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Área de Microbiología, Departamento de Biología Funcional, Facultad de Medicina, IUBA, Universidad de Oviedo, 33006 Oviedo, Spain
Correspondence
José A. Guijarro
jaga{at}fq.uniovi.es
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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The GenBank/EMBL/DDBJ accession number for the tonB operon and surrounding DNA sequence is EF408823.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). The control of outbreaks is currently based on fish management strategies and the use of antimicrobial therapy. However, these treatments are costly, antibiotic-resistant bacteria can arise (Bruun et al., 2000; Schmidt et al., 2000; Izumi & Aranishi, 2004) and, additionally, the health of human and terrestrial animals could be affected in different ways by this use of antibiotics (Cabello, 2006). An available commercially licensed vaccine has not been developed up to now. Nevertheless, several studies testing vaccination with fractions or inactivated whole cells have been done. It has been described that whole formalin-killed cells (Kondo et al., 2003), a Sarkosyl-insoluble membrane fraction (Rahman et al., 2002) and different molecular mass fractions (LaFrentz et al., 2004) of F. psychrophilum confer protection against the disease. A recent study showed that membrane vesicles contained in the stationary-phase culture supernatant had an adjuvant effect (Aoki et al., 2007). The authors of that study suggested that a combination of membrane vesicles and cells is necessary to obtain efficient protection. Additionally, an OmpH-like surface protein named P18 can induce a protective immune response in fish (Dumetz et al., 2006).
In recent years advances in cultivation (Michel et al., 1999), diagnosis (del Cerro et al., 2002), experimental infections (Garcia et al., 2000) and genetic techniques for DNA transfer (Álvarez et al., 2004) have been reported for F. psychrophilum. However, knowledge on the mechanism of pathogenesis is still limited. Iron is an essential element for the majority of bacterial life, and therefore micro-organisms have developed multiple iron-acquisition systems (Wyckoff et al., 2007). Due to the reduced availability of iron in host tissues, the ability to obtain iron in vivo by micro-organisms is considered an important factor in pathogenicity (Payne, 1993; Wooldridge & Williams, 1993; Braun, 1995). For Gram-negative bacteria, many of the iron-acquisition systems involve the presence of different kinds of outer-membrane receptors that bind a specific ligand (iron–siderophore complex, haemin, haemoglobin, transferrin or lactoferrin) (Andrews et al., 2003). These iron compounds are then introduced into the cell by means of the TonB system, which is an energy-transduction complex required for all high-affinity iron-transport systems. It is composed of TonB, ExbB and ExbD, found in the cytoplasmic membrane of many Gram-negative bacteria, where they energize iron uptake (for review see Braun & Braun, 2002; Andrews et al., 2003; Wandersman & Delepelaire, 2004). In pathogenic bacteria, these iron-acquisition systems allow the bacteria to scavenge iron and grow inside the host. Therefore, the TonB system is associated with mechanisms involved in the progress of infection in several important pathogens such as Vibrio cholerae (Seliger et al., 2001), Shigella dysenteriae (Reeves et al., 2000; Payne et al., 2006), uropathogenic Escherichia coli (Torres et al., 2001) and Bordetella pertussis (Pradel et al., 2000). In the bacteria fish pathogens Yersinia ruckeri (Fernández et al., 2004) and Vibrio anguillarum (Stork et al., 2004) a relationship between genes related to TonB systems and virulence has been reported. In the case of F. psychrophilum the production of siderophore-like molecules was indicated in a study by Møller et al. (2005).
