The stress hormone norepinephrine increases the growth and virulence of Aeromonas hydrophila

Abstract Stress is an important contributing factor in the outbreak of infectious fish diseases. To comprehensively understand the impact of catecholamine stress hormone norepinephrine (NE) on the pathogenicity of Aeromonas hydrophila, we assessed variations in bacterial growth, virulence‐related genes expression and virulence factors activity after NE addition in serum‐SAPI medium. Further, we assessed the effects of NE on A. hydrophila virulence in vivo by challenging fish with pathogenic strain AH196 and following with or without NE injection. The NE‐associated stimulation of A. hydrophila strain growth was not linear‐dose‐dependent, and only 100 μM, or higher concentrations, could stimulate growth. Real‐time PCR analyses revealed that NE notably changed 13 out of the 16 virulence‐associated genes (e.g. ompW, ahp, aha, ela, ahyR, ompA, and fur) expression, which were all significantly upregulated in A. hydrophila AH196 (p < 0.01). NE could enhance the protease activity, but not affect the lipase activity, hemolysis, and motility. Further, the mortality of crucian carp challenged with A. hydrophila AH196 was significantly higher in the group treated with NE (p < 0.01). Collectively, our results showed that NE enhanced the growth and virulence of pathogenic bacterium A. hydrophila.

In this study, we examined the effects of stress hormone NE on the growth, gene expression of selected virulence factors, lytic enzyme activity, hemolysis, and swimming motility of A. hydrophila. Moreover, we evaluated the impact of NE on the virulence of A. hydrophila in crucian carp Carassius auratus gibelio via in vivo challenge.

| Trial two
To confirm the effect of NE on the growth of A. hydrophila strains AH33, AH189, AH301, and NJ-35, the strains were inoculated in serum-SAPI medium with and without 100 μM NE. The turbidity at 600 nm was then measured at 36 hr. Trials were repeated twice and four replicates were conducted for each bacterial strain.

| Protease and hemolysis assays
A. hydrophila AH196 was grown to exponential phase (OD 600 of 0.6) in serum-SAPI media with 0 and 100 μM NE added. Broth cultures were centrifuged and the supernatants were filtered through 0.22 μm MCE membrane filters.
The protease activity of A. hydrophila AH196 was examined using azocasein (Sigma, St. Louis) as an enzyme substrate based on methods described in Chu, Zhou, Zhu, and Zhuang (2014). Briefly, 1 ml of azocasein (3 mg/ml in 50 mM Tris-HCl buffer, pH 7.5) was added to 150 μl of AH196 supernatant, and then incubated for 30 min at 37°C. The reaction was terminated by adding 10% precooled trichloroacetic acid (500 μl) and the supernatant was collected after centrifugation. The supernatant (100 μl) was neutralized with isopyknic 1 N NaOH in 96-well plates, and the absorbance was then measured at 400 nm with a Multiskan GO spectrophotometer.
The hemolysis activity of AH196 was measured using 4% sheep erythrocyte (Nanjing SenBeiJia, Nanjing, China) as a substrate based on modified methods that were previously described (Luo et al., 2016). Sheep erythrocyte ( control. All assays were repeated twice with four replicates.

| Lipase and motility assays
Lipase and motility assays followed methods described by Yang et al. (2014) with some modifications. A. hydrophila AH196 was grown in nutrient broth overnight, pelleted, washed, and diluted to 1 × 10 7 CFU/ ml. A 5 μl aliquot of bacterial suspension was spotted on the center of experimental plates. After autoclaved sterilization, two types of agar were mixed with NE (100 μM final concentration) for lipase and motility assessment. Control plate agar was mixed with equal volumes of vehicle solvent. Lipase assay plates were made by supplementing serum-SAPI agar with 1% (v/v) Tween 80 (Sinopharm, Shanghai, China).
After incubation for 48 hr at 30°C, opalescent zones and colony diameters were measured, and the ratio between both parameters was calculated to measure lipase activity. The motility assays were performed on semisolid agar plates (serum-SAPI medium + 0.5% (wt/v) agar) and diameters of swimming motility halos were determined after incubation for 24 hr at 30°C. Both lipase and motility assays were conducted twice with four technical replicates each time.

