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Vol. 118: 217–226, 2016 DISEASES OF AQUATIC ORGANISMS Published March 30 doi: 10.3354/dao02976 Dis Aquat Org

Iron assimilation and siderophore production by ordalii strains isolated from diseased Atlantic salmon Salmo salar in Chile

Pamela Ruiz1,2, Miguel Balado3, Alicia E. Toranzo3, Matías Poblete-Morales1,2, Manuel L. Lemos3,*, Ruben Avendaño-Herrera1,2,4,*

1Laboratorio de Patología de Organismos Acuáticos y Biotecnología Acuícola, Facultad de Ciencias Biológicas, Universidad Andrés Bello, Viña del Mar, Chile 2Interdisciplinary Center for Aquaculture Research (INCAR), Concepción, Chile 3Departamento de Microbiología y Parasitología. CIBUS-Facultad de Biología e Instituto de Acuicultura, Universidad de Santiago de Compostela, Santiago de Compostela 15782, Spain 4Centro de Investigación Marina Quintay (CIMARQ), Quintay, Chile

ABSTRACT: Vibrio ordalii is the causative agent of vibriosis in several cultured salmonid species worldwide. Despite its impact on aquaculture, relatively little information is available about its vir- ulence factors. The present study demonstrates for the first time that V. ordalii possesses different systems of iron acquisition, one involving siderophore synthesis and another one that uses direct binding of heme to use iron. Using 6 strains of V. ordalii from Atlantic salmon Salmo salar and the V. ordalii type strain, we could demonstrate that all strains could grow in presence of the chelating agent 2,2’-dipyridyl and produced siderophores in solid and liquid media. Cross-feeding assays among V. ordalii strains evidenced variability in the siderophores produced. Bioassays and PCR data suggest that V. ordalii could produce a siderophore with a structure similar to piscibactin, although the production of a second siderophore in certain strains cannot be discarded. Further- more, all strains were able to use hemin and hemoglobin as the only iron sources, although the cell yield was higher when using hemoglobin. A hemin-binding assay indicated the presence of con- stitutive heme-binding molecules at the cell surface of V. ordalii. Virulence tests using rainbow trout as a model of infection revealed a clear relationship between iron-uptake ability and patho- genicity in V. ordalii.

KEY WORDS: Vibrio ordalii · Siderophore · Iron sources · Virulence mechanisms · Atlantic salmon

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INTRODUCTION such as gilthead sea bream Sparus aurata in Turkey (Akayli et al. 2010). This pathogen is a Gram-nega- Vibrio ordalii, formerly biovar II tive, motile, rod-shaped bacterium that is fermenta- (Schiewe et al. 1981), is an important causative agent tive, catalase and oxidase positive, and sensitive to of vibriosis in several salmonid species cultured in the vibriostatic agent O/129 (Farmer et al. 2005). V. the USA, Japan, Australia, New Zealand and Chile ordalii causes hemorrhagic septicemia on the base of (Harrell et al. 1976, Ransom et al. 1984, Toranzo et al. the fins, exophthalmos, and injury, especially on the 1997, Colquhoun et al. 2004), although it has also area surrounding the anus, including the anal pore been reported as a pathogen for other fish species (Ransom et al. 1984).

*Corresponding authors: [email protected], © Inter-Research 2016 · www.int-res.com [email protected] 218 Dis Aquat Org 118: 217–226, 2016

Currently, the pathogenic mechanisms of V. ordalii MATERIALS AND METHODS are not yet fully understood. We have previously

copy shown that the pathogenicity is not correlated to the Bacterial strains and growth conditions hemagglutination capacity of Atlantic salmon Salmo salar red blood cells or biofilm characteristics, but it A total of 6 Vibrio ordalii strains isolated from could be associated with its hydrophobic properties Atlantic salmon from enzootics on farms in southern

