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Pseudomonas aeruginosa strain PA01 impairs enzymes of the phosphotransfer network in the gills of Rhamdia quelen
Veterinary Microbiology 201 (2017) 121–125
Contents lists available at ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Pseudomonas aeruginosa strain PA01 impairs enzymes of the phosphotransfer network in the gills of Rhamdia quelen Matheus D. Baldisseraa,* , Carine F. Souzab , Roberto C.V. Santosa , Lenita M. Stefanic, Karen Luise S. Moreirad , Marcelo L. da Veigad, Maria Izabel U.M. da Rochad , Bernardo Baldisserottob,* a
Department of Microbiology and Parasitology, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil Department of Physiology and Pharmacology, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil c Department of Animal Science, Universidade do Estado de Santa Catarina (UDESC), Chapecó, SC, Brazil d Department of Morphology, Universidade Federal de Santa Maria (UFSM), Santa Maria, RS, Brazil b
A R T I C L E I N F O
Article history: Received 21 December 2016 Received in revised form 13 January 2017 Accepted 14 January 2017 Keywords: Energy metabolism Pyruvate kinase Adenylate kinase Creatine kinase
A B S T R A C T
Integration of mitochondria with cytosolic ATP-consuming/ATP-sensing and substrate supply processes is critical for gills bioenergetics, since this tissue plays an important role in the respiratory energy metabolism. The effects of bacterial infection on gills remain poorly understood, limited only to histopathological analyses. Thus, the aim of this study was to investigate whether experimental infection by Pseudomonas aeruginosa strain PA01 alters the enzymes of the phosphoryltransfer network (adenylate kinase (AK), pyruvate kinase (PK) and cytosolic and mitochondrial creatine kinase (CK)) in gills of silver catfish (Rhamdia quelen). The animals were divided into two groups with six fish each: uninfected (negative control) and infected (positive control). On day 7 post-infection (PI), animals were euthanized and the gills collected. AK, PK, and cytosolic and mitochondrial CK activities in gills decreased in infected compared to uninfected animals. Also, severe gill damage and destruction in the primary and secondary lamellae was observed in the infected animals. Therefore, we have demonstrated, for the first time, that experimental infection by P. aeruginosa inhibits key enzymes linked to the production and utilization of metabolic energy in silver catfish, and consequently, impairs cellular energy homeostasis, which may contribute to disease pathogenesis. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Aquaculture plays an important role in food production worldwide, however bacterial diseases affect directly fish production, causing severe economic losses (Osorio et al., 2015). An important bacterial pathogen related to fish mortality is Pseudomonas aeruginosa (Mastan, 2013), a nonfermenting gram-negative bacilli, with large capacity to adapt and to survive with minimal requirements in unfavorable environmental conditions as soil, water, plant, animals and humans (Franzetti and Scarpelini, 2007). Infection by P. aeruginosa is very common in fish (Shama et al., 2000; Mastan, 2013), being characterized by petechial hemorrhage, skin darkness, abdominal ascitis, exophthalmia, ulcerative
* Corresponding authors. E-mail addresses: [email protected] (M.D. Baldissera), [email protected] (B. Baldisserotto). http://dx.doi.org/10.1016/j.vetmic.2017.01.016 0378-1135/© 2017 Elsevier B.V. All rights reserved.
syndrome, dropsy and gill rot (Thomas et al., 2014). The gills are the mostly affected organ by P. aeruginosa infection (Mastan, 2013), causing severe damage and destruction of the primary and secondary lamellae. The gills are related to ion regulation, and their lamellae are the primary sites for oxygen uptake in fish, playing an important role for respiratory energy metabolism (Sollid et al.., 2005; Nilsson et al., 2012). The maintenance of homeostasis between the release and uptake of energy is regulated by intracellular processes, and the proper functioning of the gills bioenergetic system depends on the production and delivery of energetic compounds in the gills, such as adenosine triphosphate (ATP). For this, we decided to evaluate the activities of enzymes involved in the transfer of phosphoryl group between local synthesis and utilization of ATP, such as adenylate kinase (AK), pyruvate kinase (PK) and creatine kinase (CK), that are also implicated in the regulation of KATP channels (Dzeja and Terzic, 2003). In tissues with high and sudden energy demand, such as the gills, these enzymes form the main
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pathway to secure efficient communication between subcellular compartments responsible for the production and utilization of metabolic energy (Dzeja and Terzic, 2003). Mitochondrial and cytosolic AK catalyzes reversible phosphotransfer between adenosine diphosphate (ADP) and ATP in the presence of adenosine monophosphate (AMP), being implicated in the processing of cellular signals associated with ATP utilization (Dzeja and Terzic, 1998), and damage to AK activity is linked to loss of osmoprotection (Gutierrez and Csonka, 1995). PK is a key enzyme of the glycolysis pathway that catalyzes the irreversible transphosphorylation of phosphoenolpyruvate (PEP) to ADP to form pyruvate and ATP (Wang et al., 2002) which has a fundamental role on the glycolytic pathway, the