Received: 1 September 2017 | | Revised: 19 September 2017 Accepted: 24 September 2017 DOI: 10.1111/jfd.12746 ORIGINAL ARTICLE Aeromonas caviae inhibits hepatic enzymes of the phosphotransfer network in experimentally infected silver catfish: Impairment on bioenergetics M D Baldissera1 Veiga3 | C F Souza2 | C M Verdi1 | K L M dos Santos3 | M L Da | M I U M da Rocha3 | R C V Santos1 | B S Vizzotto4 | B Baldisserotto2 1 Department of Microbiology and Parasitology, Universidade Federal de Santa Maria, Santa Maria, Brazil Abstract Several studies have been demonstrated that phosphotransfer network, through the 2 Department of Physiology and Pharmacology, Universidade Federal de Santa Maria, Santa Maria, Brazil 3 adenylate kinase (AK) and pyruvate kinase (PK) activities, allows for new perspectives leading to understanding of disease conditions associated with disturbances in Department of Morphology, Universidade Federal de Santa Maria (UFSM), Santa Maria, Brazil energy metabolism, metabolic monitoring and signalling. In this sense, the aim of 4 alters hepatic AK and PK activities of silver catfish Rhamdia quelen. Hepatic AK and Laboratory of Molecular Biology, Centro Universitario Franciscano, Santa Maria, Brazil this study was to evaluate whether experimental infection by Aeromonas caviae PK activities decreased in infected animals compared to uninfected animals, as well as the hepatic adenosine triphosphate (ATP) levels. Also, a severe hepatic damage Correspondence M D Baldissera, Department of Microbiology and Parasitology, Universidade Federal de Santa Maria, Santa Maria, Brazil. Email: matheusd.biomed@yahoo.com.br and B Baldisserotto, Department of Physiology and Pharmacology, Universidade Federal de Santa Maria, Santa Maria, Brazil. Email: bbaldisserotto@hotmail.com was observed in the infected animals due to the presence of dilation and congestion of vessels, degeneration of hepatocytes and loss of liver parenchyma architecture and sinusoidal structure. Therefore, we have demonstrated, for the first time, that experimental infection by A. caviae inhibits key enzymes linked to the communication between sites of ATP generation and ATP utilization. Moreover, the absence of a reciprocal compensatory mechanism between these enzymes contributes directly to hepatic damage and for a severe energetic imbalance, which may contribute to disease pathophysiology. KEYWORDS adenosine triphosphate, adenylate kinase, bacterial disease, energy homeostasis, pyruvate kinase 1 | INTRODUCTION Aeromonas caviae is a mesophilic species of the genus Aeromonas and occurs ubiquitously in aquatic environments (Lim, Ee, Yin, & Aquaculture plays an important role in food production worldwide, Chan, 2014), such as wastewater (Figueira, Vaz-Moreira, Silva, & as the fish and fishery products represent a source of proteins and Manaia, 2011), natural water such as rivers, lakes and estuaries essential micronutrients important for human health (Ottinger, (Pic~ao et al., 2013), aquacultures (Schmidt, Bruun, Dalsgaard, & Lar- Clauss, & Kuenzer, 2016). However, the intensive rearing in aquacul- sen, 2001) and urban drinking water (Carvalho, Martınez-Murcia, ture in order to supply the fish demand causes environmental stress Esteves, Correia, & Saavedra, 2012), and possesses the capacity to to fish, which results in increasing susceptibility to bacterial diseases, infect a vast number of hosts, such as humans and fishes (Janda & caused by Aeromonas caviae, that are considered the major impedi- Abbott, 2010). Recently, several studies have demonstrated the ment to the development of aquaculture and are often the most sig- increase in incidence of A. caviae infection in fish (Abd-El-Malek, nificant cause of economic loss (Jiang et al., 2016). 2017; Hoel, Vadstein, & Jakobsen, 2017), which is characterized by J Fish Dis. 2018;41:469–474. wileyonlinelibrary.com/journal/jfd © 2017 John Wiley & Sons Ltd | 469 470 | BALDISSERA ET AL. haemorrhagic septicaemia, hepatosplenomegaly, eye disease and  rio de Fisiologia de Peixes at the Universidade Federal de Laborato ulcerative lesions on the body surface (Thomas et al., 2013). This Santa Maria, where they were maintained in 250-L fibreglass tanks results in high mortality and economic losses in species such as rain- with continuous aeration under controlled water variables: tempera- bow trout (Oncorhynchus mykiss) (Zepeda-Velazquez et al., 2017), ture 18–20°C (maintained with air conditioner), pH 7.1–7.3 and dis- Nile tilapia (Oreochromis niloticus) (Meidong, Doolgindachbaporn, solved oxygen levels 5.9–7.5 mg/L, in freshwater for 7 days. Sakai, & Tongpim, 2017), tambaqui (Colossoma macropomum) (Mar- Dissolved oxygen and temperature were measured with a YSI oxy- ques et al., 2016), Indian catfish (Clarias batrachus) (Thomas et al., gen meter (model Y5512, OH, USA) and the pH with a DMPH-2 