Author (Ruiz et al. 2015). V. ordalii can also survive in S. salar Chile during 2003 to 2011 were studied. The mucus, which likely facilitates in vivo host coloniza- Atlantic salmon isolates were obtained from internal tion and helps to neutralize or evade the host organs (kidney or spleen) of diseased cage-cultured immune system, including bactericidal activities fish. The strains chosen for this work were selected (Ruiz et al. 2015). on the basis of their cell-surface characteristics A critical virulence factor for most Gram-negative (Ruiz et al. 2015) because a first requisite for suc- is the ability to obtain iron (Wolf & Crosa cessful colonization is the ability to adhere to the 1986). In hosts, iron is a cofactor for enzymes in- host, and hydrophobicity (water repelling) of bac - volved in general metabolism, DNA replication, and terial cells has been associated with bacterial path- the electron transport chain (Ratledge & Dover 2000), ogenicity (González-Contreras et al. 2011). Strains and it is mostly bound to proteins, such as hemoglo- Vo-LM-03 and Vo-LM-05 (Group 1) were classified bin, transferrin, lactoferrin, or ferritin (Wooldridge & as strongly hydrophobic (hydrophobicity >29.5%), Williams 1993, Ratledge & Dover 2000). Therefore, strains Vo-LM-01, Vo-LM-13, and Vo-LM-18 (Group within a host, free iron is often highly limited. Thus, 2) were classified as relatively hydrophobic (hydro - most pathogenic bacteria possess 2 main iron- phobicity ranging from 19.6 to 29.5%), and strain uptake mechanisms: (1) a direct utilization of iron Vo-LM-06 (Group 3) was classified as non-hydro - contained in host proteins, such as transferrin or phobic (hydrophobicity ranging from 9.6 to 19.5%). hemoglobin; and (2) the production of intermedi- V. ordalii type strain ATCC 33509T and V. anguilla- aries, such as hemophores and sidero phores, to rum ATCC 43307T (obtained from the American scavenge iron from heme- or iron-containing com- Type Culture Collection) were also used for com- pounds (Wooldridge & Williams 1993, Ratledge & parative purposes. Identification of each strain as V. Dover 2000, Debarbieux & Wandersman 2004, Ray- ordalii was achieved by standard phenotypic char- mond & Dertz 2004). In V. anguillarum, it has been acterization procedures (Silva-Rubio et al. 2008, demonstrated that both types of mechanisms are Poblete-Morales et al. 2013) and confirmed by the present. V. anguillarum can efficiently use heme and PCR-based analysis described by Avendaño-Her- hemoglobin as iron sources (Mazoy & Lemos 1991, rera et al. (2014). V. ordalii strains were routinely Mouriño et al. 2004) and also produces sidero - cultivated on trypticase soy agar or broth supple- phores, anguibactin in strains harboring pJM1-like mented with 1% (w/v) NaCl (TSA-1 and TSB-1, plasmids (Crosa et al. 1980, Wolf & Crosa 1986), and respectively) under aerobic conditions at 18°C for vanchrobactin in strains lacking these plasmids 48 to 72 h. Stock cultures were maintained frozen (Balado et al. 2006, Naka et al. 2008). at −80°C in Criobilles tubes (AES Laboratory) or in In V. ordalii, Pybus et al. (1994) showed that the TSB-1 containing 15% (v/v) glycerol. siderophores produced by the V. anguillarum strains VL4355 and 775 (that produce anguibactin) inhibit the growth of V. ordalii and that V. ordalii strains are Ability to grow under iron-limiting conditions unable to use anguibactin as a siderophore. These observations point to the existence of different iron- The ability of the V. ordalii strains to grow under uptake mechanisms for these 2 closely related micro- iron-restricted conditions was determined by their organisms. More recently, Naka et al. (2011) compared capacity to grow in TSB-1 supplemented with the the complete genome sequence of V. anguillarum non-assimilable iron chelator 2,2′-dipyridyl (Sigma- and V. ordalii and described the presence of several Aldrich). The minimal inhibitory concentration (MIC) genes putatively involved in iron-uptake mecha- of 2,2′-dipyridyl was determined by culturing each V. nisms in V. ordalii ATCC 33509T. Therefore, in the ordalii strain in TSB-1 containing increasing concen- present work, we sought a first insight into the mech- trations of 2,2′-dipyridyl (from 0 to 400 µM) and was anisms that V. ordalii possesses for iron assimilation defined as the lowest concentration at which no bac- and sought to determine whether iron levels affect terial growth was observed. Growth was determined

the virulence for fish of V. ordalii isolates. using optical density at 620 nm (OD620) in a Multi- Ruiz et al.: Iron acquisition by Vibrio ordalii 219

skan Ex (Thermo Scientific) spectrophotometer. All supernatant by absorbance at 488 nm for Congo red experiments were carried out in triplicate using dif- and at 400 nm for hemin. The remaining cell suspen-

copy ferent batches of media. sion was incubated with shaking at 18°C, and con- centration of unbound Congo red or hemin were measured at intervals from 15 to 240 min as Utilization of different iron sources described above. The experiment was performed in