In an attempt to discover more about the pathogenesis mechanisms of F. psychrophilum, a set of mutants deficient in growth in iron-limited conditions were previously isolated using a Tn4351-mutagenesis system (Álvarez et al., 2006). In the present work, one of these mutants, designated FP1033, was further analysed. This mutant had a transposon insertion within a gene with identity to the exbD locus from a TonB system. Sequence, complementation and physiological studies defined a TonB system in F. psychrophilum. Interestingly, this system included two exbD (exbD1 and exbD2) homologous loci. LD50 experiments revealed that FP1033 had reduced virulence, indicating that the exbD2 gene plays an important role in virulence of F. psychrophilum. Furthermore, FP1033, as an attenuated strain, was capable of inducing a protective immune response in rainbow trout fry a few weeks post-vaccination. These results open the possibility of using an attenuated live vaccine against CWD.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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pir (Simon et al., 1983), used to transfer DNA into F. psychrophilum, was grown at 37 °C in 2x TY medium (10 g tryptone, 10 g yeast extract and 5 g NaCl l–1) with 20 g agar l–1 added for solid medium. F. psychrophilum strain THC02-90 was grown at 20 °C on solid EAOS medium (Michel et al., 1999) or at 12 °C and 18 °C in nutrient broth (NB; Pronadisa), as previously described (Álvarez et al., 2004). To evaluate growth on iron-depleted conditions, the iron chelator 2,2'-dipyridyl was added to NA (nutrient agar, consisting of NB with 1.5 %, w/v, agar) or NB at a final concentration of 50 µM . For selective growth, antibiotics were added when needed at the following concentrations: 10 µg erythromycin ml–1 (to maintain the Tn4351 in the mutant); 100 µg ampicillin ml–1; and 10 µg tetracycline ml–1. The plasmids and primers used are listed in Table 1.
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). The site of Tn4351 insertion and the surrounding DNA in FP1033 was analysed by Southern blot as described by Álvarez et al. (2006).
To perform inverse PCR, digestion of chromosomal DNA of the mutant with HindIII followed by religation was carried out. The resulting circular molecules were used as template to amplify the sequences adjacent to the Tn4351 insertion site using the Tn4351-specific pair of primers TN-1/IS4351-F (Table 1
) and the Certamp long amplification kit (BIOTOOLS B&M Laboratories). DNA surrounding exbD2 was amplified from DNA of the wild-type strain THC02-90 previously digested with XbaI and religated. The following pairs of primers were used: INV-A/INV-B, INV-A/INV-C, INV-D/INV-E and INV-F/INV-G (Table 1).
Nucleic acid sequencing.
Automated fluorescence sequencing was performed at the Oviedo University DNA analysis facility using BigDye 3.1 Terminator chemistry on an ABI PRISM 3100 Genetic Analyzer platform (Applied Biosystems). Sequences were compared to databases by using BLAST from the National Center for Biotechnology Information (NCBI) website (http://www.ncbi.nlm.nih.gov/BLAST/).
RT-PCR.
Total RNA was obtained from 150 µl of late-exponential-phase cultures of the wild-type THC02-90 and the mutant FP1033 grown in NB. RNA was isolated using the RNeasy Mini kit (Qiagen) and treated twice with RNase-Free DNase (Promega) to eliminate traces of DNA. RT-PCRs were performed using Superscript One-Step RT-PCR with Platinum Taq (Invitrogen Life Technologies); 15 ng RNA was used in each reaction. PCR control reactions using GoTaq Flexi DNA polymerase (Promega) were performed to determine whether RNA was free of contaminant DNA. Primers used (RT-exbB-F, RT-exbD1-R, RT-exbD2-R, RT-exbD2-F, RT-tonB-R, RT-tonB-F, RT-orf3-R, RT-Tn) are listed in Table 1.
Complementation of the exbD2 : : Tn4351 mutation.
Different amplicons containing (i) exbD2, (ii) exbD2 and tonB, and (iii) exbD2, tonB and orf2 were cloned in pCP23 in order to complement FP1033. The exbD2 gene was amplified by PCR using the Certamp long amplification kit (BIOTOOLS B&M Laboratories) and primers ExbD2-F and ExbD2-R (Table 1). exbD2 and tonB were amplified using the primers ExbD2-F and TonB-R (Table 1), and a DNA fragment that contained exbD2, tonB and orf2 was amplified using the primers ExbD2-F and Orf2-R (Table 1). BamHI and PstI restriction sites had been introduced in the sequences of the forward primers and reverse primers, respectively, to clone the obtained PCR products digested with BamHI and PstI into pCP23 that had been digested with the same restriction enzymes. The resulting plasmids were named pJB1, which contained exbD2, pJB2, which contained exbD2 and tonB, and pJB3, which contained exbD2, tonB and orf2. For complementation analysis pJB1, pJB2 and pJB3 were first introduced into F. psychrophilum THC02-90 by conjugation. The plasmids were isolated from wild-type F. psychrophilum and transferred by electroporation into F. psychrophilum FP1033, as previously described (Álvarez et al., 2004).
