Regulatory effects of Shewanella putrefaciens isolated from shrimp Penaeus orientalis on the virulence factors of Vibrio parahaemolyticus and evaluation of the role of quorum sensing in virulence factors regulation

Regulatory effects of Shewanella putrefaciens isolated from shrimp Penaeus orientalis on the... Abstract As an aquatic pathogen widely present in aquatic food, Vibrio parahaemolyticus causes outbreaks of gastroenteritis across the globe. Virulence factors of V. parahaemolyticus increases with the amount of spoilage in aquatic organisms including shrimp, but mechanisms regulating its virulence factors are not well understood. In this study, five spoilage bacteria isolated from shrimp were investigated for their regulatory effects on the virulence factors including haemolysin and biofilm of V. parahaemolyticus. Among these isolates, Shewanella putrefaciens induced haemolytic activity in V. parahaemolyticus in a time-dose-temperature-dependent manner and we found the main component responsible for this effect to be the supernatant or cell-free extract of S. putrefaciens. Total haemolytic activity, expression of the thermostable direct haemolysin gene tdh and biofilm production of V. parahaemolyticus were significantly up-regulated by S. putrefaciens, but also by deletion of quorum-sensing luxM or luxS gene of V. parahaemolyticus. However, this regulation by S. putrefaciens was significantly impaired by deletion of the luxM gene, but not by deletion of the luxS gene. Further study showed that S. putrefaciens exhibited a strong degradation ability on the signalling molecule acylated homoserine lactone (AHL) synthesised by the LuxM enzyme. This study revealed a novel virulence regulatory mechanism that S. putrefaciens can significantly increase the virulence factors of V. parahaemolyticus via interfering with the luxM- type quorum-sensing signalling pathway through its AHL-degradation ability. Vibrio parahaemolyticus, virulence regulation, Shewanella putrefaciens, quorum sensing, luxM, luxS INTRODUCTION As an aquatic food-spoilage pathogen, Vibrio parahaemolyticus is frequently detected in spoiled fish and shrimp (Chandrasekaran, Lakshmanaperumalsamy and Chandramohan 1984; Ruangpan and Kitao 2006), and coexists with various microorganisms. The risk of acquiring gastroenteritis during consumption of these potential V. parahaemolyticus-contaminated shrimp has raised great concern among scientists and the general public (Abdullah Sani, Ariyawansa and Babji 2013). The virulence factors of V. parahaemolyticus and other vibrio species are influenced by co-cultured bacteria, especially by the spoilage bacteria (Nayak and Karunasagar 1997; Chabrillón, Rico and Arijo 2005). In order to identify the potential hazard of these spoilage bacteria with V. parahaemolyticus and to guarantee seafood security, it is necessary to evaluate the effect of these spoilage bacteria on virulence factors of V. parahaemolyticus. Virulence factors such as hemolysin and biofilm have been found to play important roles in human gastroenteritis and other seafood-borne illness infection (Daniels, MacKinnon and Bishop 2000; Takahashi, Sato and Shiomi 2000; Zhang and Orth 2013; Liao, Li and Wu 2015). The thermostable direct hemolysin (TDH) is considered as a major virulence factor of the pathogenic V. parahaemolyticus in causing human gastrointestinal disorders (Takahashi, Sato and Shiomi 2000; Raghunath 2014). Quorum sensing (QS) is a stimulus–response communication that occurs between intra- or inter-specific bacteria including V. parahaemolyticus (Zhu, Zhao and Feng 2016), and controls a majority of bacterial virulence factors through the signalling molecules, autoinducers (Miller and Bassler 2001). In V. parahaemolyticus, the main autoinducers, acylated homoserine lactone (AHL) and AI-2, produced by luxM and luxS synthase, respectively, allow intra- and inter-specific QS (Miller and Bassler 2001; Henke and Bassler 2004; Jensen, Depasquale and Harbolick 2013). It is hypothesised that spoilage bacteria regulate the virulence factors of V. parahaemolyticus through interfering with luxM- and luxS-type QS. However, to our knowledge, this hypothesis has not yet been confirmed. This study was conducted to identify the function and mechanisms regulating the virulence factors of V. parahaemolyticus in spoiled shrimp. The regulatory effects of seafood spoilage bacteria on the virulence factors of V. parahaemolyticus were explored for hemolytic activity and biofilm formation, as well as the role of luxM- and luxS-type QS in this regulation. MATERIALS AND METHODS Bacterial strains and growth conditions The strains of bacteria used were the wild-type V. parahaemolyticus strain ATCC33847 (obtained from the China Committee for Culture Collection of Microorganisms), VP∆LuxS (luxS∆::Cmr, from our lab) and VP∆LuxM (luxM∆::Cmr, from our lab). Vibrio parahaemolyticus and mutants (VP∆LuxS and VP∆LuxM, constructed by using the homologous recombination method) were grown in Luria-Bertani (LB) culture (1.0% tryptone, 1.0% yeast extract and 0.5% NaCl, w/v) supplemented without or with 10 µg mL–1 of chloramphenicol (Sigma, St Louis, MO, USA) at 37°C. Yeast extract, tryptone, agar and NaCl were purchased from Beijing Landbridge Technical Co. (Beijing, China); LB broth was purchased from Guangdong Huankai Microbial Sci. & Tech. Co. (Guangzhou, China); and DAPI (4',6-diamidino-2-phenylindole), crystal violet, sodium citrate and ammonium acetate were purchased from Guangdong Qiyun BIO-Tech. Co. (Guangzhou, China). Isolation and identification of spoilage bacteria from spoiled shrimp The isolation of spoilage bacteria from spoiled shrimp was performed as previously described (Setia and Suharjono 2015). Twenty spoiled shrimps were eviscerated and homogenised with a glass homogeniser and 0.1 mL of 10-fold step-dilutedsuspensions of shrimp homogenate were spread onto LB medium plates (LB broth supplemented with 2.0 % agar, w/v) at 37°C for 20 h. One thousand colonies were added to 3 mL sterilizing saline water and identified by auto-biochemical identification system (Vitek 2 Compact 60, Marcy l'Etoile, France) and the isolation frequencies of each bacterial species in total colonies (frequency in total) were calculated. Each colony was cultured and stored at −80°C. Fifteen samples (three random colonies from each dominant species) were sent to the Beijing Genomics Institute for further identification by 16s rRNA gene sequencing with universal primers 27F (5'-GAGAGTTTGATCCTGG CTCAG-3') and 1492R (5'-CTACGGCTACCTTGT TACGA-3'), and the 16S rRNA gene sequence was aligned against GenBank databases (Wang, Fan and Yao 2010) Extracts of culture supernatants of Shewanella putrefaciens Revived Shewanellaputrefaciens was grown in 500 mL of fresh LB culture. After 18-h incubation at 37°C and 200 r.p.m., the cultured supernatants were extracted as previously described (Aguirre-Alvarez, Rodriguez-Huezo and Hernandez-Fuentes 2011). The culture supernatants were centrifuged and condensed through an Amicon Ultra-15 membrane (10 kDa, Millipore-Sigma, USA) at 3000g at 4°C for 2 h, yielding ultrafiltered extracts of culture supernatants of S. putrefaciens. The extracts of culture supernatants were diluted with pre-cooling LB culture to 5 mL (S. putrefaciens supernatant stock solution), and the stock solution was gradually diluted with pre-cooling LB culture to 1:100, 1:125, 1:250 and 1:500 for further analysis. Vibrio parahaemolyticus and shrimp spoilage bacteria co-culture system The V. parahaemolyticus and shrimp spoilage bacteria co-culture system was established as previously described (Gehin, Cailliez and Petitdemange 1996; Nayak and Karunasagar 1997). One hundred microlitres (∼1 OD600) revived V. parahaemolyticus strain ATCC33847, VP∆LuxS and VP∆LuxM were inoculated into 10 mL of fresh LB culture in sterile dialysis bags (8–14.4 kDa). The bags were sealed and transferred into a 90-mLLB culture inoculated with revived shrimp spoilage bacteria or S. putrefaciens supernatant extracts (at the dilution of 1:500, 1:250, 1:125 and 1:100, v/v). After co-incubation for time periods (3, 9, 15, 18, 21, 24, 27, 30, 33 h) at indicated temperatures 25°C, 37°C or 42°C with shaking (at 150 r.p.m.), samples in sterile dialysis bags were harvested to analyse the influence of shrimp spoilage bacteria or S. putrefaciens supernatant extracts on total hemolytic activity, tdh expression and biofilm formation of V. parahaemolyticus. Total hemolytic activity The total hemolytic activity of V. parahaemolyticus was measured as previously described (Tang, Iida and Yamamoto 1994; Wang, Ling and Jiang 2013). Fresh erythrocytes from rabbit blood were harvested with 3500 r.p.m. centrifugation for 5 minat 4°C, and washed and diluted to 5% with saline solution. One hundred microlitres of incubated cultures of V. parahaemolyticus strains ATCC33847, VP∆LuxM or VP∆LuxS were mixed with 400 µLof 5% (v/v) rabbit blood in saline solution. Vibrio parahaemolyticus strain ATCC33847 in sterile dialysis bags co-incubated with 0 dose of S. putrefaciens supernatant was taken as the control, fresh LB culture in sterile dialysis bags without bacteria was taken as the blank for determining the total hemolytic activity of single cultivated V. parahaemolyticus strains. Fresh LB culture in sterile dialysis bags separately co-incubated with shrimp spoilage bacteria or their supernatants was taken as the blank for determining total haemolytic activity of co-cultivated V. parahaemolyticus strains. After 90 min incubation at 37°C, samples were centrifuged at 12000g for 1 min at 4°C. The absorbance of supernatants was measured using an ultraviolet spectrophotometer at 570 nm. All assays were performed at least three times. Quantitative RT-PCR Analyses of tdh mRNA levels were performed as previously described (Ma, Sun and Xu 2015) with minor modification. Vibrio parahaemolyticus was ground in liquid nitrogen, and V. parahaemolyticus membrane ruptured by using a Trizol reagent. The total RNA was purified with isopropanol (50%) and ethanol (75%), and digested with DNase I (total RNA 6 µL, DNase I 12 µL, 10× buffer2 µLand RNase-free H2O 10 µL) at 37°C for 30 min,and the reaction was terminated at 65°C for 10 min. The concentration was determined using a K2800 UV/Vis spectrophotometer (Kaiao, Beijing, China). One microgram of total RNA was reverse transcribed by using the HiScript Q™ RT SuperMix Kit (Vazyme Biotech, Nanjing, China). The PCR system was composed of 5 µLof AceQ® qPCR SYBR® Green Master Mix, 4 µLcDNA, 0.2 µL 50× ROXReference Dye 2, 0.2 µLforward primer (5'-GTCCCTTTTCCTGCCCC10-3', 10 µM), 0.2 µLreverse primer (5'-CCATAAACATCTTCGTACGGTTTTC-3', 10 µM)and 0.4 µLddH2O. Reactions were conducted using an ABI7500 real-time PCR detection system (Thermo Fisher, MA, USA). Three replicates were arranged for each treatment, and all measurements were performed in triplicate. 