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Vibrio parahaemolyticus infection impaired intestinal barrier function and nutrient absorption in Litopenaeus vannamei
Journal Pre-proof Vibrio parahaemolyticus infection impaired intestinal barrier function and nutrient absorption in Litopenaeus vannamei Le Fei Jiao, Tian Meng Dai, Sun Qian Zhong, Min Jin, Peng Sun, Qi Cun Zhou PII:
S1050-4648(20)30080-2
DOI:
https://doi.org/10.1016/j.fsi.2020.02.009
Reference:
YFSIM 6808
To appear in:
Fish and Shellfish Immunology
Received Date: 27 November 2019 Revised Date:
14 January 2020
Accepted Date: 5 February 2020
Please cite this article as: Jiao LF, Dai TM, Zhong SQ, Jin M, Sun P, Zhou QC, Vibrio parahaemolyticus infection impaired intestinal barrier function and nutrient absorption in Litopenaeus vannamei, Fish and Shellfish Immunology (2020), doi: https://doi.org/10.1016/j.fsi.2020.02.009. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.
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Vibrio parahaemolyticus infection impaired intestinal barrier function and nutrient absorption in Litopenaeus vannamei
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Le Fei Jiao1, Tian Meng Dai1, Sun Qian Zhong2, Min Jin1, Peng Sun1, Qi Cun Zhou1*
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315211, PR China;
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Ningbo, 315800, PR China
Laboratory of Fish Nutrition, School of Marine Sciences, Ningbo University, Ningbo
Ningbo Economic Technical Development Area Bolun Marine Surveyors Office,
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* Corresponding author: Qicun Zhou
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E-mail address: [email protected]
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Abstract
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The intestine is the primary target of pathogenic microbes during invasion. However,
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the interaction of Vibrio parahaemolyticus (V. parahaemolyticus) with intestinal
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epithelial cells and its effects on the intestinal function of Litopenaeus vannamei (L.
26
vannamei) are poorly studied. Therefore, the aim of this study was to investigate the
27
influence of V. parahaemolyticus infection on intestinal barrier function and nutrient
28
absorption in L. vannamei. In the present study, a total of 90 shrimp were randomly
29
divided into two groups including the control group and V. parahaemolyticus infection
30
group (final concentration of 1×105 CFU/mL), with three replicates per group. The
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result showed that compared with the control group, V. parahaemolyticus infection
32
increased (P < 0.05) serum diamine oxidase activity and endotoxin quantification, and
33
down-regulated (P < 0.05) the mRNA levels of intestinal peroxinectin, integrin,
34
midline fasciclin at 48 h and 72 h; V. parahaemolyticus infection decreased (P < 0.05)
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the mRNA expression of intestinal amino acid transporter (CAT1, EAAT3 and ASCT1)
36
and glucose transporter (SGLT-1, GLUT) at 24 h, 48 h and 72 h, and increased
37
(P < 0.05) serum glucose and amino acid (Asp, Thr, Ser, Glu, Gly, Ala, Val, Ile, Leu,
38
Tyr, Phe, Lys, His and Arg) concentration at 24 h. The results indicated that V.
39
parahaemolyticus infection increased intestinal permeability, inhibited absorption of
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glucose and amino acid in L. vannamei.
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Keywords: Vibrio parahaemolyticus; Litopenaeus vannamei; Barrier function;
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Nutrient absorption; Transporter
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1. Introduction
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The pacific white shrimp Litopenaeus vannamei (L. vannamei), accounting for
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over 70% of shrimp production, is the most important shrimp aquaculture species
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worldwide [1]. In recent years, intensification of shrimp culture has achieved high
48
production, accompanied by increased outbreak of serious bacterial disease problems,
49
resulting in significant socio-economic losses and subsequently hindering sustainable
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aquaculture development [2, 3]. Vibrio parahaemolyticus (V. parahaemolyticus) is
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opportunistically pathogenic and have been reported as the pathogen of acute
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hepatopancreatic necrosis syndrome (AHPNS) or early mortality syndrome (EMS) [4,
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5]. To date, the mechanism of V. parahaemolyticus infection in L. vannamei has not
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been completely elucidated.
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Much attention has been paid to the role of host–pathogen interactions, which is
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important in defining the mechanism of pathogenesis and could potentially lead to
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new therapeutic avenues [6]. The intestine is the primary target of pathogenic
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microbes during invasion. The intestine mucosa acts as a barrier against microbial
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invaders, whereas foreign invading bacteria interact intimately with the gut epithelium
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and subsequently impact the host’s health [7, 8]. During invading, pathogen could
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cross epithelial or endothelial host barriers and get access to internal tissues, leading
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to severe diseases [9, 10]. Moreover, hosts and their pathogens share a wide range of
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resource needs that are required to support normal metabolism and growth [11]. The
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development of an infectious disease within a host involves the processes of invasion
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and resource consumption [12]. It is becoming increasingly clear that pathogen have
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evolved specific mechanisms to access their environment and the nutrients available
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to them. Recent evidence suggests the difference in pathogens in competing with their
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hosts for nutrients [11, 13, 14]. Until now, whether V. parahaemolyticus utilizes
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nutrient competition strategy for replication and colonization is unknown in L.
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vannamei.
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The intestine is the main site of the digestive system for nutrient absorption. The
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active nutrient uptake across the apical brush border of intestinal epithelial cells is
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accomplished by membrane-anchored transporters in vertebrate [15]. Transport of
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glucose and amino acid into the enterocyte was primarily regulated by specific
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transporters, which could been influenced by pathogen infection[16, 17]. Therefore,
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we hypothesized that expression of nutrient transporters in the intestines could be
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affected by V. parahaemolyticus colonization in L. vannamei. The aim of the present
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study was to outline the influence of V. parahaemolyticus infection on intestinal
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barrier function, and absorption of glucose and amino acid in L. vannamei.
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2. Materials and Methods
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2.1. Shrimp bacterial strain and experimental design
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Healthy pacific white shrimp were obtained from Ningbo Marine fishery science
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and technology innovation base, and reared in a semi-intensive culture pond with
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running aerated water at ambient temperature (28±2°C), salinity at 20 parts per
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thousand (ppt) prior to the experiment. Shrimp were fed a commercial formulated
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pellet and the daily feeding rates was 6% of their body weight.
