ORIGINAL RESEARCH published: 27 March 2020 doi: 10.3389/fmicb.2020.00489 The Effect of the Environmental Temperature on the Adaptation to Host in the Zoonotic Pathogen Vibrio vulnificus Carla Hernández-Cabanyero 1 , Eva Sanjuán 1 , Belén Fouz 1 , David Pajuelo 1† , Eva Vallejos-Vidal 2,3 , Felipe E. Reyes-López 2 and Carmen Amaro 1* 1 Departamento de Microbiología y Ecología and Estructura de Recerca Interdisciplinar en Biotecnologia i Biomedicina, Universitat de València, Valencia, Spain, 2 Department of Cell Biology, Physiology and Immunology, Universitat Autònoma de Barcelona, Barcelona, Spain, 3 Centro de Biotecnología Acuícola, Facultad de Química y Biología, Universidad de Santiago de Chile, Santiago, Chile Edited by: Vibrio vulnificus is a zoonotic pathogen that lives in temperate, tropical and subtropical Eckhard Strauch, Federal Institute for Risk Assessment aquatic ecosystems whose geographical distribution is expanding due to global (BfR), Germany warming. The species is genetically variable and only the strains that belong to the Reviewed by: zoonotic clonal-complex can cause vibriosis in both humans and fish (being its main Craig Baker-Austin, Centre for Environment, Fisheries host the eel). Interestingly, the severity of the vibriosis in the eel and the human depends and Aquaculture Science (CEFAS), largely on the water temperature (highly virulent at 28◦ C, avirulent at 20◦ C or below) United Kingdom and on the iron content in the blood, respectively. The objective of this work was to Delphine Destoumieux-Garzon, Centre National de la Recherche unravel the role of temperature in the adaptation to the host through a transcriptomic Scientifique (CNRS), France and phenotypic approach. To this end, we obtained the transcriptome of a zoonotic *Correspondence: strain grown in a minimum medium (CM9) at 20, 25, 28, and 37◦ C, and confirmed the Carmen Amaro carmen.amaro@uv.es transcriptomic results by RT-qPCR and phenotypic tests. In addition, we compared † Present address: the temperature stimulon with those previously obtained for iron and serum (from David Pajuelo, eel and human, respectively). Our results suggest that warm temperatures activate Department of Microbiology, adaptive traits that would prepare the bacteria for host colonization (metabolism, motility, University of Alabama at Birmingham, Birmingham, AL, United States chemotaxis, and the protease activity) and fish septicemia (iron-uptake from transferrin and production of O-antigen of high molecular weight) in a generalized manner, while Specialty section: environmental iron controls the expression of a host-adapted virulent phenotype (toxins This article was submitted to Infectious Diseases, and the production of a protective envelope). Finally, our results confirm that beyond the a section of the journal effect of temperature on the V. vulnificus distribution in the environment, it also has an Frontiers in Microbiology effect on the infectious capability of this pathogen that must be taken into account to Received: 11 November 2019 Accepted: 06 March 2020 predict the real risk of V. vulnificus infection caused by global warming. Published: 27 March 2020 Keywords: V. vulnificus, temperature, host adaptation, transcriptome, microarray Citation: Hernández-Cabanyero C, Sanjuán E, Fouz B, Pajuelo D, INTRODUCTION Vallejos-Vidal E, Reyes-López FE and Amaro C (2020) The Effect of the Vibrio vulnificus is a worldwide-distributed aquatic pathogen that inhabits waters of salinity Environmental Temperature on the Adaptation to Host between 0.3 and 2.5% located in tropical, subtropical and temperate areas. The species in the Zoonotic Pathogen Vibrio can be isolated from coastal ecosystems when water temperature is between 10 and vulnificus. Front. Microbiol. 11:489. 30◦ C (Baker-Austin and Oliver, 2018). In fact, environmental temperature determines doi: 10.3389/fmicb.2020.00489 the life strategy of V. vulnificus in the aquatic ecosystem as this species enters into Frontiers in Microbiology | www.frontiersin.org 1 March 2020 | Volume 11 | Article 489 Hernández-Cabanyero et al. Vibrio vulnificus Transcriptome in Response to Temperature the viable but non-culturable (VBNC) state below 4◦ C and (MMW) O-antigen together with two iron regulated outer multiplies in the water column (if nutrients are available) above membrane proteins (IROMPs), Ftbp and Fpcrp (Pajuelo et al., 18◦ C with an optimum above 25◦ C (Oliver, 2015). There is 2016; Hernández-Cabanyero et al., 2019). These two proteins a strong scientific evidence suggesting that global warming is constitute a kit for survival in fish blood that is encoded in a increasing the sea surface temperature, which is about 1◦ C higher transmissible virulence plasmid (pVvbt2) (Pajuelo et al., 2015; than 100 years ago1 . It is also predicted that the melting of the Hernández-Cabanyero et al., 2019). ice at the poles will produce a decrease in the seawater salinity In this work, we hypothesized that environmental temperature around the world. Given that V. vulnificus grows preferentially (either water and/or host) could also contribute to the generation in warm brackish water (1–2% NaCl) at temperatures above of the virulent phenotype in V. vulnificus. In fact, V. vulnificus 18◦ C, the warming of marine environments together with the is able to cause severe septicemia at both 28◦ C (in warm reduction in salinity will result in larger populations of these fish species) and 37◦ C (in humans). Although temperature- bacteria in coastal waters and, therefore will increase the risk of dependent gene expression has been widely studied in other Vibrio infections (Le Roux et al., 2015). bacterial species (Gao et al., 2006; Wang et al., 2017; Matanza Vibrio vulnificus is a highly heterogeneous species that and Osorio, 2018; Lages et al., 2019), to our knowledge this comprises avirulent and virulent isolates for multiple hosts. The is the first time that the global transcriptomic response to species has been divided in five phylogenetic lineages and one an increase in the environmental temperature is analyzed in pathovar (pv. piscis) from a phylogenomic study based on the V. vulnificus. Nevertheless, it is well known that temperature SNPs present in the core genome (Roig et al., 2018). Within impacts V. vulnificus growth ability (Kim Y. W. et al., 2012) the lineage II, there is a worldwide distributed clonal-complex and the expression of some virulence factors such as the major with the unique ability to infect homeothermic (humans) and protease (Vvp), iron uptake systems (HupA and VuuA) and poikilothermic animals (fish and shrimps; main host the eel) quorum sensing (McDougald et al., 2001; Oh et al., 2009; Kim (Amaro et al., 2015; Oliver, 2015). This is the sole group et al., 2016; Elgaml and Miyoshi, 2017). within the Vibrio genus that has been related to true zoonotic Taken all together, the aim of this work was to find out the cases (Amaro et al., 2015; Oliver, 2015). This clonal-complex role of temperature in the generation of the virulent phenotype can infect humans by contact with seawater or fish (mainly in V. vulnificus, using a transcriptomic and phenotypic approach. diseased fish) causing severe wound infections that can lead to For this purpose, we grew a representative strain of the zoonotic septicemia and death at risk patients (mainly those that present clonal-complex (CECT4999, hereafter R99) in a minimal high levels of free iron in blood due to different predisposing medium (CM9) at three infective temperatures: 25◦ C (outbreaks underlying diseases such as hemochromatosis) (Horseman and of medium mortality in fish farms), 28◦ C (outbreaks of high Surani, 2011; Oliver, 2015; Amaro et al., 2015). The disease caused mortality), and 37◦ C (human host body temperature). A non- in fish is known as warm-water vibriosis since the most serious infective temperature, 20◦ C (control), was also assessed (Amaro outbreaks always occur at temperatures above 25◦ C (Amaro et al., et al., 1995, 2015; Oliver, 2015). Then, the bacterial transcriptome 2015). Warm-water vibriosis is an acute hemorrhagic septicemia was obtained by microarray hybridization followed by the that takes place in farms of eels and tilapia as epizootic or bioinformatics analysis (Pajuelo et al., 2016). The transcriptomes outbreaks of high and medium mortalities depending on water were compared between them and also to those obtained temperature (Fouz and Amaro, 2002; Fouz et al., 2006; Amaro in previous studies: iron stimulon and fur regulon (Pajuelo et al., 2015). Epizootiological studies performed at laboratory et al., 2016); and eel and human serum stimulons (Hernández- scale confirmed that the pathogen virulence for eels is dependent Cabanyero et al., 2019). Finally, the transcriptomic results on water temperature: maximal at 28◦ C (the maximum tested were confirmed by RT-qPCR and phenotypic tests. Our results temperature) and minimal (no virulence at all) at 20◦ C and below show that environmental temperature, either in the water or (Amaro et al., 1995). in the host body in the moment of infection, contributes In previous studies, we performed a transcriptomic analysis to the pathogen’s pre-adaptation to a within-host survival by of this pathogen grown in a minimal medium, with and activating cellular mechanisms that prepare the bacterium for without iron, and in the serum of its most susceptible hosts the subsequent invasion and survival in host blood. Therefore, (Pajuelo et al., 2016; Hernández-Cabanyero et al., 2019). Those under infective temperatures, bacterial fitness is enhanced by an studies confirmed that iron in serum is one of the external increase in metabolic abilities, motility, chemotaxis, and protease signals triggering a host-adapted virulent phenotype in the production, accompanied by the production of a partially pathogen. This response includes the production of the two protective envelope. Nevertheless, temperature is not enough to main toxins of the species, RtxA1 and VvhA, together with trigger a host-adapted virulent phenotype, for which host and a host-specific protective envelope to resist the host innate iron markers are essential. immunity (including nutritional immunity, complement system and phagocytosis). In case of human infection, the envelope is enriched in capsule; while in case of eel infection it is enriched in MATERIALS AND METHODS high molecular weight (HMW) and medium molecular weight Bacterial