Author triplicate. To test the use of different sources of iron by V. ordalii, TSB-1 medium with 2,2′-dipyridyl (at inhi- bitory concentrations for each strain) was supple- Production of siderophores mented with various iron sources: hemin (bovine), hemoglobin (bovine), ferric ammonium citrate (FAC), Siderophore activity was tested using the Chrome and ferric chloride (Sigma), and growth in each con- Azurol S (CAS) assay of Schwyn & Neilands (1987) in

dition was determined by OD620. Hemin (Sigma) solid and liquid media. This test detects the presence stock solution at 5 mM was prepared in 10 mM of compounds with iron-chelating activity using a NaOH, and hemoglobin (Becton Dickinson) was dis- ferri–chromogen complex that changes color when it solved in distilled water at 1 mM. FAC was dissolved loses Fe3+ ions. Siderophores, which have more affin- in phosphate buffered saline (PBS) and ferric chlo- ity for iron than the chromogen (CAS), can capture ride in distilled water, both at 10 mM. All stock solu- iron from the ferri–chromogen complex, resulting in tions were sterilized by filtration through 0.22 µm a color change of the dye from blue to yellow-orange. pore size membranes (Millipore) and maintained at The assays were performed by spotting 10 µl of a 4°C until use. Final concentrations of the iron sources bacterial suspension in PBS obtained from a 3 d ranged from 0.1 to 20 µM. The growth was assessed TSA-1 culture onto CAS agar plates. The sidero- in triplicate in 96-well plates (NUNC Surface) con- phore levels produced by the strains on CAS agar taining 100 µl of TSB-1, supplemented with 2,2’- were expressed as the ratio of the orange halo diam- dipyridyl and the iron source to be tested, seeded eter to the growth diameter after 48 to 72 h of incuba-

with a 1:100 (v/v) inoculum from TSB-1. The OD620 tion. Plates were also spotted with V. anguillarum was measured after 48 h of incubation at 18°C in a ATCC 43307T as a positive control. For siderophore shaker at 100 rpm. detection in liquid media, supernatants from a cul- ture in TSB-1 containing 2,2′-dipyridyl were mixed with a CAS supernatant solution, and the absorbance

Hemin-binding assays (A630) of the mixture was measured. The presence of catecholate-type and hydroxam- Once the ability to use hemin as iron source was ate-type siderophores was detected in cell-free cul- identified, the existence of possible outer membrane ture supernatants obtained from V. ordalii strains receptors for heme compounds in V. ordalii was grown under iron-limiting conditions (TSA-1 plus determined using the Congo red and hemin-binding 2,2′-dipyridyl) using the colorimetric assays de - assays in liquid media, according to the procedures scribed by Arnow (1937) and Csáky (1948), modified described by Kay et al. (1985) with some modifica- by Andrus et al. (1983), respectively. Deferoxamine tions. Congo red dye-binding ability is an indicator of mesylate (a trihydroxamate siderophore; Sigma) and heme-binding capacity because Congo red struc- 2,3-dihydroxybenzoic acid (Sigma) were used as turally resembles heme (Surgalla & Beesley 1969). positive controls. The experiments were performed Briefly, V. ordalii cells grown in (1) TSB-1 with 15 µM twice.

FeCl3, (2) TSB-1 with 20 µM hemoglobin, and (3) TSB-1 supplemented with half of the MIC of 2,2′- dipyridyl were harvested by centrifugation, washed Cross-feeding assays in sterile 0.9% saline solution, and resuspended in

20 ml of saline solution to an OD620 of 1.0. Congo red To test the ability of each V. ordalii strain to induce (Sigma) or hemin (Sigma) were added at a final con- the growth of the other strains subjected to iron star- centration of 30 and 40 µg ml−1, respectively. Samples vation, cross-feeding assays were used (Avendaño- (1 ml) were immediately removed and centrifuged at Herrera et al. 2005). Briefly, V. ordalii strains were 13 000 × g for 30 s, and binding of the chromogen was grown in iron-limiting conditions (TSB-1 supple- determined by measuring the residual dye in the mented with half of the MIC of 2,2′-dipyridyl) and 220 Dis Aquat Org 118: 217–226, 2016

centrifuged at 12 000 × g for 5 min. Table 1. Genes and primers used for PCR Cell-free supernatants from the