16S rRNA PCR products (forward primer: 5'-GGGGAATATTGCACAATGGG-3'; reverse primer 5'- TTCTTCTGGCGCTAA CGTCA -3') were used as an internal control. Analysis of biofilm formation The quantification of biofilm formation was performed as previously described (Klug, Rodler and Koller 2011; Vezzulli, Pezzati and Stauder 2015) with minor modification. Revived V. parahaemolyticus strains in LB medium on a glass slide in a test tube were incubated or co-cultivated with S. putrefaciens supernatant (dilution of 1:100) for 5 days at 37°C, after which the glass slide was gently taken out and washed three times with water. Biofilm adhering to the surface was fixed by immersing the slide in methyl alcohol for 15 min, and then stained with 2% crystal violet. After 5 min of incubation at room temperature, the residual dye was washed with water, and bound dye was extracted from stained cells by adding 33% acetic acid, and quantified by measuring the absorbance at 590 nm. For biofilm formation analysis, the fluorescence in situ hybridisation (FISH) method was performed as previously described (Klug, Rodler and Koller 2011) with minor modifications. Biofilm adhering to the glass slide surface was fixed in 4% paraformaldehyde (pH = 7.4) for 60 min at 4°C, and washed twice with phosphate-buffered saline. After cell permeabilisation by 1% Triton X-100 for 5 min, adherent biofilm was washed twice with phosphate-buffered saline, and fixed in 70%, 85%, 96% (v/v) ice-ethanol for 5 min. After air-drying at room temperature, the sample slide was co-incubated with freshly prepared hybridisation buffer (50% formamide, 10% 50× SSC (Saline Sodium Citrate), 0.5% SDS, 100 µg mL–1 Salmon Sperm DNA, 10% dextran sulfate) for 5 min at 73°C, and then cooled in ice. Then 15 µLof CY3 probe solution (Biosense, Guangdong, China) was added to the sample slide, and a coverslip placed over the sample. After heat denaturation for 3 min at 73°C and incubation for 16 h at 46°C, the sample slide was washed twice with wash buffer at 48°C followed by two washes with ice-cold SSC buffer (0.3 M NaCl, 0.03 M sodium citrate). After FISH, washing and re-dyeing with 100 µg mL–1 DAPI (4',6-diamidino-2-phenylindole), biofilm present on the sample slide was observed under a DMI4000B fluorescence microscope (Leica, Wetzlar, Germany). Measurement of acyl-homoserine lactones (AHL) by LC-MS/MS AHL of V. parahaemolyticus were extracted and prepared as previously described (Burton, Read and Pellitteri 2005). AHL was made into different dilutions [(1:250, 1:125, 1:100), and the mixture of AHL and LB culture was taken as the control] of S. putrefaciens supernatant culture and allowed to react at 37°C for 1 h as described (Morohoshi, Nakazawa and Ebata 2008). AHL samples and standards (Cayman Chemical and Sigma) were analysed by liquid chromatography (LC)-mass spectrometry (MS) using a Tandem Quadrupole LC-MS/MS system (LCMS-8030, Shimadzu) as previously described (Ortori, Dubern and Chhabra 2011). The parameters used were: flow rate 0.3 mL min–1, injection volume 10.0 µL, column temperature 33°C, stop time 25 min. The mobile phase was a mixture of methanol (A) and 2 mM ammonium acetate solution containing 0.1% formic acid (B). The ratios of LC-MS/MS peak areas of the analyte to an internal standard were measured. Statistical analysis All experiments were performed at least three times. Values are expressed as the mean ± standard deviation (SD). Statistical analysis was performed using SPSS version 19.0 and Student’s t-test. The test results were compared with control and statistical significance was determined at a P value of <0.05 or <0.01. RESULTS Effects of shrimp spoilage bacteria on total hemolytic activity of V. parahaemolyticus In addition to V. parahaemolyticus, five additional bacterial species predominating in spoiled shrimp were isolated. These bacteria were identified by 16s RNA sequencing as Oceanisphaera profunda, Proteus vulgaris, S. putrefaciens, Stenotrophomonas maltophilia and Bacillus siamensis (Table 1). To reveal the mechanisms regulating the virulence factors of V. parahaemolyticus in shrimp, the effects of these five bacterial isolates on total hemolytic activity of V. parahaemolyticus were tested. Among them, S. putrefaciens, S. maltophilia and P. vulgaris exhibited significant enhancement of total hemolytic activity, with S. putrefaciens being the highest (Fig. 1) . The order of enhancement effects of bacterial isolates is S. putrefaciens > P. vulgaris > S. maltophilia. Therefore, we focused on the S. putrefaciens for further investigation. Figure 1 View largeDownload slide . Effects of five spoilage bacterial isolates on total haemolytic activity of V. parahaemolyticus (VP). Total haemolytic activity of V. parahaemolyticus single-cultured or co-cultured with different bacterial isolates in LB medium was tested after 24-h incubation at 37°C. The data represent the means of three independent experiments. Error bars represent mean ± SD. Asterisks (*P <0.05) indicate results that differ significantly from the single-cultured V. parahaemolyticus. Figure 1 View largeDownload slide . Effects of five spoilage bacterial isolates on total haemolytic activity of V. parahaemolyticus (VP). Total haemolytic activity of V. parahaemolyticus single-cultured or co-cultured with different bacterial isolates in LB medium was tested after 24-h incubation at 37°C. The data represent the means of three independent experiments. Error bars represent mean ± SD. Asterisks (*P <0.05) indicate results that differ significantly from the single-cultured V. parahaemolyticus. Table 1. Five bacterial isolates from spoilage shrimp. Isolates Frequency in total Accession No. Identities Identity O. profunda 9.2% KX675452.1 941/952 98.84% P. vulgaris 7.5% KT887953.1 1405/1406 99.92% S. putrefaciens 7.1% KC607519.1 1368/1374 99.56% S. maltophilia 6.4% MF429096.1 912/915 99.67% B. siamensis 6.1% MF465780.1 941/942 99.89% Isolates Frequency in total Accession No. Identities Identity O. profunda 9.2% KX675452.1 941/952 98.84% P. vulgaris 7.5% KT887953.1 1405/1406 99.92% S. putrefaciens 7.1% KC607519.1 1368/1374 99.56% S. maltophilia 6.4% MF429096.1 912/915 99.67% B. siamensis 6.1% MF465780.1 941/942 99.89% View Large Table 1. Five bacterial isolates from spoilage shrimp. Isolates Frequency in total Accession No. Identities Identity O. profunda 9.2% KX675452.1 941/952 98.84% P. vulgaris 7.5% KT887953.1 1405/1406 99.92% S. putrefaciens 7.1% KC607519.1 1368/1374 99.56% S. maltophilia 6.4% MF429096.1 912/915 99.67% B. siamensis 6.1% MF465780.1 941/942 99.89% Isolates Frequency in total Accession No. Identities Identity O. profunda 9.2% KX675452.1 941/952 98.84% P. vulgaris 7.5% KT887953.1 1405/1406 99.92% S. putrefaciens 7.1% KC607519.1 1368/1374 99.56% S. maltophilia 6.4% MF429096.1 912/915 99.67% B. siamensis 6.1% MF465780.1 941/942 99.89% View Large Time, dose and temperature effects of S. putrefaciens on total hemolytic activity of V. parahaemolyticus To better understand virulence factors regulation by S. putrefaciens, first its effect on the total hemolytic activity of V. parahaemolyticus over time at 37°C was tested. At 15 h, total hemolytic activity of V. parahaemolyticus was not enhanced on co-cultivation with S. putrefaciens. However, at 18 h, the enhancement was significant and the largest effect was observed at 24 h. But this enhancement effect gradually weakened at >33 h (Fig. 2A). Figure 2. View largeDownload slide Time, dose and temperature effects of S. putrefaciens on total haemolytic activity of V. parahaemolyticus (VP). Total haemolytic activity of V. parahaemolyticus single-cultured or co-cultured with S. putrefaciens for the indicated time (3, 9, 15, 18, 21, 24, 27, 30, 33 h) (A) or co-cultured with indicated doses of S. putrefaciens supernatant (SP) (at the dilution of 1:500, 1:250, 1:125 and 1:100) (B) at 25, 37 and 42°C (C). Vibrio parahaemolyticus co-incubated with 0 dose of S. putrefaciens supernatant was taken as the control. Fresh LB culture in sterile dialysis bags separately co-incubated with S. putrefaciens supernatant (SP) was taken as the