main route that provides energy to proper tissues functioning. Finally, CK catalyzes the reversible transfer of a phosphoryl group from ATP to ADP and creatine to produce phosphocreatine (PCr), being important in energy supply required for cell division, cell motility and ion homeostasis (Kuiper et al., 2009). Recently, studies have demonstrated that alterations on these enzymes contribute directly to disease pathophysiology during many infectious diseases, such as those caused by Trypanosoma evansi (Baldissera et al., 2015a,b) and Fasciola hepatica (Baldissera et al., 2015a,b). However, the activities of these enzymes during P. aeruginosa infection were not evaluated so far. Thus, due to the importance of energy metabolism to the gills, the aim of this study was to evaluate whether experimental infection by P. aeruginosa strain PA01 alters AK, PK and CK activities in gills of silver catfish, Rhamdia quelen. 2. Materials and methods 2.1. Fish harvesting, maintenance and water quality parameters Healthy fish were collected for experimental purpose from a fish farm located in Cruz Alta, Rio Grande do Sul, South of Brazil. Alive fish were transported and maintained in 250 L fiberglass tanks with continuous aeration under controlled water parameters (21–23 C, pH 7.2–7.6, dissolved oxygen levels: 5.6–7.2 mg L 1), in fresh water for 14 days. Dissolved oxygen and temperature were measured with a YSI oxygen meter (Model Y5512, Ohio, USA). The pH was measured using a DMPH-2 pH meter (Digimed, São Paulo, Brazil). Total ammonia levels were determined according to Verdouw et al. (1978) and un-ionized ammonia (NH3) levels were calculated according to Colt (2001). The animals were fed once a day with commercial fish feed at 3% of biomass of group, and the static water was substituted once a day at same period (08:00 at 09:00 h AM). The fish health was observed through evaluation the normal swim, normal alimentation and absence of lesions. 2.2. Inoculum confirmation The pathogen was confirmed through colony characteristics (metallic aspect and colony or culture media with green pigmentation). Moreover, some tests were performed: positive oxidase, catalase positive and lactose negative colonies, as well as in Triple Iron agar (non-fermenting glucose and in addition O+/F(using Hugh and Leifson media), that confirmed P. aeruginosa. 2.3. Inoculum preparation The strain of P. aeruginosa (PA01) was kindly donated by Prof. Barbara H. Iglewski, Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642. This pathogen was maintained frozen in glycerol and in skim milk until utilization. Prior to use, this microorganism was defrosted and seeded in Nutrient broth for
24 h, and after is seeded again in Nutrient agar. After, the bacterial culture was grown on MacConkey agar and Nutrient agar for use in this experimental model. The suspensions of PA01 were washed twice in sterile saline (NaCl 0.9%), turbidity (OD600) adjusted to 0.8–1.0 (equivalent to 108 CFU mL 1) and used for the infection model. 2.4. Animal model and experimental design Twelve juveniles silver catfish (63.6 10 g; 23 4 cm) were used as the experimental model to assess enzymatic activities of the phosphoryltransfer network in gills. Fish were assigned into two groups with six animals each: uninfected animals (negative control) and experimentally infected animals (positive control) inoculated intramuscularly with 100 mL of P. aeruginosa strain PA01 (107 colony forming unit/mL: OD600 = 0.8–1.0) on the right latero-dorsal side of each fish, according the protocol stablished by Thomas et al. (2014). The negative control received the same volume of sterile saline solution by the same route. Fish were fed once a day to satiation with commercial feed. Uneaten food, other residues and feces were removed 30 min after feeding. The methodology used in this experiment was approved by the Ethical and Animal Welfare Committee of the Universidade Federal de Santa Maria under protocol number 074/2014. 2.5. Sample collection and tissue preparation On day 7 post-infection (PI), gills were collected after animal euthanasia through spinal cord section, as recommended by the Ethics Committee. In order to measure enzymes from the phosphoryltransfer network, the gills were washed in SET buffer (0.32 M sucrose, 1 mM EGTA, 10 mM Tris–HCl, pH 7.4) and homogenized (1:10 w/v) in the same SET buffer with a PotterElvehjem glass homogenizer. The homogenate was centrifuged at 800 g for 10 min at 4 C. Part of this supernatant was used to evaluate AK activity; the pellet was discarded and the supernatant was once again centrifuged at 10,000 g for 15 min at 4 C. The supernatant containing cytosol and other cellular components, as endoplasmatic reticulum, was collected for determination of PK and cytosolic CK activities. The pellet, containing mitochondria, was washed twice with the same SET buffer, resuspended in 100 mM Trizma and 15 mM MgSO4 buffer (pH 7.5) to evaluate mitochondrial CK activity. The supernatants were stored for no more than 1 week at 80 C only when the assay was not carried out immediately. 2.6. AK, PK and CK activities and protein determination Adenylate kinase activity (AK) was measured with a coupled enzymatic assay using hexokinase (HK) and glucose 6-phosphate dehydrogenase (G6PD) according to Dzeja et al. (1999). The reaction mixture contained 100 mM KCl, 20 mM HEPES, 20 mM glucose, 4 mM MgCl2, 2 mM NADP+, 1 mM EDTA, 4.5 U/mL of HK, 2 U/mL of G6PD and 20 mL of homogenate. The reaction was initiated by the addition of 2 mM ADP and the reduction of NADP+ was evaluated at 340 nm for 3 min in a spectrophotometer. ADP, NADP+, G6PD, and HK were dissolved in milli-Q water. The concentration of the reagents and assay time (3 min) were chosen to assure the linearity of the reaction. The results were expressed in pmol of ATP formed per min per mg of protein. Pyruvate kinase activity (PK) was assayed essentially as described by Leong et al. (1981). The incubation medium consisted of 0.1 M Tris/HCl buffer, pH 7.5, 10 mM MgCl2, 0.16 mM NADH, 75 mM KCl, 5.0 mM ADP, 7 U L-lactate dehydrogenase, 0.1% (v/v) Triton X-100, and 20 mL of the mitochondria-free supernatant in a final volume of 500 mL. After 10 min of pre-incubation at 37 C, the