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V. parahaemolyticus (ATCC 17802) were purchased from Beijing Solarbio
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Science and Technology Co., Ltd, China. V. parahaemolyticus was incubated at 37°C
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for 24 h on a Marine 2216E agar plate, and then single colonies were picked and
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inoculated into liquid marine 2216E medium at 37 °C overnight. The cell density
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was measured at 600 nm in UV-visible spectrophotometer and adjusted to a final
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concentration of 1×108 CFU/mL.
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The shrimp were randomly divided into two groups including control group and
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V. parahaemolyticus infection group, with three replicates per group. A total of 90
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shrimp (average weight: 8.91 g) were randomly assigned into six 100-L cylindrical
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fibre-glass tanks with 15 shrimp per tank. In this study, we added V. parahaemolyticus
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into water directly, instead of injection and immersion technique, to make a more
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natural way of establishing an infection. In. parahaemolyticus infection group,
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V.parahaemolyticus suspending in marine 2216E medium was added into tank with a
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final concentration of 1×105 CFU/mL, while an equal volume of sterilized marine
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2216E medium was added into tanks which belonged to the control group. In this
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study, the infection dosage of V. parahaemolyticus had been verified by
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pre-experiments.
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2.2. Sample collection
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Five shrimps in each tank were sampled at 24 h, 48 h, 72 h after infection.
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Hemolymph were taken from the pericardial cavity using a 1 mL syringe, sorted into
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1.5 mL Eppendorf tubes, and subjected to 10 min of centrifugation at 4000 rpm at 4°C.
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The supernatant (serum) was collected, packaged and stored at -80°C until analysis.
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An approximately 0.5 cm intestinal segment (mid-intestine) was harvested for a
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microscopic assessment of the mucosa morphology. The adjacent intestine was
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rapidly collected, rapidly frozen in liquid nitrogen, and stored at -80°C for further
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enzyme activity and PCR analysis.
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2.3. mRNA expression analysis of barrier function-related genes, amino acid
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transporter, glucose transporter by qPCR
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The mRNA levels of barrier function-related genes (peroxinectin, integrin,
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midline fasciclin), amino acid transporter (CAT1, EAAT3 and ASCT1), glucose
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transporter (SGLT-1, GLUT) were analyzed. Sequence of primers used for the qPCR
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was shown in Table 1, and β-actin was used as a house-keeping gene. Total RNA was
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extracted from intestine using TRIzol reagent (Invitrogen, Carlsbad, CA), following
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the manufacturer’s protocol. The concentration and purity of all RNA samples were
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measured using a Nano-Drop ND-2000 spectrophotometer (NanoDrop Technologies,
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Wilmington, DE, USA). The cDNA was generated from 1000 ng of DNAase treated
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RNA and synthesized by a Prime Script™ RT Reagent Kit with gDNA Eraser (perfect
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realtime) (TaKaRa Biotechnology, Dalian, China) according to the manufacturer's
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protocol, using Mastercycler nexus GSX1 PCR (Eppendorf, Germany).
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Quantitative analysis of PCR was carried out on a StepOne Plus real-time PCR
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system (Applied Biosystems, Foster City, CA) using SYBR Green Master mix
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(Promega, Madison, WI), according to the manufacturer’s specification. The
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amplifications were performed in a 20 µL reaction mixture consisting of 10 µL of 2 ×
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conc SYBR Green I Master Mix, 1.0 µL (each) gene-specific forward and reverse
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primers which concentrations were 10 µM, 6 µL DEPC-water and 2 µL of cDNA. The
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quantitative PCR program was 95 °C for 2 min, followed by 45 cycles of 95 °C for 10
133
s, 58 °C for 10 s and 72 °C for 20 s. The 2−∆∆Ct method was used to analyze the
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relative expression (fold changes) of target gene. All samples were run in triplicate.
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2.4. Analysis of Serum Glucose concentration
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Serum glucose was determined with an automatic biochemistry analyser (Hitachi
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7600-110 Ltd., Japan) from a clinical laboratory in Ningbo University Hospital.
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2.5. Serum biomarkers analysis of intestinal permeability (endotoxin and diamine
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oxidase)
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Serum diamine oxidase activity and endotoxin quantification were determined
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using commercial enzyme-linked immunosorbent assay (ELISA) kits (R & D Systems,
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Inc. Minneapolis,MN, USA) following the protocols of the manufacturer. Standard
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curves were generated to measure serum endotoxins concentration and diamine
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oxidase activity.
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2.6. Identification and quantification of serum amino acids
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Amino acid profiles of serum samples were determined using a High-speed
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Amino Acid Analyzer (L-8900, Hitachi High-Technologies Co., Tokyo, Japan).
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Briefly, approximately 0.2mL serum was added into a 15 ml glass thread screw neck
149
vial with 18 mm screw cap containing a translucent blue silicone septa gasket (CNW,
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Germany). After adding 5 mL HCl (6 N), the tube was sealed under N2 and immersed
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in a sand bath at 110 °C overnight for digestion. After cooling, the digested samples
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were washed into a 50 ml volumetric flask using ultrapure water. 1 mL of this solution
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was transferred into a 4 ml ampoule bottle (CNW, Germany), evaporated to dryness in
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a rotary evaporator (IKA RV10, Germany), resuspended in 1 ml HCl (0.02 N) and
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filtered through a 0.22 µm membrane using a hydrophilic polyether sulfone (PES)
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syringe filter (CNW, Germany) to remove any residue and impurity. Finally, 20 µL of
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the solution was used for amino acid determination. The packed column was Hitachi
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ion-exchange resin 2622 (4.6 mm × 60 mm, particle size 5 µm) and ninhydrin
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coloring solution was the reactive reagent for the detection of amino acids.
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2.7. Statistical Analysis
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Independent t-tests were conducted to compare the differences between control
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group and V. parahaemolyticus infection group using SPSS 20.0 statistical package
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(SPSS Inc., Chicago, IL). Data were expressed as mean ± SD. Differences were
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considered statistically significant at P < 0.05.
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3. Results
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3.1. Serum biomarkers analysis of intestinal permeability (endotoxin and diamine
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oxidase) after infection with V.parahaemolyticus in L. vannamei
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Serum diamine oxidase activity and endotoxin quantification is shown in Figure
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1. Compared with the control group, V.parahaemolyticus infection significantly
170
increased serum diamine oxidase activity and endotoxin quantification at 48 h and 72
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h (P < 0.05). Serum diamine oxidase activity and endotoxin quantification were not
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affected at 24 h after V.parahaemolyticus infection (P>0.05).