Growth curve and production of siderophores by THC02-90 and FP1033.
Flasks containing NB were inoculated with 1/300 volume of stationary-phase cultures of THC02-90 and FP1033 and incubated at 250 r.p.m. and 12 °C. Growth was determined by measuring OD525. To test the effect on growth of inorganic iron, FeCl3 was added to NB to a concentration of 20 µM.
Siderophore quantification during growth of THC02-90 and FP1033 in NB was carried out using the Chrome Azurol S (CAS) assay as described by Schwyn & Neilands (1987). At different incubation times 1 ml aliquots were collected by centrifugation at 15 000 g for 4 min and stored at –20 °C until required. Siderophore units (%) were defined as [(Ar–As)/Ar]x100, according to Schwyn & Neilands (1987).
LD50 determination of FP1033.
LD50 determinations were done using rainbow trout fry (Oncorhynchus mykiss; mean weight 5 g), which were kept in 40 l tanks containing dechlorinated water at 12±1 °C and acclimatized for a minimum of 3 days. F. psychrophilum cells from exponential-phase cultures of both THC02-90 and FP1033 were harvested by centrifugation and washed with PBS. Groups of 10 fish were challenged by intramuscular injection of 0.05 ml of serial dilutions in PBS containing 103–109 bacterial cells and LD50 was calculated according to the method of Reed & Muench (1938).
Vaccination.
The rainbow trout (mean weight 5 g) were kept in the same conditions as described for the LD50 experiments. They were fed a commercial feed (Proaqua) at a rate of 1 % biomass per day throughout the experiments. Vaccination trials were conducted on two 50-fish groups. Fish of the control group were injected intramuscularly with 0.05 ml PBS, whereas fish of the other group were injected with 0.05 ml PBS containing 103 FP1033 bacterial cells. After 6 weeks of immunization the fish were challenged with 1 LD50 of the parental strain. The cumulative percentage mortality (CPM) was determined after 21 days, and the relative percentage survival (RPS) was calculated using the following equation: RPS=[1–(CPM of immunized trout/CPM of PBS-injected trout)]x100.
Genetic nomenclature and nucleotide sequence accession number.
ORFs coding for proteins with amino acid sequences similar to proteins of known function were named after the corresponding genes. The remaining ORFs, which did not have high sequence similarity with known genes, were named orf2 and orf3.
The tonB operon and surrounding DNA sequence has been assigned GenBank accession number EF408823.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). Among them, strain FP1033, which lacked extracellular proteolytic activity on gelatin plates and grew poorly on iron-limited solid medium, was further analysed. Southern blot analysis revealed that FP1033 had a single Tn4351 insertion (data not shown). The DNA sequence surrounding the transposon insertion was obtained by inverse PCR as described by Álvarez et al. (2006). The disrupted sequence corresponds to an ORF of 543 nt that codes for a protein of 180 aa and 19.8 kDa (Fig. 1a). This protein exhibits sequence identity to predicted ExbD proteins from other micro-organisms such as Prosthecochloris vibrioformis (GenBank accession no. ZP_00661181; 32 %) and Chlorobium phaeobacteroides (GenBank accession no. ZP_00527754; 29 %). Nineteen nucleotides upstream of this exbD locus lies a locus that encodes a putative gene product with 28 % sequence identity to a predicted ExbD/TolR protein from Prosthecochloris aestuarii (GenBank accession no. ZP_00591804). Based on these identities the loci were named exbD2 and exbD1, respectively. The deduced amino acid sequence of the exbD1 and exbD2 loci showed 17 % identity and 44 % similarity to each other (Fig. 1b). Additional nucleotide sequence analysis revealed three contiguous ORFs downstream of the exbD2 locus (Fig. 1a). The first ORF, with the start codon located 31 nt downstream of the exbD2 stop codon, designated tonB, encodes a predicted protein of 272 aa that is 53 % identical to the TonB-like protein from Flavobacterium johnsoniae (GenBank accession no. ZP_01247260). Downstream of tonB lies orf2, which encodes a hypothetical protein of 76 aa that does not exhibit sequence identity to proteins of known function. The orf2 stop codon is followed by the orf3 start codon. The amino acid sequence of the predicted orf3 product of 301 aa was 54 % similar to a putative ABC transporter from F. johnsoniae (GenBank accession no. ZP_01247261). Finally, 65 nt upstream of the exbD1 start codon is the stop codon of the exbB locus. The protein encoded by exbB is highly similar (78 %) to the MotA/TolQ/ExbB proton channel from F. johnsoniae (GenBank accession no. ZP_01247257). Therefore, according to the identities, gene organization and the presence of characteristic elements of the TonB system, this cluster of genes is designated hereafter as the TonB system of F. psychrophilum (Fig. 1a).