Strains and Growth Conditions 1 https://www.epa.gov/climate-indicators/climate-change-indicators-sea-surface- R99 strain, a representative strain of the zoonotic clonal- temperature complex (Roig et al., 2018), was routinely grown on Tryptone Frontiers in Microbiology | www.frontiersin.org 2 March 2020 | Volume 11 | Article 489 Hernández-Cabanyero et al. Vibrio vulnificus Transcriptome in Response to Temperature Soy agar supplemented with 1% NaCl (TSA-1) and M9 TABLE 1 | Microarray validation by RT-qPCR. supplemented with 0.2% casamino acids (CM9) (Miller, 1972). Comparisona Gene Fold changeb The inoculum for assays was prepared by inoculating 5 ml of media with bacteria from overnight cultures in a ratio Array RT-qPCR 1:100 (v/v) and incubated with agitation (100 rpm) at 20, 25◦ C vs. 20◦ C ftbp 2 (+) 1.3 (=) 25, 28, and 37◦ C. Drop plate counts on TSA-1 (Hoben and cpsA 2 (+) 4.9 (+) Somasegaran, 1982) and Abs625 measurements were used to vvp – 1.5 (=) follow bacterial growth. vvhA – 1.6 (=) rtxA13 – 1.9 (= ) Transcriptomic Experiments 28◦ C vs. 20◦ C flp 14 (++) 3.6 (+) Microarray Analysis malG −3.1 (−) −1.2 (=) Bacterial transcriptome was obtained as previously described vvp – 1.7 (=) (Pajuelo et al., 2016). Briefly, total RNA from mid-log vvhA – 1.3 (=) phase cultures (Abs625 0.3: 19 h cultures grown at 20◦ C; rtxA13 – 1.9 (=) 8 h cultures grown at 25◦ C; and 6 h cultures grown 37◦ C vs. 20◦ C vpsT 5.1 (+) 3.6 (+) at 28 and 37◦ C) was extracted using NucleoZOL RNA ktrA 3.4 (+) 1.6 (=) isolation product (Macherey-Nagel). The RNA was then treated vvp – 1.9 (=) with TURBOTM DNase (Ambion) and cleaned with the vvhA – 1.7 (=) GeneJET RNA Cleanup and Concentration Micro kit (Thermo rtxA13 – 1.4 (=) Scientific). RNA concentration and integrity were measured Comparison of fold change values obtained by array and RT-qPCR using the using the Nanodrop 2000 spectrophotometer (Thermo Fisher) V. vulnificus R99 strain. In case of RT-qPCR, results were obtained using recA and the 2100 Bioanalizer (Agilent), respectively. Only those as reference gene. The fold induction (2−11Ct ) for each gene was calculated. a RNA samples were obtained from mid-log phase cultures in CM9 at 20, 25, 28, samples with a RIN ≥ 7.5 were selected to obtain labeled and 37◦ C by using NucleoZOL (Macherey-Nagel), TURBOTM DNAse (Ambion) cDNA using the Low Input Quick Amp Labeling kit. The and GeneJET RNA Cleanup and Concentration Micro kit (Thermo Scientific). resultant cDNA was hybridized with the R99-specific microarray b Qualitative classification of the fold change values: = , −2 ≤ X ≤ 2; +, 2 ≤ X ≤ 10; (Pajuelo et al., 2016) which contains probes for the 4,553 ++, 10 ≤ X ≤ 25; −, −10 ≤ X ≤ −2. –, not detected as differentially expressed by the microarray analysis. predicted coding sequences in the R99 genome (Pajuelo et al., 2016). Then, the slides were scanned with the Agilent DNA Microarray Scanner (G2505B). Raw data were obtained with Agilent’s Feature Extraction software version 10.4.0.0 In vitro Assays (Agilent Technologies). The extracted data were analyzed with Biofilm Production Genespring 14.5 GX software (Agilent technologies) as previously R99 was grown in glass tubes with 2 ml of CM9 at 20, 25, 28, and specified (Pajuelo et al., 2016). Student’s t-test (P < 0.05) 37◦ C for 24 h. Biofilm production was quantified by staining with available in Genespring software was applied to reveal the crystal violet as previously described by Jones et al. (2008). bacterial differentially expressed genes (DEGs) in CM9 at 25◦ C (compared to that at 20◦ C [25 vs. 20]), 28◦ C (compared Motility to that at 20◦ C [28 vs. 20]) and at 37◦ C (compared to Motility was assayed on motility agar (MA [CM9 0.3% agar that at 20◦ C [37 vs. 20]). Results are presented as fold (wt/vol)] plates as previously described by Pajuelo et al. (2016). change mean from three independent biological samples. Venn Briefly, plates were inoculated with 5 µl from an exponential diagrams were created using the free software available at phase culture and incubated at 20, 25, 28, and 37◦ C for 24 h. Bioinformatics and Evolutionary Genomics group at Ghent Then, the motility rate was calculated as the rate between the University2 . surface of the bacterial colony on the plate (mm2 ) and the number of bacteria forming the colony (log CFU). RT-qPCR RNA samples, obtained in the same conditions as for Proteolytic and Hemolytic Activity the microarray assays, were used to produce cDNA The extracellular products (ECPs) from R99 strain were obtained with Maxima H Minus Reverse Transcriptase (Thermo from 24 h cultures on CM9-agar plates (1.5% agar [wt/vol]) at Scientific). The qPCR was performed on the cDNA 20, 25, 28, and 37◦ C according to Biosca and Amaro (1996). to calculate the relative expression of selected genes Proteolytic and hemolytic activities in ECPs was evaluated as (Table 1) as the fold induction using the 2−11Ct previously described by Valiente et al. (2008b), either using method and recA as the reference gene (Livak and azocasein (Sigma) as substratum (protease) or seeding two-times Schmittgen, 2001). Primers used in this study are listed in dilutions on agarose-erythrocytes (5%) (bovine erythrocytes from Supplementary Table S1. Sigma) plates. Proteolytic activity was calculated as proteolytic units (PU) produced in each condition as described by Miyoshi et al. (1987) and hemolytic activity as the inverse of the 2 http://bioinformatics.psb.ugent.be/webtools/Venn/ highest dilution giving a positive result on the plate. In parallel, Frontiers in Microbiology | www.frontiersin.org 3 March 2020 | Volume 11 | Article 489 Hernández-Cabanyero et al. Vibrio vulnificus Transcriptome in Response to Temperature the hemolytic activity of live bacteria was determined as described by Hernández-Cabanyero et al. (2019). Chemotaxis Eel skin mucus was collected by placing non-anesthetized eels in empty sterile flasks, and the secreted material was recovered and filtered through 0.8 and 0.45 µm pore-size membranes (Millipore) (Esteve-Gassent et al., 2003). The chemotaxis toward eel skin mucus was determined at 20, 25, 28, and 37◦ C by using the capillary assay described by Larsen et al. (2001). Briefly, capillary tubes (100 µl pre-calibrated capillaries; BRAND) were filled with eel skin mucus or chemotaxis buffer (ChB) (PBS + 0.01 mM EDTA) (control condition). Then, the capillaries were place in eppendorfs containing 0.5 ml of 1 × 108 CFU/ml bacterial suspension in ChB from mid-log phase R99 cultures in CM9 at 20, 25, 28, or 37◦ C. Eppendorfs together with capillaries were incubated for 35 min at the corresponding temperature and FIGURE 1 | Growth of V. vulnificus in a minimal medium at different incubation the number of bacteria inside the capillaries was determined on temperature. R99 strain was inoculated in CM9 and incubated with agitation at 20, 25, 28, and 37◦ C. Growth was followed by measuring the Abs625 at TSA-1 by the drop plate method. The chemotaxis toward eel skin regular time intervals. #: significant differences (P < 0.05) in Abs625 between mucus was expressed as the chemotactic response defined as the 20 and 28◦ C. *: significant differences (P < 0.05) in Abs625 between 25 and ratio between bacterial numbers in the corresponding capillaries 28◦ C. *∗ : significant differences (P < 0.05) in Abs625 between 37 and 28◦ C. vs. control capillaries. Surface Cell-Associated Polysaccharides Crude fractions of cell-associated polysaccharides (LPS plus Transcriptomic Results capsule) were obtained from mid-log phase cultures of R99 We compared the DEGs at infective temperatures (25, 28, and strain in CM9 at 20, 25, 28, and 37◦ C as described by 37) with those expressed at the non-infective temperature (20◦ C) Hitchcock and Brown (1983). Total polysaccharide concentration (Supplementary Tables S2–S4). Figure 2 shows the number and was determined with the Total Carbohydrate Assay Kit distribution of the DEGs in the genome per temperature, Figure 3 (BioVision) and 10 µg of cell-associated polysaccharides of shows the main cellular processes modulated at each temperature, each condition were separated by SDS-PAGE, transferred onto Table 1 provides a comparison between fold change values a PVDF membrane and subjected to immunoblot analyses obtained by hybridization with the R99-specific microarray and according to Pajuelo et al. (2016). by RT-qPCR, and Table 2 shows the selected DEGs as relevant per temperature. Statistical Analysis The temperature stimulon, defined as the set of DEGs at the All the results are represented as mean ± standard deviation from analyzed temperatures, totalized 1,375 genes (30.2% of genes in three independent biological experiments. Statistical differences the genome) (Supplementary Table S5). This value was similar were tested with GraphPad software (version 7) by using one-way to that found in the iron stimulon (25.78%), Fur regulon (35.91%) ANOVA. A P < 0.05 was considered significant. and host-serum stimulons (26.35% for eel serum; 20.32% for risk patient serum) (Pajuelo et al., 2016; Hernández-Cabanyero et al., 2019) (Figure 2). In addition, around 40% of the chromosomal genes that belonged to temperature stimulon were also found in RESULTS the rest of stimulons and/or regulons (Supplementary Figure S1), while less of 1% of the plasmid DEGs in the temperature Bacterial Growth stimulon belonged to another stimulon/regulon (results not Figure 1 shows the growth curves obtained at the different shown). Globally, this result suggests that plasmid genes involved tested temperatures. The pathogen presented growth patterns in response to temperature neither are controlled by Fur nor statistically different at 20 vs. 25◦ C, 20 vs. 28◦ C, and 25 vs. belong to the bacterial networks involved in response to iron 28◦ C but not at 28 vs. 37◦ C. Temperature mainly affected the or serum. The principal component analysis (PCA) showed the lag phase of growth, whose duration was longer at 20◦ C (14 h) transcriptomic profiles clearly grouped in accordance to different followed by 25◦ C (5 h), and shorter and similar at 28 and 37◦ C temperatures (Figure 4), being those obtained at 20 and 25◦ C (around 2 h). The bacterial numbers achieved at 24 h post- more similar to each other than those obtained at 28 and 37◦ C. incubation were around 1 × 109 CFU/ml at 20, 25, and 28◦ C, and In addition, the PCA showed that (i) the biological replicates around 1 × 108 CFU/ml