copy strains to be tested were pipetted Gene Primer Nucleotide sequence 5’–3’ Source onto sterile filter paper disks and, after being dried, were placed on irp1 irp1_ang_1 TGA TGC ATT AGC CCA TCA GG Present study irp1_ang_2 AAA GCA AGG GTC GAG AGT GT the agar surface of TSA-1 plates vabF pMB5_R AAT CAC CGC CAC TTC ACC TG Balado et al. (2008) Author seeded with the strain to be used pMB5_-R TAG ATG GCA ACG TGT TGG AC as an indicator and containing an huvA P3-1 GGA ATG TTG TCG CAG CAC TA Mouriño et al. (2005) inhibitory concentration of 2,2′- HuvA-Short 3′ CAT GGA ACA ACA AAG CCA GC dipyridyl for that strain. The de - huvS huvS3 CGA AGA CCA GCG GTT AAT AT Mouriño et al. (2005) tection of a growth halo around huvS6-rev GCT CTC GCA GAA GAA GTT TC the disks indicated the production tonB1 cDNA-tonB1 AAA CCC CAC CTC CAG AAA AG Stork et al. (2004) by the tested strain of a diffusible cDNA-tonB2 TGG GCG AGT AAC AAA AGA CG siderophore that can be utilized tonB2 TonB2-1 TTA CTG CTT GCT TTG CCA A Stork et al. (2004) TonB2-2 TTT GTC CCG GCT GCT CAA T by the indicator strain. Purified siderophores vanchro - bactin (from V. anguillarum) and 33509T grown under iron-limitation (TSB-1 with half piscibactin (from Photobacterium damselae subsp. of the MIC of 2,2′-dipyridyl) and under an excess of piscicida), obtained as previously described (Soengas iron (TSB-1). et al. 2006, Souto et al. 2012), were also used to test Groups of 10 fishes (rainbow trout weighing 28 to the ability of V. ordalii strains to utilize them as iron 30 g) were inoculated by intraperitoneal injection sources. Different concentrations of each sidero - with the V. ordalii strains grown in both conditions phore were added to liquid cultures of each strain in using a dose of 4 × 105 colony-forming units per fish. TSB-1 with an inhibitory concentration of 2,2′- Control fishes received 0.1 ml of sterile TSB-1 with or dipyridyl for each particular strain. Growth was without the iron-chelating agent. All fish were main-

measured by determining the OD600. tained in tanks at 16 ± 1°C with aeration during the course of the experiments (21 d), with 50% of the tank water refreshed daily. Dead fish were removed DNA isolation and PCR assays from each tank daily, and the cause of death was con- firmed by streaking kidney, liver, and spleen samples To test the possible presence in V. ordalii strains directly onto TSA-1 plates that were incubated at of genes known in V. anguillarum to be involved in 18°C for 1 wk. Pure or mixed cultures from these iron assimilation, PCR assays were performed to plates, as well as fish tissues (kidney, liver, and test the presence of genes vabF (siderophore van - spleen) samples, were tested by PCR using a specific chrobactin biosynthesis), irp1 (piscibactin biosyn- protocol (Avendaño-Herrera et al. 2014) to confirm thesis), huvA and huvS (encoding heme receptors), the presence of V. ordalii. In addition, the identity of and tonb1 and tonb2 (encoding TonB systems). each isolate was confirmed by standard phenotyping Genomic DNA from each V. ordalii strain was procedures (Silva- Rubio et al. 2008, Poblete-Morales extracted using the Genome® DNA Kit (Q-BIO- et al. 2013). gene) following the manufacturer’s instructions. Primers employed for detection of each gene are shown in Table 1. PCR reactions were performed RESULTS AND DISCUSSION following standard protocols as described in the sources shown in Table 1. Growth under iron-limiting conditions and utilization of different iron sources

Effect of iron availability on the lethality The ability to grow under iron-limiting conditions of V. ordalii strains for fish was tested in all strains examined. The results show that all Vibrio ordalii strains, including the type strain The influence of iron levels on the pathogenicity of ATCC 33509T, were able to grow under severe iron- V. ordalii strains from different virulence categories limiting growth conditions in TSB-1 medium supple- (Ruiz et al. 2015) was tested by comparing the viru- mented with the iron-chelating agent 2,2’-dipyridyl. lence of strain LM-Vo-18 and the type strain ATCC The MICs of 2,2’-dipyridyl ranged between 90 and Ruiz et al.: Iron acquisition by Vibrio ordalii 221

