory chain in gills of R. quelen experimentally infected by P. aeruginosa causes an increase on ROS production, that may contribute directly to disease pathogenesis. Therefore, the aim of this study was to evaluate whether an inhibition on complexes I-III, II and IV of the respiratory chain causes an increase on ROS levels in gills during P. aeruginosa infection. 2. Materials and methods 2.1. Animals and water quality variables Healthy fish were harvested for experimental purpose from a fish farm located at Rio Grande do Sul state (Brazil). The fish were transported in live condition and maintained in 250 L fiberglass tanks of continuous aeration with fresh water for 14 days under controlled water parameters, such as: temperature 21e23 C (maintained with air conditioner), pH 7.2e7.6 and dissolved oxygen 5.6e7.2 mg L1. Dissolved oxygen and temperature were measured with a YSI oxygen meter (Model Y5512, Ohio, USA). The pH was ~o Paulo, Brazil). measured using a DMPH-2 pH meter (Digimed, Sa
Total ammonia levels were determined according to Verdouw et al. [12] and un-ionized ammonia (NH3) levels were calculated according to Colt [13]. The animals were fed once a day with commercial fish feed. Water quality variables were maintained as follows: temperature 23 ± 1 C, pH 7.4 ± 0.05, dissolved oxygen at 6.86 ± 0.22 mg L1, total ammonia 0.93 ± 0.07 mg L1 and nonionized ammonia 0.004 ± 0.0003 mg L1. 2.2. Inoculum preparation The strain of P. aeruginosa (PAO1) was kindly donated by Professor Barbara H. Iglewski (Department of Microbiology and Immunology, School of Medicine and Dentistry, University of Rochester, Rochester, New York 14642). The bacterial culture was grown on MacConkey agar and Nutrient agar. The suspensions of PAO1 were washed twice in sterile saline (NaCl 0.9%), turbidity (OD600) adjusted to 0.8e1.0 (equivalent to 1.5 108 CFU mL1) and used for the infection model. 2.3. Animal model and experimental design Twelve juvenile silver catfish (63.6 ± 10 g; 23 ± 4 cm) were used as the experimental model for the assessment of the enzymatic activities of the respiratory chain complexes in the gills. The silver catfish were assigned into two groups with six animals each: uninfected animals (the negative control group); and experimentally infected animals (the positive control group) inoculated intramuscularly with 100 mL of P. aeruginosa strain PAO1 (108 CFU: OD600 ¼ 0.8e1.0) on the right latero-dorsal side of each fish, according to the protocol established by Baldissera et al. [3]. The negative control received the same volume of sterile saline by the same route. Fish were fed once daily to apparent satiation with commercial feed. Uneaten food, other residues and feces were removed 30 min after feeding. The methodology used in the experiment was approved by the Ethical and Animal Welfare Committee of the Universidade Federal de Santa Maria under protocol number 074/2014. 2.4. Sampling and tissue preparation On day 7 post-infection (PI), all animals were euthanized by spinal cord section according to the Ethics Committee recommendations, and the gills were collected and dissected in a glass over ice. In order to measure the activities involved in the electron transport chain and ROS levels, the gills were washed in SETH buffer (0.250 mM sucrose, 2 mM EDTA, 10 mM Trizma base, 50 UI/ mL heparin, pH 7.4) and homogenized (1:10 w/v) in the same SETH buffer with a Potter-Elvehjen glass homogenizer. The homogenate was centrifuged at 800g for 10 min at 4 C, and the supernatants were stored at 80 C until used. 2.5. Assessment of mitochondrial respiratory chain enzymatic complexes Enzymatic activities (complexes I-III, II and IV) of the mitochondrial respiratory chain were measured in branchial homogenates. The activity of NADH: cytochrome c oxidoreductase (complex I-III) was assayed in gills according to the method described by Schapira et al. [14]. The activity of succinate: DCIPoxidoreductase (complex II) was determined according to the method of Fischer et al. [15], and that of cytochrome c oxidase (complex IV) was measured according to Rustin et al. [16]. These methods were slightly modified, as described in details in a previous report by Baldissera et al. [17]. The activities of the respiratory chain complexes were calculated as nmol.min1.mg protein1.