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). In this bacterium ExbD1 is involved in iron uptake whereas ExbD2 is not (Wiggerich & Pühler, 2000). As for X. campestris, in F. psychrophilum the inactivation of the exbD2 gene demonstrated that it is involved in the iron uptake, probably by the TonB system. This more complex TonB system with an additional exbD locus could be related to an increase in its scope. The TonB system may be involved in the import of other compounds into the bacteria in addition to iron uptake (Postle, 1990; Braun, 1995; Köster, 2001). Further investigation is needed to determine the specific role of each of the two exbD genes in F. psychrophilum.
RT-PCR analysis and complementation studies
Transcription analysis of the loci exbB, exbD1, exbD2, tonB, orf2 and orf3 by RT-PCR in F. psychrophilum THC02-90 was carried out. Amplicons corresponding to overlapping transcripts from exbB to exbD1, exbB to exbD2, exbD2 to tonB and tonB to orf3 were obtained (Fig. 1a, lanes 2, 3, 4 and 5). These results confirmed that all these genes form part of an operon that was named the tonB operon. In the case of FP1033 no RT-PCR product was obtained from exbB to exbD2 (Fig. 1a, lane 8). However, an amplicon from FP1033 RNA was produced using a specific primer at the end of Tn4351 (RT-Tn) and the primer RT-exbD2-R, indicating that a promoter within the transposon drives the transcription of tonB, orf2 and orf3 (Fig. 1a, lane 9). Therefore, the insertion of the transposon in exbD2 caused a non-polar mutation and this result also demonstrates that orf2 and orf3 are indeed part of the tonB operon. As was previously indicated by experiments using Tn4351 in Bacteroides fragilis (Smith et al., 1992) and F. johnsoniae (Hunnicutt & McBride, 2000), our result confirms that Tn4351 possesses, at least at one end, an outwards-orientated promoter activity since DNA was generated when using primers situated inside Tn4351 and in the middle of the exbD2 gene.
Using the pCP23 expression vector (Álvarez et al., 2004, 2006), three plasmids, pJB1, pJB2 and pJB3, were constructed as described in Methods to complement FP1033. Introduction of pJB1, which contained only exbD2, into FP1033 restored growth on iron-depleted medium (Fig. 1c). The same results were obtained with plasmids pJB2, which contained exbD2 and tonB, and pJB3, which contained exbD2, tonB and fpo2. The complementation studies are in agreement with the RT-PCR results and indicate that the defects of FP1033 in growth on iron-depleted medium were caused by the lack of the ExbD2 protein.
Phenotypic characterization of the FP1033 mutant
In the presence of 50 µM of the iron chelator 2,2'-dipyridyl, growth of FP1033 in NB was inhibited, whereas a minor effect was found in the parental strain (data not shown). When the growth of the two strains was compared, it was clear that there is a significant difference between them in growth rate but not in the final yield (Fig. 2a). FP1033 showed a reduced growth rate in the exponential phase and a longer lag phase, delaying the growth by approximately 20 h in comparison with the parental strain (Fig. 2a). However, when the NB medium was supplemented with iron (FeCl3) complete growth restoration was observed in the FP1033 strain (Fig. 2a).
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Quantitative analysis of siderophores in the supernatants from the culture media throughout the growth curve from the parental and mutant strains showed that siderophore production was necessary for growth in NB and was growth stage dependent. In both strains the amount of siderophores increased during growth, reaching maximum values at the beginning of the stationary phase. However, a subsequent decrease in the level of siderophores occurred in the parental strain whereas no changes were found in the culture supernatant of FP1033 (Fig. 2b), suggesting that FP1033 has problems in the uptake of the iron–siderophore complex due to the interruption of the exbD2 gene, which generates a dysfunction in the TonB system.