180 µM (Table 2), which suggests that V. ordalii pos-

test sess at least 1 high-affinity iron uptake system. Based copy + + on these results, concentrations of 45 to 90 µM were used as the iron-restrictive conditions for subsequent experiments. To identify the iron sources that could be used by

Author V. ordalii, we first tested the ability of strains to use – – heme and hemoglobin, 2 iron sources present in tis- Use of siderophores

) values of supernatants at sues of all animal hosts. Under high iron-restricted Vanchrobactin Piscibactin 630 conditions in TSB-1 medium, all strains A V. ordalii e of growth, (+): weak growth, T could utilize hemoglobin as the only iron source with

0 nm ( high efficiency: cell yield was 3-fold higher (increase + + in the OD620 values from 0.5 to 1.6) in relation to con- trol grown in plain TSB-1 (Fig. 1A). Growth values were dependent on the hemoglobin concentration, with the highest values obtained when using 20 µM hemoglobin (Fig. 1A). There were no differences among the 3 groups of strains (based on their hydro - phobicity) analyzed, suggesting that the mechanisms of iron uptake are independent of this surface char- acteristic (Fig. 1A). All strains could also use hemin as the only iron source, although the utilization was much less effi- cient, since the cell yield reached was 4-fold lower than that obtained when using hemoglobin and lower than the yield obtained in TSB-1 (Fig. 1B). This Cross-feeding assays is accordance with the results obtained in V. anguil- larum in which all strains could utilize hemin and hemoglobin as sole iron sources (Mazoy & Lemos of the CAS dye). Cross-feeding assays between strains and for utilization of Use of siderophores produced by strain −: negative results 1991, Mouriño et al. 2005).

630 To test the genetic relationship among the heme A uptake systems between V. anguillarum and V. ordalii, we checked by PCR the presence in V. ordalii strains of the genes that encode the outer membrane −−+−+ – + +−−−− −+−−−(+)− + –+−−−− − + +−−−− + − + – – + + +−−−− − + – + heme receptors in V. anguillarum: huvA and huvS. As shown in Table 3, all V. ordalii strains tested, Vo-LM-01 Vo-LM-03 Vo-LM-05 Vo-LM-06 Vo-LM-13 Vo-LM-18 33509 ATCC including the type strain ATCC 33509T, were positive for the presence of huvS, an alternative receptor gene to huvA that is present in many strains of V. anguillarum (Mouriño et al. 2005). When searching genomes of other V. ordalii strains deposited in Gen- Bank, we found that some strains harbor huvA gene strains under iron-limiting conditions; siderophore production, cross-feeding assays, and use of pure siderophores. The chemical while others harbor huvS, a situation similar to what has been previously reported for V. anguillarum (Mouriño et al. 2005). We also demonstrated that V. ordalii strains could also grow using FAC or ferric chloride (Fig. 1C,D) 90 2.97 ± 0.08 −0.164 ± 0.0014 (µM) CAS agar CAS in liquid as iron sources when these compounds were T added to TSB-1 supplemented with inhibitory con- centrations of 2,2’-dipyridyl. When using ferric chloride, all V. ordalii strains exhibited identical growth than when they were grown in plain TSB-1 Vo-LM-05Vo-LM-06 150Vo-LM-18 180 2.13 ± 0.08 33509 ATCC −0.086 ± 0.0021 2.22 ± 0.13 −0.196 ± 0.0042 120 1.73 ± 0.06 −0.229 ± 0.0028 + (+) (+) (+) (+) (+) Strain MICVo-LM-01Vo-LM-03 180 180 Chemical test 2.75 ± 0.09Vo-LM-13 −0.226 ± 0.0042 2.53 ± 0.03 −0.116 ± 0.0021 180 + 2.37 ± 0.12 −0.215 ± 0.0042 (+) (+) (+) (+) (+) was the Chrome Azurol S (CAS) assay of Schwyn & Neilands (1987) in solid and liquid media. Siderophore production on CAS plates was expressed as the ratio of diameter of orange halo to the growth after 48 h. CAS values in liquid medium are expressed as the absorbance at 63 exogenous siderophores were performed as described in ‘Materials and methods’. MIC: minimum inhibitory concentration; +: presenc 48 h post-inoculation (a positive reaction gives a decrease in Vibrio ordalii 2. Growth Table of Vibrio (Fig. 1D). 222 Dis Aquat Org 118: 217–226, 2016

1.8 1.8 A B Group 1 1.6 1.6 Group 2 copy Group 3 1.4 1.4 MIC 2,2‘-dipyridyl group 1 MIC 2,2‘-dipyridyl group 2 1.2 1.2 MIC 2,2‘-dipyridyl group 3