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2.6. Branchial ROS levels The 20 ,7’-dichloroflurescein diacetate (DCF-DA) levels were determined as an index based on the amount of peroxide produced in the cellular components via DCFH production on the oxidation assay. This experimental method of analysis is based on the deacetylation of the DCFH-DA probe, that is subsequently oxidized by reactive species into DCF, a highly fluorescent compound [18]. The branchial ROS levels were determined by using the DCFH-DA method described by Lawler et al. [19]. The gills homogenate was added to a medium containing Tris-HCl buffer (10 mM; pH 7.4). The absorbance of the samples was measured by a spectrophotometer (wavelength of 522 nm). The ROS levels were expressed as fluorescence unit per mg of protein1 (x109). 2.7. Protein determination Protein determination was measured in the gills homogenate by the method of Lowry et al. [20] using bovine serum albumin as standard. 2.8. Histopathology Gill samples were fixed in 10% formaldehyde and submitted to histological sections (6 mm), and Hematoxylin-Eosin (HE) staining. All slides were examined by two pathologists in a double blinded manner to detect microarchitecture and lamellae alterations. 2.9. Statistical analysis Normality and homoscedasticity were analyzed through the Kolmogorov-Smirnov and Levene tests, respectively, and the data were transformed when necessary. Significant differences between groups were analyzed and detected by two tailed Student's t-test for independent samples. The differences were considered to be statistically significant at p < 0.05. The effect size (r2) was described and scored as follows: 0.1 (small), 0.1 to 0.3 (medium), and 0.5 (large). The Pearson correlation was used to verify the correlation between ROS levels and mitochondrial respiratory chain complex activities. The correlation coefficient (r) and the coefficient of determination (r2) were used to determine the intensity of the association. 3. Results 3.1. Mitochondrial respiratory chain complex activities Complex I-III (cytochrome c oxidoreductase) decreased 44% [t(10) ¼ 2.09; p < 0.05; r2 ¼ 0.38], complex II (DCIP-oxidoreductase) decreased 11% [t(10) ¼ 4.43; p < 0.001; r2 ¼ 0.79] and complex IV (cytochrome c oxidase) decreased 17% [t(10) ¼ 4.75; p < 0.001; r2 ¼ 0.88] in gills of animals infected by P. aeruginosa compared to uninfected animals (Fig. 1). 3.2. Branchial ROS levels ROS levels increased 180% [t(10) ¼ 6.19; p < 0.001; r2 ¼ 0.88] in gills of animals infected by P. aeruginosa compared to uninfected animals (Fig. 2). 3.3. Study correlation A significant negative correlation was observed between
Fig. 1. Mean and standard error of complex I-III (cytochrome c oxidoreductase) [A], complex II (DCIP-oxidoreductase) [B] and complex IV (cytochrome c oxidase) [C] activities in gills of silver catfish experimentally infected by Pseudomonas aeruginosa strain PAO1 compared to the uninfected control group on day 7 post-infection (PI). Bars with different letters are statistically different (p < 0.05; n ¼ 6 per group) according to the two-tailed Student's t-test for independent samples.
branchial ROS levels and branchial activity of complex I-III (r ¼ 0.947; r2 ¼ 0.887; p ¼ 0.004) and for branchial ROS levels and branchial activity of complex IV (r ¼ 0.926; r2 ¼ 0.922; p ¼ 0.002) (Fig. 3). No correlation was observed between branchial ROS levels and branchial activity of complex II (r ¼ 0.345; r2 ¼ 0.311; p ¼ 0.089) (data not shown).
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3.4. Histopathology The uninfected animals did not show any pathological alteration in gill tissue (Fig. 4A). Infected animals showed severe gill damage and destruction of the primary and secondary lamellae. Moreover, edema with epithelial hyperplasia, severe desquamation, epithelial lifting and telangiectasia were observed in the gills. Hyperplasia, leukocytic infiltration and dilation of the central venous sinus were observed in the secondary lamellae (Fig. 4B). 4. Discussion
Fig. 2. Mean and standard error of reactive oxygen species (ROS) in gills of silver catfish experimentally infected by Pseudomonas aeruginosa strain PAO1 compared to the uninfected control group on day 7 post-infection (PI). Bars with different letters are statistically different (p < 0.05; n ¼ 6 per group) according to the two-tailed Student's t-test for independent samples.
Fig. 3. Pearson's correlation between reactive oxygen species (ROS) levels and the activity of complex I-III (cytochrome c oxidoreductase) [A], and between reactive oxygen species (ROS) levels and the activity of complex IV (cytochrome c oxidase) in gills of silver catfish experimentally infected by Pseudomonas aeruginosa strain PAO1. The r (correlation coefficient), r2 (coefficient of determination) and p values were used to verify the intensity of the association between the variables and the level of significance.