at 37◦ C, suggesting that the death phase obtained at each temperature were very similar to each other, is accelerated at this temperature. Interestingly the maximum with the exception of those obtained at 37◦ C, which showed population size was similar regardless the incubation temperature greater variability; and (ii) the samples obtained at 28◦ C were (around 109 CFU/ml). the furthest from the rest. Precisely, the highest number of Frontiers in Microbiology | www.frontiersin.org 4 March 2020 | Volume 11 | Article 489 Hernández-Cabanyero et al. Vibrio vulnificus Transcriptome in Response to Temperature FIGURE 2 | Graphical representation of DEGs distribution among V. vulnificus genome in response to temperature. Figure shows the DEGs according to their distribution per regulation category (upregualted, downregulated, or non-regulated) and replicon (two chromosomes and one plasmid). The different categories are represented by color. DEGs was obtained at 28◦ C (Supplementary Figure S1A and and fatty acid metabolism and transport were upregulated at Figure 3), temperature at which the highest number of plasmid infective temperatures (Table 2). Accordingly, fabR, a repressor DEGs was detected with 21 genes upregulated (including ftbp, of unsaturated fatty acid metabolism was downregulated at a gene encoding a fish transferrin binding protein essential for 37◦ C. Finally, we found some genes involved in vulnibactin fish virulence, Pajuelo et al., 2015) and none downregulated siderophore biosynthesis and iron transport (either ferrous or (Figure 2). pVvBt2 is a plasmid essential for virulence given that it ferric iron), including the plasmid gene ftbp, upregulated at confers resistance to the innate immunity (nutritional immunity all the infective temperatures (Table 2). In accordance, crp and complement system) of teleost fish (Lee et al., 2008; (cyclic AMP [c-AMP] receptor protein) was upregulated at 28 Hernández-Cabanyero et al., 2019). Therefore, this difference and 37◦ C, a regulator reported to be responsible for positive in plasmid gene expression could help to explain why the control of iron uptake systems (Choi et al., 2006; Oh et al., pathogen is more virulent for fish at 28 than at 25 or 20◦ C 2009; Kim et al., 2016). Altogether, our results show that an (Amaro et al., 1995, 2015). increase in temperature leads to a metabolic shift as it is Nutrient uptake and metabolism were among the cellular suggested by the upregulation of multiple genes involved in processes most strongly activated by an increase in temperature biosynthesis, degradation and transport of amino acids, fatty (Table 2 and Supplementary Tables S2–S4). For example, dns, acids, lipids and iron, which would ensure bacterial growth and encoding an extracellular nuclease (Blokesch and Schoolnik, constitute an advantage for V. vulnificus at host physiological 2008), together with nupC, which encodes a nucleoside uptake temperature during the infection. Curiously, and as an exception, protein involved in V. cholerae fitness in nutrient limited we found transcriptomic evidence of maltose/maltodextrin environments (Gumpenberger et al., 2016), and multiple genes transport downregulation at 25 and 28◦ C [a process previously related to amino acid biosynthesis and transport (including found upregulated by iron and when the bacterium was grown genes for oligopeptide transport [opp genes]) as well as lipid in iron-overloaded human serum at 37◦ C (Pajuelo et al., Frontiers in Microbiology | www.frontiersin.org 5 March 2020 | Volume 11 | Article 489 Hernández-Cabanyero et al. Vibrio vulnificus Transcriptome in Response to Temperature FIGURE 3 | Cellular processes modulated by temperature. Categories of cellular processes affected at each temperature. Cellular processes differentially expressed at iron stimulon, fur regulon and serum regulons are also included. 2016; Hernández-Cabanyero et al., 2019)], together with the pilus (msh and tad genes) (Watnick et al., 1999; Pu and Rowe- upregulation of a gene for the maltose regulon repressor Magnus, 2018) were upregulated when temperature rises, as they (mall) (Table 2). were in iron deficiency and in eel serum (Table 2). Contrary, Transcriptomic signs of stress response activated by vpsT, encoding a repressor for biofilm production (Krasteva V. vulnificus were obtained at all the assayed temperatures et al., 2010) was upregulated by temperature. This modulation (Table 2). Although the DEGs involved in the stress response suggests the activation of regulatory mechanisms in response were not exactly the same at each of the assayed temperatures, to the upregulation of genes associated to attachment and the global response was very similar among them, and mostly biofilm formation. enhanced at 28◦ C (Table 2). This stress response included Finally, we also found that multiple external envelope the upregulation of defense mechanisms such as membrane biosynthesis genes (LPS and capsule production), involved in protection and regeneration (phospholipid biosynthesis), resistance to host complement system (Amaro et al., 1994, resistance to oxidative and nitrosative environments (aphF 1997), were upregulated by an increase in temperature (Table 2), [Alkyl hydroperoxide reductase protein F], a superoxide although not as many as those previously observed to be dismutase precursor, glutathione-related genes and nitrate regulated by iron and host serum (Pajuelo et al., 2016; reductase-related genes), together with the repression of ohr Hernández-Cabanyero et al., 2019). Interestingly, we did not (encoding a repressor for oxidative stress resistance) as well as find any of the major toxins or virulence master regulators genes involved in resistance to microcidal compounds (Table 2). differentially expressed in any of the comparisons performed, All these processes together comprise the bacterial ability to resist with the exception of the upregulation of quorum sensing inside the host during the infection (Kaufmann and Dorhoi, regulators (luxU and luxO at 28◦ C and smcR at 37◦ C) and 2016; Lim et al., 2017; Uribe-Querol and Rosales, 2017). tetR, encoding a master regulator involved in transcriptional Motility and chemotaxis are bacterial virulence factors regulation of multidrug efflux pumps, antibiotics biosynthesis, required for host colonization (Lee et al., 2004; Kim S. M. response to osmotic stress, control of catabolic pathways and et al., 2012). Our transcriptomic results showed several flagella- pathogenicity (Ramos et al., 2005), at 25 and 28◦ C (Table 2). related genes (including genes encoding flagellar hook proteins, flagellar basal-body proteins and flagellar motor activity proteins) and chemotaxis-related genes upregulated in all the assayed Phenotypic Results conditions (Table 2). Curiously, all these motility-related genes Biofilm Production found upregulated by an increase in temperature were previously Our transcriptomic results suggested changes in biofilm found downregulated in iron stimulon and eel serum, but formation with an increase in temperature (Table 2). Therefore, upregulated in iron-overloaded human serum (Pajuelo et al., we phenotypically tested the bacterial ability to form biofilm 2016; Hernández-Cabanyero et al., 2019). by staining biofilms with crystal violet after 24 h of growth Concerning attachment and biofilm formation, our and measuring Abs540 . No significant differences were found transcriptomic results showed that bacterial MSHA and Flp for biofilm production between conditions although a slight Frontiers in Microbiology | www.frontiersin.org 6 March 2020 | Volume 11 | Article 489 Hernández-Cabanyero et al. Vibrio vulnificus Transcriptome in Response to Temperature TABLE 2 | Selected DEGs for V. vulnificus grown at infective temperatures (25, 28, and 37◦ C) vs. non-infective temperature (20◦ C). Gene(s)a 25◦ Cb 28◦ Cc 37◦ Cd Putative function/processe Metabolic and nutrient transport and utilization genes (including iron) fadH 11.3 – 2.3 Unsatturated fatty acids metabolism Peptide ABC transporters# 10.4-2.4 – 3.9 Peptide transport ilvE, ilvG, ilvI 7.8-2.5 – 2.1 Amino acid biosynthesis trpCD 6.1-4 – 2.8 Amino acid biosynthesis (tryptophan) C4-dicarboxylate transporters# 6-2.4 – 20.7-2.9 Dicarboxilate transport argG, argI 5-2.1 2.1 – Amino acid metabolism Ferric iron ABC transporter*# 3.7 3.4-2.3 2.6 Ferric iron transport Long-chain fatty acid transport protein 3.3 – – Fatty acids transport Vulnibactin biosynthetic genes*# 3-2.3 2.9-2.5 2.3 Vulnibactin biosynthesis and transport hisBD 2.7 −2.1 – Amino acid biosynthesis fabGH 2.4 – 2.4 Fatty acids metabolism Amino acid ABC transporter* 2.3 5.5-2.8 13.8-6.2 Amino acid transport metB, metK, metL 2.3-2.1 8.5 2.8 Amino acid biosynthesis L-serine dehydratase 2.3 – – Amino acid metabolism potB, potD*# 2.2 2 – Polyamine transport PTS system, N-acetylglucosamine-specific IIB, IID 2.1 – 3.5 Aminosugar transport components*# Nitrate ABC transporter 2.1 – – Nitrogen metabolism ftbp*# 2 2 2.8 Fish transferrin binding protein rbsABCD# −2.6 −(3.4-3.7) 6.6-3.4 Ribose ABC transport system Short chain fatty acids transporter −2.7 −2.4 – Fatty acids transport malG −2.9 −3.1 – Maltose/maltodextrin ABC transporter prsA −3.1 −2.6 – Ribose metabolism Chitinase proteins – 22.3-2.5 8.8 Chitinase activity serB – 10.6 9.8 Amino acid biosynthesis pflA – 8.4 – Glucose metabolism Succinate-semialdehyde dehydrogenase [NADP+] – 8.4 – Amino acid degradation nupC*# – 6.7 8.1 Permease for nucleoside uptake dns*# – 5.7 – Degradation of DNA for nutrient uptake (competence related) Zinc ABC transporter – 4.5-3.5 – Zinc transport Lipase-related proteins – 4.2-2.7 8-5.3 Extracellular lipid utilization Phosphate ABC transporter# – 3.8-2.6 6.6 Phosphate transport trkA, trkH – 3.1 3.9-2.4 Potassium uptake oppBCD, oppF – 3-2.7 7.9-2.1 Oligopeptide transport system permease ktrA# – 2.8 3.4 Potassium uptake thrC – 2.7 – Amino acid iosynthesis dctQ# – 2.3 6.5 TRAP dicarboxylate transporters proA – 2.3 – Amino acid biosynthesis Ferrous iron transporter B – 2 – Ferrous iron transport acp12 – −(2.6-3.7) – Fatty acids biosynthesis glgX# – – 12.8 Glycogen debranching enzyme hmgA – – 10-4 Amino acid degradation citAB – – 8.3-2.8 Citrate metabolism Anaerobic respiration Nitrate reductase cytochrome c550-type subunit* 2.7 – 3 Nitrite reductase complex subunit napC, napE, napGH*# – 22.5-6.7 3.9-2.2 Subunit of the periplasmic nitrite reductase complex Nitrite reductase subunits – 3.5-2.8 – Nitrite reductase complex nrfF – – 23.3 Formate-dependent nitrite reductase complex Stress response and defense mechanisms Anaerobic glycerol-3-phosphate dehydrogenase 4.6-2.2 – 4.4-3.7 Phospholipid biosynthesis/membrane regeneration subunits (B, C)# (Continued) Frontiers in Microbiology | www.frontiersin.org 7 March 2020 | Volume 11 | Article 489 Hernández-Cabanyero et al. Vibrio vulnificus Transcriptome in Response to Temperature TABLE 2 | Continued Gene(s)a 25◦ Cb 28◦ Cc 37◦ Cd Putative function/processe S-(hydroxymethyl)glutathione dehydrogenase# 3 2.3 – Resistance to oxidative stress aphF 2.8 – – Alkyl hydroperoxide reductase protein F. Resistance to oxidative stress Glutathione S-transferase# 2.7 4.5 – Resistance to oxidative stress uspA 2.7 – – Universal stress protein A, involved in DA-damage resistance mutS* 2.5 – – DNA mismatch repair protein Superoxide dismutase [Cu–Zn] precursor 2.3 – – Resistance to oxidative stress plsXY # 2.2 4.9 3.3 Phospholipid biosynthesis/membrane regeneration Phosphoglycerol transferase I*# 2.2 4.2 – Phospholipid biosynthesis/membrane regeneration YfgC precursor* 2.1 – – Outer membrane integrity cydD 2.1 – – Glutathione transport Permease of the drug/metabolite transporters (DMT)# 2 4.8-2.1 11.5-2.3 Resistance to microcidal compounds Glycerophosphoryl diester phosphodiesterase# 2 – 3.1 Phospholipid biosynthesis/membrane regeneration sspAB −(2-2.3) −2.1 – Stringent starvation proteins rseABC*# – 9.5-6.2 – Negative regulatory proteins for RpoE, a sigma factor for envelope stress response nsrR*# – 8.2 – Repressor for resistance to nitrosative stress cmeB – 7.2 – Drug efflux system clpB*# – 5.7 – Stress-induced chaperone Formate efflux transporter*# – 4.8 – Resistance to microcidal peptides cspD* – 3.7 – Cold shock proteins, involved in stress caused by membrane damage Manganese superoxide dismutase – 3.5 11 Resistance to oxidative stress norR* – 3.4 – Anaerobic nitric oxide reductase transcription regulator uvrC*# – 3.2 4.5 Excinuclease ABC subunit C for DNA repair marR – 3 6.6 Multiple antibiotic resistance protein htpG – 3 – Chaperone protein msrAB# – 2.7 2.3 Peptide methionine sulfoxide reductase involved in reparation of oxidized proteins ohr* – −2.6 -3.65 Repressor for organic hydroperoxidase resistance degQ# – – 2.1 Outer membrane integrity Flagellum, pili and chemotaxis Methyl-accepting chemotaxis protein*# 8.5-2.4 12.8-2.5 15.5-2 Chemotaxis mshH, mshJK, mshOP*# 2.7 3.2-2.1 4.2-2.1 Pili MSHA biosynthesis fleQ, fleS 2.4-2.2 2 4.1-3.1 Flagellar regulatory protein flgA, flgHI, flgL, flgT*# 2.2-2.1 4.1 11.9-3.2 Flagellar basal-body rod proteins tadBCD, tadZ* 2.2-2 14.1-3.2 10 Flp pili assembly fliF, fliM*# 2 2.2-2.1 3.6-2.6 Flagellar motor activity Probable type IV pilus assembly FimV-related −2.4 −2.4 – Pili MSHA rpoN*# – 27.1 3.8 RNA polymerase sigma factor flgN – 8.7 2.1 Flagellar biosynthesis protein flgK, flgM*# – 8.5 16 Hook associated protein motAB*# – 7.5-2.8 3.6 Flagellar motor rotation protein fleN*# – 5.8 2.1 Flagellar synthesis regulator RNA polymerase sigma factor for flagellar operon – 5.4 – Flagellar biosynthesis acfD – 4 – Accessory colonization factor, putatively involved in motility fliL – 3.7 – Controls rotational direction of flagella during chemotaxis flaD. flaFG*# – 3.6-2.3 6.5-4 Flagellin protein cheD – 3.3 – Chemotaxis protein Chemotaxis regulator# – 3.1 5.8 Transmits chemoreceptor signals to flagellar motor (Continued) Frontiers in Microbiology | www.frontiersin.org 8 March 2020 | Volume 11 | Article 489 Hernández-Cabanyero et al. Vibrio vulnificus Transcriptome in Response to Temperature TABLE 2 | Continued Gene(s)a 25◦ Cb 28◦ Cc 37◦ Cd Putative function/processe LPS and capsule cpsABC# 2 5.5-3.3 – Capsule biosynthesis sypAB, sypR – 5.1-2.3 15.8-5.2 Capsule biosynthesis wza# – −2.8 – LPS biosynthesis galE – – 4.7 O-antigen biosynthesis lptA – – 2.3 LPS biosynthesis Transcriptional regulators Nitrogen regulation protein NR(I)* 3.1 −2.7 – Nitrogen starvation tetR*# 2.6 7.9 – Involved in transcriptional control of multidrug efflux pumps, pathways for the biosynthesis of antibiotics, response to osmotic stress and toxic chemicals, control of catabolic pathways, differentiation processes, and pathogenicity rpoS 2.5 – – Stress and metabolism management arcA# −2.4 −2.6 – Repressor for aerobic metabolism uxuR*# – 9 – Repressor for oligogalacturonide metabolism mall*# – 4.3 – Maltose regulon repressor protein deoR – 4 – Repressor of deoxyribose operon vpsT*# – 3.7 5.1 Repressor of biofilm and biofilm related polysaccharide formation luxO*# – 3.6 – Involved in quorum sensing pathways phoR*# – 2.9 – Histidine kinase for PhoB, involved in phosphate metabolism luxU*# – 2.9 – Phosphorelay protein involved in quorum sensing CRP* – 2.9 5 cAMP receptor protein, regulatory protein argR – 2.5 – Repressor of arginine metabolism luxQ*# – −2.6 – Autoinducer 2 sensor kinase/phosphatase, involved in quorum sensing smcR# – – 2.2 Involved in quorum sensing pathways fabR*# – – −2.7 Repressor for unsaturated fatty acid biosynthesis The value of fold change per gene at each infective temperature (25, 28, and 37◦ C vs. non-infective temperature [20◦ C]) is shown. Only genes with values of fold change −2 ≤ X ≤ 2 with a p-value cut-off of 0.05 were considered. a Identified DEGs are indicated. b−d Fold change value or range of values for each identified gene or group of related genes at 25◦ Cb , 28◦ Cc , and 37◦ Cd . See Supplementary Tables S2–S4 for specific gene and fold change value. e Putative function for selected genes and related process. *Differentially expressed in iron stimulon (Pajuelo et al., 2016). #Differentially expressed in eel or iron-overloaded human serum (Hernández-Cabanyero et al., 2019). –, not detected as differentially expressed. FIGURE 4 | Principal component analysis (PCA) 3-D plot of temperature samples. The transcriptomic profile is represented by different colors. Frontiers in Microbiology | www.frontiersin.org 9 March 2020 | Volume 11 | Article 489 Hernández-Cabanyero et al. Vibrio vulnificus Transcriptome in Response to Temperature increase in biofilm quantity with rising temperature was observed (Table 3). Motility Flagella-related genes were the most abundant DEGs in our transcriptomic study, upregulated when incubation temperature increases (Table 2). We performed a motility assay and confirm that the motility rate of V. vulnificus significantly increases when the environmental temperature increases, being non-motile at 20◦ C (non-infective temperature), intermediate-motile at 25 and 28◦ C (without significant differences between these two FIGURE 5 | Chemotaxis toward eel skin mucus in V. vulnificus. Chemotaxis temperatures) and high-motile at 37◦ C (Table 3). was measured as Chemotactic response (ratio of bacterial numbers in eel skin mucus-capillaries vs. control-capillaries [containing Chemotaxis buffer (ChB)]). Proteolytic and Hemolytic Activity Horizontal line marks the borderline between positive and negative chemotaxis. *: significant differences in chemotaxis response toward eel skin Vvp, the major exoprotease of V. vulnificus, is involved in mucus at each temperature vs. 20◦ C (P < 0.05). #: significant differences in adhesion and mucosal colonization and therefore, is considered chemotaxis response toward eel skin mucus between each temperature and an important virulence factor for both fish and human 28◦ C (P < 0.05). colonization (Amaro et al., 1995; Paranjpye and Strom, 2005; Valiente et al., 2008b; Jones and Oliver, 2009). Although we did not find vvp differentially expressed in any of the (Tables 2, 3), and motile V. vulnificus cells which produce Vvp are transcriptomic comparisons performed, we found upregulated positively chemo-attracted by eel mucus (Valiente et al., 2008b), at optimal temperatures for eel and human vibriosis (28 and we tested V. vulnificus chemo-attraction toward eel skin mucus 37◦ C, respectively) crp, encoding c-AMP receptor protein, and at 20, 25, 28, and 37◦ C. Interestingly, the pathogen only exhibits quorum sensing regulators, both described as inducers of Vvp positive attraction to eel mucus at 28 and 37◦ C but not at 20 or activity (Elgaml and Miyoshi, 2017) (Table 2). Therefore, we 25◦ C (Figure 5), which agrees with the efficient temperatures for checked the effect of temperature on protease production by protease activity (28◦ C) and motility (37◦ C) (Table 3). V. vulnificus through the measuring of azocasein proteolysis at 20, 25, 28, and 37◦ C. Our results revealed that at 20◦ C Surface Cell-Associated Polysaccharides there was no proteolytic activity and this activity increases We determined in previous studies that V. vulnificus external with temperature, achieving the major point of activity at 28◦ C envelope production is regulated by iron concentration and and again decreasing at 37◦ C (Table 3). Our results are in seems to confer resistance to innate immunity in a host- accordance with previous studies of temperature dependent dependent manner (Pajuelo et al., 2016; Hernández-Cabanyero regulation of Vvp as previous studies described that this protease et al., 2019). A few genes related to O-antigen (galE) and activity is efficient at 26◦ C (Elgaml and Miyoshi, 2017). On the capsule biosynthesis (cps and syp genes) were upregulated contrary, hemolytic activity was not affected by temperature, in response to an increase in temperature, albeit they did as we did not find differences in neither live bacteria nor not correspond to those previously obtained in response ECPs hemolytic activity among the analyzed temperatures to iron and/or host sera (Table 2). In order to better (results not shown). understand how temperature could influence external envelope formation, cell-associated polysaccharides of V. vulnificus grown Chemotaxis at 20, 25, 28, and 37◦ C were extracted, quantified and Among the most upregulated genes in response to temperature analyzed. The quantity of cellular-associated polysaccharides (µg were those related to chemotactic response (Table 2). Given that per 108 cells) significantly increased with temperature until both motility and proteolytic activity are temperature-dependent 28◦ C and from that on (37◦ C) the increase in temperature TABLE 3 | Phenotypic characterization of V. vulnificus grown at different temperatures. Condition Biofilm (Abs540 )a Motility rateb Cellular-associated polysaccharides (µg/108 cells)c Proteolytic activity (PU)d 