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3.2. Expression of barrier-related genes after infection with V.parahaemolyticus in L.
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vannamei
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Expression of barrier-related genes is shown in Figure 2. Compared with the
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control group, V.parahaemolyticus infection significantly down-regulated the mRNA
177
levels of peroxinectin, integrin, midline fasciclin at 48 h and 72 h (P < 0.05). The
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mRNA levels of peroxinectin, integrin, midline fasciclin were not affected at 24h after
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infection with V.parahaemolyticus (P > 0.05).
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3.3. Serum glucose concentration after V. parahaemolyticus infection in L. vannamei
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Serum glucose concentration is shown in Figure 3. Compared with the control
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group, V.parahaemolyticus infection increased the serum glucose concentration at 24
183
h and 72h (P < 0.05). Serum glucose concentration were not affected at 48 h after
184
V.parahaemolyticus infection (P>0.05).
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3.4. Relative gene expression of intestinal glucose transporters (SGLT-1, GLUT) after
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V. parahaemolyticus infection in L. vannamei
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The relative gene expression of glucose transporters is shown in Figure 4.
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Compared with the control group, V.parahaemolyticus infection significantly
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decreased the relative mRNA expression of SGLT-1 and GLUT in the intestine, and
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increased serum glucose concentration at 24 h, 48 h and 72 h (P < 0.05).
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3.5. Amino acid concentration in serum after V. parahaemolyticus infection in L.
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vannamei
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Serum amino acid concentration is shown in Figure 5. Compared with the control
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group, V.parahaemolyticus infection significantly increased the concentration of Asp,
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Thr, Ser, Glu, Gly, Ala, Val, Ile, Leu, Tyr, Phe, Lys, His and Arg in serum at 24 h
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(P < 0.05). No significant difference was observed in amino acid concentrationat 48 h
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after V.parahaemolyticus infection (P > 0.05). Serum amino acid concentration (Asp,
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Thr, Ser, Glu, Gly, Ala, Val, Ile, Leu, Tyr, Phe, Lys, His and Arg) has a tendency to be
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increased at 72 h after V.parahaemolyticus infection (P > 0.05).
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3.6. Relative gene expression of intestinal amino acid transporter (CAT1, EAAT3 and
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ASCT1 after V. parahaemolyticus infection in L. vannamei
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Relative gene expression of amino acid transporter (CAT1, EAAT3 and ASCT1)
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is shown in Figure 6. Compared with the control group, V.parahaemolyticus infection
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significantly decreased relative mRNA expression of CAT1, EAAT3 and ASCT1 in
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intestinal tissues at 24 h, 48 h and 72 h (P < 0.05).
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4. Discussion
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The intestinal epithelial barrier serves as an infectious foothold for many
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bacterial pathogens and as an entry port for pathogens to disseminate into deeper
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tissues [18, 19]. Enteric bacterial pathogens have evolved a wide range of strategies
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that finally allowing bacteria to cross epithelial or impair endothelial host barriers and
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get access to internal tissues, leading to severe diseases [20]. Transcriptomic profiling
212
revealed that genes expression associated with various physical, chemical and
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immunological intestinal barrier functions were affected by V. paraheamolyticus
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challenge in L.vannamei [21]. Among them, cell adhesion genes such as peroxinectin,
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integrin, midline fasciclin had been identified that reflecting the intestinal barrier
216
function in L.vannamei. Moreover, intestinal barrier function can be commonly
217
assessed by permeability biomarkers such as serum diamine oxidase activity and
218
endotoxin
concentration
[22].
The
current
study
demonstrated
that
V.
219
paraheamolyticus infection increased the serum diamine oxidase activity and
220
endotoxin concentration, and decreased the mRNA levels of intestinal barrier-related
221
genes (peroxinectin, integrin, midline fasciclin) at 48 h and 72 h, indicating the
222
impaired intestinal barrier function. This observation is similar with results from other
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aquatic animals, such as grass carp and turbot. It has been reported that grass carp
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Ctenopharyngodon idella infection with Aeromonas hydrophila impaired the structure
225
and function of the intestinal mucosal barrier through regulating the expression of
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tight junction protein [23]. Vibrio anguillarum challenge induced the signatures of
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intestinal barrier alteration and pathogen entry in Turbot (Scophthalmus maximus)
228
[24].
229
In the recent years, investigations on the relationships between host and invading
230
pathogen are crucial for the development of new effective preventive and curative
231
strategies for enteric infections [12, 25]. In addition to strict pro quo virulence factors,
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nutrient scavenging by pathogenic bacteria from the intestine is an emerging theme in
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bacterial pathogenesis [11, 26]. The intestine generates a complex nutrient
234
environment by breaking down indigestible food products into metabolites that are
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used by both the host and the microbiota [26]. Both the invading intestinal pathogen
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and the microbiota compete for these metabolites. For instance, Enterohemorrhagic
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Escherichia coli competed with commensal Escherichia coli for the same carbon
238
sources during growth in the mammalian intestine [27, 28]. In Vibrio cholerae,
239
passage through the intestinal tract induces genes involved in succinate, glycine, and
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chitin utilization that enhance the ability of the pathogen to persist within aquatic
241
environments, an important trait that enhances transmission and propagation of this
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water-borne pathogen [29]. Energy generation and colonization by the food-borne
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bacterial pathogen Campylobacter jejuni, which resides in the gastrointestinal tract of
244
its avian reservoir, depends upon scavenging of free amino and keto acids and
245
chemotaxis towards the carbon sources asparagine, formate, lactate and chicken
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mucus [30, 31]. Our present work demonstrated that competition for nutrient sources
247
is an important determinant of the outcome of bacterial infections of the intestine in
248
shrimp. The result showed that V. paraheamolyticus infection inhibited gene
249
expression of amino acid transporters (CAT1, EAAT3 and ASCT1) and glucose
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transporters (SGLT1, GLUT) in L.vannamei. The down-regulation of mRNA
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expression of glucose and amino acid transporters may result in accumulation of
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nutrients in the intestinal lumen, which may favor V. paraheamolyticus replication and
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colonization. Similar result was reported by Awad et al. (2014) who reported that
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Campylobacter jejuni infection down-regulated the expression of amino acid
255
transporters (CAT-2, EAAT-3, y+LAT-2), glucose transporter (SGLT-1, GLUT-2) and
256
peptide transporter (PepT-1) in the intestine of chickens [16]. Miska et al. (2017)
257
found that Eimeria infection significantly decreased the amounts of amino acids and
258
sugars that enter the gut epithelial cells at the brush border membrane, and this effect
259
can be prolonged past the height of the infection [32]. To our knowledge, this is the
260
first study to address the effects of V. paraheamolyticus infection on intestinal nutrient
261
transporters gene expression in L.vannamei.