The crucial involvement of mitochondria in branchial bioenergetic regulation, as well as in the balance of oxidant and antioxidant agents, suggest that these organelles are deeply involved in the pathophysiology of gills [21]. In this study, we demonstrated, for the first time, that P. aeruginosa infection impairs the activities of respiratory chain leading to increased ROS levels in gills, which may contribute to disease pathophysiology. Mitochondria are vital organelles to maintain biological functions of a cell by providing energy, playing an important role in cell survival [22]. However, under pathological conditions such as during infectious diseases, mitochondria release cell deathinducing molecules and contribute to the progression of pathology and cell death [17], in accordance to the observations of this present study. We found that complex I-III, II and IV activities were inhibited by P. aeruginosa infection, which may result in decreased availability of ATP and impairment of energy supply, in accordance to what was observed during Trypanosoma cruzi [23,24], and T. evansi [9,17] infections. According to these authors, impairment of respiratory chain enzymatic complexes contributes directly to pathophysiology of the disease, contributing to animal death. Of particular interest, a recent study conducted by Garrabou et al. [11] demonstrated that mitochondrial complexes I, III and IV were significantly inhibited during bacterial sepsis, leading to impaired oxygen supply and an oxidative stress frame with excessive ROS production, which contribute directly to disease pathogenesis. Thus, we can conclude that inhibition of the activities of mitochondrial complexes IeIII, II and IV caused by P. aeruginosa may contribute to decreased availability of ATP and impairment of energy supply, which is supported by the impairment on enzymes of the phosphotransfer network (adenylate kinase, pyruvate kinase, and creatine kinase), that are linked to the transfer of phosphoryl group between local synthesis and utilization of ATP [3]. Under pathologic conditions, diverse pathways result in excessive ROS production, including in the mitochondria, since this organelle is one of the main intracellular site of O2 generation [6]. In this study, we observed a significant increase of branchial ROS levels during P. aeruginosa infection, which may be linked to the inhibition of the enzymatic activities of mitochondrial complexes IIII and IV. According to Quijano et al. [6], the mitochondrial complexes I, III and IV are considered the main sites of oxidant formation in mitochondria, since the transfer of electrons to oxygen generates superoxide radical, which leads to secondary oxidant formation (anion superoxide) by dismutation reaction with metals or by reaction with nitric oxide, that is corroborated by significantly negative correlation between ROS formation and mitochondrial complex I-III and IV activities. Moreover, it is important to emphasize that ROS produced due to inhibition on mitochondrial complexes may also contribute to impairment of other complexes, such as complexes I and II of the respiratory chain [25]. Thus, the inhibition on mitochondrial complexes I-III and IV of respiratory chain may be considered an important pathway of ROS production, which may contribute to disease pathogenesis. In summary, we have demonstrated for the first time that
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Fig. 4. Gill histopathology of Rhamdia quelen experimentally infected by Pseudomonas aeruginosa. (A) Uninfected fish showed normal histology with normal distribution of cellular constituents and normal organization of primary and secondary lamellae. (B) Fish infected by P. aeruginosa showed severe gill damage and destruction of primary and secondary lamellae.
experimental infection by P. aeruginosa inhibits the activities of mitochondrial respiratory chain complexes and consequently impairs cellular energy homeostasis. Moreover, the inhibition on mitochondrial complexes I-III and IV is linked to ROS production, which contributes to disease pathogenesis. Declaration of interest The authors report no conflict of interest. References [1] J. Thomas, S. Thanigaivel, S. Vijayakumar, K. Acharya, D. Shinge, T.S.J. Seelan, et al., Pathogenicity of Pseudomonas aeruginosa in Oreochromis mossambicus and treatment using lime oil nanoemulsion, Colloids Surf. B 116 (2014) 372e377. [2] P. Mishra, R.S. Sk, J. Jerobin, J. Thomas, A. Mukherjee, N. Chandrasekaran, Study on antimicrobial potential of neem oil nanoemulsion against Pseudomonas aeruginosa infection in Labeo rohita, Biotechnol. Appl. Biochem. 