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reaction was started with the addition of 1 mM phosphoenol pyruvate. All assays were performed in duplicate at 25 C. Results were expressed as pmol of pyruvate formed per min per mg of protein. Creatine kinase activity (CK) was assayed in the reaction mixture containing the following final concentrations: 65 mM Tris–HCl buffer, pH 7.5, 7 mM phosphocreatine, 9 mM MgSO4, and 1 mg of protein in a final volume of 150 mL. After 10 min of preincubation at 37 C, the reaction was started by the addition of 0.3 mmol of ADP and stopped after 10 min by the addition of 1 mmol of r-hydroxymercuribenzoic acid. Concentration of the reagents and the incubation time were chosen to assure linearity of the enzymatic reaction. Appropriate controls were carried out to measure chemical hydrolysis of phosphocreatine. The creatine was estimated according to the colorimetric method of Hughes (1962). The color was developed by the addition of 0.1 mL of 2% a-naphthol and 0.1 mL of 0.05% diacetyl in a final volume of 1 mL, and read after 20 min at 540 nm. Results were expressed as nmol of creatine formed per min per mg of protein. Protein content in gills homogenate was determined by the method of Bradford (1976), using serum bovine albumin as the standard. 2.7. Feed consumption In order to evaluate the feed consumption, we used a specific protocol for R. quelen, recently published in details by Souza et al. (2015). 2.8. Gills histopathology Samples of gills were fixed in 10% formaldehyde solution and submitted to histological routine. Histological section (6 mm) were cut and stained with hematoxylin-eosin (HE). All slides were examined by two pathologists in a double blinded manner to detect microarchitecture and gills alterations, in magnification of 40 (objective lens) using an optical microscope (Zeiss, model Primo Star PB-4138-L).
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animals with P. aeruginosa when compared to uninfected animals, that is compatible to loss of appetite. 3.3. AK, PK and CK activities AK activity decreased 66.6% [t(10) = 9.55; p < 0.001; r2 = 0.96] in gills of animals infected by P. aeruginosa compared to the uninfected control group (Fig. 1A). PK activity decreased 58% [t (10) = 7.19; p < 0.001; r2 = 0.76] in gills of animals infected by P. aeruginosa compared to the uninfected control group (Fig. 1B). Cytosolic CK activity decreased 62.5% [t(10) = 8.60; p < 0.001; r2 = 0.95] and mitochondrial CK activity decreased 32% [t (10) = 5.60; p < 0.001; r2 = 0.65] in gills of animals infected by P. aeruginosa compared to the uninfected control group (Fig. 2). 3.4. Gills histopathology The uninfected animals did not show pathological alteration in gill tissue (Fig. 3A). Infected animals showed severe gill damage and destruction in the primary and secondary lamellae. Also, edema with epithelial hyperplasia, severe desquamation, epithelial lifting and telangiectasia was observed in the gills. In the secondary lamellae were observed hyperplasia, leukocytic infiltration and dilation of the central venous sinus. 4. Discussion The present study is novel since it evaluates important alterations in the phosphoryltransfer network in gills of animals experimentally infected by P. aeruginosa strain PA01. Our findings clearly shows the inhibition of the AK, PK and the cytosolic and mitochondrial CK, indicating an imbalance of energy homeostasis in gills of infected animals.
2.9. Statistical analyses Normality and homoscedasticity were analyzed through the Shapiro-Wilk and Levene test, respectively. Significant differences between groups were analyzed and detected by two tailed Student’s t test for independent samples. Logarithmic transformation of the data was performed when necessary. 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). 3. Results 3.1. Water quality parameters The water quality parameters remained stable within acceptable limits throughout the experimental period. The temperature was maintained at 23 1 C, the pH 7.4 0.05 and the dissolved oxygen at 6.86 0.22 mg L 1, total ammonia 0.93 0.07 mg L 1 and non-ionized ammonia 0.004 0.0003 mg L 1. 3.2. Course of infection None infected animal with P. aeruginosa died during the experimental period. From fifth day PI, a significantly decrease of feed consumption (approximately 40%) was verified in the infected
Fig. 1. Mean and standard deviation of adenylate kinase (AK) and pyruvate kinase (PK) activities in silver catfish experimentally infected by Pseudomonas aeruginosa strain PA01 compared to the uninfected control group on day 7 post-infection (PI). Groups with *** are statistically different (p < 0.001; n = 6 per group) using the twotailed Student’s t-test for independent samples.