blank. The data represent the means of three independent experiments. Error bars represent mean ± SD. Asterisks (*P <0.05, **P <0.01) indicate results that differ significantly from the single-cultured V. parahaemolyticus. Figure 2. View largeDownload slide Time, dose and temperature effects of S. putrefaciens on total haemolytic activity of V. parahaemolyticus (VP). Total haemolytic activity of V. parahaemolyticus single-cultured or co-cultured with S. putrefaciens for the indicated time (3, 9, 15, 18, 21, 24, 27, 30, 33 h) (A) or co-cultured with indicated doses of S. putrefaciens supernatant (SP) (at the dilution of 1:500, 1:250, 1:125 and 1:100) (B) at 25, 37 and 42°C (C). Vibrio parahaemolyticus co-incubated with 0 dose of S. putrefaciens supernatant was taken as the control. Fresh LB culture in sterile dialysis bags separately co-incubated with S. putrefaciens supernatant (SP) was taken as the blank. The data represent the means of three independent experiments. Error bars represent mean ± SD. Asterisks (*P <0.05, **P <0.01) indicate results that differ significantly from the single-cultured V. parahaemolyticus. Next, the dose effect of S. putrefaciens supernatant on total hemolytic activity of V. parahaemolyticus was tested following 18 h co-cultivation at 37°C. Compared with the control (V. parahaemolyticus alone), co-cultivation with a 1:500 dilution of S. putrefaciens supernatant did not exhibit significant enhancement. However, at low dilutions (<1:250), S. putrefaciens supernatant exhibited significant dose-dependent enhancement on total hemolytic activity of V. parahaemolyticus (Fig. 2B). However, S. putrefaciens supernatant itself (blank) did not induce obvious hemolytic activity. Lastly, the temperature effect on this regulation by S. putrefaciens supernatant was tested after 18 h co-cultivation at a dilution of 1:100 (similarly hereinafter). At 37°C, S. putrefaciens supernatant exhibited higher enhancement than at room temperature (25°C). However, a higher temperature (42°C) did not significantly increase the regulatory effect of S. putrefaciens supernatant on the total hemolytic activity of V. parahaemolyticus (Fig. 2C). Roles of luxM- and luxS-type QS in regulation of S. putrefaciens Because luxM- and luxS-type QS play an important role in virulence factors regulation of V. parahaemolyticus, we presumed that S. putrefaciens supernatant would enhance the total hemolytic activity of V. parahaemolyticus through luxM and/or luxS. To test this hypothesis, the QS autoinducer-synthesis enzyme encoding genes luxM and/or luxS defect strains of V. parahaemolyticus (namely, VP∆LuxM and VP∆LuxS) were employed, and the enhancement effects of S. putrefaciens supernatant on total hemolytic activity of VP∆LuxM and VP∆LuxS were determined. The deletion of luxM but not luxS (VP∆LuxS) significantly (P < 0.05) impaired the enhancement effect of S. putrefaciens (Fig. 3A), confirming that S. putrefaciens supernatant enhanced the total hemolytic activity of V. parahaemolyticus through luxM. In addition, compared with the wild-type VP, VP∆LuxM and VP∆LuxS exhibited higher total hemolytic activity. Deletion of luxM or luxS significantly increased the total hemolytic activity of V. parahaemolyticus, indicating that total hemolytic activity was negatively regulated by luxM and luxS. Figure 3. View largeDownload slide Effects of deletion of luxM and luxS on total hemolytic activity and tdh expression regulation by S. putrefaciens. (A) Total hemolytic activity of V. parahaemolyticus (VP) or mutants, single-cultured and co-cultured with S. putrefaciens in LB medium for 24 h at 37°C. (B) The relative tdh mRNA level of V. parahaemolyticus (VP) or mutants single-cultured and co-cultured with S. putrefaciens in LB medium for 24 h at 37°C. The data represent the means of three independent experiments. Error bars represent mean ± SD. Asterisks (*P <0.05, **P <0.01) indicate results that differ significantly from their corresponding single-cultured V. parahaemolyticus strains respectively, and hash signs (#P <0.05, ##P <0.01) indicate results that differ significantly from the single-cultured VP. Figure 3. View largeDownload slide Effects of deletion of luxM and luxS on total hemolytic activity and tdh expression regulation by S. putrefaciens. (A) Total hemolytic activity of V. parahaemolyticus (VP) or mutants, single-cultured and co-cultured with S. putrefaciens in LB medium for 24 h at 37°C. (B) The relative tdh mRNA level of V. parahaemolyticus (VP) or mutants single-cultured and co-cultured with S. putrefaciens in LB medium for 24 h at 37°C. The data represent the means of three independent experiments. Error bars represent mean ± SD. Asterisks (*P <0.05, **P <0.01) indicate results that differ significantly from their corresponding single-cultured V. parahaemolyticus strains respectively, and hash signs (#P <0.05, ##P <0.01) indicate results that differ significantly from the single-cultured VP. Role of luxM in induction expression of tdh by S. putrefaciens TDH is considered to be a major virulence factor in gastrointestinal disorders and possesses marked hemolytic activity (Fabbri, Falzano and Frank 1999; Yanagihara, Nakahira and Yamane 2010). We hypothesised that S. putrefaciens may promote tdh gene expression and thereby increase the total hemolytic activity of V. parahaemolyticus. To test this, the tdh expression of V. parahaemolyticus before and after co-cultivation with S. putrefaciens supernatant was quantified. The tdh expression was significantly up-regulated after co-cultivation with S. putrefaciens supernatant (Fig. 3B), which confirmed that S. putrefaciens increased the total hemolytic activity through tdh. Compared with the tdh of wild-type V. parahaemolyticus, the tdh of V. parahaemolyticus with defective luxM (VP∆LuxM) was not significantly induced by S. putrefaciens supernatant. Deletion of luxM from the wild-type V. parahaemolyticus significantly impaired the induction by S. putrefaciens supernatant. However, deletion of luxS did not significantly impair this induction. In addition, like S. putrefaciens supernatant, the deletion of luxM also increased the tdh expression level (Fig. 3B), which confirmed the negative regulation of tdh by luxM. These results suggest that S. putrefaciens induces tdh expression to enhance hemolytic activity by negatively affecting luxM. Important role of luxM in induction of biofilm formation by S. putrefaciens Biofilm formation was also negatively regulated by luxM-type QS but increased when the autoinducer concentration was low at low cell density (Hammer and Bassler 2003). To further confirm the relationship between S. putrefaciens supernatant and luxM on biofilm formation by V. parahaemolyticus, the biofilm formation of VP∆LuxM and VP∆LuxS were determined before and after co-cultivation with S. putrefaciens supernatant. Biofilm production, as observed under a fluorescence microscopy, increased after deletion of luxM or luxS (Fig. 4A). Co-cultivation with S. putrefaciens supernatant further increased the biofilm production of VP∆LuxS. However, co-cultivation with S. putrefaciens supernatant did not further increase the production of biofilm of VP∆LuxM (Fig. 4A). Biofilm production was also quantified with the crystal violet staining method. Co-cultivation with S. putrefaciens supernatant further significantly increased the biofilm production of VP and VP∆LuxS, but not VP∆LuxM (Fig.4B). Deletion of luxM impaired the regulation of S. putrefaciens supernatant on the biofilm formation of V. parahaemolyticus, which is consistent with the tdh expression results. These results together suggest that S. putrefaciens supernatant has regulatory effects on the hemolytic activity and biofilm formation of V. parahaemolyticus through its supernatant. Based on the above, we suggest that S. putrefaciens supernatant may induce tdh and biofilm formation by degrading the autoinducer AHL synthesised by luxM. However, this hypothesis has not been confirmed in V. parahaemolyticus and S. putrefaciens before. Figure 4. View largeDownload slide Effects of luxM and luxS deletion on biofilm formation regulation by S. putrefaciens. (A) Vibrio parahaemolyticus (VP) mutants single-cultured or co-cultured with S. putrefaciens supernatant were stained and visualised by fluorescence microscopy as described in the Materials and methods section (scale 100 µm; left cells stained with DAPI dye; right cells, with the bound CY3 fluorescent probe). (B) Quantification of biofilm formation of indicated strains in (A) by measuring its absorbance at 590 nm. Error bars represent mean ± SD. Asterisks (*P <0.05, **P <0.01) indicate results that differ significantly from their corresponding single-cultured strains, and hash signs (#P <0.05, ##P <0.01) indicate results that differ significantly from the single-cultured VP. Figure 4. View largeDownload slide Effects of luxM and luxS deletion on biofilm formation regulation by S. putrefaciens. (A) Vibrio parahaemolyticus (VP) mutants single-cultured or co-cultured with S. putrefaciens supernatant were stained and visualised by fluorescence microscopy as described in the Materials and methods section (scale 100 µm; left cells stained with DAPI dye; right cells, with the bound CY3 fluorescent probe). (B) Quantification of biofilm formation of indicated strains in (A) by measuring its absorbance at 590 nm. Error bars represent mean ± SD. Asterisks (*P <0.05, **P <0.01) indicate results that differ significantly from their