20◦ C 0.027 ± 0.002 0.24 ± 0.02* 87.67 ± 0.37* 12.09 ± 6.65* 25◦ C 0.034 ± 0.005 1.56 ± 0.08# 95.37 ± 1.32*# 6216.17 ± 250.55# 28◦ C 0.035 ± 0.016 1.40 ± 0.31# 103.55 ± 2.77# 6498.76 ± 210.18# 37◦ C 0.066 ± 0.048 2.46 ± 0.60*# 108.68 ± 5.92# 4021.47 ± 181.49*# a For biofilm quantification bacteria were grown in glass tubes for 24 h. Then, planktonic bacteria were eliminated and biofilm was quantified after staining with crystal violet by measuring absorbance at 540 nm (Abs540 ). b Motility was measured as motility rate (colony surface in mm2 /log of colony bacterial number in CFU). The assay was performed on plates of CM9-agar (0.3%) as described by Pajuelo et al. (2016). c Total polysaccharide concentration was determined with Total Carbohydrate Assay Kit (BioVision) as described by the manufacturers. d Proteolytic activity in ECPs, obtained according to Biosca and Amaro (1996), was evaluated using azocasein (Sigma) as substratum (Valiente et al., 2008b). Proteolytic activity is expressed as proteolytic units (PU) produced in each condition (Miyoshi et al., 1987). In all cases results are presented as the averages ± standard deviation of three independent experiments. *: significant differences (P < 0.05) at each temperature vs. 28◦ C. #: significant differences (P < 0.05) at each temperature vs. 20◦ C. Frontiers in Microbiology | www.frontiersin.org 10 March 2020 | Volume 11 | Article 489 Hernández-Cabanyero et al. Vibrio vulnificus Transcriptome in Response to Temperature humans and fish that has been registered in these new areas in the last years (Baker-Austin and Oliver, 2018). In fact, the water temperature is a risk factor that enhances the occurrence of disease outbreaks caused by Vibrio species (Le Roux et al., 2015). There are numerous field data that relate water temperature with outbreak severity of V. vulnificus vibriosis in fish farms (Rodolfo Barrera, personal communication), data that have been probed at laboratory scale by infecting eels at different water temperatures (Amaro et al., 1995). These experiments demonstrated that the pathogen was avirulent at 20◦ C (or below) and caused septicemia in fish maintained over 22◦ C being moderately virulent at 25◦ C and highly virulent at 28◦ C (Amaro et al., 1995). Moreover, V. vulnificus, as a multi-host pathogen, is also able to infect humans and cause septicemia at 37◦ C (Horseman and Surani, 2011). On the other hand, we have demonstrated in previous FIGURE 6 | Vibrio vulnificus LPS and capsule profiles in response to studies that V. vulnificus upregulates a high virulent host-adapted temperature. R99 strain was grown until mid-log phase in CM9 at 20, 25, 28, phenotype in order to face the drastic change in environmental and 37◦ C. Then, cell-associated polysaccharides (LPS + capsule) were conditions upon host encounter (i.e., iron availability [low in extracted with the method of Hitchcock and Brown (1983) and quantified using the Total Carbohydrate Assay Kit (BioVision). Ten µg of cell healthy eels and high in risk human patients] and innate associated-polysaccharides were separated by SDS-PAGE on discontinuous immunity [serum complement proteins]) (Pajuelo et al., 2016; gels (4% stacking gel, 10% separating gel), transferred to a PVDF membrane, Hernández-Cabanyero et al., 2019). subjected to immunoblot analysis with antibodies against cell-envelopes of In this work, we hypothesized that temperature is one of the R99 according to Pajuelo et al. (2016). MMW, medium molecular weight. external signals, which together with iron, triggers virulence in V. vulnificus. In fact, an increase in environmental temperature activates virulence factors expression in other bacteria that are decreased polysaccharide concentration (Table 3). Accordingly, also accidental pathogens (Konkel and Tilly, 2000). To test our the cell-associated polysaccharide pattern also showed changes hypothesis, we analyzed the global transcriptome of V. vulnificus with temperature. This pattern can be only visualized by grown at infective (25, 28, and 37◦ C) vs. non-infective (20◦ C) immunoblotting because neither the LPS nor the capsule are temperature and confirmed the results by phenotypic assays. stained with silver (Amaro et al., 1992). Given that the quantity Finally, we compared the obtained temperature stimulon with the of polysaccharide per well was the same, the obtained results previously described in iron and host serum stimulons (Pajuelo suggest that the immunogenicity of external polysaccharides et al., 2016; Hernández-Cabanyero et al., 2019). seems to change with temperature: no band was immunostained Figure 7 proposes a model on the role of environmental at 20◦ C while when the O-antigen pattern at 25, 28, and 37◦ C temperature in the life cycle and host-adaptation in V. vulnificus was compared, we found the MMW O-antigen portion clearly as well as highlights several genes that belong to the temperature- increased at 28◦ C (Figure 6). Interestingly, MMW is the portion stimulon as putatively involved in some processes. This model of the O-antigen directly involved in resistance to eel innate integrates the results obtained in this work with those previously immunity (Valiente et al., 2008a). Similar changes affecting obtained about the role of iron and the host serum in the life O-antigen pattern were also observed when the LPS from cells strategy of this species inside and outside its main hosts (Pajuelo grown in eel serum (28◦ C) was compared with that obtained in et al., 2016; Hernández-Cabanyero et al., 2019). iron-overloaded human serum (37◦ C), while the change observed in capsule production in presence or absence of iron was not reproduced by changes in temperature (Pajuelo et al., 2016; Motility, Chemotaxis and Protease Hernández-Cabanyero et al., 2019). Altogether, these results Activity suggest that temperature influences the LPS biosynthesis while Motility and chemotaxis have been previously related to capsule production is controlled by iron, among other factors, in fish (mucus and blood) and human (blood) colonization by V. vulnificus. V. vulnificus (Valiente et al., 2008b). We found that both activities were controlled by the environmental temperature, as the bacteria were significantly more motile and chemo-attracted DISCUSSION toward the eel mucus at the warmest temperatures. Attraction to mucus and blood by V. vulnificus is also dependent on vvp, V. vulnificus is an opportunistic pathogen from brackish water a gene encoding the main exoprotease of this species, as the ecosystems located in tropical, subtropical and temperate areas mutants deficient in this gene are unable to be attracted by that is spreading to traditionally cold areas, such as the Baltic mucine (the main component of mucus), fish mucus or blood Sea or Greenland, due to the global warming (Motes et al., (from humans and fish) (Valiente et al., 2008b). In fact, the 1998; Baker-Austin et al., 2012; Oliver, 2015). This expansion is protease is involved in mucine and hemoglobin degradation responsible for an unusually high number of infections affecting (Miyoshi et al., 1997; Valiente et al., 2008b) and therefore, Frontiers in Microbiology | www.frontiersin.org 11 March 2020 | Volume 11 | Article 489 Hernández-Cabanyero et al. Vibrio vulnificus Transcriptome in Response to Temperature FIGURE 7 | Life cycle of the zoonotic pathogen V. vulnificus: role of iron and temperature. This figure summarizes the role of iron levels and temperature in the surrounding environment in determining the life strategy of V. vulnificus. The main steps and processes as well as some genes involved are shown. (1) Resuscitation and induction of the viable but non-culturable state (VBNC). As a free living form, the pathogen swifts between a VBNC and a vegetative state depending on nutrient availability as well as on water temperature and salinity. (2) Capsule and flagellum production in the environment. Vegetative bacteria produce a capsule and a polar flagellum when iron, and probably other nutrients, are available. The flagellum production is also controlled by temperature. (Continued) Frontiers in Microbiology | www.frontiersin.org 12 March 2020 | Volume 11 | Article 489 Hernández-Cabanyero et al. Vibrio vulnificus Transcriptome in Response to Temperature FIGURE 7 | Continued (3) Host colonization. Motile/unmotile bacteria could be attracted by blood/mucus (chemotaxis) from their susceptible hosts (eels and humans with high iron-levels in the blood) and colonize a wound or fish mucus. Alternatively, bacteria can be uptaken by filtering organisms and infect humans by ingestion and colonize the intestine or can infect humans by diseased fish handling. (4) Septicemia. From the wound or mucosal tissue, the pathogen arrives to the bloodstream; in case of humans with high iron levels, the pathogen produces a capsule, multiplies and secretes the toxins VvhA and RtxA1 that cause the death by a toxic sepsis; in case of an eel, only the cells with the plasmid produce two iron-regulated outer membrane proteins, Fpcrp (fish phagocytosis complement resistance protein) and Ftbp (fish transferrin binding protein) that protect against innate immunity (in addition to an envelope enriched in O-antigen), multiply and secrete VvhA, which lyses erythrocytes, increases iron levels and, indirectly, actives the production of RtxA1, which causes the death of the fish by a toxic sepsis. (5) Shedding bacteria to water. Diseased fish liberate bacteria to water. If water is rich in iron, bacteria can infect humans (zoonosis). (6) Biofilm formation and dispersion. Bacteria could be attached to surfaces (including fish mucosae) and to form biofilms under iron restriction. Under iron excess, bacteria will be dispersed from the biofilms as capsulated motile bacteria. Figure modified from Pajuelo et al. (2016). could facilitate chemo-attraction toward mucosal surfaces and temperature clearly influenced the entrance of V. vulnificus in wounds. We phenotypically confirmed that protease activity the log phase of growth, which was very slow at 20◦ C, medium was increased with temperature with a maximum at 28◦ C, and 25◦ C and fast at 28 and 37◦ C; while did not affect the followed by 37◦ C. This increase in activity should be related maximum population size, which was similar at all the assayed to a post-transcriptional activation rather than an increase in temperatures. Therefore, once V. vulnificus attached to the host transcription