pH  nior 2013) and silver catfish (Rhamdia quelen) (Baldissera, Souza, Ju meter (S~ao Paulo, Brazil). The total ammonia and non-ionized ammo- et al., 2017). The liver is one of the organs most affected by nia levels were determined according to Verdouw et al. (1978) and A. caviae (Igbinosa, Igumbor, Aghdasi, Tom, & Okoh, 2012), but the Colt (2001), respectively, as recently published in detail by Baldis- effects of bacterial infection on hepatic tissue remain poorly under- nior et al. (2017). The animals were fed to apparent sera, Souza, Ju stood. Thus, more studies are needed to understand the mechanism satiation with commercial feed once a day. Any uneaten food, faeces of disease pathophysiology, such as the involvement of enzymes and other residues were removed daily 1 hr after feeding. belonging to phosphotransfer network: adenylate kinase (AK) and pyruvate kinase (PK), essential enzymes for the maintenance of bioenergetics homeostasis. 2.2 | Inoculum confirmation and preparation Delivery of metabolic signals to intracellular compartments plays The pathogen was confirmed through colony morphology and physi- a critical role in cellular homeostasis, as an efficient communication ological characteristics, as well as using polymerase chain reaction between cellular energetics and membrane metabolic sensors is (PCR) by the analysis of the 16S rRNA gene of A. caviae using the required to adequately regulate cell functions (Carrasco et al., 2001). primers 50 TCG TTG GGT TGG GAT GTG 30 (forward) and 50 TGT In this sense, cellular phosphotransfer reactions catalyse nucleotide TAC CGC GGT GAA AGG 30 (reverse), according to the methodology exchange facilitating communication between sites of adenosine  nior et al. (2017). described in detail by Baldissera, Souza, Ju triphosphate (ATP) generation and ATP utilization (Saupe, Spindler, The bacterial isolate was grown on nutrient agar for use in this Hopkins, Shen, & Ingwall, 2000). In this way, the AK catalyses the experimental model. The suspension of A. caviae was washed twice reversible transfer of the c-phosphate group from a phosphate donor in sterile saline (NaCl 0.9%), and turbidity (OD600) was adjusted to (normally ATP) to adenosine monophosphate (AMP), releasing two 0.9–1.1 (equivalent to 106 CFU/ml) and the suspension used for the molecules of adenosine diphosphate (ADP) (ATP + AMP ↔ 2 ADP), infection model. that is, molecules involved in the processing of metabolic signals associated with cellular energy utilization (Dzeja & Terzic, 1998). Moreover, the PK is a key enzyme of the glycolysis pathway that 2.3 | Animals and experimental design catalyses the irreversible phosphotransfer of phosphoenolpyruvate Twenty adult silver catfish (118  21 g; 30  3 cm) were used as (PEP) to ADP to form pyruvate and ATP (PEP + ADP ? ATP + Pyr) the experimental model to assess the hepatic AK and PK activities, (Wang et al., 2002), the main route that provides energy to proper as well as the hepatic ATP levels. The animals were divided into two tissue functioning. In this sense, Baldissera, Souza, Santos et al. groups with 10 animals each: uninfected animals (negative control (2017) demonstrated that inhibition of branchial AK and PK activities group) and experimentally infected animals (positive control group) in silver catfish experimentally infected with Pseudomonas aeruginosa inoculated intramuscularly with 100 ll of a bacterial suspension con- impairs the cellular energy homeostasis, contributing to disease taining 55 9 106 viable cells of A. caviae, according to the protocol pathogenesis. Thus, our hypothesis is that impairment of enzymes nior et al. (2017). The negative established by Baldissera, Souza, Ju belonging to phosphotransfer network contributes to disease patho- control group received the same dose of sterile saline through the physiology of hepatic tissue. same route. Based on the fact that hepatic tissue plays a central role in sus- The methodology used in the experiment was approved by the taining energetic homeostasis by maintaining a constant supply of Ethical and Animal Welfare Committee of the Universidade Federal energy to fuel body tissues, the aim of this study was to evaluate de Santa Maria under protocol number 074/2014. whether experimental infection by A. caviae alters hepatic AK and PK activities of silver catfish. 