262
Furthermore, it was notable that amino acid (Asp, Thr, Ser, Glu, Gly, Ala, Val, Ile,
263
Leu, Tyr, Phe, Lys, His, Arg) and glucose concentration was significantly increased at
264
24 h, instead of decreased in serum. This result might indicate the loss of nutrient
265
stores characteristic in L.vannamei. As infected shrimp has an impaired ability to
266
absorb amino acid and glucose, serum glucose and amino acid were compensatively
267
increased for maintaining physiological activities. The conjecture is supported that
268
infection and malnutrition have always been intricately linked [33]. It was reported
269
that during infection, protein catabolism in muscle was increased as a source of energy,
270
leading to the increased serum amino acids during infection in human [34]. Saiki et al.
271
(2013) found that Plasmodium yoelii infection increased ten amino acid (valine,
272
leucine, tyrosine, phenylalanine, EOHNH2, histidine, proline, aspartate, glutamate,
273
alanine) and decreased in five amino acids (citrulline, cysteine, methionine, 1-MeHis,
274
and arginine) in plasma of infected mice [35].
275
In conclusion, the present study certificated that V. parahaemolyticus infection
276
increased intestinal permeability, and inhibited absorption of glucose and amino acid
277
in L. vannamei. The down-regulation of mRNA expression of glucose and amino acid
278
transporters might favor V. parahaemolyticus colonization of the L. vannamei
279
intestine due to increased luminal nutrient availability. Increased of serum glucose and
280
amino acid concentration may be used for maintaining physiological activities, which
281
indicates the loss of nutrient stores characteristic in L.vannamei.
282
Acknowledgments
283 284
This research was supported by the National Key R&D Program of China (2018YFD0900400).
285
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29. Schild S, Tamayo R, Nelson EJ, Qadri F, Calderwood SB, Camilli A. Genes
366
induced late in infection increase fitness of Vibrio cholerae after release into the
367
environment. Cell host & microbe. 2007 2:264-77.
368
30. Velayudhan J, Jones MA, Barrow PA, Kelly DJ. L-serine catabolism via an
369
oxygen-labile L-serine dehydratase is essential for colonization of the avian gut
370
by Campylobacter jejuni. Infection and immunity. 2004 72:260-8.
371
31. Vegge CS, Brondsted L, Li YP, Bang DD, Ingmer H. Energy Taxis Drives
372
Campylobacter jejuni toward the Most Favorable Conditions for Growth.
373
Applied and Environmental Microbiology. 2009 75:5308-14.
374
32. Miska KB, Fetterer RH. The mRNA expression of amino acid and sugar
375
transporters, aminopeptidase, as well as the di- and tri-peptide transporter PepT1
376
in the intestines of Eimeria infected broiler chickens. Poultry Science.2017
377
96(2):465-473.
378 379 380 381
33. Keusch GT. The history of nutrition: malnutrition, infection and immunity. The Journal of nutrition. 2003 133:336S-40S. 34. Wannemacher RW. Key role fo various individual amino acids in host response to infection. American Journal of Clinical Nutrition. 1977 30: 1269-1280.
382
35. Saiki E, Nagao K, Aonuma H, Fukumoto S, Xuan X, Bannai M, Kanuka, H.
383
Multivariable analysis of host amino acids in plasma and liver during infection of
384
malaria parasite Plasmodium yoelii. Malaria Journal. 2013 12:19.
385 386 387 388 389 390 391 392 393 394
395
Table 1. Real-time quantitative PCR primers Gene
Nucleotide sequence (from 5′ to 3′) F:CCTGTCTCCAGCCTGTTTGAAGTTG
CAT1 R: CCAGTCAATGCTCCCAAATGTGCTC F:GCTCTTTGGGCGACTTTCTGTTGGT SGLT1 R: CGCCCGTTCTCCTGGCACTGCTGAT F:TTGGTCTTCTGTGTCCTGGTGGTGC GLUT R:ACTCTACCATCTCATTTTCTCGGAC F:TGGAGCCCAATGACTCAAGGATAGA EAAT3 R:AGGCGTCAGAGAGTGAGTGGAAAAC F:AAGTTGAGGTAGTTCCAGCCCCCGT ASCT1 R:GCGGATTCAGACCCTATTTACTGGC F:CAAGAAAGGAGACCGATAC peroxinectin R:GCTGGACGGCTTGGATC F:CCTGAAGGACGAGCCGCTGAGTGAT integrin R: TGTAGGTCACGCTGATGTGTTGGGA F:CTCCAGCTCTGGGATACTGC midline fasciclin R:TGCTTTTCGTTCACCTTCCT F:CCACGAGACCACCTACAAC β-actin R:AGC GAG GGC AGT GAT TTC 396 397 398 399 400 401 402
403
404
Figure 1. Effect of V.parahaemolyticus infection on the endotoxin concentration and
405
diamine oxidase activities in L. vannamei. Values are means and SD represented by
406
vertical bars. * Asterisks indicate significant differences (P < 0.05).
407 408 409 410 411 412 413 414 415 416
417 418
419 420
Figure 2. Effect of V.parahaemolyticus infection on the intestinal barrier-related
421
genes expression (peroxinectin, integrin, midline fasciclin) in L. vannamei. Values are
422
means and SD represented by vertical bars. * Asterisks indicate significant differences
423
(P < 0.05).
424 425 426 427 428 429 430 431 432 433
434
435 436 437
Figure 3. Effect of V.parahaemolyticus infection on serum glucose concentration in L.
438
vannamei. Values are means and SD represented by vertical bars. * Asterisks indicate
439
significant differences (P < 0.05).
440 441 442 443 444 445 446 447
448
Figure 4. Effect of V.parahaemolyticus infection on expression of intestinal glucose
449
transporter (SGLT-1, GLUT) in L. vannamei. Values are means and SD represented by
450
vertical bars. * Asterisks indicate significant differences (P < 0.05).