corresponding single-cultured strains, and hash signs (#P <0.05, ##P <0.01) indicate results that differ significantly from the single-cultured VP. AHL degradation ability of the S. putrefaciens supernatant Some proteins secreted by bacteria, such as the lactonase enzyme, will degrade the extracellular autoinducer (Wang, Weng and Dong 2004). We hypothesised that the S. putrefaciens supernatant may degrade the luxM product (AHL) to increase the virulence factors of V. parahaemolyticus. To test this, the effect of S. putrefaciens supernatant on AHL was investigated and the AHL content significantly and exponentially decreased in a dose-dependent manner following S. putrefaciens supernatant treatment (Fig. 5). Shewanella putrefaciens supernatant exhibited a strong degradation ability on signalling molecule AHL. This demonstrated that S. putrefaciens extracellular supernatant increase the virulence factors by degrading the AHL of V. parahaemolyticus. Figure 5. View largeDownload slide Degradation of AHL by S. putrefaciens supernatant. Effect of S. putrefaciens supernatant on AHL following incubation for 1 h at 37°C, measured with LC-MS/MS. The mixture of AHL and blank culture without treatment was taken as the no treatment control. The data represent the means of three independent experiments. Error bars represent mean ± SD. Asterisks (*P <0.05, **P <0.01) indicate results that differ significantly from the no-treatment control. Figure 5. View largeDownload slide Degradation of AHL by S. putrefaciens supernatant. Effect of S. putrefaciens supernatant on AHL following incubation for 1 h at 37°C, measured with LC-MS/MS. The mixture of AHL and blank culture without treatment was taken as the no treatment control. The data represent the means of three independent experiments. Error bars represent mean ± SD. Asterisks (*P <0.05, **P <0.01) indicate results that differ significantly from the no-treatment control. DISCUSSION In this study, we obtained five bacterial species from spoiled shrimp. Among them, S. putrefaciens exhibited the highest enhancement of total hemolytic activity and was chosen for further investigation. A study of the effect or regulatory mechanism of S. putrefaciens on virulence factors of V. parahaemolyticus has not been previously reported. Previous studies have reported that S. putrefaciens is a major microbial cause of spoilage in seafood, frequently detected in marine environments and organisms, especially in spoiled shrimp and fish (Wright, Matthews and Arnold 2016). It is usually non-pathogenic to humans except under particular circumstances (Pagani, Lang and Vedovelli 2003). Shewanella was found to affect the spoilage of Pseudosciaena crocea through the QS system (Zhu, Zhao and Feng 2016) and Shewanella algae show an inhibitory effect against V. parahaemolyticus (Shakibazadeh, Saad and Christianus 2008). Some members of the human gut microbiota also affect Vibrio cholerae colonisation through autoinducers (Hsiao, Ahmed and Subramanian 2014). Our data suggest that S. putrefaciens increases the virulence factors of V. parahaemolyticus through interfering with QS. The S. putrefaciens exhibited a time-dependent enhancement effect (at >18 h) on total hemolytic activity of V. parahaemolyticus (Fig. 2A). Similar to that, the S. putrefaciens supernatant exhibited this enhancement effect only at dilutions <1:250(Fig.2B). Such time- and dose-dependent effects of S. putrefaciens on V. parahaemolyticus partly follow the patterns of QS activity in other Vibrio species (Defoirdt and Sorgeloos 2012). Vibrio harveyi showed about a 200-fold higher maximal QS-regulated bioluminescence when associated with shrimp larvae, especially dead shrimp, than in the culture water in early 14 to 24-h infection (Defoirdt and Sorgeloos 2012). The results further suggest QS may be involved in the regulation of virulence factors by S. putrefaciens. Temperature influences virulence factors of pathogens, especially the Vibrio species (Kimes, Grim and Johnson 2012). In our study, temperature also influenced the enhancement effect of S. putrefaciens on total hemolytic activity of V. parahaemolyticus (Fig. 2C). Compared with room temperature (25°C) or extreme temperature (42°C), the normal temperature (37°C) of the human body is an optimal co-cultivation temperature to increase the total hemolytic activity of V. parahaemolyticus by S. putrefaciens supernatant, suggesting that the storage temperature of S. putrefaciens-contaminated seafood may influence the risk of acquiring gastroenteritis. Generally, the virulence factors of bacteria are controlled by the QS system (Shih and Huang 2002; Hammer and Bassler 2003), such as in Pseudomonas aeruginosa (Bjarnsholt, Jensen and Burmolle 2005) and some Vibrio species (Hammer and Bassler 2003). The hemolytic activity-related genes including hlyA are negatively regulated by the QS system in V. cholerae (Tsou and Zhu 2010). Similar virulence factors regulation may also exist in V. parahaemolyticus, but the mechanism for this is unclear. There has been no prior explanation as to why S. putrefaciens supernatant may increase the total hemolytic activity of V. parahaemolyticus. From our study, the regulation effect of S. putrefaciens supernatant was impaired after deletion of luxM and not luxS, which indicated that the regulation of V. parahaemolyticus by S. putrefaciens supernatant is mainly dependent on luxM. This is in contrast to the more important role luxS plays in the virulence inhibition of vibrio by gut microbiota (Hsiao, Ahmed and Subramanian 2014). TDH possesses the main hemolytic activity (Fabbri, Falzano and Frank 1999; Yanagihara, Nakahira and Yamane 2010) and the tdh expression results (Fig. 3B) from our study confirmed that luxM instead of luxS plays an important role in the virulence factors regulation of V. parahaemolyticus by S. putrefaciens. luxM produces AHL (AI-1) by catalysing the AHL precursor, and that plays an important role in intra-species cell-to-cell communication (Miller and Bassler 2001). Therefore, the evidence is clear and compelling that the luxM-type QS of V. parahaemolyticus is regulated by S. putrefaciens. Biofilm formation is negatively controlled by the QS system, and also is negatively correlated with the AI concentrations (Hammer and Bassler 2003). From our study, the production of biofilm increased after deletion of luxM or luxS as expected. More importantly, the production of biofilm increased after co-cultivation with S. putrefaciens, and deletion of luxM impaired this induction by S. putrefaciens. These results confirmed that luxM is associated with the virulence factors regulation of V. parahaemolyticus by S. putrefaciens. From the results of tdh and biofilm, both S. putrefaciens supernatant and deletion of luxM increased tdh and biofilm levels. It would be reasonable to believe that S. putrefaciens supernatant degrades the extracellular products of luxM to inhibit the activity of luxM. In our assessment of degradation of the S. putrefaciens supernatant, it showed a strong degradation ability on AHL synthesised by luxM. We checked all the known proteins of S. putrefaciens from NCBI. It was apparent that beta-lactamase VPA0477 is highly conserved with N-acylhomoserine lactone-acylase of Shewanella, which is reported to markedly degrade the AHL production of Vibrio anguillarum (Morohoshi, Nakazawa and Ebata 2008). In this case, the S. putrefaciens supernatant contributed to reducing AHL in V. parahaemolyticus. In conclusion, the data we have generated support that the AHL degradation activity of S. putrefaciens may serve to increase the V. parahaemolyticus virulence factors. This discovery provides useful evidence for revealing the virulence factors up-regulation mechanism of V. parahaemolyticus in aquatic food spoilage. ACKNOWLEDGEMENTS The Tandem Quadrupole LC-MS/MS system was provided by the National Marine Products Quality Supervision & Inspection Center. We thank Jianmeng Liao for technical assistance with measurement of acyl-homoserine lactones. FUNDING This work was supported by the National Natural Science Foundation of China (grant number 31371746, 31371777, 31701706) and Higher Educational Cultivation Program for Major Scientific Research Projects of Guangdong Ocean University (grant number GDOU2013050205, 2014050203). Conflict of interest. None declared. REFERENCES Abdullah Sani N , Ariyawansa S , Babji AS . The risk assessment of Vibrio parahaemolyticus in cooked black tiger shrimps (Penaeus monodon) in Malaysia . Food Control . 2013 ; 31 : 546 – 52 . Google Scholar CrossRef Search ADS Aguirre-Alvarez G , Rodriguez-Huezo ME , Hernandez-Fuentes AD . Characterization of a yeast culture extract compound stimulating the growth of an anaerobic cellulolytic consortium . J Anim Physiol Anim Nutr (Berl) . 2011 ; 95 : 434 – 9 . Google Scholar CrossRef Search ADS PubMed Bjarnsholt T , Jensen PO , Burmolle M . 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Int J Food Microbiol . 2016 ; 217 : 146 – 55 . Google Scholar CrossRef Search ADS PubMed © FEMS 2018. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png FEMS Microbiology Ecology Oxford University Press

Regulatory effects of Shewanella putrefaciens isolated from shrimp Penaeus orientalis on the virulence factors of Vibrio parahaemolyticus and evaluation of the role of quorum sensing in virulence factors regulation

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Blackwell
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© FEMS 2018.