2 | MATERIAL AND METHODS 2.4 | Sample collection and tissue preparation On day 4 post-infection (PI), all animals were anaesthetized with natural anaesthetic (Cymbopogon flexuosus essential oil) followed by 2.1 | Fish harvesting, maintenance of animals and water quality variables spinal cord section according to the Ethics Committee recommenda- Healthy fish were collected for experimental purposes from a fish ment of AK and PK activities and ATP levels, while the other portion farm located in southern Brazil. The fish were transported to the for histopathological analysis. tions. Thereafter, the liver was removed and dissected in a glass dish over ice and divided into two portions: one portion for the measure- BALDISSERA | ET AL. 471 For the measurement of the enzymes of phosphoryl transfer transverse sections of 4 lm thickness and stained with haematoxylin network, the hepatic tissue was washed in SET buffer (0.32 M and eosin (HE) for identification of the standard structures. The sucrose, 1 mM EGTA, 10 mM Tris–HCl, pH 7.4) and homogenized slides were analysed by two histopathologists in a double-blinded (1:10 w/v) in the same SET buffer with a Potter-Elvehjem glass manner using an optical microscope. homogenizer. The homogenate was centrifuged at 800 g for 10 min at 4°C, and part of the supernatant was used for the determination of AK activity. The pellet was discarded and the rest of the super- 2.9 | Statistical analysis natant was centrifuged at 10,000 g for 15 min at 4°C. The super- Normality and homoscedasticity were analysed by the Shapiro–Wilk natant of this second centrifugation, containing cytosol and other and Levene tests, respectively. Significant differences between cellular components, was collected for the determination of PK groups were analysed and detected by two-tailed Student’s t-tests activity. for independent samples. The differences were considered to be statistically significant at p < .05. The effect size (r2) was described 2.5 | Hepatic AK and PK activities and scored as follows: ≤.1 (small), ≥.1 to ≤.3 (medium) and ≥.5 (large). Hepatic AK activity was measured with a coupled enzyme assay with hexokinase (HK) and glucose 6-phosphate dehydrogenase (G6PD), according to Dzeja, Vitkevicius, Redfield, Burnettm, and Terzic (1999). The reaction mixture contained 100 mM of KCl, 20 mM of HEPES, 20 mM of glucose, 4 mM of MgCl2, 2 mM of NADP+, 1 mM 3 | RESULTS 3.1 | Hepatic AK and PK activities of EDTA, 4.5 U/ml of HK, 2 U/ml of G6PD and 20 ll of hepatic Adenylate kinase activity decreased by 62% [t(18) = 5.23; p = .0002; homogenate. The reaction was initiated by the addition of 2 mM of r2 = .78] (Figure 1) and PK activity decreased by 34% [t(18) = 3.11; + ADP and the reduction of NADP was followed at 340 nm for 3 min in a spectrophotometer. The results were expressed in pmol p = .001; r2 = .52] in the liver of animals infected with A. caviae compared to the uninfected control group (Figure 2). ATP formed/min/mg of protein. Hepatic PK activity was assayed according to the protocol established by Leong, Lai, Lim, and Clark (1981). The incubation medium 3.2 | Hepatic ATP levels consisted of 0.1 M Tris–HCl buffer, pH 7.5, 10 mM of MgCl2, Hepatic ATP levels decreased by 50% [t(18) = 4.16; p = .005; 0.16 mM of NADH, 75 mM of KCl, 5.0 mM of ADP, 7 U of lactate r2 = .61] in animals infected with A. caviae compared to the unin- dehydrogenase, 0.1% of Triton X-100 and 10 ll of the mitochon- fected control group (Figure 3). dria-free supernatant in a final volume of 500 ll. After 10 min of preincubation at 37°C, the reaction was started by the addition of 1 mM of PEP. The results were expressed as lmol pyruvate formed/ min/mg of protein. 3.3 | Hepatic histopathology The uninfected animals did not show pathological alterations in hepatic tissue (Figure 4a). Infected animals showed dilation and 2.6 | Hepatic ATP levels The ATP levels in hepatic homogenates were measured by Firefly congestion of vessels (asterisk), degeneration of hepatocytes and loss of liver parenchyma architecture and sinusoidal structure (Figure 4b). Luciferase ATP assay kit (Beyotime, China), according to the manufacturer’s protocol, based on luciferase requirements for ATP to produce light (emission maximum at 560 nm), as recently published in detail by Wen et al. (2015). ATP levels are reported as pmol/mg of protein. 2.7 | Protein determination Hepatic protein content was determined by the method of Lowry, Rosebrough, Farr, and Randall (1951), using bovine albumin serum as standard. 2.8 | Hepatic histopathology After euthanasia, fragments of the liver were fixed in Bouin solution, processed by the usual routine method, embedded in paraffin for F I G U R E 1 Hepatic adenylate kinase (AK) activity in silver catfish experimentally infected with Aeromonas caviae compared to the uninfected control group on day 4 post-infection (PI). Bars with different letters are statistically different (p < .05; n = 10 per group) using the two-tailed Student’s t test for independent samples 472 | BALDISSERA ET AL. bioenergetics of infected animals, compromising the ATP synthesis and the communication between sites of ATP generation and ATP utilization. The liver plays an essential role in the physiological regulation of whole-body energy homeostasis, and the enzymes belonging to phosphotransfer network are considered to be mainly involved in the