451 452 453 454 455 456 457 458 459 460 461 462 463
,
464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483
Figure 5. Effect of V.parahaemolyticus infection on serum amino acid concentration
484
in
485
indicate significant differences (P < 0.05)
L. vannamei. Values are means and SD represented by vertical bars. * Asterisks
486 487
Figure 6. Effect of V.parahaemolyticus infection on expression of intestinal amino
488
acid transporter (CAT1, EAAT3 and ASCT1) in L. vannamei. Values are means and
489
SD represented by vertical bars. * Asterisks indicate significant differences (P < 0.05).
490 491 492 493 494 495 496 497 498 499 500 501
Highlights V. parahaemolyticus infection increased intestinal permeability in L. vannamei. V. parahaemolyticus infection inhibited intestinal absorption of glucose and amino acid in L. vannamei.
S1050-4648(20)30080-2
DOI:
https://doi.org/10.1016/j.fsi.2020.02.009
Reference:
YFSIM 6808
To appear in:
Fish and Shellfish Immunology
Received Date: 27 November 2019 Revised Date:
14 January 2020
Accepted Date: 5 February 2020
Please cite this article as: Jiao LF, Dai TM, Zhong SQ, Jin M, Sun P, Zhou QC, Vibrio parahaemolyticus infection impaired intestinal barrier function and nutrient absorption in Litopenaeus vannamei, Fish and Shellfish Immunology (2020), doi: https://doi.org/10.1016/j.fsi.2020.02.009. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.
1
Vibrio parahaemolyticus infection impaired intestinal barrier function and nutrient absorption in Litopenaeus vannamei
2 3
Le Fei Jiao1, Tian Meng Dai1, Sun Qian Zhong2, Min Jin1, Peng Sun1, Qi Cun Zhou1*
4
5
1
6
315211, PR China;
7
2
8
Ningbo, 315800, PR China
Laboratory of Fish Nutrition, School of Marine Sciences, Ningbo University, Ningbo
Ningbo Economic Technical Development Area Bolun Marine Surveyors Office,
9
10
* Corresponding author: Qicun Zhou
11
E-mail address: [email protected]
12 13 14 15 16 17 18 19 20 21
22
Abstract
23
The intestine is the primary target of pathogenic microbes during invasion. However,
24
the interaction of Vibrio parahaemolyticus (V. parahaemolyticus) with intestinal
25
epithelial cells and its effects on the intestinal function of Litopenaeus vannamei (L.
26
vannamei) are poorly studied. Therefore, the aim of this study was to investigate the
27
influence of V. parahaemolyticus infection on intestinal barrier function and nutrient
28
absorption in L. vannamei. In the present study, a total of 90 shrimp were randomly
29
divided into two groups including the control group and V. parahaemolyticus infection
30
group (final concentration of 1×105 CFU/mL), with three replicates per group. The
31
result showed that compared with the control group, V. parahaemolyticus infection
32
increased (P < 0.05) serum diamine oxidase activity and endotoxin quantification, and
33
down-regulated (P < 0.05) the mRNA levels of intestinal peroxinectin, integrin,
34
midline fasciclin at 48 h and 72 h; V. parahaemolyticus infection decreased (P < 0.05)
35
the mRNA expression of intestinal amino acid transporter (CAT1, EAAT3 and ASCT1)
36
and glucose transporter (SGLT-1, GLUT) at 24 h, 48 h and 72 h, and increased
37
(P < 0.05) serum glucose and amino acid (Asp, Thr, Ser, Glu, Gly, Ala, Val, Ile, Leu,
38
Tyr, Phe, Lys, His and Arg) concentration at 24 h. The results indicated that V.
39
parahaemolyticus infection increased intestinal permeability, inhibited absorption of
40
glucose and amino acid in L. vannamei.
41
Keywords: Vibrio parahaemolyticus; Litopenaeus vannamei; Barrier function;
42
Nutrient absorption; Transporter
43
44
1. Introduction
45
The pacific white shrimp Litopenaeus vannamei (L. vannamei), accounting for
46
over 70% of shrimp production, is the most important shrimp aquaculture species
47
worldwide [1]. In recent years, intensification of shrimp culture has achieved high
48
production, accompanied by increased outbreak of serious bacterial disease problems,
49
resulting in significant socio-economic losses and subsequently hindering sustainable
50
aquaculture development [2, 3]. Vibrio parahaemolyticus (V. parahaemolyticus) is
51
opportunistically pathogenic and have been reported as the pathogen of acute
52
hepatopancreatic necrosis syndrome (AHPNS) or early mortality syndrome (EMS) [4,
53
5]. To date, the mechanism of V. parahaemolyticus infection in L. vannamei has not
54
been completely elucidated.
55
Much attention has been paid to the role of host–pathogen interactions, which is
56
important in defining the mechanism of pathogenesis and could potentially lead to
57
new therapeutic avenues [6]. The intestine is the primary target of pathogenic
58
microbes during invasion. The intestine mucosa acts as a barrier against microbial
59
invaders, whereas foreign invading bacteria interact intimately with the gut epithelium
60
and subsequently impact the host’s health [7, 8]. During invading, pathogen could
61
cross epithelial or endothelial host barriers and get access to internal tissues, leading
62
to severe diseases [9, 10]. Moreover, hosts and their pathogens share a wide range of
63
resource needs that are required to support normal metabolism and growth [11]. The
64
development of an infectious disease within a host involves the processes of invasion
65
and resource consumption [12]. It is becoming increasingly clear that pathogen have
66
evolved specific mechanisms to access their environment and the nutrients available
67
to them. Recent evidence suggests the difference in pathogens in competing with their
68
hosts for nutrients [11, 13, 14]. Until now, whether V. parahaemolyticus utilizes
69
nutrient competition strategy for replication and colonization is unknown in L.
70
vannamei.
71
The intestine is the main site of the digestive system for nutrient absorption. The
72
active nutrient uptake across the apical brush border of intestinal epithelial cells is
73
accomplished by membrane-anchored transporters in vertebrate [15]. Transport of
74
glucose and amino acid into the enterocyte was primarily regulated by specific
75
transporters, which could been influenced by pathogen infection[16, 17]. Therefore,
76
we hypothesized that expression of nutrient transporters in the intestines could be
77
affected by V. parahaemolyticus colonization in L. vannamei. The aim of the present
78
study was to outline the influence of V. parahaemolyticus infection on intestinal
79
barrier function, and absorption of glucose and amino acid in L. vannamei.
