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1 Área de Microbiología, Departamento de Biología Funcional, Facultad de Medicina, IUBA, Universidad de Oviedo, 33006 Oviedo, Asturias, Spain
2 Laboratorio de Sanidad Animal de Jove, Serida, 33299 Gijón, Asturias, 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 sequences reported in this paper is AY576533.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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; Fouz et al., 2006). Therefore, this pathology continues to cause important economic losses in the aquaculture industry in many countries.
Early work showed that some extracellular products of Y. ruckeri, including several enzymic activities such as protease, lipase and haemolysin, may play an important role in the development of pathogenesis. Romalde & Toranzo (1993) observed that the injection of these products into fish reproduced some characteristic symptoms of yersiniosis such as haemorrhages in the mouth and intestine. Nevertheless, it was only recently that several studies started to shed light on the specific pathogenicity mechanisms of the enteric redmouth bacterium. For example, it has been demonstrated that the protease Yrp1, produced by the so-called Azo+ strains, as well as ruckerbactin, a catecholate siderophore, are involved in virulence (Fernández et al., 2002, 2003, 2004).
With the aim of achieving a better understanding of the precise virulence factors possessed by this micro-organism, Fernández et al. (2004) used an in vivo expression technology (IVET) system to identify genes induced during the infection of fish by Y. ruckeri, which would very likely be related to virulence. This technique allowed the identification of 14 different in vivo-induced (ivi) genetic loci. One of these ivi genes encodes a protein putatively involved in the secretion and activation of a Serratia-type haemolysin (Fernández et al., 2004). It is well known that haemolysins participate in the pathogenicity of Gram-positive and Gram-negative bacteria and that they sometimes also show a cytolytic activity against different types of nucleated cells. For this reason, it seemed interesting to carry out further analyses on this in vivo-induced haemolysin.
This paper reports the study of two genes required for the production of an in vivo-induced extracellular cytolysin named YhlA, concerning its regulation by temperature and iron, transcriptional analysis, implication in virulence and cytotoxicity, and presence in Y. ruckeri strains of different origins.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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. Escherichia coli strains were routinely grown in 2x TY (tryptone/yeast extract) broth and agar, and Y. ruckeri strains in nutrient broth (NB) and agar (NA) (Pronadisa). When required, the following compounds were added to the media: 100 µg ampicillin ml–1, 50 µg rifampicin ml–1, 100 µg 2,2'-dipyridyl ml–1 or 100 µg FeCl3 ml–1, all of them from Sigma Aldrich. Liquid cultures were incubated at 37 °C for E. coli and 18 °C or 28 °C for Y. ruckeri in orbital shakers at 250 r.p.m. Growth was monitored by determining the OD600 at different times during incubation and bacterial concentrations were determined by serial dilutions and plate counts. Experiments on regulation of gene expression were performed using minimal medium M9, prepared as described by Romalde et al. (1991). All the glassware needed for the preparation of this medium was previously treated with 1 M HCl to remove iron traces and rinsed with double-distilled water.
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. Phage T4 DNA ligase and calf intestinal alkaline phosphatase were purchased from Roche, restriction enzymes from Amersham Pharmacia Biotech, DNA polymerase from Biotools, and oligonucleotides from Sigma Aldrich. Nucleic acid sequencing was performed by the dideoxy chain-termination method with the DR Terminator kit (Applied Biosystems) in an ABI Prism 310A automated DNA sequencer from Perkin-Elmer at the Universidad de Oviedo facilities. The sequences obtained were analysed with the computer program BLASTX.
The sequence adjacent to the partial fragment of yhlB present in clone iviV was obtained by inverse PCR. Briefly, genomic DNA from Y. ruckeri 150 was digested with ClaI or SalI, and the generated fragments were religated. The ligation mixture was then used as template DNA for a PCR, using the Long Amplification kit (Biotools) and oligonucleotides corresponding to the known DNA sequence. The reaction was performed in a Perkin-Elmer 9700 GeneAmp thermocycler.
RT-PCR.
Total RNA was obtained from 3 ml late-exponential-phase cultures of parental strain 150R and mutant 150RyhlB grown in M9 supplemented with 2,2'-dipyridyl. RNA was isolated using an RNeasy mini kit (Qiagen) and was treated with RNase-free DNase (Promega) to eliminate traces of DNA. Reverse transcription (RT)-PCRs were performed using Superscript One-Step with Platinum Taq (Invitrogen Life Technologies); 20 ng RNA was used in each reaction. Control PCRs using DNA polymerase (Biotools) were performed to determine whether RNA was free of contaminant DNA. The primers used were: A1, 5'-ATATCCGGGCCGAAGGC-3' (nt 911 to 927 of yhlA) and A2, 5'-ATTGTCGATCAATAAGC-3' (nt 1848 to 1832 of yhlA), for yhlA (938 bp); B1, 5'-ATAACCGGTGGAGATCA-3' (nt 472 to 488 of yhlB) and B2, 5'-CAGGTTATGAGTGCGGT-3' (nt 1206 to 1190 of yhlB), for yhlB (735 bp); BA1, 5'-GCAGAATACTTTATCGC-3' (nt 1430 to 1447 of yhlB) and BA2, 5'-GCTGAAGGTGTCACAAT-3' (nt 185 to 169 of yhlA), for a region overlapping yhlB and yhlA (510 bp); and O1, 5'-ACAGGCAAATTATGGAC-3' (nt 8 to 24 of orf1) and O2, 5'-TTATCAACTGGGGTTCA-3' (nt 298 to 282 of orf1), for the ORF located downstream of yhlA (293 bp).
