Aeromonas caviae inhibits hepatic enzymes of the phosphotransfer network in experimentally infected silver catfish: Impairment on bioenergetics

Marcelo Leite da Veiga

Aeromonas caviae inhibits hepatic enzymes of the phosphotransfer network in experimentally infected silver catfish: Impairment on bioenergetics

Marcelo Leite da Veiga

2017, Journal of fish diseases

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Several studies have been demonstrated that phosphotransfer network, through the adenylate kinase (AK) and pyruvate kinase (PK) activities, allows for new perspectives leading to understanding of disease conditions associated with disturbances in energy metabolism, metabolic monitoring and signalling. In this sense, the aim of this study was to evaluate whether experimental infection by Aeromonas caviae alters hepatic AK and PK activities of silver catfish Rhamdia quelen. Hepatic AK and PK activities decreased in infected animals compared to uninfected animals, as well as the hepatic adenosine triphosphate (ATP) levels. Also, a severe hepatic damage 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 betw...

| MATERIAL AND METHODS

| Fish harvesting, maintenance of animals and water quality variables

Healthy fish were collected for experimental purposes from a fish farm located in southern Brazil. The fish were transported to the Laborat orio de Fisiologia de Peixes at the Universidade Federal de Santa Maria, where they were maintained in 250-L fibreglass tanks with continuous aeration under controlled water variables: temperature 18-20°C (maintained with air conditioner), pH 7.1-7.3 and dissolved oxygen levels 5.9-7.5 mg/L, in freshwater for 7 days.

Dissolved oxygen and temperature were measured with a YSI oxygen meter (model Y5512, OH, USA) and the pH with a DMPH-2 pH meter (São Paulo, Brazil). The total ammonia and non-ionized ammonia levels were determined according to Verdouw et al. (1978) and Colt (2001), respectively, as recently published in detail by Baldissera, Souza, J unior et al. (2017). The animals were fed to apparent satiation with commercial feed once a day. Any uneaten food, faeces and other residues were removed daily 1 hr after feeding.

| Inoculum confirmation and preparation

The pathogen was confirmed through colony morphology and physiological characteristics, as well as using polymerase chain reaction (PCR) by the analysis of the 16S rRNA gene of A. caviae using the primers 5 0 TCG TTG GGT TGG GAT GTG 3 0 (forward) and 5 0 TGT TAC CGC GGT GAA AGG 3 0 (reverse), according to the methodology described in detail by Baldissera, Souza, J unior et al. (2017).

The bacterial isolate was grown on nutrient agar for use in this experimental model. The suspension of A. caviae was washed twice in sterile saline (NaCl 0.9%), and turbidity (OD600) was adjusted to 0.9-1.1 (equivalent to 10 6 CFU/ml) and the suspension used for the infection model.

| Animals and experimental design

Twenty adult silver catfish (118 AE 21 g; 30 AE 3 cm) were used as the experimental model to assess the hepatic AK and PK activities, as well as the hepatic ATP levels. The animals were divided into two groups with 10 animals each: uninfected animals (negative control group) and experimentally infected animals (positive control group) inoculated intramuscularly with 100 ll of a bacterial suspension containing 55 9 10 6 viable cells of A. caviae, according to the protocol established by Baldissera, Souza, J unior et al. (2017). The negative control group received the same dose of sterile saline through the same route.

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.

| Sample collection and tissue preparation

On day 4 post-infection (PI), all animals were anaesthetized with natural anaesthetic (Cymbopogon flexuosus essential oil) followed by spinal cord section according to the Ethics Committee recommendations. Thereafter, the liver was removed and dissected in a glass dish over ice and divided into two portions: one portion for the measurement of AK and PK activities and ATP levels, while the other portion for histopathological analysis.

For the measurement of the enzymes of phosphoryl transfer network, the hepatic tissue was 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 Potter-Elvehjem glass 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 supernatant was centrifuged at 10,000 g for 15 min at 4°C. The supernatant of this second centrifugation, containing cytosol and other cellular components, was collected for the determination of PK activity.

| Hepatic AK and PK activities

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 MgCl 2 , 2 mM of NADP + , 1 mM of EDTA, 4.5 U/ml of HK, 2 U/ml of G6PD and 20 ll of hepatic homogenate. The reaction was initiated by the addition of 2 mM of ADP and the reduction of NADP + was followed at 340 nm for 3 min in a spectrophotometer. The results were expressed in pmol ATP formed/min/mg of protein.

Hepatic PK activity was assayed according to the protocol established by Leong, Lai, Lim, and Clark (1981)

Adenylate kinase activity decreased by 62% [t(18) = 5.23; p = .0002; r 2 = .78] (Figure 1) and PK activity decreased by 34% [t(18) = 3.11; p = .001; r 2 = .52] in the liver of animals infected with A. caviae compared to the uninfected control group (Figure 2).

Figure 1

Figure 2

| Hepatic ATP levels

The ATP levels in hepatic homogenates were measured by Firefly 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.

Hepatic ATP levels decreased by 50% [t(18) = 4.16; p = .005; r 2 = .61] in animals infected with A. caviae compared to the uninfected control group (Figure 3).

Figure 3

| Protein determination

Hepatic protein content was determined by the method of Lowry, Rosebrough, Farr, and Randall (1951), using bovine albumin serum as standard.

| Hepatic histopathology

After euthanasia, fragments of the liver were fixed in Bouin solution, processed by the usual routine method, embedded in paraffin for transverse sections of 4 lm thickness and stained with haematoxylin and eosin (HE) for identification of the standard structures. The slides were analysed by two histopathologists in a double-blinded manner using an optical microscope.

The uninfected animals did not show pathological alterations in hepatic tissue (Figure 4a). Infected animals showed dilation and congestion of vessels (asterisk), degeneration of hepatocytes and loss of liver parenchyma architecture and sinusoidal structure (Figure 4b).

Figure 4

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 The present study is novel as it evaluates important alterations in the hepatic phosphoryl transfer network of animals experimentally infected by A. caviae. Our findings clearly show the inhibition of the hepatic AK and PK activities, indicating an imbalance of hepatic 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 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 pathophysiology.

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 cellular ATP consumption and production in an attempt to preserve the energetic homeostasis (Janssen et al., 2000). Studies also revealed a remarkable plasticity of the cellular phosphotransfer network system, where a deficiency in an individual enzyme is compensated through the remodelling of the whole energetics at enzymatic, architectural and genomic levels (Dzeja, Terzic, & Wieringa, 2004); that is, a 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 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 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.

Based on these evidences, the inhibition of hepatic AK and PK activities by A. caviae caused an impairment in hepatic energy homeostasis, decreasing the hepatic ATP availability. Moreover, the absence of a reciprocal compensatory mechanism between these enzymes directly contributes to hepatic damage and a severe energetic imbalance, which may contribute to disease pathophysiology.

| Statistical analysis

Normality and homoscedasticity were analysed by the Shapiro-Wilk and Levene tests, respectively. Significant differences between groups were analysed and detected by two-tailed Student's t-tests for independent samples. The differences were considered to be statistically significant at p < .05. The effect size (r 2 ) was described and scored as follows: ≤.1 (small), ≥.1 to ≤.3 (medium) and ≥.5 (large).

| RESULTS

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Aeromonas caviae inhibits hepatic enzymes of the phosphotransfer network in experimentally infected silver catfish: Impairment on bioenergetics

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