level, as vvp was not differentially expressed with tissues it could be easily eliminated by the local immunity before an increase in temperature. In any case, during host infection achieving the population size needed to produce fish vibriosis at warm temperatures (28◦ C) V. vulnificus would produce more when fish are kept bellow 20◦ C, while it could multiply and cause active protease, which would favor colonization of mucosal a local inflammation that would facilitate invasion of the blood epithelial tissues, and consequently, the infection. This high stream (Callol et al., 2015) when fish are kept at 28◦ C. In case of level of active protease, together with the chemo-attraction to humans, the bacterial survival in a wound could also be favored mucus at 28◦ C but not at 20◦ C either at 25◦ C would provide at temperatures above 25◦ C. evidence why the highly virulent phenotype for eels is expressed When we analyzed the global transcriptome from cells by V. vulnificus at 28◦ C. Several regulators related to motility, obtained in the mid-log phase of growth, we found that most chemotaxis and protease production were also found to belong of the DEGs in response to temperature were involved in to the temperature stimulon and were regulated accordingly with metabolism and transport of nutrients. Thus, an increase in the observed phenotype: i.e., upregulation of vpsT, encoding temperature upregulated genes related to amino acids, lipids, a repressor of biofilm formation, that promotes motility from fatty acids, nucleoside and iron metabolism and ferrous/ferric biofilm dispersion (Krasteva et al., 2010); upregulation of crp, iron- and vulnibactin dependent transport, most of these nutrient which encodes for a global regulator involved in the activation being present in the blood and mucosae that surrounds fish tissue of Vvp (Elgaml and Miyoshi, 2017); and the overtranscription (Reverter et al., 2018; Chernyavskikh et al., 2019). Similar results of several quorum sensing-related regulators that activate the were previously reported in other gram negative species such expression of vvp (Kim et al., 2013; Elgaml and Miyoshi, as Pdp and Pseudomonas putida (Fonseca et al., 2011; Matanza 2017). Similar results were reported in Photobacterium damselae and Osorio, 2018) including V. vulnificus (Kim et al., 2016). In subsp. piscicida (Pdp) and V. harveyi (Matanza and Osorio, agreement, crp, a positive regulator for iron uptake (Kim et al., 2018; Montánchez et al., 2019), two multi-host pathogens like 2016), was also upregulated at 28 and 37◦ C. Remarkably, and V. vulnificus, which suggests a general role of temperature on contrarily to previous data in other Vibrionaceae species (Liu bacterial colonization in Vibrionaceae. Our model in Figure 7 et al., 2017; Matanza and Osorio, 2018), oligopeptide uptake also shows that an environment rich in free iron would also and metabolism (opp operon) was also activated by temperature contribute to activate a motile and chemotactic phenotype. Thus, suggesting a putative role of this kind of metabolism in bacterial iron and temperature could act synergistically facilitating the first fitness in serum, a medium particularly rich in oligopeptides step in host colonization in the environment. (Samant et al., 2008). In regard to biofilm, temperature did not affect its formation, Attachment, Multiplication and Biofilm unless in the conditions assayed. In fact, some studies Formation reported that this process is controlled by iron, among Once attracted to the epithelium, V. vulnificus has to attach and other external factors (Phippen and Oliver, 2015; Pajuelo multiply to colonize successfully the host tissues. In case of eels, et al., 2016; Chodur et al., 2018; Chodur and Rowe-Magnus, V. vulnificus attaches to the gill epithelium, multiplies on it and 2018) (Figure 7). forms biofilms (Valiente et al., 2008b). With the exception of the flagellum, temperature did not seem to control the expression Resistance to Innate Defenses of any of the attachment appendages described in the species, V. vulnificus needs to overcome host innate defenses in although all of them are controlled by iron levels (Pajuelo et al., mucus and blood to cause septicemia. We found evidence 2016). In this sense, the flagellum could contribute to host tissue that temperature enhances bacterial ability to cope with stress attachment, and this initial adhesion could be enhanced by caused by the host innate immunity as an upregulation of an increase in temperature. Regarding bacterial multiplication, genes involved in membrane regeneration, and resistance to the Frontiers in Microbiology | www.frontiersin.org 13 March 2020 | Volume 11 | Article 489 Hernández-Cabanyero et al. Vibrio vulnificus Transcriptome in Response to Temperature oxidative and nitrosative stress were found upregulated with an Pdp virulence plasmid are upregulated with an increase in increase in temperature. We tested resistance to oxidative stress temperature, including ftbp- and fpcrp-like genes (Matanza and and microcidal peptides (colystin and lysozime) and we only Osorio, 2018). Our results only show ftbp but not fpcrp as confirmed this relationship for microcidal peptides resistance genes regulated by temperature. This could be explained since (data not shown). Therefore, temperature could indirectly in V. vulnificus these two genes are located in different operons, enhance V. vulnificus virulence, preparing the bacterium for while in Pdp they belong to the same operon (results not shown). resisting the microcidal peptides, ROS and/or NO produced Finally, most of these genes also belonged to iron stimulon as by host phagocytes and neutrophils (Kaufmann and Dorhoi, they were upregulated under iron starvation, which stressed that 2016; Uribe-Querol and Rosales, 2017). These processes are also temperature and iron could control synergistically the entire life controlled by iron although the genes involved are not the cycle of the pathogen (Figure 7). same (Pajuelo et al., 2016). In previous studies, we demonstrated that iron also controls the production of a protective envelope, either rich in capsule in iron-overloaded human blood or rich CONCLUSION in HMW/MMW O-antigen portion of LPS together with two plasmid IROMPs in eel blood (Pajuelo et al., 2016; Hernández- Our results show that the capability of V. vulnificus to cause Cabanyero et al., 2019). These two IROMPs constitute a disease and its severity is multifactorial, revealing that the previously described protein kit for survival in fish blood environmental temperature acts as an external signal that, that confers the bacterium the specific ability to resist to fish together with other signals (i.e., iron), controls virulence and complement and phagocytosis (Hernández-Cabanyero et al., adaptation to host mechanisms in the pathogen V. vulnificus. 2019). We found transcriptomic and phenotypic evidence that Thus, environmental temperatures over 25◦ C activate motility, temperature could also control the LPS production; in particular, chemotaxis and protease production, shorten the lag phase, the MMW O-antigen production, as well as the protein of activate metabolism, iron uptake, and the production of a the kit that acts as a specific receptor for fish transferrin partially protective external envelope (at least against fish innate (Ftbp), and, therefore it could act synergistically with iron immunity), all these processes being involved in host colonization contributing to the highly resistant phenotype to fish innate and fish septicemia. However, the main virulence factors for immunity in the blood. this pathogen, the toxin RtxA1 and the hemolysin VvhA, both involved in death by sepsis, are not controlled by temperature. Sepsis According to these results, in iron rich environments, like Notably, in our study none of the V. vulnificus major toxins or those found in fish farms, and at infective temperatures, their regulators were controlled by temperature. These toxins V. vulnificus would be motile, which in turn would result in act synergistically in vivo being RtxA1 the toxin responsible attraction to mucus and colonization of the fish host. In the for death by sepsis in both mice and eels (Lee et al., same way, in seawater the bacterium would increase motility 2013; Callol et al., 2015). This result is in accordance with with temperature (in summer, or warm months) being able previous studies that suggest that virulence-related genes are to be attracted by the human blood and colonize wounds. controlled, directly or indirectly, by other external signals such From gills and human wounds, the pathogen would enter as iron and/or presence of eukaryotic cells in the environment occasionally to the bloodstream where it should produce some (Pajuelo et al., 2016; Murciano et al., 2017; Hernández- of the factors involved in resistance to complement system Cabanyero et al., 2019). In fact, we found in a previous and phagocytosis. From that point, other signals such as iron study performed in serum that both toxins are subjected to a and contact with eukaryotic cells would trigger the formation complex regulation in which iron is a relevant signal but not of a truly protective barrier together with the expression of the only one (Hernández-Cabanyero et al., 2019). Considering a toxic phenotype involved in septicemia. Although most of the importance of the toxins RtxA1 and VvhA in V. vulnificus the genes belonging to temperature stimulon do not belong virulence, we expected to find them upregulated at 28 and to iron stimulon, many of them are involved in the same 37◦ C. However, we did not find any of them differentially processes. In this sense, both temperature and iron would expressed at the assayed conditions (either by microarray, act synergistically to trigger virulence and survival in and nor by RT-qPCR). We did not observe any phenotypic between hosts. Finally, our results confirm that beyond the difference in hemolyses capability among temperatures either. effect of temperature on the distribution of V. vulnificus These results are supported by previous studies in Pdp in the environment, there is a real effect on the infectious (Matanza and Osorio, 2018). capacity of this species that must be taken into account Finally, our results highlight that the external temperature is to predict the real risk of V. vulnificus infection caused a major factor controlling pVvBt2 gene transcription, as