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Fig. 2. Mean and standard deviation of cytosolic and mitochondrial CK activities in silver catfish experimentally infected by Pseudomonas aeruginosa strain PA01 compared to the uninfected control group on day 7 post-infection (PI). Groups with *** are statistically different (p < 0.001; n = 6 per group) using the two-tailed Student’s t-test for independent samples.
The systemic integration of energetic and metabolic signaling networks ensure cellular energy homeostasis and an adequate response to stress conditions, such as those caused by infectious diseases (Saks et al., 2006). Thus, the measurement of phosphoryltransfer network provides new perspectives for understanding the alterations in energy metabolism due to diseases. According to Dzeja et al. (2000), the coupling of spatially separated intracellular ATP-producing and ATP-consuming is a fundamental process to the
proper energetic metabolism physiology. Therefore, an enzymatic network is necessary, catalyzed by AK, CK, and in special by PK, because they support high-energy phosphoryltransfer between ATP-generating and ATP-consuming process (Dzeja and Terzic, 1998). We observed that AK, PK and cytosolic and mitochondrial CK activities were inhibited by P. aeruginosa infection, which may result in decreased availability of ATP and impairment of energy supply. These enzymes are intimately related in such a way, that a decrease in one enzyme may lead to an increase of the other, a mechanism known as energy compensation. This mechanism contributes to efficient intracellular energetic communication, maintaining the balance between cellular ATP consumption and production in attempt to preserve the energetic homeostasis (Alekssev et al., 2012). On the other hand, a concomitant decrease on these enzymes in the gills was observed in infected animals with P. aeruginosa strain PA01, contributing directly to the impairment of synthesis and release of ATP on the gills, which may be related to disease pathophysiology. Recently, a study conducted by Baldissera et al. (2015a,b) demonstrated that inhibition on hepatic, cardiac and cerebral AK, PK and CK activities contributes directly to disease pathophysiology during T. evansi infection. Recently, studies have demonstrated that impairment on enzymes of energy metabolism is linked to the appearance of clinical signs and disease evolution, such as observed during other infectious diseases (Baldissera et al., 2015a,b). Impairment on AK activity has been associated to disturbances on cellular functions, loss of osmoprotection activity, as well as focal hemorrhage, one of the main clinical symptom caused by P. aeruginosa (Toren et al., 1994). Also, inhibition of CK activity may lead to an important dysfunction in fish respiration, since mitochondrial CK activity is a modulator of the metabolic potential localized in mitochondria connected to the respiratory chain (Brdicza et al., 1994). In summary, we have demonstrated, for the first time, that experimental infection by P. aeruginosa inhibits key enzymes linked to the production and utilization of metabolic energy in silver catfish, and consequently, impairs the cellular energy homeostasis, contributing to disease pathogenesis.
Fig. 3. Gill histopathology of Rhamdia quelen experimentally infected by Pseudomonas aeruginosa. (A) Uninfected fish showed normal histology with regular distribution of cellular constituents and regular 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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Kuiper, J.W., van Horssen, R., Oerlemans, F., Peters, W., van Dommelen, M.M., te Lindert, M.M., ten Hagen, T.L., Janssen, E., Fransen, J.A., Wieringa, B., 2009. Local ATP generation by brain-type creatine kinase (CK-B) facilitates cell motility. PLoS One 4, e5030. Leong, S.F., Lai, J.K.F., Lim, L., Clark, J.B., 1981. Energy-metabolism in brain regions of adult and aging rats. J. Neurochem. 37, 1548–1556. Mastan, A.S., 2013. Pseudomonas septicemia in Labeo rohita (Ham.) and Cyprinus carpio (Linn.) in Andhra Pradesh-natural occurrence and artificial challenge. Int. J. Pharm. Sci. 5, 564–568. Nilsson, G.E., Dymowska, A., Stecyk, J.A.W., 2012. New insights into the plasticity of gill structure. Respir. Physiol. Neurobiol. 184, 214–222. Osorio, C.R., Rivas, A.J., Balado, M., Fuentes-Monteverde, J.C., Rodríguez, J., Jiménez, C., Lemos, M.L., Waldor, M.K., 2015. A transmissible plasmid-borne pathogenicity island confers piscibactin biosynthesis in the fish pathogen Photobacterium damselae subsp. piscicida. Appl. Environ. Microbiol. 81, 5867– 5879. Saks, V., Dzeja, P., Schlattner, U., Vendelin, M., Terzic, A., Wallimann, T., 2006. Cardiac system bioenergetics basis of Frank-Starling law. J. Physiol. 