61 (2014) 611e619. [3] M.D. Baldissera, C.F. Souza, R.C.V. Santos, L.M. Stefani, K.L.S. Moreira, M.L. da Veiga, et al., Pseudomonas aeruginosa strain PAO1 impairs enzymes of the phosphotransfer network in the gills of Rhamdia quelen, Vet. Microbiol. 201 (2017) 121e125. [4] S.H.M. Ahmed, A.A.M. Shoreit, Bacterial hemorrhagic septicemia in Oreochromis niloticus at Aswan fish hatcheries, J. Vet. Med. Sci. 45 (2001) 89. [5] M.D. Baldissera, C.F. Souza, R.C.V. Santos, L.V.C. da Rosa, D.B. Rosemberg, B. Baldisserotto, Pseudomonas aeruginosa strain PAO1 infection impairs locomotor activity in experimentally infected Rhamdia quelen: interplay between a stress response and brain neurotransmitters, Aquaculture 473 (2017) 74e79. [6] C. Quijano, M. Trujillo, L. Castro, A. Trostchansky, Interplay between oxidant species and energy metabolism, Redox Biol. 8 (2016) 28e42. [7] D.C. Wallace, Mitochondrial diseases in man and mouse, Science 283 (1999) 1482e1487. [8] L. Plecit a-Hlavat a, P. Jezek, Integration of superoxide formation and cristae morphology for mitochondrial redox signaling, Int. J. Biochem. Cell Biol. 80 (2016) 31e50. [9] M.D. Baldissera, V.C. Rech, M. Grings, J. Kolling, A.S. Da Silva, L.T. Gressler, et al., Relationship between pathological findings and enzymes of the energy metabolism in liver of rats infected by Trypanosoma evansi, Parasitol. Int. 64 (2015) 547e552. [10] P. Haegler, D. Grunig, B. Berger, S. Krahenbuhl, J. Bouitbir, Impaired mitochondrial function in HepG2 cells treated with hydroxy-cobalamin[c-lactam]:
a cell model for idiosyncratic toxicity, Toxicology 336 (2015) 48e58. Miro n, S. Lo pez, E. Tobías, F. Cardellach, O. , et al., The [11] G. Garrabou, C. More effects of sepsis on mitochondria, J. Infect. Dis. 205 (2012) 392e400. [12] H. Verdouw, C.J.A. Vanechteld, E.M.J. Deckkers, Ammonia determinations based on indophenol formation with sodium salicylate, Water Res. 12 (1978) 399e402. [13] J. Colt, List of spreadsheets prepared as a complement, in: G.A. Wedemeyer (Ed.), Fish Hatchery Management, second ed., American Fisheries Society., 2002. [14] A.H.V. Schapira, V.M. Mann, J.M. Cooper, D. Dexter, S.E. Daniel, P. Jenner, et al., Anatomic and disease specificity of NADH CoQ1 reductase (Complex I) deficiency in Parkinson's disease, J. Neurochem. 55 (1990) 2142e2145. [15] J.C. Fisher, W. Ruitenbeek, J.A. Berden, J.M. Trijbels, J.H. Veerkamp, A.M. Stadhouders, et al., Differential investigation of the capacity of succinate oxidation in human skeletal muscle, Clin. Chim. Acta 153 (1985) 23e26. rard, A. Rotig, J.M. Saudubray, et al., [16] P. Rustin, D. Chretien, T. Bourgeron, B. Ge Biochemical and molecular investigations in respiratory chain deficiencies, Clin. Chim. Acta 228 (1994) 35e51. [17] M.D. Baldissera, V.C. Rech, M. Grings, L.T. Gressler, R.A. Vaucher, C.E. Schwertz, et al., Enzymatic activities linked to cardiac energy metabolism of Trypanosoma evansi-infected rats and their possible functional correlations to disease pathogenesis, Parasitology 142 (2015) 1163e1170. [18] B. Halliwell, J.M.C. Gutteridge, Free Radicals in Biology and Medicine, fourth ed., Oxford University Press, New York, 2005. [19] J.M. Lawler, W. Song, S.R. Demaree, Hind limb unloading increases oxidative stress and disrupts antioxidant capacity in skeletal muscle, Free Radic. Biol. Med. 35 (2003) 9e16. [20] O.H. Lowry, N. Rosebrough, A.L. Farr, R.J. Randal, Protein measurement with a Folin phenol reagent, J. Biol. Chem. 193 (1951) 265e275. [21] S. Dey, U. Rajguru, D.C. Pathak, U.C. Goswami, Analysis of gill structure from a fresh water fish (Heteropneustes fossilis) exposed to bleached sulfite pulp mill effluents, Microsc. Microanal. 21 (2015) 385e391. [22] F. Xiang, S.Y. Ma, D.X. Zhang, Q. Zhang, Y.S. Huang, Tumor necrosis factor receptor-associated protein 1 improves hypoxia-impaired energy production in cardiomyocytes through increasing activity of cytochrome c oxidase subunit II, Int. J. Biochem. Cell Biol. 79 (2016) 239e248. [23] S. Gupta, V. Bhatia, J.J. Wen, Y. Wu, M.H. Huang, N.J. Garg, Trypanosoma cruzi infection disturbs mitochondrial membrane potential and ROS production rate in cardiomyocytes, Free Radic. Biol. Med. 47 (2009) 1414e1421. z, M.S.L. Presti, R. Fretes, C. Díaz, P. Pons, P.C. Baza n, et al., Chronic [24] A.L. Bae indeterminate phase of Chaga's disease: mitochondrial involvement in infection with two strains, Parasitology 140 (2013) 414e421. [25] E. Maranzana, G. Barbero, A.I. Falasca, G. Lenaz, M.L. Genova, Mitochondrial respiratory supercomplex association limits production of reactive oxygen species from complex I, Antioxid. Redox Signal 19 (2013) 1469e1480.