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0168-6496
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1574-6941
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10.1093/femsec/fiy097
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Abstract

Abstract As an aquatic pathogen widely present in aquatic food, Vibrio parahaemolyticus causes outbreaks of gastroenteritis across the globe. Virulence factors of V. parahaemolyticus increases with the amount of spoilage in aquatic organisms including shrimp, but mechanisms regulating its virulence factors are not well understood. In this study, five spoilage bacteria isolated from shrimp were investigated for their regulatory effects on the virulence factors including haemolysin and biofilm of V. parahaemolyticus. Among these isolates, Shewanella putrefaciens induced haemolytic activity in V. parahaemolyticus in a time-dose-temperature-dependent manner and we found the main component responsible for this effect to be the supernatant or cell-free extract of S. putrefaciens. Total haemolytic activity, expression of the thermostable direct haemolysin gene tdh and biofilm production of V. parahaemolyticus were significantly up-regulated by S. putrefaciens, but also by deletion of quorum-sensing luxM or luxS gene of V. parahaemolyticus. However, this regulation by S. putrefaciens was significantly impaired by deletion of the luxM gene, but not by deletion of the luxS gene. Further study showed that S. putrefaciens exhibited a strong degradation ability on the signalling molecule acylated homoserine lactone (AHL) synthesised by the LuxM enzyme. This study revealed a novel virulence regulatory mechanism that S. putrefaciens can significantly increase the virulence factors of V. parahaemolyticus via interfering with the luxM- type quorum-sensing signalling pathway through its AHL-degradation ability. Vibrio parahaemolyticus, virulence regulation, Shewanella putrefaciens, quorum sensing, luxM, luxS INTRODUCTION As an aquatic food-spoilage pathogen, Vibrio parahaemolyticus is frequently detected in spoiled fish and shrimp (Chandrasekaran, Lakshmanaperumalsamy and Chandramohan 1984; Ruangpan and Kitao 2006), and coexists with various microorganisms. The risk of acquiring gastroenteritis during consumption of these potential V. parahaemolyticus-contaminated shrimp has raised great concern among scientists and the general public (Abdullah Sani, Ariyawansa and Babji 2013). The virulence factors of V. parahaemolyticus and other vibrio species are influenced by co-cultured bacteria, especially by the spoilage bacteria (Nayak and Karunasagar 1997; Chabrillón, Rico and Arijo 2005). In order to identify the potential hazard of these spoilage bacteria with V. parahaemolyticus and to guarantee seafood security, it is necessary to evaluate the effect of these spoilage bacteria on virulence factors of V. parahaemolyticus. Virulence factors such as hemolysin and biofilm have been found to play important roles in human gastroenteritis and other seafood-borne illness infection (Daniels, MacKinnon and Bishop 2000; Takahashi, Sato and Shiomi 2000; Zhang and Orth 2013; Liao, Li and Wu 2015). The thermostable direct hemolysin (TDH) is considered as a major virulence factor of the pathogenic V. parahaemolyticus in causing human gastrointestinal disorders (Takahashi, Sato and Shiomi 2000; Raghunath 2014). Quorum sensing (QS) is a stimulus–response communication that occurs between intra- or inter-specific bacteria including V. parahaemolyticus (Zhu, Zhao and Feng 2016), and controls a majority of bacterial virulence factors through the signalling molecules, autoinducers (Miller and Bassler 2001). In V. parahaemolyticus, the main autoinducers, acylated homoserine lactone (AHL) and AI-2, produced by luxM and luxS synthase, respectively, allow intra- and inter-specific QS (Miller and Bassler 2001; Henke and Bassler 2004; Jensen, Depasquale and Harbolick 2013). It is hypothesised that spoilage bacteria regulate the virulence factors of V. parahaemolyticus through interfering with luxM- and luxS-type QS. However, to our knowledge, this hypothesis has not yet been confirmed. This study was conducted to identify the function and mechanisms regulating the virulence factors of V. parahaemolyticus in spoiled shrimp. The regulatory effects of seafood spoilage bacteria on the virulence factors of V. parahaemolyticus were explored for hemolytic activity and biofilm formation, as well as the role of luxM- and luxS-type QS in this regulation. MATERIALS AND METHODS Bacterial strains and growth conditions The strains of bacteria used were the wild-type V. parahaemolyticus strain ATCC33847 (obtained from the China Committee for Culture Collection of Microorganisms), VP∆LuxS (luxS∆::Cmr, from our lab) and VP∆LuxM (luxM∆::Cmr, from our lab). Vibrio parahaemolyticus and mutants (VP∆LuxS and VP∆LuxM, constructed by using the homologous recombination method) were grown in Luria-Bertani (LB) culture (1.0% tryptone, 1.0% yeast extract and 0.5% NaCl, w/v) supplemented without or with 10 µg mL–1 of chloramphenicol (Sigma, St Louis, MO, USA) at 37°C. Yeast extract, tryptone, agar and NaCl were purchased from Beijing Landbridge Technical Co. (Beijing, China); LB broth was purchased from Guangdong Huankai Microbial Sci. & Tech. Co. (Guangzhou, China); and DAPI (4',6-diamidino-2-phenylindole), crystal violet, sodium citrate and ammonium acetate were purchased from Guangdong Qiyun BIO-Tech. Co. (Guangzhou, China). Isolation and identification of spoilage bacteria from spoiled shrimp The isolation of spoilage bacteria from spoiled shrimp was performed as previously described (Setia and Suharjono 2015). Twenty spoiled shrimps were eviscerated and homogenised with a glass homogeniser and 0.1 mL of 10-fold step-dilutedsuspensions of shrimp homogenate were spread onto LB medium plates (LB broth supplemented with 2.0 % agar, w/v) at 37°C for 20 h. One thousand colonies were added to 3 mL sterilizing saline water and identified by auto-biochemical identification system (Vitek 2 Compact 60, Marcy l'Etoile, France) and the isolation frequencies of each bacterial species in total colonies (frequency in total) were calculated. Each colony was cultured and stored at −80°C. Fifteen samples (three random colonies from each dominant species) were sent to the Beijing Genomics Institute for further identification by 16s rRNA gene sequencing with universal primers 27F (5'-GAGAGTTTGATCCTGG CTCAG-3') and 1492R (5'-CTACGGCTACCTTGT TACGA-3'), and the 16S rRNA gene sequence was aligned against GenBank databases (Wang, Fan and Yao 2010) Extracts of culture supernatants of Shewanella putrefaciens Revived Shewanellaputrefaciens was grown in 500 mL of fresh LB culture. After 18-h incubation at 37°C and 200 r.p.m., the cultured supernatants were extracted as previously described (Aguirre-Alvarez, Rodriguez-Huezo and Hernandez-Fuentes 2011). The culture supernatants were centrifuged and condensed through an Amicon Ultra-15 membrane (10 kDa, Millipore-Sigma, USA) at 3000g at 4°C for 2 h, yielding ultrafiltered extracts of culture supernatants of S. putrefaciens. The extracts of culture supernatants were diluted with pre-cooling LB culture to 5 mL (S. putrefaciens supernatant stock solution), and the stock solution was gradually diluted with pre-cooling LB culture to 1:100, 1:125, 1:250 and 1:500 for further analysis. Vibrio parahaemolyticus and shrimp spoilage bacteria co-culture system The V. parahaemolyticus and shrimp spoilage bacteria co-culture system was established as previously described (Gehin, Cailliez and Petitdemange 1996; Nayak and Karunasagar 1997). One hundred microlitres (∼1 OD600) revived V. parahaemolyticus strain ATCC33847, VP∆LuxS and VP∆LuxM were inoculated into 10 mL of fresh LB culture in sterile dialysis bags (8–14.4 kDa). The bags were sealed and transferred into a 90-mLLB culture inoculated with revived shrimp spoilage bacteria or S. putrefaciens supernatant extracts (at the dilution of 1:500, 1:250, 1:125 and 1:100, v/v). After co-incubation for time periods (3, 9, 15, 18, 21, 24, 27, 30, 33 h) at indicated temperatures 25°C, 37°C or 42°C with shaking (at 150 r.p.m.), samples in sterile dialysis bags were harvested to analyse the influence of shrimp spoilage bacteria or S. putrefaciens supernatant extracts on total hemolytic activity, tdh expression and biofilm formation of V. parahaemolyticus. Total hemolytic activity The total hemolytic activity of V. parahaemolyticus was measured as previously described (Tang, Iida and Yamamoto 1994; Wang, Ling and Jiang 2013). Fresh erythrocytes from rabbit blood were harvested with 3500 r.p.m. centrifugation for 5 minat 4°C, and washed and diluted to 5% with saline solution. One hundred microlitres of incubated cultures of V. parahaemolyticus strains ATCC33847, VP∆LuxM or VP∆LuxS were mixed with 400 µLof 5% (v/v) rabbit blood in saline solution. Vibrio parahaemolyticus strain ATCC33847 in sterile dialysis bags co-incubated with 0 dose of S. putrefaciens supernatant was taken as the control, fresh LB culture in sterile dialysis bags without bacteria was taken as the blank for determining the total hemolytic activity of single