571, 253–273. Shama, S., Brandão, D.A., de Vargas, A.C., da Costa, M.M., Pedrozo, A.F., 2000. Pathogenic bacterials in kidney and external lesions of jundiá (Rhamdia quelen) in semi-intensive fish culture. Cienc. Rural 30, 293–298. Sollid, J., Kjernsli, A., De Angelis, P.M., Rohr, A.K., Nilsson, G.E., 2005. Cell proliferation and gill morphology in anoxic crucian carp. Am. J. Physiol. Regul. Integr. Comp. Physiol. 289, R1196–R1201. Souza, C.F., Salbego, J., Gressler, L.T., Golombieski, J.I., Ferst, J.G., Cunha, M.A., Heinzmann, B.M., Caron, B.O., Glanzner, W.G., Gonçalves, P.B.D., Baldisserotto, B., 2015. Neotrop. Ichthyol. 13, 707–714. Thomas, J., Thanigaivel, S., Vijayakumar, S., Acharya, D., Seelan, T.S., Mukherjee, A., Chandrasekaran, N., 2014. Pathogenecity of Pseudomonas aeruginosa in Oreochromis mossambicus and treatment using lime oil nanoemulsion. Colloids Surf. B: Biointerfaces 116, 372–377. Toren, A., Brok-Simoni, F., Ben-Bassat, I., Holtzman, F., Mandel, M., Neumann, Y., Ramot, B., Rechavi, G., Kende, G., 1994. Congenital hemolytic anaemia associated with adenylate kinase deficiency. Br. J. Haemotol. 87, 376–380. Verdouw, H., Vanechteld, C.J.A., Deckkers, E.M.J., 1978. Ammonia determinations based on indophenol formation with sodium salicylate. Water Res. 12, 399–402. Wang, H., Chu, W., Das, S.K., Ren, Q., Hasstedt, S.J., Elbein, S.C., 2002. Liver pyruvate kinase polymorphisms are associated with type 2 diabetes in Northern European Caucasians. Diabetes 51, 2861–2865.
Contents lists available at ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Pseudomonas aeruginosa strain PA01 impairs enzymes of the phosphotransfer network in the gills of Rhamdia quelen Matheus D. Baldisseraa,* , Carine F. Souzab , Roberto C.V. Santosa , Lenita M. Stefanic, Karen Luise S. Moreirad , Marcelo L. da Veigad, Maria Izabel U.M. da Rochad , Bernardo Baldisserottob,* a
Department of Microbiology and Parasitology, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil Department of Physiology and Pharmacology, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil c Department of Animal Science, Universidade do Estado de Santa Catarina (UDESC), Chapecó, SC, Brazil d Department of Morphology, Universidade Federal de Santa Maria (UFSM), Santa Maria, RS, Brazil b
A R T I C L E I N F O
Article history: Received 21 December 2016 Received in revised form 13 January 2017 Accepted 14 January 2017 Keywords: Energy metabolism Pyruvate kinase Adenylate kinase Creatine kinase
A B S T R A C T
Integration of mitochondria with cytosolic ATP-consuming/ATP-sensing and substrate supply processes is critical for gills bioenergetics, since this tissue plays an important role in the respiratory energy metabolism. The effects of bacterial infection on gills remain poorly understood, limited only to histopathological analyses. Thus, the aim of this study was to investigate whether experimental infection by Pseudomonas aeruginosa strain PA01 alters the enzymes of the phosphoryltransfer network (adenylate kinase (AK), pyruvate kinase (PK) and cytosolic and mitochondrial creatine kinase (CK)) in gills of silver catfish (Rhamdia quelen). The animals were divided into two groups with six fish each: uninfected (negative control) and infected (positive control). On day 7 post-infection (PI), animals were euthanized and the gills collected. AK, PK, and cytosolic and mitochondrial CK activities in gills decreased in infected compared to uninfected animals. Also, severe gill damage and destruction in the primary and secondary lamellae was observed in the infected animals. Therefore, we have demonstrated, for the first time, that experimental infection by P. aeruginosa inhibits key enzymes linked to the production and utilization of metabolic energy in silver catfish, and consequently, impairs cellular energy homeostasis, which may contribute to disease pathogenesis. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Aquaculture plays an important role in food production worldwide, however bacterial diseases affect directly fish production, causing severe economic losses (Osorio et al., 2015). An important bacterial pathogen related to fish mortality is Pseudomonas aeruginosa (Mastan, 2013), a nonfermenting gram-negative bacilli, with large capacity to adapt and to survive with minimal requirements in unfavorable environmental conditions as soil, water, plant, animals and humans (Franzetti and Scarpelini, 2007). Infection by P. aeruginosa is very common in fish (Shama et al., 2000; Mastan, 2013), being characterized by petechial hemorrhage, skin darkness, abdominal ascitis, exophthalmia, ulcerative
* Corresponding authors. E-mail addresses: [email protected] (M.D. Baldissera), [email protected] (B. Baldisserotto). http://dx.doi.org/10.1016/j.vetmic.2017.01.016 0378-1135/© 2017 Elsevier B.V. All rights reserved.
