Kumar et al. Vet Res (2019) 50:54 https://doi.org/10.1186/s13567-019-0673-8 RESEARCH ARTICLE Open Access Modulation of posterior intestinal mucosal proteome in rainbow trout (Oncorhynchus mykiss) after Yersinia ruckeri infection Gokhlesh Kumar1* , Karin Hummel2, Ebrahim Razzazi‑Fazeli2 and Mansour El‑Matbouli1 Abstract Yersinia ruckeri is the causative agent of enteric redmouth disease in salmonids. In fsh, the intestine represents an important site of nutrient uptake, host–pathogen interactions, and defense. The posterior intestine can be infamed, reddened, and flled with an opaque, yellowish fuid during Y. ruckeri infection. Herein, we report an investigation on the proteome alteration in the posterior intestinal mucosa of rainbow trout (Oncorhynchus mykiss) after exposure to Y. ruckeri. The intestinal mucosal proteins were identifed and quantifed by a shotgun proteomic approach by applying data‑independent quantifcation with sequential windowed acquisition of all theoretical mass spectra (SWATH). A total of 437 proteins were found to be diferentially up‑ or downregulated in the posterior intestine. Gene ontology of upregulated proteins pointed to their involvement into exopeptidase, endopeptidase, and hydrolase activities, while the downregulated proteins were involved in lipid metabolism, actin binding, and translation processes. Additionally, upregulated proteins were predicted to be involved in lysosome, oxidative phosphorylation, and metabolic pathways, while downregulated proteins were implicated in focal adhesion, regulation of actin cytoskeleton, protein digestion and absorption pathways. This study showed that Y. ruckeri infection can alter protein abundance involved in serine‑ type carboxypeptidase, cysteine and aspartic‑type endopeptidases, metallopeptidases, antioxidant defense, calcium ion binding, glycolytic and carbohydrate metabolic processes in the proteome of the intestinal mucosa of rainbow trout. Introduction in their posterior intestines [4]. Y. ruckeri appeared to Enteric redmouth disease (ERM) is one of the major dis- adhere or invade gills and gut tissue of rainbow trout [3, eases of mainly salmonid fsh. It was frst described in 5]. Y. ruckeri can also be seen in the lumen of the intes- the 1950s in rainbow trout (Oncorhynchus mykiss) in the tine, adhering to mucus and invading villi within 30 min Hagerman Valley of Idaho, USA. Te disease is caused after exposure and can be detected 7 days post-exposure by Yersinia ruckeri, a Gram-negative rod shaped entero- (dpe) in the liver, spleen, brain, and heart [6]. bacterium, and prevalent in Europe, North and South Te strains of Y. ruckeri have been classifed on the America, Australia, South Africa, the Middle East, and basis of biotypes, serotypes (O1, O2, O3, and O4), and China [1, 2]. Te signs of the disease include exophthal- outer-membrane protein types [7]. Te majority of epizo- mia, subcutaneous hemorrhages, and splenomegaly [1]. otics in salmonids are caused by the motile serotype O1a In most cases, the intestine is infamed, reddened, and [1]. Tere are two biotypes of Y. ruckeri; biotype 1 is posi- flled with an opaque, yellowish fuid [1, 3]. In rainbow tive for motility and lipase secretion, whereas biotype 2 trout, up to 25% of infected fsh could carry Y. ruckeri is negative for both tests [8]. Te outbreaks of ERM were recorded in biotype 1-vaccinated salmonids and the iso- lates associated with these outbreaks were identifed as Y. *Correspondence: [email protected] ruckeri biotype 2 [9–12]. 1 Clinical Division of Fish Medicine, University of Veterinary Medicine, In fsh, the gastrointestinal (GI) tract is a multifunc- Vienna, Austria Full list of author information is available at the end of the article tional organ, which serves a diverse range of functions © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/ publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Kumar et al. Vet Res (2019) 50:54 Page 2 of 14 from nutrient absorption to ionic and osmotic regula- recovered from the head kidney and confrmed using the tion and even air breathing [13–19]. Gut epithelial cells MONO-Yr kit (Bionor, Skien, Norway) or PCR [31]. No are protected by a mucus layer, which creates a physical morbid and moribund fsh were sampled for the study. and chemical barrier against an intruder and acts as an Te fsh were maintained in starvation 48 h before the important mechanism of innate defense that maintains sampling. Nine fsh from each group were sampled at tissue homeostasis [20]. Te GI tract is continuously 3, 9, and 28 dpe. Te posterior intestine of each sample challenged with food antigens as well as pathogens enter- was dissected and placed in cold sterile PBS (Sigma- ing the body via feed and water intake, and acts