FP1033 was also found to be defective in extracellular proteolytic activity. Up- and downregulation of extracellular enzymes by iron limitation has been described for different bacteria (Tokuda et al., 1998; Enard & Expert, 2000; Maunsell et al., 2006). Although in some cases this regulation is part of a more complex pathway related to virulence (Beare et al., 2003), the function of this association remains unclear. F. psychrophilum is able to degrade haemoglobin (Nematollahi et al., 2003); thus the production of certain proteases related to haemolysis could perhaps be regulated by the levels of iron.
LD50 determination and vaccination studies
In order to elucidate the effect of the exbD2 mutation on the virulence of F. psychrophilum, LD50 determinations were carried out by infecting rainbow trout fry with the parental and the FP1033 strains. Ten days post-infection, LD50 values were 2.7x108 and 6x105, respectively. Thus, the mutant strain was approximately 450-fold attenuated compared to the parent, indicating that inside the fish there is an iron-limited condition and a functional ExbD2 is required for full virulence in F. psychrophilum. The results obtained when in vivo competition assays between THC02-90 and FP1033 cells were carried out were inconsistent due to the low and poorly reproducible recovery of both types of cells from fish. This is one of the problems of working with this fastidious bacterium.
The high level of attenuation of FP1033 indicates that it could be a good candidate for the development of a live vaccine. Therefore, a protection challenge was carried out in rainbow trout fry to determine whether FP1033 cells can induce an immune response against CWD. At 6 weeks post-vaccination with FP1033 the fish were injected with 1 LD50 of the parental strain. As can be observed in Fig. 3, 22 out of 50 fish in the non-vaccinated group fish died within 7 days, whereas only 4 out of 50 fish in the immunized group died. Taking these data, the RPS value was 81.8 %, indicating that FP1033 is capable of inducing high protection in rainbow trout fry against the disease. Moreover, after injecting the immunized fish with the parental strain, some of them developed a skin lesion that, in contrast to the fatal development of the lesion in the control fish, gradually healed. To our knowledge, this is the first time that an attenuated strain of F. psychrophilum has been used in vaccination experiments. In previous studies, the vaccines were based on fractions (Rahman et al., 2002; LaFrentz et al., 2004; Dumetz et al., 2006) or inactivated whole cells (Kondo et al., 2003; Madetoja et al., 2006). Although it is difficult to compare results from different vaccination studies due to the heterogeneity of the conditions used (fish species, fish weight and vaccine preparation), FP1033 cells conferred a similar level of protection to fish as other vaccines. It is also notable that, after the challenge, the control fish developed a skin lesion around the injection site prior to death, whereas only some of the vaccinated fish presented such a lesion, which cicatrized in a few days, indicating that an immune response had been generated. Live attenuated vaccines have been developed for other fish pathogens such as V. anguillarum (Norqvist et al., 1989), Aeromonas salmonicida (Vaughan et al., 1993), Aeromonas hydrophila (Hernanz Moral et al., 1998) and Y. ruckeri (Temprano et al., 2005). The use of an attenuated vaccine against F. psychrophilum may offer many advantages in comparison with classical vaccine preparations, because the attenuated strain can survive in the host, stimulating its immune system without causing the disease. Thus, this vaccine may generate a prolonged immune response in fry which have an immature immune system. The results presented here establish the basis for the future development of a live attenuated vaccine that will be able to protect against CWD. Vaccine doses, bath immunization and cross-protection experiments using different strains are the next steps to carry out.
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ACKNOWLEDGEMENTS |
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Edited by: S. C. Andrews
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Received 21 July 2007;
revised 2 January 2008;
accepted 4 January 2008.
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) and FP1033 (
). The bacteria were grown in NB at 12 °C. FP1033 was also grown in NB supplemented with 20 µM FeCl3 (
). Growth was monitored by determining the OD525. No effect on THC02-90 growth was observed in NB supplemented with iron. The growth curve of FP1033 complemented with pJB1 was similar to that shown for the parental strain. (b) Siderophore levels in the supernatant of the culture media of THC02-90 and FP1033 at different times of growth. 
, 
, Siderophore units, detemined as described by Schwyn & Neilands (1987)