syndrome, dropsy and gill rot (Thomas et al., 2014). The gills are the mostly affected organ by P. aeruginosa infection (Mastan, 2013), causing severe damage and destruction of the primary and secondary lamellae. The gills are related to ion regulation, and their lamellae are the primary sites for oxygen uptake in fish, playing an important role for respiratory energy metabolism (Sollid et al.., 2005; Nilsson et al., 2012). The maintenance of homeostasis between the release and uptake of energy is regulated by intracellular processes, and the proper functioning of the gills bioenergetic system depends on the production and delivery of energetic compounds in the gills, such as adenosine triphosphate (ATP). For this, we decided to evaluate the activities of enzymes involved in the transfer of phosphoryl group between local synthesis and utilization of ATP, such as adenylate kinase (AK), pyruvate kinase (PK) and creatine kinase (CK), that are also implicated in the regulation of KATP channels (Dzeja and Terzic, 2003). In tissues with high and sudden energy demand, such as the gills, these enzymes form the main
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pathway to secure efficient communication between subcellular compartments responsible for the production and utilization of metabolic energy (Dzeja and Terzic, 2003). Mitochondrial and cytosolic AK catalyzes reversible phosphotransfer between adenosine diphosphate (ADP) and ATP in the presence of adenosine monophosphate (AMP), being implicated in the processing of cellular signals associated with ATP utilization (Dzeja and Terzic, 1998), and damage to AK activity is linked to loss of osmoprotection (Gutierrez and Csonka, 1995). PK is a key enzyme of the glycolysis pathway that catalyzes the irreversible transphosphorylation of phosphoenolpyruvate (PEP) to ADP to form pyruvate and ATP (Wang et al., 2002) which has a fundamental role on the glycolytic pathway, the main route that provides energy to proper tissues functioning. Finally, CK catalyzes the reversible transfer of a phosphoryl group from ATP to ADP and creatine to produce phosphocreatine (PCr), being important in energy supply required for cell division, cell motility and ion homeostasis (Kuiper et al., 2009). Recently, studies have demonstrated that alterations on these enzymes contribute directly to disease pathophysiology during many infectious diseases, such as those caused by Trypanosoma evansi (Baldissera et al., 2015a,b) and Fasciola hepatica (Baldissera et al., 2015a,b). However, the activities of these enzymes during P. aeruginosa infection were not evaluated so far. Thus, due to the importance of energy metabolism to the gills, the aim of this study was to evaluate whether experimental infection by P. aeruginosa strain PA01 alters AK, PK and CK activities in gills of silver catfish, Rhamdia quelen. 2. Materials and methods 2.1. Fish harvesting, maintenance and water quality parameters Healthy fish were collected for experimental purpose from a fish farm located in Cruz Alta, Rio Grande do Sul, South of Brazil. Alive fish were transported and maintained in 250 L fiberglass tanks with continuous aeration under controlled water parameters (21–23 C, pH 7.2–7.6, dissolved oxygen levels: 5.6–7.2 mg L 1), in fresh water for 14 days. Dissolved oxygen and temperature were measured with a YSI oxygen meter (Model Y5512, Ohio, USA). The pH was measured using a DMPH-2 pH meter (Digimed, São Paulo, Brazil). Total ammonia levels were determined according to Verdouw et al. (1978) and un-ionized ammonia (NH3) levels were calculated according to Colt (2001). The animals were fed once a day with commercial fish feed at 3% of biomass of group, and the static water was substituted once a day at same period (08:00 at 09:00 h AM). The fish health was observed through evaluation the normal swim, normal alimentation and absence of lesions. 2.2. Inoculum confirmation The pathogen was confirmed through colony characteristics (metallic aspect and colony or culture media with green pigmentation). Moreover, some tests were performed: positive oxidase, catalase positive and lactose negative colonies, as well as in Triple Iron agar (non-fermenting glucose and in addition O+/F(using Hugh and Leifson media), that confirmed P. aeruginosa. 2.3. Inoculum preparation The strain of P. aeruginosa (PA01) was kindly donated by Prof. Barbara H. Iglewski, Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642. This pathogen was maintained frozen in glycerol and in skim milk until utilization. Prior to use, this microorganism was defrosted and seeded in Nutrient broth for