TSB-1

Author 1.0 1.0 620

OD 0.8 0.8

0.6 0.6

0.4 0.4

0.2 0.2

0.0 0.0 0.1 5 10 15 20 0.1 5 10 15 20 Hemoglobin (µM) Hemin (µM) 1.8 C 1.8 D 1.6 1.6

1.4 1.4

1.2 1.2

1.0 1.0 620

OD 0.8 0.8

0.6 0.6

0.4 0.4

0.2 0.2

0.0 0.0 0.15 101520 0.1 5 10 15 20 Ferric ammonium citrate (µM) Ferric chloride (µM)

Fig. 1. Utilization of (A) hemoglobin, (B) hemin, (C) ferric ammonium, and (D) ferric chloride as iron sources by 5 Vibrio ordalii strains and type strain ATCC 33509T grown in trypticase soy broth supplemented with 1% (w/v) NaCl (TSB-1) after 48 h post- inoculation. Group 1 corresponds to V. ordalii Vo-LM-05, classified as strongly hydrophobic; Group 2 corresponds to Vo-LM- 01, Vo-LM-13, and Vo-LM-18, classified as relatively hydrophobic; and Group 3 is the strain Vo-LM-06, classified as quasi- hydrophilic. As negative control each V. ordalii strain was cultured into TSB-1 with 2,2’-dipyridyl at a concentration higher than the minimal inhibitory concentration (MIC) value. Growth in TSB-1 (measured as optical density [OD]) corresponds to the mean values from all V. ordalii strains tested. Error bars = 1 SD

Table 3. Results of PCR assays to determine the presence of genes related Heme-binding activity to synthesis and transport of siderophores and heme in the Vibrio ordallii strains tested To determine whether the V. ordalii cells display iron-regulated heme- Strains Synthesis of Heme TonB systems binding activity, tested strains were siderophores receptors cultured under iron excess (TSB-1 vabF irp1 huvA huvS tonB1 tonB2 plus ferric chloride or hemoglobin) Vo-LM-01 + + – + + + and under iron-restricted conditions Vo-LM-03 + + – + + + (TSB-1 plus 2,2’dipyridyl), and their Vo-LM-05 + + – + + + hemin and Congo red-binding abili- Vo-LM-06 + + – + + + ties were measured (Fig. 2). None of Vo-LM-13 + + – + + + the strains showed an increase in Vo-LM-18 + + – + + + Congo red absorption when cells were ATCC 33509T ++ –+ ++ V. anguillarum RV22++ +– ++ grown in TSB-1 with 2,2’-dipyridyl (Fig. 2A). However, when the strains Ruiz et al.: Iron acquisition by Vibrio ordalii 223

1.2 A 1.2 B 1.2 C

1.0 1.0 1.0 copy

0.8 0.8 0.8

0.6 Author 0.6 0.6 488 nm

0.4 0.4 0.4

0.2 0.2 0.2

0.0 0.0 0.0 15 30 60 90 120 150 180 210 240 15 30 60 90 120 150 180 210 240 15 30 60 90 120 150 180 210 240 1.6 D 1.6 E 1.6 F

1.4 1.4 1.4

1.2 1.2 1.2

1.0 1.0 1.0 400 nm

0.8 0.8 0.8

0.6 0.6 0.6

0.4 0.4 0.4 15 30 60 90 120 150 180 210 240 15 30 60 90 120 150 180 210 240 15 30 60 90 120 150 180 210 240 Time (min) Time (min) Time (min)

V. ordalii ATCC 33509 Vo-LM-18 Vo-LM-06 Vo-LM-03 Vo-LM-05 Vo-LM-01 Control Vo-LM-13

Fig. 2. Binding of (A,B,C) Congo red and (D,E,F) hemin by Vibrio ordalii strains, based on absorbance at 488 and 400 nm, respectively. Cells were cultured under conditions of (A,D) iron-restriction, (B,E) iron-replete with hemoglobin, or (C,F) iron- replete with FeCl3. Negative control, Congo red, and hemin data are from a cell-free flask. The uptake of Congo red and hemin is expressed here as depletion of the dye from solution as a function of time. Error bars are the SD of the data from 2 different experiments

were grown in TSB-1 with hemoglobin, they rapidly ent of the iron levels in the medium, suggesting bound most of the Congo red during the first 15 min that constitutive outer membrane proteins play a after adding the dye (Fig. 2B), with no additional role in this function (Mazoy & Lemos 1996, Mouriño binding observed after that time. The same effect et al. 2005). This could also be the case for V. was noted when we measured the hemin-binding ordalii. ability (Fig. 2E). These results suggest that binding In V. anguillarum, it has also been demonstrated could be due to cell surface components that are that the presence of a heme uptake system is not a induced in presence of hemoglobin or heme groups. marker of virulence because all strains regardless of Furthermore, when V. ordalii cells were grown in their virulence potential have the same ability to use