cultivated V. parahaemolyticus strains. Fresh LB culture in sterile dialysis bags separately co-incubated with shrimp spoilage bacteria or their supernatants was taken as the blank for determining total haemolytic activity of co-cultivated V. parahaemolyticus strains. After 90 min incubation at 37°C, samples were centrifuged at 12000g for 1 min at 4°C. The absorbance of supernatants was measured using an ultraviolet spectrophotometer at 570 nm. All assays were performed at least three times. Quantitative RT-PCR Analyses of tdh mRNA levels were performed as previously described (Ma, Sun and Xu 2015) with minor modification. Vibrio parahaemolyticus was ground in liquid nitrogen, and V. parahaemolyticus membrane ruptured by using a Trizol reagent. The total RNA was purified with isopropanol (50%) and ethanol (75%), and digested with DNase I (total RNA 6 µL, DNase I 12 µL, 10× buffer2 µLand RNase-free H2O 10 µL) at 37°C for 30 min,and the reaction was terminated at 65°C for 10 min. The concentration was determined using a K2800 UV/Vis spectrophotometer (Kaiao, Beijing, China). One microgram of total RNA was reverse transcribed by using the HiScript Q™ RT SuperMix Kit (Vazyme Biotech, Nanjing, China). The PCR system was composed of 5 µLof AceQ® qPCR SYBR® Green Master Mix, 4 µLcDNA, 0.2 µL 50× ROXReference Dye 2, 0.2 µLforward primer (5'-GTCCCTTTTCCTGCCCC10-3', 10 µM), 0.2 µLreverse primer (5'-CCATAAACATCTTCGTACGGTTTTC-3', 10 µM)and 0.4 µLddH2O. Reactions were conducted using an ABI7500 real-time PCR detection system (Thermo Fisher, MA, USA). Three replicates were arranged for each treatment, and all measurements were performed in triplicate. 16S rRNA PCR products (forward primer: 5'-GGGGAATATTGCACAATGGG-3'; reverse primer 5'- TTCTTCTGGCGCTAA CGTCA -3') were used as an internal control. Analysis of biofilm formation The quantification of biofilm formation was performed as previously described (Klug, Rodler and Koller 2011; Vezzulli, Pezzati and Stauder 2015) with minor modification. Revived V. parahaemolyticus strains in LB medium on a glass slide in a test tube were incubated or co-cultivated with S. putrefaciens supernatant (dilution of 1:100) for 5 days at 37°C, after which the glass slide was gently taken out and washed three times with water. Biofilm adhering to the surface was fixed by immersing the slide in methyl alcohol for 15 min, and then stained with 2% crystal violet. After 5 min of incubation at room temperature, the residual dye was washed with water, and bound dye was extracted from stained cells by adding 33% acetic acid, and quantified by measuring the absorbance at 590 nm. For biofilm formation analysis, the fluorescence in situ hybridisation (FISH) method was performed as previously described (Klug, Rodler and Koller 2011) with minor modifications. Biofilm adhering to the glass slide surface was fixed in 4% paraformaldehyde (pH = 7.4) for 60 min at 4°C, and washed twice with phosphate-buffered saline. After cell permeabilisation by 1% Triton X-100 for 5 min, adherent biofilm was washed twice with phosphate-buffered saline, and fixed in 70%, 85%, 96% (v/v) ice-ethanol for 5 min. After air-drying at room temperature, the sample slide was co-incubated with freshly prepared hybridisation buffer (50% formamide, 10% 50× SSC (Saline Sodium Citrate), 0.5% SDS, 100 µg mL–1 Salmon Sperm DNA, 10% dextran sulfate) for 5 min at 73°C, and then cooled in ice. Then 15 µLof CY3 probe solution (Biosense, Guangdong, China) was added to the sample slide, and a coverslip placed over the sample. After heat denaturation for 3 min at 73°C and incubation for 16 h at 46°C, the sample slide was washed twice with wash buffer at 48°C followed by two washes with ice-cold SSC buffer (0.3 M NaCl, 0.03 M sodium citrate). After FISH, washing and re-dyeing with 100 µg mL–1 DAPI (4',6-diamidino-2-phenylindole), biofilm present on the sample slide was observed under a DMI4000B fluorescence microscope (Leica, Wetzlar, Germany). Measurement of acyl-homoserine lactones (AHL) by LC-MS/MS AHL of V. parahaemolyticus were extracted and prepared as previously described (Burton, Read and Pellitteri 2005). AHL was made into different dilutions [(1:250, 1:125, 1:100), and the mixture of AHL and LB culture was taken as the control] of S. putrefaciens supernatant culture and allowed to react at 37°C for 1 h as described (Morohoshi, Nakazawa and Ebata 2008). AHL samples and standards (Cayman Chemical and Sigma) were analysed by liquid chromatography (LC)-mass spectrometry (MS) using a Tandem Quadrupole LC-MS/MS system (LCMS-8030, Shimadzu) as previously described (Ortori, Dubern and Chhabra 2011). The parameters used were: flow rate 0.3 mL min–1, injection volume 10.0 µL, column temperature 33°C, stop time 25 min. The mobile phase was a mixture of methanol (A) and 2 mM ammonium acetate solution containing 0.1% formic acid (B). The ratios of LC-MS/MS peak areas of the analyte to an internal standard were measured. Statistical analysis All experiments were performed at least three times. Values are expressed as the mean ± standard deviation (SD). Statistical analysis was performed using SPSS version 19.0 and Student’s t-test. The test results were compared with control and statistical significance was determined at a P value of <0.05 or <0.01. RESULTS Effects of shrimp spoilage bacteria on total hemolytic activity of V. parahaemolyticus In addition to V. parahaemolyticus, five additional bacterial species predominating in spoiled shrimp were isolated. These bacteria were identified by 16s RNA sequencing as Oceanisphaera profunda, Proteus vulgaris, S. putrefaciens, Stenotrophomonas maltophilia and Bacillus siamensis (Table 1). To reveal the mechanisms regulating the virulence factors of V. parahaemolyticus in shrimp, the effects of these five bacterial isolates on total hemolytic activity of V. parahaemolyticus were tested. Among them, S. putrefaciens, S. maltophilia and P. vulgaris exhibited significant enhancement of total hemolytic activity, with S. putrefaciens being the highest (Fig. 1) . The order of enhancement effects of bacterial isolates is S. putrefaciens > P. vulgaris > S. maltophilia. Therefore, we focused on the S. putrefaciens for further investigation. Figure 1 View largeDownload slide . Effects of five spoilage bacterial isolates on total haemolytic activity of V. parahaemolyticus (VP). Total haemolytic activity of V. parahaemolyticus single-cultured or co-cultured with different bacterial isolates in LB medium was tested after 24-h incubation at 37°C. The data represent the means of three independent experiments. Error bars represent mean ± SD. Asterisks (*P <0.05) indicate results that differ significantly from the single-cultured V. parahaemolyticus. Figure 1 View largeDownload slide . Effects of five spoilage bacterial isolates on total haemolytic activity of V. parahaemolyticus (VP). Total haemolytic activity of V. parahaemolyticus single-cultured or co-cultured with different bacterial isolates in LB medium was tested after 24-h incubation at 37°C. The data represent the means of three independent experiments. Error bars represent mean ± SD. Asterisks (*P <0.05) indicate results that differ significantly from the single-cultured V. parahaemolyticus. Table 1. Five bacterial isolates from spoilage shrimp. Isolates Frequency in total Accession No. Identities Identity O. profunda 9.2% KX675452.1 941/952 98.84% P. vulgaris 7.5% KT887953.1 1405/1406 99.92% S. putrefaciens 7.1% KC607519.1 1368/1374 99.56% S. maltophilia 6.4% MF429096.1 912/915 99.67% B. siamensis 6.1% MF465780.1 941/942 99.89% Isolates Frequency in total Accession No. Identities Identity O. profunda 9.2% KX675452.1 941/952 98.84% P. vulgaris 7.5% KT887953.1 1405/1406 99.92% S. putrefaciens 7.1% KC607519.1 1368/1374 99.56% S. maltophilia 6.4% MF429096.1 912/915 99.67% B. siamensis 6.1% MF465780.1 941/942 99.89% View Large Table 1. Five bacterial isolates from spoilage shrimp. Isolates Frequency in total Accession No. Identities Identity O. profunda 9.2% KX675452.1 941/952 98.84% P. vulgaris 7.5% KT887953.1 1405/1406 99.92% S. putrefaciens 7.1% KC607519.1 1368/1374 99.56% S. maltophilia 6.4% MF429096.1 912/915 99.67% B. siamensis 6.1% MF465780.1 941/942 99.89% Isolates Frequency in total Accession No. Identities Identity O. profunda 9.2% KX675452.1 941/952 98.84% P. vulgaris 7.5% KT887953.1 1405/1406 99.92% S. putrefaciens 7.1% KC607519.1 1368/1374 99.56% S. maltophilia 6.4% MF429096.1 912/915 99.67% B. siamensis 6.1% MF465780.1 941/942 99.89% View Large Time, dose and temperature effects of S. putrefaciens on total hemolytic activity of V. parahaemolyticus To better understand virulence factors regulation by S. putrefaciens, first its effect on the total hemolytic activity of V. parahaemolyticus over time at 37°C was tested. At 15 h, total hemolytic activity of V. parahaemolyticus was not enhanced on co-cultivation with S. putrefaciens. However, at 18 h, the enhancement was significant and the largest effect was observed at 24 h. But this enhancement effect gradually weakened at >33 h (Fig. 2A). Figure 2. View largeDownload slide Time, dose and temperature effects of S. putrefaciens on total haemolytic activity