24 h, and after is seeded again in Nutrient agar. After, the bacterial culture was grown on MacConkey agar and Nutrient agar for use in this experimental model. The suspensions of PA01 were washed twice in sterile saline (NaCl 0.9%), turbidity (OD600) adjusted to 0.8–1.0 (equivalent to 108 CFU mL 1) and used for the infection model. 2.4. Animal model and experimental design Twelve juveniles silver catfish (63.6 10 g; 23 4 cm) were used as the experimental model to assess enzymatic activities of the phosphoryltransfer network in gills. Fish were assigned into two groups with six animals each: uninfected animals (negative control) and experimentally infected animals (positive control) inoculated intramuscularly with 100 mL of P. aeruginosa strain PA01 (107 colony forming unit/mL: OD600 = 0.8–1.0) on the right latero-dorsal side of each fish, according the protocol stablished by Thomas et al. (2014). The negative control received the same volume of sterile saline solution by the same route. Fish were fed once a day to satiation with commercial feed. Uneaten food, other residues and feces were removed 30 min after feeding. The methodology used in this experiment was approved by the Ethical and Animal Welfare Committee of the Universidade Federal de Santa Maria under protocol number 074/2014. 2.5. Sample collection and tissue preparation On day 7 post-infection (PI), gills were collected after animal euthanasia through spinal cord section, as recommended by the Ethics Committee. In order to measure enzymes from the phosphoryltransfer network, the gills were washed in SET buffer (0.32 M sucrose, 1 mM EGTA, 10 mM Tris–HCl, pH 7.4) and homogenized (1:10 w/v) in the same SET buffer with a PotterElvehjem glass homogenizer. The homogenate was centrifuged at 800 g for 10 min at 4 C. Part of this supernatant was used to evaluate AK activity; the pellet was discarded and the supernatant was once again centrifuged at 10,000 g for 15 min at 4 C. The supernatant containing cytosol and other cellular components, as endoplasmatic reticulum, was collected for determination of PK and cytosolic CK activities. The pellet, containing mitochondria, was washed twice with the same SET buffer, resuspended in 100 mM Trizma and 15 mM MgSO4 buffer (pH 7.5) to evaluate mitochondrial CK activity. The supernatants were stored for no more than 1 week at 80 C only when the assay was not carried out immediately. 2.6. AK, PK and CK activities and protein determination Adenylate kinase activity (AK) was measured with a coupled enzymatic assay using hexokinase (HK) and glucose 6-phosphate dehydrogenase (G6PD) according to Dzeja et al. (1999). The reaction mixture contained 100 mM KCl, 20 mM HEPES, 20 mM glucose, 4 mM MgCl2, 2 mM NADP+, 1 mM EDTA, 4.5 U/mL of HK, 2 U/mL of G6PD and 20 mL of homogenate. The reaction was initiated by the addition of 2 mM ADP and the reduction of NADP+ was evaluated at 340 nm for 3 min in a spectrophotometer. ADP, NADP+, G6PD, and HK were dissolved in milli-Q water. The concentration of the reagents and assay time (3 min) were chosen to assure the linearity of the reaction. The results were expressed in pmol of ATP formed per min per mg of protein. Pyruvate kinase activity (PK) was assayed essentially as described by Leong et al. (1981). The incubation medium consisted of 0.1 M Tris/HCl buffer, pH 7.5, 10 mM MgCl2, 0.16 mM NADH, 75 mM KCl, 5.0 mM ADP, 7 U L-lactate dehydrogenase, 0.1% (v/v) Triton X-100, and 20 mL of the mitochondria-free supernatant in a final volume of 500 mL. After 10 min of pre-incubation at 37 C, the
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reaction was started with the addition of 1 mM phosphoenol pyruvate. All assays were performed in duplicate at 25 C. Results were expressed as pmol of pyruvate formed per min per mg of protein. Creatine kinase activity (CK) was assayed in the reaction mixture containing the following final concentrations: 65 mM Tris–HCl buffer, pH 7.5, 7 mM phosphocreatine, 9 mM MgSO4, and 1 mg of protein in a final volume of 150 mL. After 10 min of preincubation at 37 C, the reaction was started by the addition of 0.3 mmol of ADP and stopped after 10 min by the addition of 1 mmol of r-hydroxymercuribenzoic acid. Concentration of the reagents and the incubation time were chosen to assure linearity of the enzymatic reaction. Appropriate controls were carried out to measure chemical hydrolysis of phosphocreatine. The creatine was estimated according to the colorimetric method of Hughes (1962). The color was developed by the addition of 0.1 mL of 2% a-naphthol and 0.1 mL of 0.05% diacetyl in a final volume of 1 mL, and read after 20 min at 540 nm. Results were expressed as nmol of creatine formed per min per mg of protein. Protein content in gills homogenate was determined by the method of Bradford (1976), using serum bovine albumin as the standard. 2.7. Feed consumption In order to evaluate the feed consumption, we used a specific protocol for R. quelen, recently published in details by Souza et al. (2015). 2.8. Gills histopathology Samples of gills were fixed in 10% formaldehyde solution and submitted to histological routine. Histological section (6 mm) were cut and stained with hematoxylin-eosin (HE). All slides were examined by two pathologists in a double blinded manner to detect microarchitecture and gills alterations, in magnification of 40 (objective lens) using an optical microscope (Zeiss, model Primo Star PB-4138-L).