regulation of hepatic bioenergetics (Yegutkin, Wieringa, Robson, & Jalkanen, 2012). Moreover, a network and circuit view of the bioenergetics system allows for new perspectives leading to understanding of disease conditions associated with disturbances in F I G U R E 2 Hepatic pyruvate kinase (PK) activity in silver catfish experimentally infected with Aeromonas caviae compared to the uninfected control group on day 4 post-infection (PI). Bars with different letters are statistically different (p < .05; n = 10 per group) using the two-tailed Student’s t test for independent samples energy metabolism, metabolic monitoring and signalling (Dzeja & Terzic, 2009). Thus, the evaluation of phosphotransfer network provides new information for understanding the alterations in hepatic energetic metabolism during A. caviae infection. We observed that AK and PK activities were inhibited by A. caviae, which results in decreased availability of hepatic ATP and impairment of communication between sites of ATP generation and ATP utilization, in accordance with the results observed in the liver of experimentally infected rats with the parasite Trypanosoma evansi (Baldissera et al., 2015). Of particular interest, a recent study conducted by Baldissera, Souza, Santos et al. (2017) demonstrated that inhibition of branchial AK and PK activities by P. aeruginosa decreases the ATP availability, impairs the energy supply of experimentally infected silver catfish and contributes to disease pathogenesis, in accordance with the results observed in the present study. In summary, the inhibition of hepatic AK and PK activities leads to an impairment of energy metabolism during A. caviae infection, contributing to disease pathophysi- F I G U R E 3 Hepatic adenosine triphosphate (ATP) levels in silver catfish experimentally infected with Aeromonas caviae compared to the uninfected control group on day 4 post-infection (PI). Bars with different letters are statistically different (p < .05; n = 10 per group) using the two-tailed Student’s t test for independent samples ology. It is important to emphasize that a reciprocal compensatory relationship exists between these enzymes in order to safeguard cellular energy economy, which in turn contributes to an efficient intracellular energetic communication to maintain the balance between cellu- 4 | DISCUSSION lar ATP consumption and production in an attempt to preserve the energetic homeostasis (Janssen et al., 2000). Studies also revealed a The present study is novel as it evaluates important alterations in remarkable plasticity of the cellular phosphotransfer network system, the hepatic phosphoryl transfer network of animals experimentally where a deficiency in an individual enzyme is compensated through infected by A. caviae. Our findings clearly show the inhibition of the the remodelling of the whole energetics at enzymatic, architectural hepatic AK and PK activities, indicating an imbalance of hepatic and genomic levels (Dzeja, Terzic, & Wieringa, 2004); that is, a (a) (b) F I G U R E 4 Hepatic histopathology of Rhamdia quelen experimentally infected by Aeromonas caviae. (a) Uninfected fish showed normal hepatic architecture. (b) Fish infected by A. caviae showed dilation and congestion of vessels (asterisk), degeneration of hepatocytes and loss of liver parenchyma architecture and sinusoidal structure. Bars = 100 lm BALDISSERA | ET AL. decrease in one enzyme may lead to an increase in the other. Recently, a study conducted by Baldissera, Souza, Santos et al. (2017) demonstrated a compensatory mechanism between the cytosolic and mitochondrial creatine kinase (another important enzyme belonging to phosphotransfer network) activities in kidney tissue of experimentally infected silver catfish with A. caviae. However, this compensatory mechanism between AK and PK activities was not observed in hepatic tissue of experimentally infected silver catfish, which directly contributes to impairment of hepatic homeostasis. In this sense, the absence of energetic compensation through the enzymes of phosphotransfer network may contribute to the appearance of clinical signs of disease and hepatic lesions, as evaluated in the histopathological analyses due to the presence of dilation and congestion of vessels, degeneration of hepatocytes and loss of liver parenchyma architecture and sinusoidal structure. 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Pathogenicity of Mexican isolates of Aeromonas sp. in immersion experimentally-infected rainbow trout (Oncorhynchus mykiss, Walbaum 1792). Acta Tropica, 169, 122–124. How to cite this article: Baldissera MD, Souza CF, Verdi CM, et al. Aeromonas caviae inhibits hepatic enzymes of the phosphotransfer network in experimentally infected silver catfish: Impairment on bioenergetics. J Fish Dis. 2018;41:469– 474. https://doi.org/10.1111/jfd.12746