TSB-1 with 2,2’-dipyridyl or FeCl3, a gradual in - heme (Mouriño et al. 2005). However, this does not crease of hemin-binding was observed, reaching rule out a possible role for heme utilization during the highest value after 240 min of exposure to the infective process of V. ordalii. The ability of a hemin (Fig. 2D,F). This observation suggests that bacterial pathogen to acquire iron from heme com- binding is not iron-regulated and could be non- pounds present in host tissues could constitute an specific. It has been demonstrated that in V. anguil- advantage for colonization and invasion, as has been larum, hemin binding is mediated by cell surface demonstrated in other pathogens (Lemos & Osorio proteins and that heme-binding ability is independ- 2007, Contreras et al. 2014). 224 Dis Aquat Org 118: 217–226, 2016

Production of siderophores the ABC transporters fvtB-fvtE, necessary for ferric van chrobactin internalization, as well as the fetAB

copy The ability to grow under severely iron-restricted genes, necessary for enterobactin (a xenosidero - conditions (see ‘Heme-binding activity’) suggests that phore) transport (Naka et al. 2011). V. ordalii may possess high-affinity iron uptake mech- To test if vanchrobactin and/or piscibactin could be anisms. In siderophore production CAS test, all V. actually used by V. ordalii as iron sources, we per-

Author ordalii strains and the type strain ATCC 33509T gave formed a bioassay with pure siderophores. The positive reactions on CAS agar, showing an orange results (Table 2) indicate that none of the strains halo on the blue agar plates, clearly indicating the could utilize vanchrobactin, but all of them could use production of siderophores. The V. ordalii isolates piscibactin as a siderophore. This observation is in showed halo/growth diameter ratios ranging from agreement with the absence of fvtB–fvtE ABC trans- 1.73 to 2.97 (Table 2). As expected, a positive reaction porter for vanchrobactin in the genome of V. ordalii. was also found for V. anguillarum ATCC 43307T used We also checked by PCR the presence of TonB as a control. No differences among the groups of sytems (which transduce energy for siderophore strains (based on their hydrophobicity) were observed, transport) in V. ordalii strains. All of them were suggesting that the mechanisms of iron uptake are in- positive for the presence of tonb1 and tonb2 genes dependent of this surface characteristic. Furthermore, (Table 3), which are necessary for siderophore trans- the cross-feeding assays among the V. ordalii strains port across the outer membrane in many Gram- showed that all them were able to be cross-fed by the negative bacteria, although in V. anguillarum, only type strain ATCC 33509T and by strain Vo-LM-01. tonB2 is essential for siderophore transport and viru- However, the remaining 5 strains were unable to pro- lence (Stork et al. 2004). mote the growth of any other strain or just gave a These results altogether suggest that V. ordalii small weak growth halo (Table 2). This result points to likely produces a siderophore with a structure similar the existence of an intraspecific variability in V. to piscibactin, which is in agreement with the chemi- ordalii regarding siderophore production. cal tests results described above. However, the pro- Although all V. ordalii strains seem to produce duction of a second siderophore in certain strains siderophores, the chemical assays performed with cannot be ruled out. Further studies are currently cell-free supernatants showed that they do not con- underway to elucidate the precise chemical structure tain typical hydroxamate or catecholate-type com- of the siderophores produced by V. ordalii. pounds, suggesting that V. ordalii could produce a ‘mixed type’ siderophore (Hu et al. 1986). However, according to in silico searches and to the genomic Effect of iron availability on the lethality data reported by Naka et al. (2011), V. ordalii ATCC of V. ordalii strains for fish 33509T contains the vab cluster genes, which encode the synthesis of vanchrobactin, a typical catechol To identify the effect of iron on the virulence of V. siderophore identified in V. anguillarum (Balado et ordalii, the virulence of strains LM-Vo-18 and ATCC al. 2006). In addition, the type strain harbors a gene 33509T grown under iron limitation (TSB-1 with 2,2′- cluster that is homologous to the piscibactin (a mixed dipyridyl) and under an excess of iron (TSB-1) was type siderophore) biosynthesis genes described in tested in rainbow trout. The results show that both Photobacteria damselae subsp. piscicida (Osorio et strains increased in virulence degree when cultured al. 2006, Souto et al. 2012), genes that seem well con- under iron-restriction. Strain LM-Vo-18 grown under served in several Vibrio species (M. Balado & M. L. iron restriction (TSB-1 with 2,2’-dipyridyl) was able Lemos pers. obs.). Thus, we performed a PCR assay to kill 65% of the challenged fish, while this strain to test for the presence of both gene clusters in the 6 when cultured in TSB-1 killed 55% of fishes. When strains of V. ordalii tested. Interestingly, all strains fish were intraperitoneally injected with the type were positive for the 2 markers used, vabF and irp1 strain ATCC 33509T, the iron effect was more pro- (Table 3). The PCR result does not mean that all nec- found: the mortality increased from 15% (strain essary genes for synthesis and transport of vanchro - grown in TSB-1) to 35% (strain grown under iron- bactin and piscibactin are present in V. ordalii or that restriction). In all cases, V. ordalii was recovered and all them are actually expressed. In fact, an in silico identified from all dead fish, which showed typical analysis suggests that the vanchrobactin transport symptoms of a V. ordalii infection. These results sug- system in strain ATCC 33509T, although having the gest that the virulent strain Vo-LM-18 could constitu- outer membrane receptor gene fvtA, seems to lack tively express most of its virulence factors, while the Ruiz et al.: Iron acquisition by Vibrio ordalii 225