Contents lists available at ScienceDirect
Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
Inhibition of the mitochondrial respiratory chain in gills of Rhamdia quelen experimentally infected by Pseudomonas aeruginosa: Interplay with reactive oxygen species Matheus D. Baldissera a, *, Carine F. Souza b, Mateus Grings c, Belisa S. Parmeggiani c, Guilhian Leipnitz d, Karen L.S. Moreira e, Maria Izabel U.M. da Rocha e, Marcelo L. da Veiga e, Roberto C.V. Santos a, Lenita M. Stefani f, Bernardo Baldisserotto b, ** a
Department of Microbiology and Parasitology, Universidade Federal de Santa Maria (UFSM), Santa Maria, RS, Brazil Department of Physiology and Pharmacology, Universidade Federal de Santa Maria (UFSM), Santa Maria, RS, Brazil c ~o em Ci^ sicas da Saúde, Universidade Federal do Rio Grande do Sul s Graduaça gicas: Bioquímica, Instituto de Ci^ Programa de Po encias Biolo encias Ba (UFRGS), Porto Alegre, RS, Brazil d ~o em Ci^ sicas da Saúde, Universidade s Graduaça gicas: Bioquímica, Departamento de Bioquímica, Instituto de Ci^ Programa de Po encias Biolo encias Ba Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil e Department of Morphology, Universidade Federal de Santa Maria (UFSM), Santa Maria, RS, Brazil f , SC, Brazil Graduate School of Animal Science, Universidade do Estado de Santa Catarina (UDESC), Chapeco b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 31 March 2017 Received in revised form 10 April 2017 Accepted 11 April 2017 Available online 13 April 2017
It has long been recognized that there are several infectious diseases linked to the impairment of enzymatic complexes of the mitochondrial respiratory chain, with consequent production of reactive oxygen species (ROS), that contribute to disease pathogenesis. In this study, we determined whether the inhibition on mitochondrial respiratory chain might be considered a pathway involved in the production of ROS in gills of Rhamdia quelen experimentally infected by P. aeruginosa. The animals were divided into two groups with six fish each: uninfected (the negative control group) and infected (the positive control group). On day 7 post-infection (PI), animals were euthanized and the gills were collected to assess the activities of complexes I-III, II and IV of the respiratory chain, as well as ROS levels. The activities of complexes I-III, II and IV of the respiratory chain in gills decreased, while the ROS levels increased in infected compared to uninfected animals. Moreover, a significant negative correlation was found between enzymatic activity of the complexes I-III and IV related to ROS levels in P. aeruginosa infected animals, corroborating to our hypothesis that inhibition on complexes of respiratory chain leads to ROS formation. Also, microscopic severe gill damage and destruction of primary and secondary lamellae were observed in infected animals, with the presence of hyperplasia, leukocytic infiltration and telangiectasia. In summary, we have demonstrated, for the first time, that experimental infection by P. aeruginosa inhibits the activities of mitochondrial complexes of respiratory chain and, consequently, impairs the cellular energy homeostasis. Moreover, the inhibition on mitochondrial complexes I-III and IV are linked to the ROS production, contributing to disease pathogenesis. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Branchial tissue Cytochrome c oxidoreductase DCIP-oxidoreductase Cytochrome c oxidase Fish pathogen
1. Introduction Pseudomonas aeruginosa is a Gram-negative opportunist
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] [email protected] (B. Baldisserotto). http://dx.doi.org/10.1016/j.micpath.2017.04.017 0882-4010/© 2017 Elsevier Ltd. All rights reserved.
(M.D.