most by global warming. of the genes were maximally upregulated at 28◦ C. The plasmid encodes the protein kit for survival in fish blood together with a series of uncharacterized proteins probably involved in growth DATA AVAILABILITY STATEMENT in sepsis (Lee et al., 2008; Pajuelo et al., 2015; Hernández- Cabanyero et al., 2019). In a similar way, Matanza and Osorio All datasets generated for this study are included in the have recently described that most of the genes encoded by article/Supplementary Material. Frontiers in Microbiology | www.frontiersin.org 14 March 2020 | Volume 11 | Article 489 Hernández-Cabanyero et al. Vibrio vulnificus Transcriptome in Response to Temperature AUTHOR CONTRIBUTIONS stimulon and eel serum. (D) Number of DEGs at 37◦ C in common with iron stimulon and iron-overloaded human serum. CH-C, ES, and CA conceived and designed the study. CH-C, BF, TABLE S1 | Primers used for RT-qPCR analysis. DP, and FR-L performed the lab experiments. CH-C and EV-V TABLE S2 | Differentially expressed genes by V. vulnificus at 25◦ C vs. 20◦ C in analyzed the data. CH-C wrote the first draft of the manuscript. CM9. The fold change value for each gene is indicated with also the inclusion of ES, BF, and CA corrected the draft. CH-C and CA built the final those previously described to be differentially expressed in iron stimulon, fur version of the manuscript. All authors contributed to manuscript regulon and eel serum (Pajuelo et al., 2016; Hernández-Cabanyero et al., 2019). ∗ : revision, and read and approved the submitted version. present in iron stimulon, fur regulon or eel-serum but with upside downregulation. ∗∗ : only genes with values of fold change −2 ≤ X ≤ 2 with a p-value cut-off of 0.05 at 25◦ C vs. 20◦ C were considered. +: gene upregulated at 25◦ C; −: gene downregulated at 25◦ C. FUNDING TABLE S3 | Differentially expressed genes by V. vulnificus at 28◦ C vs. 20◦ C in This work has been funded by grants AGL2017-87723-P CM9. The fold change value for each gene is indicated with also the inclusion of [Ministerio de Ciencia, Innovación y Universidades (Spain) those previously described to be differentially expressed in iron stimulon, fur regulon and eel serum (Pajuelo et al., 2016; Hernández-Cabanyero et al., 2019). ∗ : and FEDER funds] and AICO/2018/123 (Generalitat Valenciana, present in iron stimulon, fur regulon or eel serum but with upside downregulation. Spain). CH-C has been funded by the FPI grant BES-2015-073117 ∗∗ : only genes with values of fold change −2 ≤ X ≤ 2 with a p-value cut-off of (Ministerio de Ciencia, Innovación y Universidades, Spain). 0.05 at 28◦ C vs. 20◦ C were considered. +: gene upregulated at 28◦ C; −: gene EV-V was granted with DICYT-USACH Postdoctoral Fellowship downregulated in at 28◦ C. (No. 022043IB). TABLE S4 | Differentially expressed genes by V. vulnificus at 37◦ C vs. 20◦ C in CM9. The fold change value for each gene is indicated with also the inclusion of those previously described to be differentially expressed in iron stimulon, fur SUPPLEMENTARY MATERIAL regulon and eel serum (Pajuelo et al., 2016; Hernández-Cabanyero et al., 2019). ∗ : present in iron stimulon, fur regulon or eel serum but with upside downregulation. ∗∗ : only genes with values of fold change −2 ≤ X ≤ 2 with a p-value cut-off of The Supplementary Material for this article can be found 0.05 at 37◦ C vs. 20◦ C were considered. +: gene upregulated at 37◦ C; -: gene online at: https://www.frontiersin.org/articles/10.3389/fmicb. downregulated at 37◦ C. 2020.00489/full#supplementary-material TABLE S5 | V. vulnificus R99 strain temperature stimulon. The temperature FIGURE S1 | Venn diagram for the number of DEGs found at each temperature in stimulon consists on the set of DEGs by V. vulnificus in response to an increase in common with iron stimulon (previously described by Pajuelo et al., 2016) or with temperature. The value of fold change per gene at each infective temperature (25, DEGs in host serum (previously described by Hernández-Cabanyero et al., 2019). 28, and 37◦ C vs. non-infective temperature [20◦ C]) is shown. ∗ : only genes with (A) Number of DEGs in common between the three infective temperatures values of fold change −2 ≤ X ≤ 2 with a p-value cut-off of 0.05 considered. +: assayed (25, 28, and 37◦ C). (B) Number of DEGs at 25◦ C in common with iron gene upregulated at infective temperature; −: gene downregulated in at infective stimulon and eel serum. (C) Number of DEGs at 28◦ C in common with iron temperature. NS, non-significant. REFERENCES Biosca, E. G., and Amaro, C. (1996). Toxic and enzymatic activities of Vibrio vulnificus biotype 2 with respect to host specificity. Appl. Environ. Microbiol. Amaro, C., Biosca, E. G., Fouz, B., Alcaide, E., and Esteve, C. (1995). Evidence 62, 2331–2337. doi: 10.1128/aem.62.7.2331-2337.1996 that water transmits Vibrio vulnificus Biotype 2 infections to eels. Appl. Environ. Blokesch, M., and Schoolnik, G. K. (2008). The extracellular nuclease Dns and its Microbiol. 61, 1133–1137. doi: 10.1128/aem.61.3.1133-1137.1995 role in natural transformation of Vibrio cholerae. J. Bacteriol. 190, 7232–7240. Amaro, C., Biosca, E. G., Fouz, B., and Garay, E. (1992). Electrophoretic analysis doi: 10.1128/JB.00959-08 of heterogeneus lipopolysaccharides from various strains of Vibrio vulnificus Callol, A., Pajuelo, D., Ebbesson, L., Teles, M., MacKenzie, S., and Amaro, C. biotypes 1 and 2 by silver staining and immunoblotting. Curr. Microbiol. 25, (2015). Early steps in the European eel (Anguilla anguilla)-Vibrio vulnificus 99–104. doi: 10.1007/bf01570967 interaction in the gills: role of the RtxA13 toxin. Fish Shellf. Immunol. 43, Amaro, C., Biosca, E. G., Fouz, B., Toranzo, A. E., and Garay, E. (1994). Role of 502–509. doi: 10.1016/j.fsi.2015.01.009 iron, capsule and toxins in the pathogenicity of Vibrio vulnificus biotype 2 for Chernyavskikh, S. D., Borodaeva, Z., Borisovskiy, I. P., Ostapenko, S. I., and mice. Infect. Immun. 62, 759–763. doi: 10.1128/iai.62.2.759-763.1994 Galtseva, O. A. (2019). Blood protein spectrum in representatives of the fish Amaro, C., Fouz, B., Biosca, E. G., Marco-Noales, E., and Collado, R. (1997). The superclass. EurAsian J. Biosci. 13, 979–981. lipopolysaccharide o side chain of Vibrio vulnificus serogroup E is a virulence Chodur, D. M., Coulter, P., Isaacs, J., Pu, M., Fernandez, N., Waters, C. M., determinant for eels. Infect. Immun. 65, 2475–2479. doi: 10.1128/iai.65.6.2475- et al. (2018). Environmental calcium initiates a feed-forward signaling circuit 2479.1997 that regulates biofilm formation and rugosity in Vibrio vulnificus. MBio 9, Amaro, C., Sanjuán, E., Fouz, B., Pajuelo, D., Lee, C., Hor, L., et al. (2015). The fish doi: 10.1128/mBio.01377-18. pathogen Vibrio vulnificus Biotype 2: epidemiology, phylogeny and virulence Chodur, D. M., and Rowe-Magnus, D. A. (2018). Complex control of a genomic factors involved in warm-water vibriosis. Microbiol. Spectr. 3, 0005–2014. doi: island governing biofilm and rugose colony development in Vibrio vulnificus. 10.1128/microbiolspec.VE-0005-2014 J. Bacteriol. 200:e190-18. doi: 10.1128/JB.00190-18 Baker-Austin, C., and Oliver, J. D. (2018). Vibrio vulnificus: new insights into a Choi, M. H., Sun, H. Y., Park, R. Y., Kim, C. M., Bai, Y. H., Kim, Y. R., et al. (2006). deadly opportunistic pathogen. Environ. Microbiol. 20, 423–430. doi: 10.1111/ Effect of the crp mutation on the utilization of transferrin-bound iron by Vibrio 1462-2920.13955 vulnificus. FEMS Microbiol. Lett. 257, 285–292. doi: 10.1111/j.1574-6968.2006. Baker-Austin, C., Trinanes, J. A., Taylor, N. G. H., Hartnell, R., Siitonen, A., 00183.x and Martinez-Urtaza, J. (2012). Emerging Vibrio risk at high latitudes in Elgaml, A., and Miyoshi, S. I. (2017). Regulation systems of protease and hemolysin response to ocean warming. Nat. Clim. Change. 3:73. doi: 10.1038/nclimate production in Vibrio vulnificus. Microbiol. Immunol. 61, 1–11. doi: 10.1111/ 1628 1348-0421.12465 Frontiers in Microbiology | www.frontiersin.org 15 March 2020 | Volume 11 | Article 489 Hernández-Cabanyero et al. Vibrio vulnificus Transcriptome in Response to Temperature Esteve-Gassent, M. D., Nielsen, M. E., and Amaro, C. (2003). The kinetics of Lages, M. A., Balado, M., and Lemos, M. L. (2019). The expression of antibody production in mucus and serum of European eel (Anguilla anguilla virulence factors in Vibrio anguillarum is dually regulated by iron levels L.) after vaccination against Vibrio vulnificus: Development of a new method and temperature. Front. Microbiol. 10:2335. doi: 10.3389/FMICB.2019. for antibody quantification in skin mucus. Fish Shellf. Immunol. 15, 51–61. 02335 doi: 10.1016/S1050-4648(02)00138-9 Larsen, M., Larsen, J., and Olsen, J. (2001). Chemotaxis of Vibrio anguillarum Fonseca, P., Moreno, R., and Rojo, F. (2011). Growth of Pseudomonas putida at low to fish mucus: role of the origin of the fish mucus, the fish species and the temperature: global transcriptomic and proteomic analyses. Environ. Microbiol. serogroup of the pathogen. FEMS Microbiol. Ecol. 38, 77–80. doi: 10.1111/j. Rep. 3, 329–339. doi: 10.1111/j.1758-2229.2010.00229.x 1574-6941.2001.tb00884.x Fouz, B., and Amaro, C. (2002). Isolation of a new serovar of Vibrio vulnificus Le Roux, F., Wegner, K. M., Baker-Austin, C., Vezzulli, L., Osorio, C. R., Amaro, C., pathogenic for eels cultured in freshwater farms. Aquaculture 217, 677–682. et al. (2015). The emergence of Vibrio pathogens in Europe: ecology, evolution doi: 10.1016/S0044-8486(02)00200-4 and pathogenesis (Paris, 11-12 March 2015). Front. Microbiol. 6:830. doi: 10. Fouz, B., Larsen, J. L., and Amaro, C. (2006). Vibrio vulnificus serovar A: an 3389/fmicb.2015.00830 emerging pathogen in European anguilliculture. J. Fish Dis. 29, 285–291. doi: Lee, C. T., Amaro, C., Wu, K. M., Valiente, E., Chang, Y. F., Tsai, S. F., et al. 10.1111/j.1365-2761.2006.00719.x (2008). A common virulence plasmid in biotype 2 Vibrio vulnificus and its Gao, H., Yang, Z. K., Wu, L., Thompson, D. K., and Zhou, J. (2006). Global dissemination aided by a conjugal plasmid. J. Bacteriol. 190, 1638–1648. doi: transcriptome analysis of the cold shock response of Shewanella oneidensis MR- 10.1128/JB.01484-07 1 and mutational analysis of its classical cold shock proteins. J. Bacteriol. 188, Lee, C. T., Pajuelo, D., Llorens, A., Chen, Y. H., Leiro, J. M., Padrós, F., et al. (2013). 