as the Aldrich, Neustadt, Germany). Te intestine was opened frst line of defense against pathogen attachment and along the mesenteric border and washed three times with invasion [20–22]. Te posterior portion of the GI tract cold sterile PBS containing a cocktail of mammalian pro- is a site where uptake of macromolecules and foreign tease inhibitors (Sigma-Aldrich) to remove digesta and antigens occurs, which leads to antigen uptake and pro- fecal matter. Intestinal mucosa was scraped with a ster- cessing as well as the initiation of a systemic immune ile large scalpel blade. Each sample was divided into two response [20, 23]. Te disease has been of a major con- parts, one immediately snap-frozen in liquid nitrogen for cern in salmonids, which infuences the physiological proteomic analysis, and one fxed in RNALater (Sigma- response of fsh. Rainbow trout is highly susceptible to Aldrich) for molecular analysis and stored at −80 °C. ERM [1]. Te efect of natural herbal products on growth performance, hematological and biochemical values and Protein extraction resistance to Y. ruckeri infection was studied as a feed additives for rainbow trout [24, 25]. Equal amounts (30 mg) of nine individual intestinal In ERM, hyperemia and hemorrhages throughout the mucosal samples from each group were pooled randomly intestinal mucosa in rainbow trout have been observed as three pools of three samples each in order to mini- [1]. Te intestinal response of rainbow trout has been mize the efects of individual variation. It refers to N = 3 mainly studied at the transcription level, especially for the biological replicates per time point (3, 9 and 28 regarding immune gene expression [26–29]. It is still dpe) and per exposed group (control, Y. ruckeri biotype largely unknown how the gut mucosal proteome of the 1 and biotype 2). Each pool was ground using a sterilized rainbow trout responds to alternations in the luminal mortar and pestle in the presence of liquid nitrogen to a environment triggered by Y. ruckeri. Terefore, in the fne powder, which was then mixed with 800 μL of pre- current study, we aimed to evaluate the possible efects cooled denaturing lysis bufer (7 M urea, 2 M thiourea, of Y. ruckeri exposure on the modulation of the proteome 4% CHAPS, and 1% DTT) containing mammalian pro- profle in the posterior intestine of rainbow trout. In tease inhibitor cocktail (Sigma-Aldrich). Te samples order to meet this aim, we applied a label-free shotgun were subjected to ultrasonic disruption for 10 cycles of proteomic approach. 10 s pulse-on and 30 s pulse-of. Te samples were then centrifuged at 12 000 rpm for 20 min at 4 °C to remove Materials and methods any cellular debris. Te supernatant was collected. Te protein content was measured using the Pierce 660 nm Collecting posterior intestine samples Protein Assay (Termo Scientifc, Vienna, Austria). Te details of the experimental setup have been described in our previous study [30]. Briefy, specifc pathogen free Protein digestion rainbow trout (15 ± 1 cm) were allocated to 9 aquaria, 18 fsh per aquarium. In order to monitor the efect of Te protein digestion was performed following a stand- Y. ruckeri exposure, the following three treatments were ard enhanced, flter-aided sample preparation protocol given in parallel—Treatment 1: an exposure with biotype (FASP) [32]. Briefy, after washing, proteins were reduced 1 (CSF007–82), Treatment 2: an exposure with biotype 2 and alkylated. On-flter digestion was performed with (A7959–11), and Treatment 3: a mock exposure to sterile 1.2 µg Trypsin/Lys-C mix (Promega, Madison, USA) for tryptic soya broth. In both the test groups, the fsh were 14 h at 37 °C. Digested peptides were recovered, dried, 6 challenged by bath exposing to 2 × 10 colony forming and redissolved in 0.1% aqueous trifuoroacetic acid. units (CFU) of Y. ruckeri biotype 1 (CSF007-82) and Y. After desalting and sample cleanup by C18 ZipTips ruckeri biotype 2 (A7959-11) strains for 2 h. Te fsh were (Sigma-Aldrich), all samples were spiked with standard- maintained in a fow-through system supplied with UV- ized indexed retention time reference peptides for facili- treated ground water at 19 ± 1 °C, monitored daily, and tation of retention time alignment (iRT kit, Biognosys, morbid, moribund, and dead fsh were taken out imme- Switzerland). For each liquid chromatography (LC)–mass diately from the aquaria. Te mortality of fsh was only spectrometer (MS) analysis, 3 µg of digested protein was considered to be caused by Y. ruckeri if the bacteria were loaded onto the LC column. Kumar et al. Vet Res (2019) 50:54 Page 3 of 14 Microfow high‑performance liquid search, and 0.0011 Da in MS and 0.01 Da in MSMS mode chromatography electrospray‑ionisation for the fnal search.