non-virulent isolate ATCC 33509T under iron- K (2004) First description of Vibrio ordalii from Chile. limiting conditions can induce expression of genes Bull Eur Assoc Fish Pathol 24: 185−188 Contreras H, Chim N, Credali A, Goulding C (2014) Heme copy in volved not only in the synthesis of siderophores but uptake in bacterial pathogens. Curr Opin Chem Biol 19: also in other iron-regulated factors. Similar results 34−41 have been described in P. damselae subsp. piscicida, Crosa JH, Hodges LL, Schiewe MH (1980) Curing of a plas- which has been classically considered a non- mid is correlated with an attenuation of virulence in the

Author marine fish pathogen Vibrio anguillarum. Infect Immun proteolytic bacterium in normal culture conditions 27: 897−902 (Magariños et al. 1992), but low-iron environments Csáky TZ (1948) On the estimation of bound hydroxylamine within fish trigger the expression of these enzymatic in biological materials. Acta Chem Scand 2:450−454 activities (Magariños et al. 1994). Debarbieux L, Wandersman C (2004) Hemophore-depen- In summary, the investigation reported here clearly dent heme acquisition systems. In: Crosa JH, Mey AR, Payne SM (eds) Iron transport in bacteria. ASM Press, demonstrated for first time that V. ordalii possesses Washington, DC, p 38−47 different systems of iron acquisition, one involving Farmer JJ, Janda M, Brenner FW, Cameron DN, Birkhead the synthesis of siderophores and another that allows KM (2005) Genus I. Vibrio Pacini 1854. In: Brenner DJ, the utilization of heme groups as an iron source by Krieg NR, Staley JT (eds) Bergey’s manual of systematic bacteriology, 2nd edn, Vol 2. The . Part B: direct binding. In addition, our results establish a the . Springer, New York, NY, p clear relationship between iron uptake ability and 495−545 the pathogenicity of V. ordalii. González-Contreras A, Magariños B, Godoy M, Irgang R, Toranzo AE, Avendaño-Herrera R (2011) Surface proper- ties of Streptococcus phocae strains isolated from dis- Acknowledgements. This work was supported by Grant eased Atlantic salmon, Salmo salar L. J Fish Dis 34: CONICYT/FONDAP/15110027 and FONDECYT No. 1150695 203−215 from the Comisión Nacional de Investigación Científica y Harrell LW, Etlinger HM, Hodgins HO (1976) Humoral fac- Tecnológica (CONICYT, Chile). A.E.T. and M.L.L. thank tors important in resistance of salmonid fish to bacterial Xunta de Galicia (Spain) for the financial support for the disesase. II. 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Editorial responsibility: David Bruno, Submitted: August 4, 2015; Accepted: February 3, 2016 Aberdeen, UK Proofs received from author(s): March 14, 2016