Baldissera),
pathogen widely distributed in aquaculture farms, responsible for considerable economic losses for fish producers [1]. This bacterium leads to the development of the so called red skin disease in fish, which occurs during a stressful condition, such as inappropriate handling or during transportation [2], affecting mainly the Nile tilapia (Oreochromis niloticus), Mozambique tilapia (O. mossambicus) [1] and the silver catfish (Rhamdia quelen) [3]. This disease is characterized by petechial hemorrhages, darkness of the skin, abdominal ascitis, ulcerative syndrome, rotten fish gills,
350
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septicemia and behavioral alterations [4,5], with severe histopathological lesions of the primary and secondary lamellae of gills [1]. Recently, a study conducted by Baldissera et al. [5] demonstrated that infections caused by P. aeruginosa leads to oxidative stress due to the excessive production of reactive oxygen species (ROS) as an attempt to control the disease, but the effects of ROS on mitochondrial synthesis of adenosine triphosphate (ATP) during this bacterial infection remains unknown. Mitochondria are vital intracellular organelles that accommodate a large amount of four different oxidative phosphorylation complexes (complexes I-IV) in their inner or cristae membranes, that are involved in biological and physiological energy for intracellular metabolic pathways [6]. These complexes accept and transfer electrons through oxidation-reduction reactions and translocation of protons across the inner membrane. Also, the proton efflux generated in the respiratory chain drives the formation of ATP from adenosine diphosphate (ADP), and inorganic phosphate through the enzyme ATP synthase, known as complex V [7]. Recently, studies have demonstrated that redox reactions are linked to dysfunctions on the electron transport chain due to the formation of ROS and superoxide anions, that are considered products of metabolic redox reactions [6,8]. Also, mitochondria are considered the primary sites of intracellular ROS formation, and depending on the formation rates, ROS may contribute to signaling events and can mediate mitochondrial dysfunction in pathologies through oxidative modifications of mitochondrial components [6]. During mitochondrial ATP synthesis, the main sites of oxidant formation in mitochondria include mitochondrial electron transport chain complexes I and III, as well as the transfer of electrons that generates superoxide radical. Moreover, mitochondrial targets of oxidant species include the enzyme succinate dehydrogenase (SDH), and the complexes I and IV of the respiratory chain. Mitochondrial involvement in diseases has been linked to its central role on ROS production and to the harmful effects of ROS on this organelle, as observed during other pathological conditions, such as parasitic infections [9]. According to Haegler et al. [10], impaired mitochondrial respiratory chain function results in an oxidative phosphorylation disorder and decreased ATP synthesis associated to cell death. In this sense, a recent study conducted by Garrabou et al. [11] demonstrated that sepsis is associated with mitochondrial dysfunction and impaired oxygen consumption, and consequently, inhibition of the mitochondrial complexes I, III and IV, that contributes to disease evolution. Based on these evidences, our hypothesis is that the impairment of mitochondrial complexes of the respiratory chain in gills of R. quelen experimentally infected by P. aeruginosa causes an increase on ROS production, that may contribute directly to disease pathogenesis. Therefore, the aim of this study was to evaluate whether an inhibition on complexes I-III, II and IV of the respiratory chain causes an increase on ROS levels in gills during P. aeruginosa infection. 2. Materials and methods 2.1. Animals and water quality variables Healthy fish were harvested for experimental purpose from a fish farm located at Rio Grande do Sul state (Brazil). The fish were transported in live condition and maintained in 250 L fiberglass tanks of continuous aeration with fresh water for 14 days under controlled water parameters, such as: temperature 21e23 C (maintained with air conditioner), pH 7.2e7.6 and dissolved oxygen 5.6e7.2 mg L1. Dissolved oxygen and temperature were measured with a YSI oxygen meter (Model Y5512, Ohio, USA). The pH was ~o Paulo, Brazil). measured using a DMPH-2 pH meter (Digimed, Sa
Total ammonia levels were determined according to Verdouw et al. [12] and un-ionized ammonia (NH3) levels were calculated according to Colt [13]. The animals were fed once a day with commercial fish feed. Water quality variables were maintained as follows: temperature 23 ± 1 C, pH 7.4 ± 0.05, dissolved oxygen at 6.86 ± 0.22 mg L1, total ammonia 0.93 ± 0.07 mg L1 and nonionized ammonia 0.004 ± 0.0003 mg L1. 2.2. Inoculum preparation The strain of P. aeruginosa (PAO1) was kindly donated by Professor Barbara H. Iglewski (Department of Microbiology and Immunology, School of Medicine and Dentistry, University of Rochester, Rochester, New York 14642). The bacterial culture was grown on MacConkey agar and Nutrient agar. The suspensions of PAO1 were washed twice in sterile saline (NaCl 0.9%), turbidity (OD600) adjusted to 0.8e1.0 (equivalent to 1.5 108 CFU mL1) and used for the infection model. 