80
2. Materials and Methods
81
2.1. Shrimp bacterial strain and experimental design
82
Healthy pacific white shrimp were obtained from Ningbo Marine fishery science
83
and technology innovation base, and reared in a semi-intensive culture pond with
84
running aerated water at ambient temperature (28±2°C), salinity at 20 parts per
85
thousand (ppt) prior to the experiment. Shrimp were fed a commercial formulated
86
pellet and the daily feeding rates was 6% of their body weight.
87
V. parahaemolyticus (ATCC 17802) were purchased from Beijing Solarbio
88
Science and Technology Co., Ltd, China. V. parahaemolyticus was incubated at 37°C
89
for 24 h on a Marine 2216E agar plate, and then single colonies were picked and
90
inoculated into liquid marine 2216E medium at 37 °C overnight. The cell density
91
was measured at 600 nm in UV-visible spectrophotometer and adjusted to a final
92
concentration of 1×108 CFU/mL.
93
The shrimp were randomly divided into two groups including control group and
94
V. parahaemolyticus infection group, with three replicates per group. A total of 90
95
shrimp (average weight: 8.91 g) were randomly assigned into six 100-L cylindrical
96
fibre-glass tanks with 15 shrimp per tank. In this study, we added V. parahaemolyticus
97
into water directly, instead of injection and immersion technique, to make a more
98
natural way of establishing an infection. In. parahaemolyticus infection group,
99
V.parahaemolyticus suspending in marine 2216E medium was added into tank with a
100
final concentration of 1×105 CFU/mL, while an equal volume of sterilized marine
101
2216E medium was added into tanks which belonged to the control group. In this
102
study, the infection dosage of V. parahaemolyticus had been verified by
103
pre-experiments.
104
2.2. Sample collection
105
Five shrimps in each tank were sampled at 24 h, 48 h, 72 h after infection.
106
Hemolymph were taken from the pericardial cavity using a 1 mL syringe, sorted into
107
1.5 mL Eppendorf tubes, and subjected to 10 min of centrifugation at 4000 rpm at 4°C.
108
The supernatant (serum) was collected, packaged and stored at -80°C until analysis.
109
An approximately 0.5 cm intestinal segment (mid-intestine) was harvested for a
110
microscopic assessment of the mucosa morphology. The adjacent intestine was
111
rapidly collected, rapidly frozen in liquid nitrogen, and stored at -80°C for further
112
enzyme activity and PCR analysis.
113
2.3. mRNA expression analysis of barrier function-related genes, amino acid
114
transporter, glucose transporter by qPCR
115
The mRNA levels of barrier function-related genes (peroxinectin, integrin,
116
midline fasciclin), amino acid transporter (CAT1, EAAT3 and ASCT1), glucose
117
transporter (SGLT-1, GLUT) were analyzed. Sequence of primers used for the qPCR
118
was shown in Table 1, and β-actin was used as a house-keeping gene. Total RNA was
119
extracted from intestine using TRIzol reagent (Invitrogen, Carlsbad, CA), following
120
the manufacturer’s protocol. The concentration and purity of all RNA samples were
121
measured using a Nano-Drop ND-2000 spectrophotometer (NanoDrop Technologies,
122
Wilmington, DE, USA). The cDNA was generated from 1000 ng of DNAase treated
123
RNA and synthesized by a Prime Script™ RT Reagent Kit with gDNA Eraser (perfect
124
realtime) (TaKaRa Biotechnology, Dalian, China) according to the manufacturer's
125
protocol, using Mastercycler nexus GSX1 PCR (Eppendorf, Germany).
126
Quantitative analysis of PCR was carried out on a StepOne Plus real-time PCR
127
system (Applied Biosystems, Foster City, CA) using SYBR Green Master mix
128
(Promega, Madison, WI), according to the manufacturer’s specification. The
129
amplifications were performed in a 20 µL reaction mixture consisting of 10 µL of 2 ×
130
conc SYBR Green I Master Mix, 1.0 µL (each) gene-specific forward and reverse
131
primers which concentrations were 10 µM, 6 µL DEPC-water and 2 µL of cDNA. The
132
quantitative PCR program was 95 °C for 2 min, followed by 45 cycles of 95 °C for 10
133
s, 58 °C for 10 s and 72 °C for 20 s. The 2−∆∆Ct method was used to analyze the
134
relative expression (fold changes) of target gene. All samples were run in triplicate.
135
2.4. Analysis of Serum Glucose concentration
136
Serum glucose was determined with an automatic biochemistry analyser (Hitachi
137
7600-110 Ltd., Japan) from a clinical laboratory in Ningbo University Hospital.
138
2.5. Serum biomarkers analysis of intestinal permeability (endotoxin and diamine
139
oxidase)
140
Serum diamine oxidase activity and endotoxin quantification were determined
141
using commercial enzyme-linked immunosorbent assay (ELISA) kits (R & D Systems,
142
Inc. Minneapolis,MN, USA) following the protocols of the manufacturer. Standard
143
curves were generated to measure serum endotoxins concentration and diamine
144
oxidase activity.
145
2.6. Identification and quantification of serum amino acids
146
Amino acid profiles of serum samples were determined using a High-speed
147
Amino Acid Analyzer (L-8900, Hitachi High-Technologies Co., Tokyo, Japan).
148
Briefly, approximately 0.2mL serum was added into a 15 ml glass thread screw neck
149
vial with 18 mm screw cap containing a translucent blue silicone septa gasket (CNW,
150
Germany). After adding 5 mL HCl (6 N), the tube was sealed under N2 and immersed
151
in a sand bath at 110 °C overnight for digestion. After cooling, the digested samples
152
were washed into a 50 ml volumetric flask using ultrapure water. 1 mL of this solution
153
was transferred into a 4 ml ampoule bottle (CNW, Germany), evaporated to dryness in
154
a rotary evaporator (IKA RV10, Germany), resuspended in 1 ml HCl (0.02 N) and
155
filtered through a 0.22 µm membrane using a hydrophilic polyether sulfone (PES)
156
syringe filter (CNW, Germany) to remove any residue and impurity. Finally, 20 µL of
157
the solution was used for amino acid determination. The packed column was Hitachi
158
ion-exchange resin 2622 (4.6 mm × 60 mm, particle size 5 µm) and ninhydrin
159
coloring solution was the reactive reagent for the detection of amino acids.