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animals with P. aeruginosa when compared to uninfected animals, that is compatible to loss of appetite. 3.3. AK, PK and CK activities AK activity decreased 66.6% [t(10) = 9.55; p < 0.001; r2 = 0.96] in gills of animals infected by P. aeruginosa compared to the uninfected control group (Fig. 1A). PK activity decreased 58% [t (10) = 7.19; p < 0.001; r2 = 0.76] in gills of animals infected by P. aeruginosa compared to the uninfected control group (Fig. 1B). Cytosolic CK activity decreased 62.5% [t(10) = 8.60; p < 0.001; r2 = 0.95] and mitochondrial CK activity decreased 32% [t (10) = 5.60; p < 0.001; r2 = 0.65] in gills of animals infected by P. aeruginosa compared to the uninfected control group (Fig. 2). 3.4. Gills histopathology The uninfected animals did not show pathological alteration in gill tissue (Fig. 3A). Infected animals showed severe gill damage and destruction in the primary and secondary lamellae. Also, edema with epithelial hyperplasia, severe desquamation, epithelial lifting and telangiectasia was observed in the gills. In the secondary lamellae were observed hyperplasia, leukocytic infiltration and dilation of the central venous sinus. 4. Discussion The present study is novel since it evaluates important alterations in the phosphoryltransfer network in gills of animals experimentally infected by P. aeruginosa strain PA01. Our findings clearly shows the inhibition of the AK, PK and the cytosolic and mitochondrial CK, indicating an imbalance of energy homeostasis in gills of infected animals.
2.9. Statistical analyses Normality and homoscedasticity were analyzed through the Shapiro-Wilk and Levene test, respectively. Significant differences between groups were analyzed and detected by two tailed Student’s t test for independent samples. Logarithmic transformation of the data was performed when necessary. 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). 3. Results 3.1. Water quality parameters The water quality parameters remained stable within acceptable limits throughout the experimental period. The temperature was maintained at 23 1 C, the pH 7.4 0.05 and the dissolved oxygen at 6.86 0.22 mg L 1, total ammonia 0.93 0.07 mg L 1 and non-ionized ammonia 0.004 0.0003 mg L 1. 3.2. Course of infection None infected animal with P. aeruginosa died during the experimental period. From fifth day PI, a significantly decrease of feed consumption (approximately 40%) was verified in the infected
Fig. 1. Mean and standard deviation of adenylate kinase (AK) and pyruvate kinase (PK) activities in silver catfish experimentally infected by Pseudomonas aeruginosa strain PA01 compared to the uninfected control group on day 7 post-infection (PI). Groups with *** are statistically different (p < 0.001; n = 6 per group) using the twotailed Student’s t-test for independent samples.
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Fig. 2. Mean and standard deviation of cytosolic and mitochondrial CK activities in silver catfish experimentally infected by Pseudomonas aeruginosa strain PA01 compared to the uninfected control group on day 7 post-infection (PI). Groups with *** are statistically different (p < 0.001; n = 6 per group) using the two-tailed Student’s t-test for independent samples.
The systemic integration of energetic and metabolic signaling networks ensure cellular energy homeostasis and an adequate response to stress conditions, such as those caused by infectious diseases (Saks et al., 2006). Thus, the measurement of phosphoryltransfer network provides new perspectives for understanding the alterations in energy metabolism due to diseases. According to Dzeja et al. (2000), the coupling of spatially separated intracellular ATP-producing and ATP-consuming is a fundamental process to the
proper energetic metabolism physiology. Therefore, an enzymatic network is necessary, catalyzed by AK, CK, and in special by PK, because they support high-energy phosphoryltransfer between ATP-generating and ATP-consuming process (Dzeja and Terzic, 1998). We observed that AK, PK and cytosolic and mitochondrial CK activities were inhibited by P. aeruginosa infection, which may result in decreased availability of ATP and impairment of energy supply. These enzymes are intimately related in such a way, that a decrease in one enzyme may lead to an increase of the other, a mechanism known as energy compensation. This mechanism contributes to efficient intracellular energetic communication, maintaining the balance between cellular ATP consumption and production in attempt to preserve the energetic homeostasis (Alekssev et al., 2012). On the other hand, a concomitant decrease on these enzymes in the gills was observed in infected animals with P. aeruginosa strain PA01, contributing directly to the impairment of synthesis and release of ATP on the gills, which may be related to disease pathophysiology. Recently, a study conducted by Baldissera et al. (2015a,b) demonstrated that inhibition on hepatic, cardiac and cerebral AK, PK and CK activities contributes directly to disease pathophysiology during T. evansi infection. Recently, studies have demonstrated that impairment on enzymes of energy metabolism is linked to the appearance of clinical signs and disease evolution, such as observed during other infectious diseases (Baldissera et al., 2015a,b). Impairment on AK activity has been associated to disturbances on cellular functions, loss of osmoprotection activity, as well as focal hemorrhage, one of the main clinical symptom caused by P. aeruginosa (Toren et al., 1994). Also, inhibition of CK activity may lead to an important dysfunction in fish respiration, since mitochondrial CK activity is a modulator of the metabolic potential localized in mitochondria connected to the respiratory chain (Brdicza et al., 1994). In summary, we have demonstrated, for the first time, that experimental infection by P. aeruginosa inhibits key enzymes linked to the production and utilization of metabolic energy in silver catfish, and consequently, impairs the cellular energy homeostasis, contributing to disease pathogenesis.
Fig. 3. Gill histopathology of Rhamdia quelen experimentally infected by Pseudomonas aeruginosa. (A) Uninfected fish showed normal histology with regular distribution of cellular constituents and regular 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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