| Statistical analysis
All data are presented as the mean ± SD. The growth assay data were analyzed by one-way ANOVA followed by Tukey's post hoc tests. Data from the gene expression profiles, protease, hemolysis, lipase, and motility assays were analyzed by Welch's t test. The survival of crucian carp was analyzed and expressed as a Kaplan-Meier survival curve with a log-rank (Mantel-Cox) test. A probability (p) value < 0.05 was considered as statistically significant, and a probability (p) value < 0.01 was considered as extremely significant. All figures were plotted using the GraphPad Prism program version 7 (https://www.graphpad.com/, RRID: SCR_002798).

| Growth response of Aeromonas hydrophila to NE
To investigate the response of A. hydrophila AH196 growth with NE in vitro, minimal nutrient, low-iron SAPI medium that was supplemented with 10% FBS was used to imitate host environment ( Figure 1). Based on preliminary tests, we observed that all concentrations of NE could not stimulate growth of AH196 in serum-SAPI medium when initial inoculum densities were 10 3 −10 5 CFU/ ml (data not shown). There were no significant differences in OD 600 among the groups with 0, 12.5, 25, and 50 μM NE additions.
When compared to control cultures, the maximum cell density of  (Figure 2).

| Virulence-associated genes expression
Variation in gene expression of A. hydrophila AH196 with and without NE addition is shown in Figure 3.

| Protease activity, lipase activity, hemolysis, and swimming motility
The protease activity, lipase activity, hemolysis, and swimming motility of Aeromonas hydrophila AH196 were shown in

NE(12.5 µM)
Control F I G U R E 2 Growth of Aeromonas hydrophila strains that were isolated from distinct organs of cyprinid fish after exposure to norepinephrine (NE) for 36 hr in serum-SAPI medium containing 10% fetal bovine serum. Four Aeromonas hydrophila strains were examined and exposed to 100 μM NE or equivalent volumes of normal saline in the experimental and control groups, respectively (**p < 0.01) and Pseudomonas aeruginosa (Lyte & Ernst, 1992 (Halang et al., 2015), Aeromonas hydrophilia (Dong et al., 2016), Campylobacter jejuni (Xu et al., 2015), and Vibrio parahaemolyticus .
Indeed, NE was effective to promote proteinase activity and alter the expression of ahp and ela of A. hydrophila, which suggested that NE facilitated the infection process and virulence of A. hydrophila.
The theromstable cytotonic enterotoxin (ast) and hemolysin (hly) are vital exotoxins of A. hydrophila, and can promote the hemolysis, cytotoxicity, and enterotoxigenesis (Chopra et al., 1993). Our results also indicated that NE enhanced ast and hly gene expression of A. hydrophila.
Fur, an predominant iron-regulating factor in Gram-negative bacteria, regulates iron metabolism-related genes and cellular processes by sensing iron availability in the surrounding environment, such as acid resistance, oxidative and nitrosative stress, chemotaxis, and the expression of virulence factors (Escolar, Pérez-martín, & De Lorenzo, 1999;Salvail & Massé, 2012). Our results indicated that NE considerably upregulated fur and sodB gene expression in A. hydrophila. To maintain intracellular iron homeostasis, fur activity is activated in iron-rich environments, while the repression of fur activity is alleviated in low-iron conditions, which then promotes the synthesis of siderophores to uptake iron (Porcheron & Dozois, 2015 (Holmes et al., 2005;Oglesby, Murphy, Iyer, & Payne, 2005).
Hydroxyl radicals may be produced by fenton chemistry reactions that then result in oxidative stress during iron metabolism (Touati, Jacques, Tardat, Bouchard, & Despied, 1995). Miura, Muraoka, Fujimoto, and Zhao (2000) showed that DNA damage could be induced by catecholamine hormones in the presence of iron.
Therefore, the upregulation of sodB could result in catalytic conversion of superoxide radicals, thereby promote tolerance to the extremely toxic and oxidative compounds and ultimately enhance A. hydrophila viability. This explanation agrees well with previous research that the effect of NE on sodB gene expression (Graziano et al., 2014). Sha, Lu, and Chopra (2001) showed that the repression of act at the transcriptional level was relieved in fur isogenic mutants. Conversely, the upregulated fur could repress act gene expression, which may explain the downregulation of act in NE-

CO N FLI C T O F I NTE R E S T
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.