160
2.7. Statistical Analysis
161
Independent t-tests were conducted to compare the differences between control
162
group and V. parahaemolyticus infection group using SPSS 20.0 statistical package
163
(SPSS Inc., Chicago, IL). Data were expressed as mean ± SD. Differences were
164
considered statistically significant at P < 0.05.
165
3. Results
166
3.1. Serum biomarkers analysis of intestinal permeability (endotoxin and diamine
167
oxidase) after infection with V.parahaemolyticus in L. vannamei
168
Serum diamine oxidase activity and endotoxin quantification is shown in Figure
169
1. Compared with the control group, V.parahaemolyticus infection significantly
170
increased serum diamine oxidase activity and endotoxin quantification at 48 h and 72
171
h (P < 0.05). Serum diamine oxidase activity and endotoxin quantification were not
172
affected at 24 h after V.parahaemolyticus infection (P>0.05).
173
3.2. Expression of barrier-related genes after infection with V.parahaemolyticus in L.
174
vannamei
175
Expression of barrier-related genes is shown in Figure 2. Compared with the
176
control group, V.parahaemolyticus infection significantly down-regulated the mRNA
177
levels of peroxinectin, integrin, midline fasciclin at 48 h and 72 h (P < 0.05). The
178
mRNA levels of peroxinectin, integrin, midline fasciclin were not affected at 24h after
179
infection with V.parahaemolyticus (P > 0.05).
180
3.3. Serum glucose concentration after V. parahaemolyticus infection in L. vannamei
181
Serum glucose concentration is shown in Figure 3. Compared with the control
182
group, V.parahaemolyticus infection increased the serum glucose concentration at 24
183
h and 72h (P < 0.05). Serum glucose concentration were not affected at 48 h after
184
V.parahaemolyticus infection (P>0.05).
185
3.4. Relative gene expression of intestinal glucose transporters (SGLT-1, GLUT) after
186
V. parahaemolyticus infection in L. vannamei
187
The relative gene expression of glucose transporters is shown in Figure 4.
188
Compared with the control group, V.parahaemolyticus infection significantly
189
decreased the relative mRNA expression of SGLT-1 and GLUT in the intestine, and
190
increased serum glucose concentration at 24 h, 48 h and 72 h (P < 0.05).
191
3.5. Amino acid concentration in serum after V. parahaemolyticus infection in L.
192
vannamei
193
Serum amino acid concentration is shown in Figure 5. Compared with the control
194
group, V.parahaemolyticus infection significantly increased the concentration of Asp,
195
Thr, Ser, Glu, Gly, Ala, Val, Ile, Leu, Tyr, Phe, Lys, His and Arg in serum at 24 h
196
(P < 0.05). No significant difference was observed in amino acid concentrationat 48 h
197
after V.parahaemolyticus infection (P > 0.05). Serum amino acid concentration (Asp,
198
Thr, Ser, Glu, Gly, Ala, Val, Ile, Leu, Tyr, Phe, Lys, His and Arg) has a tendency to be
199
increased at 72 h after V.parahaemolyticus infection (P > 0.05).
200
3.6. Relative gene expression of intestinal amino acid transporter (CAT1, EAAT3 and
201
ASCT1 after V. parahaemolyticus infection in L. vannamei
202
Relative gene expression of amino acid transporter (CAT1, EAAT3 and ASCT1)
203
is shown in Figure 6. Compared with the control group, V.parahaemolyticus infection
204
significantly decreased relative mRNA expression of CAT1, EAAT3 and ASCT1 in
205
intestinal tissues at 24 h, 48 h and 72 h (P < 0.05).
206
4. Discussion
207
The intestinal epithelial barrier serves as an infectious foothold for many
208
bacterial pathogens and as an entry port for pathogens to disseminate into deeper
209
tissues [18, 19]. Enteric bacterial pathogens have evolved a wide range of strategies
210
that finally allowing bacteria to cross epithelial or impair endothelial host barriers and
211
get access to internal tissues, leading to severe diseases [20]. Transcriptomic profiling
212
revealed that genes expression associated with various physical, chemical and
213
immunological intestinal barrier functions were affected by V. paraheamolyticus
214
challenge in L.vannamei [21]. Among them, cell adhesion genes such as peroxinectin,
215
integrin, midline fasciclin had been identified that reflecting the intestinal barrier
216
function in L.vannamei. Moreover, intestinal barrier function can be commonly
217
assessed by permeability biomarkers such as serum diamine oxidase activity and
218
endotoxin
concentration
[22].
The
current
study
demonstrated
that
V.
219
paraheamolyticus infection increased the serum diamine oxidase activity and
220
endotoxin concentration, and decreased the mRNA levels of intestinal barrier-related
221
genes (peroxinectin, integrin, midline fasciclin) at 48 h and 72 h, indicating the
222
impaired intestinal barrier function. This observation is similar with results from other
223
aquatic animals, such as grass carp and turbot. It has been reported that grass carp
224
Ctenopharyngodon idella infection with Aeromonas hydrophila impaired the structure
225
and function of the intestinal mucosal barrier through regulating the expression of
226
tight junction protein [23]. Vibrio anguillarum challenge induced the signatures of
227
intestinal barrier alteration and pathogen entry in Turbot (Scophthalmus maximus)
228
[24].