2.3. Animal model and experimental design Twelve juvenile silver catfish (63.6 ± 10 g; 23 ± 4 cm) were used as the experimental model for the assessment of the enzymatic activities of the respiratory chain complexes in the gills. The silver catfish were assigned into two groups with six animals each: uninfected animals (the negative control group); and experimentally infected animals (the positive control group) inoculated intramuscularly with 100 mL of P. aeruginosa strain PAO1 (108 CFU: OD600 ¼ 0.8e1.0) on the right latero-dorsal side of each fish, according to the protocol established by Baldissera et al. [3]. The negative control received the same volume of sterile saline by the same route. Fish were fed once daily to apparent satiation with commercial feed. Uneaten food, other residues and feces were removed 30 min after feeding. The methodology used in the experiment was approved by the Ethical and Animal Welfare Committee of the Universidade Federal de Santa Maria under protocol number 074/2014. 2.4. Sampling and tissue preparation On day 7 post-infection (PI), all animals were euthanized by spinal cord section according to the Ethics Committee recommendations, and the gills were collected and dissected in a glass over ice. In order to measure the activities involved in the electron transport chain and ROS levels, the gills were washed in SETH buffer (0.250 mM sucrose, 2 mM EDTA, 10 mM Trizma base, 50 UI/ mL heparin, pH 7.4) and homogenized (1:10 w/v) in the same SETH buffer with a Potter-Elvehjen glass homogenizer. The homogenate was centrifuged at 800g for 10 min at 4 C, and the supernatants were stored at 80 C until used. 2.5. Assessment of mitochondrial respiratory chain enzymatic complexes Enzymatic activities (complexes I-III, II and IV) of the mitochondrial respiratory chain were measured in branchial homogenates. The activity of NADH: cytochrome c oxidoreductase (complex I-III) was assayed in gills according to the method described by Schapira et al. [14]. The activity of succinate: DCIPoxidoreductase (complex II) was determined according to the method of Fischer et al. [15], and that of cytochrome c oxidase (complex IV) was measured according to Rustin et al. [16]. These methods were slightly modified, as described in details in a previous report by Baldissera et al. [17]. The activities of the respiratory chain complexes were calculated as nmol.min1.mg protein1.
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2.6. Branchial ROS levels The 20 ,7’-dichloroflurescein diacetate (DCF-DA) levels were determined as an index based on the amount of peroxide produced in the cellular components via DCFH production on the oxidation assay. This experimental method of analysis is based on the deacetylation of the DCFH-DA probe, that is subsequently oxidized by reactive species into DCF, a highly fluorescent compound [18]. The branchial ROS levels were determined by using the DCFH-DA method described by Lawler et al. [19]. The gills homogenate was added to a medium containing Tris-HCl buffer (10 mM; pH 7.4). The absorbance of the samples was measured by a spectrophotometer (wavelength of 522 nm). The ROS levels were expressed as fluorescence unit per mg of protein1 (x109). 2.7. Protein determination Protein determination was measured in the gills homogenate by the method of Lowry et al. [20] using bovine serum albumin as standard. 2.8. Histopathology Gill samples were fixed in 10% formaldehyde and submitted to histological sections (6 mm), and Hematoxylin-Eosin (HE) staining. All slides were examined by two pathologists in a double blinded manner to detect microarchitecture and lamellae alterations. 2.9. Statistical analysis Normality and homoscedasticity were analyzed through the Kolmogorov-Smirnov and Levene tests, respectively, and the data were transformed when necessary. Significant differences between groups were analyzed and detected by two tailed Student's t-test for independent samples. The differences were considered to be statistically significant at p < 0.05. The effect size (r2) was described and scored as follows: 0.1 (small), 0.1 to 0.3 (medium), and 0.5 (large). The Pearson correlation was used to verify the correlation between ROS levels and mitochondrial respiratory chain complex activities. The correlation coefficient (r) and the coefficient of determination (r2) were used to determine the intensity of the association. 3. Results 3.1. Mitochondrial respiratory chain complex activities Complex I-III (cytochrome c oxidoreductase) decreased 44% [t(10) ¼ 2.09; p < 0.05; r2 ¼ 0.38], complex II (DCIP-oxidoreductase) decreased 11% [t(10) ¼ 4.43; p < 0.001; r2 ¼ 0.79] and complex IV (cytochrome c oxidase) decreased 17% [t(10) ¼ 4.75; p < 0.001; r2 ¼ 0.88] in gills of animals infected by P. aeruginosa compared to uninfected animals (Fig. 1). 3.2. Branchial ROS levels ROS levels increased 180% [t(10) ¼ 6.19; p < 0.001; r2 ¼ 0.88] in gills of animals infected by P. aeruginosa compared to uninfected animals (Fig. 2). 3.3. Study correlation A significant negative correlation was observed between
Fig. 1. Mean and standard error of complex I-III (cytochrome c oxidoreductase) [A], complex II (DCIP-oxidoreductase) [B] and complex IV (cytochrome c oxidase) [C] activities in gills of silver catfish experimentally infected by Pseudomonas aeruginosa strain PAO1 compared to the uninfected control group on day 7 post-infection (PI). Bars with different letters are statistically different (p < 0.05; n ¼ 6 per group) according to the two-tailed Student's t-test for independent samples.
branchial ROS levels and branchial activity of complex I-III (r ¼ 0.947; r2 ¼ 0.887; p ¼ 0.004) and for branchial ROS levels and branchial activity of complex IV (r ¼ 0.926; r2 ¼ 0.922; p ¼ 0.002) (Fig. 3). No correlation was observed between branchial ROS levels and branchial activity of complex II (r ¼ 0.345; r2 ¼ 0.311; p ¼ 0.089) (data not shown).