4560–4569. doi: 10.1128/JB.01908-05 MARTX of Vibrio vulnificus biotype 2 is a virulence and survival factor. Environ. Gumpenberger, T., Vorkapic, D., Zingl, F. G., Pressler, K., Lackner, S., Seper, A., Microbiol. 15, 419–432. doi: 10.1111/j.1462-2920.2012.02854.x et al. (2016). Nucleoside uptake in Vibrio cholerae and its role in the transition Lee, J. H., Rho, J. B., Park, K. J., Kim, C. B., Han, Y. S., Choi, S. H., et al. (2004). Role fitness from host to environment. Mol. Microbiol. 99, 470–483. doi: 10.1111/ of flagellum and motility in pathogenesis of Vibrio vulnificus. Infect. Immun. 72, mmi.13143 4905–4910. doi: 10.1128/IAI.72.8.4905-4910.2004 Hernández-Cabanyero, C., Lee, C.-T., Tolosa-Enguis, V., Sanjuán, E., Pajuelo, D., Lim, J. J., Grinstein, S., and Roth, Z. (2017). Diversity and versatility of Reyes-López, F., et al. (2019). Adaptation to host in Vibrio vulnificus, a zoonotic phagocytosis: roles in innate immunity, tissue remodeling, and homeostasis. pathogen that causes septicemia in fish and humans. Environ. Microbiol. 21, Front. Cell. Infect. Microbiol. 7:1–12. doi: 10.3389/fcimb.2017.00191 3118–3139. doi: 10.1111/1462-2920.14714 Liu, W., Huang, L., Su, Y., Qin, Y., Zhao, L., and Yan, Q. (2017). Contributions Hitchcock, P. J., and Brown, T. M. (1983). Morphological heterogeneity of the oligopeptide permeases in multistep of Vibrio alginolyticus pathogenesis. among Salmonella lipopolysaccharide chemotypes in silver-stained Microbiologyopen 6:e511. doi: 10.1002/mbo3.511 polyacrylamide gels. J. Bacteriol. 154, 269–277. doi: 10.1128/jb.154.1.269-277. Livak, K. J., and Schmittgen, T. D. (2001). Analysis of relative gene expression data 1983 using real-time quantitative PCR and. Methods 25, 402–408. doi: 10.1006/meth. Hoben, H. J., and Somasegaran, P. (1982). Comparison of the pour, spread, and 2001.1262 drop plate methods for enumeration of Rhizobium spp. in inoculants made Matanza, X. M., and Osorio, C. R. (2018). Transcriptome changes in response to from presterilized peat. Appl. Environ. Microbiol. 44, 1246–1247. doi: 10.1128/ temperature in the fish pathogen Photobacterium damselae subsp. damselae: aem.44.5.1246-1247.1982 Clues to understand the emergence of disease outbreaks at increased seawater Horseman, M. A., and Surani, S. (2011). A comprehensive review of Vibrio temperatures. PLoS ONE 13:e0210118. doi: 10.1371/journal.pone.0210118 vulnificus: an important cause of severe sepsis and skin and soft-tissue infection. McDougald, D., Rice, S. A., and Kjelleberg, S. (2001). SmcR-dependent regulation Int. J. Infect. Dis. 15:e157-66. doi: 10.1016/j.ijid.2010.11.003 of adaptive phenotypes in Vibrio vulnificus. J. Bacteriol. 183, 758–762. doi: Jones, M. K., and Oliver, J. D. (2009). Vibrio vulnificus: disease and pathogenesis. 10.1128/JB.183.2.758-762.2001 Infect. Immun. 77, 1723–1733. doi: 10.1128/IAI.01046-08 Miller, J. H. (1972). Experiments in Molecular Genetics. Cold Spring Harbor, NY: Jones, M. K., Warner, E. B., and Oliver, J. D. (2008). csrA inhibits the formation Cold Spring Harbor Laboratory Press. of biofilms by Vibrio vulnificus. Appl. Environ. Microbiol. 74, 7064–7066. doi: Miyoshi, N., Shimizu, C., Miyoshi, S. I., and Shinoda, S. (1987). Purification and 10.1128/AEM.01810-08 characterization of Vibrio vulnificus protease. Microbiol. Immunol. 31, 13–25. Kaufmann, S. H. E., and Dorhoi, A. (2016). Molecular determinants in phagocyte- doi: 10.1111/j.1348-0421.1987.tb03064.x bacteria interactions. Immunity 44, 476–491. doi: 10.1016/j.immuni.2016. Miyoshi, S., Wakae, H., Tomochika, K., and Shinoda, S. (1997). Functional 02.014 domains of a zinc metalloprotease from Vibrio vulnificus. J. Bacteriol. 179, Kim, C. M., Ahn, Y. J., Kim, S. J., Yoon, D. H., and Shin, S. H. (2016). Temperature 7606–7609. doi: 10.1128/jb.179.23.7606-7609.1997 change induces the expression of vuuA encoding Vulnibactin receptor and crp Montánchez, I., Ogayar, E., Plágaro, A. H., Esteve-Codina, A., Gómez-Garrido, encoding cyclic AMP receptor protein in Vibrio vulnificus. Curr. Microbiol. 73, J., Orruño, M., et al. (2019). Analysis of Vibrio harveyi adaptation in sea 54–64. doi: 10.1007/s00284-016-1026-8 water microcosms at elevated temperature provides insights into the putative Kim, I. H., Wen, Y., Son, J. S., Lee, K. H., and Kim, K. S. (2013). The fur-iron mechanisms of its persistence and spread in the time of global warming. Sci. complex modulates expression of the quorum-sensing master regulator, smcr, Rep. 9:289. doi: 10.1038/s41598-018-36483-0 to control expression of virulence factors in Vibrio vulnificus. Infect. Immun. 81, Motes, M. L., DePaola, A., Cook, D. W., Veazey, J. E., Hunsucker, J. C., Garthright, 2888–2898. doi: 10.1128/IAI.00375-13 W. E., et al. (1998). Influence of water temperature and salinity on Vibrio Kim, S. M., Lee, D. H., and Choi, S. H. (2012). Evidence that the Vibrio vulnificus in Northern Gulf and atlantic coast oysters (Crassostrea virginica). vulnificus flagellar regulator FlhF is regulated by a quorum sensing Appl. Environ. Microbiol. 64, 1459–1465. doi: 10.1128/aem.64.4.1459-1465.1998 master regulator SmcR. Microbiology 158, 2017–2025. doi: 10.1099/mic.0.05 Murciano, C., Lee, C. T., Fernández-Bravo, A., Hsieh, T. H., Fouz, B., Hor, L. I., 9071-0 et al. (2017). MARTX toxin in the zoonotic serovar of Vibrio vulnificus triggers Kim, Y. W., Lee, S. H., Hwang, I. G., and Yoon, K. S. (2012). Effect of temperature an early cytokine storm in mice. Front. Cell. Infect. Microbiol. 7:332. doi: 10. on growth of Vibrio paraphemolyticus and Vibrio vulnificus in flounder, salmon 3389/fcimb.2017.00332 sashimi and oyster meat. Int. J. Environ. Res. Public Health 9, 4662–4675. Oh, M. H., Lee, S. M., Lee, D. H., and Choi, S. H. (2009). Regulation of the doi: 10.3390/ijerph9124662 Vibrio vulnificus hupA gene by temperature alteration and cyclic AMP receptor Konkel, M. E., and Tilly, K. (2000). Temperature-regulated expression of bacterial protein and evaluation of its role in virulence. Infect. Immun. 77, 1208–1215. virulence genes. Microbes Infect. 2, 157–166. doi: 10.1016/S1286-4579(00) doi: 10.1128/IAI.01006-08 00272-0 Oliver, J. D. (2015). The biology of Vibrio vulnificus. Microbiol. Spectr. 3, 1–10. Krasteva, P. V., Jiunn, J. C., Shikuma, N. J., Beyhan, S., Navarro, M. V. A. S., Yildiz, Pajuelo, D., Hernández-Cabanyero, C., Sanjuan, E., Lee, C. T., Silva-Hernández, F. H., et al. (2010). Vibrio cholerae vpst regulates matrix production and motility F. X., Hor, L. I., et al. (2016). Iron and Fur in the life cycle of the zoonotic by directly sensing cyclic di-GMP. Science 327, 866–868. doi: 10.1126/science. pathogen Vibrio vulnificus. Environ. Microbiol. 18, 4005–4022. doi: 10.1111/ 1181185 1462-2920.13424 Frontiers in Microbiology | www.frontiersin.org 16 March 2020 | Volume 11 | Article 489 Hernández-Cabanyero et al. Vibrio vulnificus Transcriptome in Response to Temperature Pajuelo, D., Lee, C. T., Roig, F. J., Hor, L. I., and Amaro, C. (2015). Novel host- Valiente, E., Jiménez, N., Merino, S., Tomas, J. M., and Amaro, C. (2008a). Vibrio specific iron acquisition system in the zoonotic pathogen Vibrio vulnificus. vulnificus biotype 2 serovar E gne but not galE is essential for lipopolysaccharide Environ. Microbiol. 17, 2076–2089. doi: 10.1111/1462-2920.12782 biosynthesis and virulence. Infect. Immun. 76, 1628–1638. doi: 10.1128/IAI. Paranjpye, R. N., and Strom, M. S. (2005). A Vibrio vulnificus type IV pilin 01393-07 contributes to biofilm formation, adherence to epithelial cells and virulence. Valiente, E., Lee, C., Hor, L., Fouz, B., and Amaro, C. (2008b). Role of the Infect. Immun. 73, 1411–1422. doi: 10.1128/IAI.73.3.1411-1422.2005 metalloprotease Vvp and the virulence plasmid pR99 of Vibrio vulnificus serovar Phippen, B. L., and Oliver, J. D. (2015). Role of anaerobiosis in capsule production E in surface colonization and fish virulence. Environ. Microbiol. 10, 328–338. and biofilm formation in Vibrio vulnificus. Infect. Immun. 83, 551–559. doi: doi: 10.1111/j.1462-2920.2007.01454.x 10.1128/IAI.02559-14 Wang, Z., Li, Y., and Lin, X. (2017). Transcriptome analysis of the Pu, M., and Rowe-Magnus, D. A. (2018). A Tad pilus promotes the establishment Antarctic psychrotrophic bacterium Psychrobacter sp. G in response to and resistance of Vibrio vulnificus biofilms to mechanical clearance. Npj Biofilms temperature stress. Acta Oceanol. Sin. 36, 78–87. doi: 10.1007/s13131-016-0 Microbiomes 4:10. 956-0 Ramos, J. L., Martı, M., Molina-henares, A. J., Tera, W., Brennan, R., and Tobes, Watnick, P. I., Fullner, K. J., and Kolter, R. (1999). A role for the mannose-sensitive R. (2005). The TetR family of transcriptional. Microbiol. Mol. Biol. Rev. 69, hemagglutinin in biofilm formation by Vibrio cholerae El Tor. J. Bacteriol. 181, 326–356. doi: 10.1128/MMBR.69.2.326 3606–3609. doi: 10.1128/jb.181.11.3606-3609.1999 Reverter, M., Tapissier-Bontemps, N., Lecchini, D., Banaigs, B., and Sasal, P. (2018). Biological and ecological roles of external fish mucus: a review. Fishes 3:41. Conflict of Interest: The authors declare that the research was conducted in the doi: 10.3390/fishes3040041 absence of any commercial or financial relationships that could be construed as a Roig, F. J., González-Candelas, F., Sanjuán, E., Fouz, B., Feil, E. J., Llorens, C., potential conflict of interest. et al. (2018). Phylogeny of Vibrio vulnificus from the analysis of the core- genome: implications for intra-species taxonomy. Front. Microbiol. 8:2613. doi: Copyright © 2020 Hernández-Cabanyero, Sanjuán, Fouz, Pajuelo, Vallejos-Vidal, 10.3389/fmicb.2017.02613 Reyes-López and Amaro. This is an open-access article distributed under the terms Samant, S., Lee, H., Ghassemi, M., Chen, J., Cook, J. L., Mankin, A. S., et al. (2008). of the Creative Commons Attribution License (CC BY). The use, distribution or Nucleotide biosynthesis is critical for growth of bacteria in human blood. PLoS reproduction in other forums is permitted, provided the original author(s) and the Pathog. 4:e37. doi: 10.1371/journal.ppat.0040037 copyright owner(s) are credited and that the original publication in this journal Uribe-Querol, E., and Rosales, C. (2017). Control of phagocytosis by microbial is cited, in accordance with accepted academic practice. No use, distribution or pathogens. Front. Immunol. 8:1368. doi: 10.3389/fimmu.2017.01368 reproduction is permitted which does not comply with these terms. Frontiers in Microbiology | www.frontiersin.org 17 March 2020 | Volume 11 | Article 489