229
In the recent years, investigations on the relationships between host and invading
230
pathogen are crucial for the development of new effective preventive and curative
231
strategies for enteric infections [12, 25]. In addition to strict pro quo virulence factors,
232
nutrient scavenging by pathogenic bacteria from the intestine is an emerging theme in
233
bacterial pathogenesis [11, 26]. The intestine generates a complex nutrient
234
environment by breaking down indigestible food products into metabolites that are
235
used by both the host and the microbiota [26]. Both the invading intestinal pathogen
236
and the microbiota compete for these metabolites. For instance, Enterohemorrhagic
237
Escherichia coli competed with commensal Escherichia coli for the same carbon
238
sources during growth in the mammalian intestine [27, 28]. In Vibrio cholerae,
239
passage through the intestinal tract induces genes involved in succinate, glycine, and
240
chitin utilization that enhance the ability of the pathogen to persist within aquatic
241
environments, an important trait that enhances transmission and propagation of this
242
water-borne pathogen [29]. Energy generation and colonization by the food-borne
243
bacterial pathogen Campylobacter jejuni, which resides in the gastrointestinal tract of
244
its avian reservoir, depends upon scavenging of free amino and keto acids and
245
chemotaxis towards the carbon sources asparagine, formate, lactate and chicken
246
mucus [30, 31]. Our present work demonstrated that competition for nutrient sources
247
is an important determinant of the outcome of bacterial infections of the intestine in
248
shrimp. The result showed that V. paraheamolyticus infection inhibited gene
249
expression of amino acid transporters (CAT1, EAAT3 and ASCT1) and glucose
250
transporters (SGLT1, GLUT) in L.vannamei. The down-regulation of mRNA
251
expression of glucose and amino acid transporters may result in accumulation of
252
nutrients in the intestinal lumen, which may favor V. paraheamolyticus replication and
253
colonization. Similar result was reported by Awad et al. (2014) who reported that
254
Campylobacter jejuni infection down-regulated the expression of amino acid
255
transporters (CAT-2, EAAT-3, y+LAT-2), glucose transporter (SGLT-1, GLUT-2) and
256
peptide transporter (PepT-1) in the intestine of chickens [16]. Miska et al. (2017)
257
found that Eimeria infection significantly decreased the amounts of amino acids and
258
sugars that enter the gut epithelial cells at the brush border membrane, and this effect
259
can be prolonged past the height of the infection [32]. To our knowledge, this is the
260
first study to address the effects of V. paraheamolyticus infection on intestinal nutrient
261
transporters gene expression in L.vannamei.
262
Furthermore, it was notable that amino acid (Asp, Thr, Ser, Glu, Gly, Ala, Val, Ile,
263
Leu, Tyr, Phe, Lys, His, Arg) and glucose concentration was significantly increased at
264
24 h, instead of decreased in serum. This result might indicate the loss of nutrient
265
stores characteristic in L.vannamei. As infected shrimp has an impaired ability to
266
absorb amino acid and glucose, serum glucose and amino acid were compensatively
267
increased for maintaining physiological activities. The conjecture is supported that
268
infection and malnutrition have always been intricately linked [33]. It was reported
269
that during infection, protein catabolism in muscle was increased as a source of energy,
270
leading to the increased serum amino acids during infection in human [34]. Saiki et al.
271
(2013) found that Plasmodium yoelii infection increased ten amino acid (valine,
272
leucine, tyrosine, phenylalanine, EOHNH2, histidine, proline, aspartate, glutamate,
273
alanine) and decreased in five amino acids (citrulline, cysteine, methionine, 1-MeHis,
274
and arginine) in plasma of infected mice [35].
275
In conclusion, the present study certificated that V. parahaemolyticus infection
276
increased intestinal permeability, and inhibited absorption of glucose and amino acid
277
in L. vannamei. The down-regulation of mRNA expression of glucose and amino acid
278
transporters might favor V. parahaemolyticus colonization of the L. vannamei
279
intestine due to increased luminal nutrient availability. Increased of serum glucose and
280
amino acid concentration may be used for maintaining physiological activities, which
281
indicates the loss of nutrient stores characteristic in L.vannamei.
282
Acknowledgments
283 284
This research was supported by the National Key R&D Program of China (2018YFD0900400).
285
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Table 1. Real-time quantitative PCR primers Gene
Nucleotide sequence (from 5′ to 3′) F:CCTGTCTCCAGCCTGTTTGAAGTTG
CAT1 R: CCAGTCAATGCTCCCAAATGTGCTC F:GCTCTTTGGGCGACTTTCTGTTGGT SGLT1 R: CGCCCGTTCTCCTGGCACTGCTGAT F:TTGGTCTTCTGTGTCCTGGTGGTGC GLUT R:ACTCTACCATCTCATTTTCTCGGAC F:TGGAGCCCAATGACTCAAGGATAGA EAAT3 R:AGGCGTCAGAGAGTGAGTGGAAAAC F:AAGTTGAGGTAGTTCCAGCCCCCGT ASCT1 R:GCGGATTCAGACCCTATTTACTGGC F:CAAGAAAGGAGACCGATAC peroxinectin R:GCTGGACGGCTTGGATC F:CCTGAAGGACGAGCCGCTGAGTGAT integrin R: TGTAGGTCACGCTGATGTGTTGGGA F:CTCCAGCTCTGGGATACTGC midline fasciclin R:TGCTTTTCGTTCACCTTCCT F:CCACGAGACCACCTACAAC β-actin R:AGC GAG GGC AGT GAT TTC 396 397 398 399 400 401 402
403
404
Figure 1. Effect of V.parahaemolyticus infection on the endotoxin concentration and
405
diamine oxidase activities in L. vannamei. Values are means and SD represented by
406
vertical bars. * Asterisks indicate significant differences (P < 0.05).
407 408 409 410 411 412 413 414 415 416
417 418
419 420
Figure 2. Effect of V.parahaemolyticus infection on the intestinal barrier-related
421
genes expression (peroxinectin, integrin, midline fasciclin) in L. vannamei. Values are
422
means and SD represented by vertical bars. * Asterisks indicate significant differences
423
(P < 0.05).
424 425 426 427 428 429 430 431 432 433
434
435 436 437
Figure 3. Effect of V.parahaemolyticus infection on serum glucose concentration in L.
438
vannamei. Values are means and SD represented by vertical bars. * Asterisks indicate
439
significant differences (P < 0.05).
440 441 442 443 444 445 446 447
448
Figure 4. Effect of V.parahaemolyticus infection on expression of intestinal glucose
449
transporter (SGLT-1, GLUT) in L. vannamei. Values are means and SD represented by
450
vertical bars. * Asterisks indicate significant differences (P < 0.05).
451 452 453 454 455 456 457 458 459 460 461 462 463
,
464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483
Figure 5. Effect of V.parahaemolyticus infection on serum amino acid concentration
484
in
485
indicate significant differences (P < 0.05)
L. vannamei. Values are means and SD represented by vertical bars. * Asterisks
486 487
Figure 6. Effect of V.parahaemolyticus infection on expression of intestinal amino
488
acid transporter (CAT1, EAAT3 and ASCT1) in L. vannamei. Values are means and
489
SD represented by vertical bars. * Asterisks indicate significant differences (P < 0.05).
490 491 492 493 494 495 496 497 498 499 500 501
Highlights V. parahaemolyticus infection increased intestinal permeability in L. vannamei. V. parahaemolyticus infection inhibited intestinal absorption of glucose and amino acid in L. vannamei.