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3.4. Histopathology The uninfected animals did not show any pathological alteration in gill tissue (Fig. 4A). Infected animals showed severe gill damage and destruction of the primary and secondary lamellae. Moreover, edema with epithelial hyperplasia, severe desquamation, epithelial lifting and telangiectasia were observed in the gills. Hyperplasia, leukocytic infiltration and dilation of the central venous sinus were observed in the secondary lamellae (Fig. 4B). 4. Discussion
Fig. 2. Mean and standard error of reactive oxygen species (ROS) in gills of silver catfish experimentally infected by Pseudomonas aeruginosa strain PAO1 compared to the uninfected control group on day 7 post-infection (PI). Bars with different letters are statistically different (p < 0.05; n ¼ 6 per group) according to the two-tailed Student's t-test for independent samples.
Fig. 3. Pearson's correlation between reactive oxygen species (ROS) levels and the activity of complex I-III (cytochrome c oxidoreductase) [A], and between reactive oxygen species (ROS) levels and the activity of complex IV (cytochrome c oxidase) in gills of silver catfish experimentally infected by Pseudomonas aeruginosa strain PAO1. The r (correlation coefficient), r2 (coefficient of determination) and p values were used to verify the intensity of the association between the variables and the level of significance.
The crucial involvement of mitochondria in branchial bioenergetic regulation, as well as in the balance of oxidant and antioxidant agents, suggest that these organelles are deeply involved in the pathophysiology of gills [21]. In this study, we demonstrated, for the first time, that P. aeruginosa infection impairs the activities of respiratory chain leading to increased ROS levels in gills, which may contribute to disease pathophysiology. Mitochondria are vital organelles to maintain biological functions of a cell by providing energy, playing an important role in cell survival [22]. However, under pathological conditions such as during infectious diseases, mitochondria release cell deathinducing molecules and contribute to the progression of pathology and cell death [17], in accordance to the observations of this present study. We found that complex I-III, II and IV activities were inhibited by P. aeruginosa infection, which may result in decreased availability of ATP and impairment of energy supply, in accordance to what was observed during Trypanosoma cruzi [23,24], and T. evansi [9,17] infections. According to these authors, impairment of respiratory chain enzymatic complexes contributes directly to pathophysiology of the disease, contributing to animal death. Of particular interest, a recent study conducted by Garrabou et al. [11] demonstrated that mitochondrial complexes I, III and IV were significantly inhibited during bacterial sepsis, leading to impaired oxygen supply and an oxidative stress frame with excessive ROS production, which contribute directly to disease pathogenesis. Thus, we can conclude that inhibition of the activities of mitochondrial complexes IeIII, II and IV caused by P. aeruginosa may contribute to decreased availability of ATP and impairment of energy supply, which is supported by the impairment on enzymes of the phosphotransfer network (adenylate kinase, pyruvate kinase, and creatine kinase), that are linked to the transfer of phosphoryl group between local synthesis and utilization of ATP [3]. Under pathologic conditions, diverse pathways result in excessive ROS production, including in the mitochondria, since this organelle is one of the main intracellular site of O2 generation [6]. In this study, we observed a significant increase of branchial ROS levels during P. aeruginosa infection, which may be linked to the inhibition of the enzymatic activities of mitochondrial complexes IIII and IV. According to Quijano et al. [6], the mitochondrial complexes I, III and IV are considered the main sites of oxidant formation in mitochondria, since the transfer of electrons to oxygen generates superoxide radical, which leads to secondary oxidant formation (anion superoxide) by dismutation reaction with metals or by reaction with nitric oxide, that is corroborated by significantly negative correlation between ROS formation and mitochondrial complex I-III and IV activities. Moreover, it is important to emphasize that ROS produced due to inhibition on mitochondrial complexes may also contribute to impairment of other complexes, such as complexes I and II of the respiratory chain [25]. Thus, the inhibition on mitochondrial complexes I-III and IV of respiratory chain may be considered an important pathway of ROS production, which may contribute to disease pathogenesis. In summary, we have demonstrated for the first time that
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Fig. 4. Gill histopathology of Rhamdia quelen experimentally infected by Pseudomonas aeruginosa. (A) Uninfected fish showed normal histology with normal distribution of cellular constituents and normal organization of primary and secondary lamellae. (B) Fish infected by P. aeruginosa showed severe gill damage and destruction of primary and secondary lamellae.
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