of V. parahaemolyticus (VP). Total haemolytic activity of V. parahaemolyticus single-cultured or co-cultured with S. putrefaciens for the indicated time (3, 9, 15, 18, 21, 24, 27, 30, 33 h) (A) or co-cultured with indicated doses of S. putrefaciens supernatant (SP) (at the dilution of 1:500, 1:250, 1:125 and 1:100) (B) at 25, 37 and 42°C (C). Vibrio parahaemolyticus co-incubated with 0 dose of S. putrefaciens supernatant was taken as the control. Fresh LB culture in sterile dialysis bags separately co-incubated with S. putrefaciens supernatant (SP) was taken as the blank. The data represent the means of three independent experiments. Error bars represent mean ± SD. Asterisks (*P <0.05, **P <0.01) indicate results that differ significantly from the single-cultured V. parahaemolyticus. Figure 2. View largeDownload slide Time, dose and temperature effects of S. putrefaciens on total haemolytic activity of V. parahaemolyticus (VP). Total haemolytic activity of V. parahaemolyticus single-cultured or co-cultured with S. putrefaciens for the indicated time (3, 9, 15, 18, 21, 24, 27, 30, 33 h) (A) or co-cultured with indicated doses of S. putrefaciens supernatant (SP) (at the dilution of 1:500, 1:250, 1:125 and 1:100) (B) at 25, 37 and 42°C (C). Vibrio parahaemolyticus co-incubated with 0 dose of S. putrefaciens supernatant was taken as the control. Fresh LB culture in sterile dialysis bags separately co-incubated with S. putrefaciens supernatant (SP) was taken as the blank. The data represent the means of three independent experiments. Error bars represent mean ± SD. Asterisks (*P <0.05, **P <0.01) indicate results that differ significantly from the single-cultured V. parahaemolyticus. Next, the dose effect of S. putrefaciens supernatant on total hemolytic activity of V. parahaemolyticus was tested following 18 h co-cultivation at 37°C. Compared with the control (V. parahaemolyticus alone), co-cultivation with a 1:500 dilution of S. putrefaciens supernatant did not exhibit significant enhancement. However, at low dilutions (<1:250), S. putrefaciens supernatant exhibited significant dose-dependent enhancement on total hemolytic activity of V. parahaemolyticus (Fig. 2B). However, S. putrefaciens supernatant itself (blank) did not induce obvious hemolytic activity. Lastly, the temperature effect on this regulation by S. putrefaciens supernatant was tested after 18 h co-cultivation at a dilution of 1:100 (similarly hereinafter). At 37°C, S. putrefaciens supernatant exhibited higher enhancement than at room temperature (25°C). However, a higher temperature (42°C) did not significantly increase the regulatory effect of S. putrefaciens supernatant on the total hemolytic activity of V. parahaemolyticus (Fig. 2C). Roles of luxM- and luxS-type QS in regulation of S. putrefaciens Because luxM- and luxS-type QS play an important role in virulence factors regulation of V. parahaemolyticus, we presumed that S. putrefaciens supernatant would enhance the total hemolytic activity of V. parahaemolyticus through luxM and/or luxS. To test this hypothesis, the QS autoinducer-synthesis enzyme encoding genes luxM and/or luxS defect strains of V. parahaemolyticus (namely, VP∆LuxM and VP∆LuxS) were employed, and the enhancement effects of S. putrefaciens supernatant on total hemolytic activity of VP∆LuxM and VP∆LuxS were determined. The deletion of luxM but not luxS (VP∆LuxS) significantly (P < 0.05) impaired the enhancement effect of S. putrefaciens (Fig. 3A), confirming that S. putrefaciens supernatant enhanced the total hemolytic activity of V. parahaemolyticus through luxM. In addition, compared with the wild-type VP, VP∆LuxM and VP∆LuxS exhibited higher total hemolytic activity. Deletion of luxM or luxS significantly increased the total hemolytic activity of V. parahaemolyticus, indicating that total hemolytic activity was negatively regulated by luxM and luxS. Figure 3. View largeDownload slide Effects of deletion of luxM and luxS on total hemolytic activity and tdh expression regulation by S. putrefaciens. (A) Total hemolytic activity of V. parahaemolyticus (VP) or mutants, single-cultured and co-cultured with S. putrefaciens in LB medium for 24 h at 37°C. (B) The relative tdh mRNA level of V. parahaemolyticus (VP) or mutants single-cultured and co-cultured with S. putrefaciens in LB medium for 24 h at 37°C. The data represent the means of three independent experiments. Error bars represent mean ± SD. Asterisks (*P <0.05, **P <0.01) indicate results that differ significantly from their corresponding single-cultured V. parahaemolyticus strains respectively, and hash signs (#P <0.05, ##P <0.01) indicate results that differ significantly from the single-cultured VP. Figure 3. View largeDownload slide Effects of deletion of luxM and luxS on total hemolytic activity and tdh expression regulation by S. putrefaciens. (A) Total hemolytic activity of V. parahaemolyticus (VP) or mutants, single-cultured and co-cultured with S. putrefaciens in LB medium for 24 h at 37°C. (B) The relative tdh mRNA level of V. parahaemolyticus (VP) or mutants single-cultured and co-cultured with S. putrefaciens in LB medium for 24 h at 37°C. The data represent the means of three independent experiments. Error bars represent mean ± SD. Asterisks (*P <0.05, **P <0.01) indicate results that differ significantly from their corresponding single-cultured V. parahaemolyticus strains respectively, and hash signs (#P <0.05, ##P <0.01) indicate results that differ significantly from the single-cultured VP. Role of luxM in induction expression of tdh by S. putrefaciens TDH is considered to be a major virulence factor in gastrointestinal disorders and possesses marked hemolytic activity (Fabbri, Falzano and Frank 1999; Yanagihara, Nakahira and Yamane 2010). We hypothesised that S. putrefaciens may promote tdh gene expression and thereby increase the total hemolytic activity of V. parahaemolyticus. To test this, the tdh expression of V. parahaemolyticus before and after co-cultivation with S. putrefaciens supernatant was quantified. The tdh expression was significantly up-regulated after co-cultivation with S. putrefaciens supernatant (Fig. 3B), which confirmed that S. putrefaciens increased the total hemolytic activity through tdh. Compared with the tdh of wild-type V. parahaemolyticus, the tdh of V. parahaemolyticus with defective luxM (VP∆LuxM) was not significantly induced by S. putrefaciens supernatant. Deletion of luxM from the wild-type V. parahaemolyticus significantly impaired the induction by S. putrefaciens supernatant. However, deletion of luxS did not significantly impair this induction. In addition, like S. putrefaciens supernatant, the deletion of luxM also increased the tdh expression level (Fig. 3B), which confirmed the negative regulation of tdh by luxM. These results suggest that S. putrefaciens induces tdh expression to enhance hemolytic activity by negatively affecting luxM. Important role of luxM in induction of biofilm formation by S. putrefaciens Biofilm formation was also negatively regulated by luxM-type QS but increased when the autoinducer concentration was low at low cell density (Hammer and Bassler 2003). To further confirm the relationship between S. putrefaciens supernatant and luxM on biofilm formation by V. parahaemolyticus, the biofilm formation of VP∆LuxM and VP∆LuxS were determined before and after co-cultivation with S. putrefaciens supernatant. Biofilm production, as observed under a fluorescence microscopy, increased after deletion of luxM or luxS (Fig. 4A). Co-cultivation with S. putrefaciens supernatant further increased the biofilm production of VP∆LuxS. However, co-cultivation with S. putrefaciens supernatant did not further increase the production of biofilm of VP∆LuxM (Fig. 4A). Biofilm production was also quantified with the crystal violet staining method. Co-cultivation with S. putrefaciens supernatant further significantly increased the biofilm production of VP and VP∆LuxS, but not VP∆LuxM (Fig.4B). Deletion of luxM impaired the regulation of S. putrefaciens supernatant on the biofilm formation of V. parahaemolyticus, which is consistent with the tdh expression results. These results together suggest that S. putrefaciens supernatant has regulatory effects on the hemolytic activity and biofilm formation of V. parahaemolyticus through its supernatant. Based on the above, we suggest that S. putrefaciens supernatant may induce tdh and biofilm formation by degrading the autoinducer AHL synthesised by luxM. However, this hypothesis has not been confirmed in V. parahaemolyticus and S. putrefaciens before. Figure 4. View largeDownload slide Effects of luxM and luxS deletion on biofilm formation regulation by S. putrefaciens. (A) Vibrio parahaemolyticus (VP) mutants single-cultured or co-cultured with S. putrefaciens supernatant were stained and visualised by fluorescence microscopy as described in the Materials and methods section (scale 100 µm; left cells stained with DAPI dye; right cells, with the bound CY3 fluorescent probe). (B) Quantification of biofilm formation of indicated strains in (A) by measuring its absorbance at 590 nm. Error bars represent mean ± SD. Asterisks (*P <0.05, **P <0.01) indicate results that differ significantly from their corresponding single-cultured strains, and hash signs (#P <0.05, ##P <0.01) indicate results that differ significantly from the single-cultured VP. Figure 4. View largeDownload slide Effects of luxM and luxS deletion on biofilm formation regulation by S. putrefaciens. (A) Vibrio parahaemolyticus (VP) mutants single-cultured or co-cultured with S. putrefaciens supernatant were stained and visualised by fluorescence microscopy as described in the Materials and methods section (scale 100 µm; left cells stained with DAPI dye; right cells, with the bound CY3 fluorescent probe). (B) Quantification of biofilm formation of indicated strains in (A) by measuring its absorbance at 590 nm. Error bars represent mean ± SD. Asterisks (*P <0.05, **P <0.01) indicate results that differ significantly from their corresponding single-cultured strains, and hash signs (#P <0.05, ##P <0.01) indicate results that differ significantly from the single-cultured VP. AHL degradation ability of the S. putrefaciens supernatant Some proteins secreted by bacteria, such as the lactonase enzyme, will degrade the extracellular autoinducer (Wang, Weng and Dong 2004). We hypothesised that the S. putrefaciens supernatant may degrade the luxM product (AHL) to increase the virulence factors of V. parahaemolyticus. To test this, the effect of S. putrefaciens supernatant on AHL was investigated and the AHL content significantly and exponentially decreased in a dose-dependent manner following S. putrefaciens supernatant treatment (Fig. 5). Shewanella putrefaciens supernatant exhibited a strong degradation ability on signalling molecule AHL. This demonstrated that S. putrefaciens extracellular supernatant increase the virulence factors by degrading the AHL of V. parahaemolyticus. Figure 5. View largeDownload slide Degradation of AHL by S. putrefaciens supernatant. Effect of S. putrefaciens supernatant on AHL following incubation for 1 h at 37°C, measured with LC-MS/MS. The mixture of AHL and blank culture without treatment was taken as the no treatment control. The data represent the means of three independent experiments. Error bars represent mean ± SD. Asterisks (*P <0.05, **P <0.01) indicate results that differ significantly from the no-treatment control. Figure 5. View largeDownload slide Degradation of AHL by S. putrefaciens supernatant. Effect of S. putrefaciens supernatant on AHL following incubation for 1 h at 37°C, measured with LC-MS/MS. The mixture of AHL and blank culture without treatment was taken as the no treatment control. The data represent the means of three independent experiments. Error bars represent mean ± SD. Asterisks (*P <0.05, **P <0.01) indicate results that differ significantly from the no-treatment control. DISCUSSION In this study, we obtained five bacterial species from spoiled shrimp. Among them, S. putrefaciens exhibited the highest enhancement of total hemolytic activity and was chosen for further investigation. A study of the effect or regulatory mechanism of S. putrefaciens on virulence factors of V. parahaemolyticus has not been previously reported. Previous studies have reported that S. putrefaciens is a major microbial cause of spoilage in seafood, frequently detected in marine environments and organisms, especially in spoiled shrimp and fish (Wright, Matthews and Arnold 2016). It is usually non-pathogenic to humans except under particular circumstances (Pagani, Lang and Vedovelli 2003). Shewanella was found to affect the spoilage of Pseudosciaena crocea through the QS system (Zhu, Zhao and Feng 2016) and Shewanella algae show an inhibitory effect against V. parahaemolyticus (Shakibazadeh, Saad and Christianus 2008). Some members of the human gut microbiota also affect Vibrio cholerae colonisation through autoinducers (Hsiao, Ahmed and Subramanian 2014). Our data suggest that S. putrefaciens increases the virulence factors of V. parahaemolyticus through interfering with QS. The S. putrefaciens exhibited a time-dependent enhancement effect (at >18 h) on total hemolytic activity of V. parahaemolyticus (Fig. 2A). Similar to that, the S. putrefaciens supernatant exhibited this enhancement effect only at dilutions <1:250(Fig.2B). Such time- and dose-dependent effects of S. putrefaciens on V. parahaemolyticus partly follow the patterns of QS activity in other Vibrio species (Defoirdt and Sorgeloos 2012). Vibrio harveyi showed about a 200-fold higher maximal QS-regulated bioluminescence when associated with shrimp larvae, especially dead shrimp, than in the culture water in early 14 to 24-h infection (Defoirdt and Sorgeloos 2012). The results further suggest QS may be involved in the regulation of virulence factors by S. putrefaciens. Temperature influences virulence factors of pathogens, especially the Vibrio species (Kimes, Grim and Johnson 2012). In our study, temperature also influenced the enhancement effect of S. putrefaciens on total hemolytic activity of V. parahaemolyticus (Fig. 2C). Compared with room temperature (25°C) or extreme temperature (42°C), the normal temperature (37°C) of the human body is an optimal co-cultivation temperature to increase the total hemolytic activity of V. parahaemolyticus by S. putrefaciens supernatant, suggesting that the storage temperature of S. putrefaciens-contaminated seafood may influence the risk of acquiring gastroenteritis. Generally, the virulence factors of bacteria are controlled by the QS system (Shih and Huang 2002; Hammer and Bassler 2003), such as in Pseudomonas aeruginosa (Bjarnsholt, Jensen and Burmolle 2005) and some Vibrio species (Hammer and Bassler 2003). The hemolytic activity-related genes including hlyA are negatively regulated by the QS system in V. cholerae (Tsou and Zhu 2010). Similar virulence factors regulation may also exist in V. parahaemolyticus, but the mechanism for this is unclear. There has been no prior explanation as to why S. putrefaciens supernatant may increase the total hemolytic activity of V. parahaemolyticus. From our study, the regulation effect of S. putrefaciens supernatant was impaired after deletion of luxM and not luxS, which indicated that the regulation of V. parahaemolyticus by S. putrefaciens supernatant is mainly dependent on luxM. This is in contrast to the more important role luxS plays in the virulence inhibition of vibrio by gut microbiota (Hsiao, Ahmed and Subramanian 2014). TDH possesses the main hemolytic activity (Fabbri, Falzano and Frank 1999; Yanagihara, Nakahira and Yamane 2010) and the tdh expression results (Fig. 3B) from our study confirmed that luxM instead of luxS plays an important role in the virulence factors regulation of V. parahaemolyticus by S. putrefaciens. luxM produces AHL (AI-1) by catalysing the AHL precursor, and that plays an important role in intra-species cell-to-cell communication (Miller and Bassler 2001). Therefore, the evidence is clear and compelling that the luxM-type QS of V. parahaemolyticus is regulated by S. putrefaciens. Biofilm formation is negatively controlled by the QS system, and also is negatively correlated with the AI concentrations (Hammer and Bassler 2003). From our study, the production of biofilm increased after deletion of luxM or luxS as expected. More importantly, the production of biofilm increased after co-cultivation with S. putrefaciens, and deletion of luxM impaired this induction by S. putrefaciens. These results confirmed that luxM is associated with the virulence factors regulation of V. parahaemolyticus by S. putrefaciens. From the results of tdh and biofilm, both S. putrefaciens supernatant and deletion of luxM increased tdh and biofilm levels. It would be reasonable to believe that S. putrefaciens supernatant degrades the extracellular products of luxM to inhibit the activity of luxM. In our assessment of degradation of the S. putrefaciens supernatant, it showed a strong degradation ability on AHL synthesised by luxM. We checked all the known proteins of S. putrefaciens from NCBI. It was apparent that beta-lactamase VPA0477 is highly conserved with N-acylhomoserine lactone-acylase of Shewanella, which is reported to markedly degrade the AHL production of Vibrio anguillarum (Morohoshi, Nakazawa and Ebata 2008). In this case, the S. putrefaciens supernatant contributed to reducing AHL in V. parahaemolyticus. In conclusion, the data we have generated support that the AHL degradation activity of S. putrefaciens may serve to increase the V. parahaemolyticus virulence factors. This discovery provides useful evidence for revealing the virulence factors up-regulation mechanism of V. parahaemolyticus in aquatic food spoilage. ACKNOWLEDGEMENTS The Tandem Quadrupole LC-MS/MS system was provided by the National Marine Products Quality Supervision & Inspection Center. 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Int J Food Microbiol . 2016 ; 217 : 146 – 55 . Google Scholar CrossRef Search ADS PubMed © FEMS 2018. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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FEMS Microbiology EcologyOxford University Press

Published: May 24, 2018

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