In vitro regulation analysis.
For promoter expression studies, Y. ruckeri 150RiviV was grown in M9 supplemented with either FeCl3 or 2,2'-dipyridyl. To determine the influence of temperature, cells were incubated at 18 °C or 28 °C. Samples from stationary-phase cultures, under these conditions, were collected and their 
-galactosidase activity measured as described by Miller (1972). The results were then submitted to an analysis of variance test and P values <0.05 were considered significant.
Construction of insertion mutants.
Internal fragments of the predicted ORFs of yhlB (735 bp) and yhlA (938 bp) were amplified by PCR with the following primers: forward primers yhlB1 and yhlA1 (5'-GCAGAGATCTATAACCGGTGGAGATCA-3', nt 472 to 488 in bold type, and 5'-GTCAAGATCTATATCCGGGCCGAAGGC-3', nt 911 to 927 in bold type), respectively, and reverse primers yhlB2 and yhlA2 (5'-CTGTAGATCTCAGGTTATGAGTGCGGT-3', nt 1206 to 1190 in bold type, and 5'-GACCAGATCTATTGTCGATCAATAAGC-3', nt 1848 to 1832 in bold type), respectively. All primers contained a BglII site (in italics) and four additional bases at their 5' end. The generated amplicons were digested with BglII and ligated into pIVET8, previously digested with the same enzyme and dephosphorylated. The ligation mixture was utilized to transform by electroporation competent cells of E. coli SM10
pir.
Clones containing the vector with each insert were used to transfer the recombinant plasmids to Y. ruckeri 150R by filter mating, as described by Fernández et al. (2002). The mutation of the target gene in the transconjugants was verified by Southern blot analysis. Probe labelling, hybridization and developing were performed with the DIG DNA labelling and detection kit from Roche, following the manufacturer's instructions. To check that gene interruption had taken place as expected, DNA from the mutant and parental strains was prepared and digested with BamHI or ClaI for yhlB and yhlA, respectively. After separation of the restriction fragments in an agarose gel, these were transferred to a nylon membrane and hybridized with probes corresponding to each gene. This digestion produced bands of approximately 10 and 4 kb in the yhlA mutant strain and 14 and 4.5 kb in the yhlB mutant when hybridizing with the respective digoxigenin-labelled probes, instead of the 4 and 16 kb single fragments that appear, respectively, in the parental strain. Since pIVET8 contains internal BamHI and ClaI sites (Fig. 1), these patterns of hybridization demonstrated that plasmid pLPY5 or pLPY6 (Table 1
) harbouring internal fragments of yhlA or yhlB, respectively, was integrated in the chromosome by a single crossover event and, therefore, that the desired mutations had occurred.
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), as well as for their ability to grow in M9 supplemented with either FeCl3 or 2,2'-dipyridyl. To determine the involvement of haemolysin production in virulence, 50 % lethal dose (LD50) experiments with the parental and the mutant strains were carried out as described by Fernández et al. (2002). The doses injected ranged from 10 to 107 c.f.u. per fish and mortalities were followed up for 7 days. The LD50 values were calculated by the method of Reed & Muench (1938). Additionally, the cytotoxicity of the mutant and parental strains was studied using cultures of the BF-2 (blue gill fry) fish cell line. Cell cultures were grown in 24-well plates at 16 °C in Tagle medium (Sigma) supplemented with 1 % (w/v) NaHCO3 and 10 % (v/v) bovine fetal serum. Bacterial cultures were grown in M9 medium with 2,2'-dipyridyl. Before inoculation, bacterial cells were washed three times with PBS. The inoculation was carried out in a 1 : 1 proportion (5x105 bacterial cells to each well containing 5x105 eukaryotic cells, approximately, as determined with a haemocytometer). Control wells were inoculated with PBS. After 48 h incubation at 18 °C, the cultures were observed with a Zeiss Axiovert 200M inverted microscope.
PCR detection of yhlB and yhlA in different Y. ruckeri strains.
The production of haemolytic activity in cultures of 12 Y. ruckeri strains was examined following the method described above (Brillard et al., 2002). The primers used for the detection of yhlB and yhlA genes were as above.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). The analysis of one of these clones, Y. ruckeri 150RiviV, revealed the presence of the initial 292 nt of an ORF, encoding a protein putatively involved in the activation and secretion of a Serratia-type haemolysin (Fig. 1). In order to examine more closely the function of this toxin throughout the infectious process by Y. ruckeri, the adjacent region of this genetic locus was amplified by means of inverse PCR. This technique, and subsequent DNA sequencing, allowed the identification of two genes thought necessary for the production of the haemolysin (Fig. 1).
The upstream gene, named yhlB, consists of 1686 bp and encodes a protein of 561 amino acids which shares a high identity with ShlB from Serratia marcescens (65 %) (Poole et al., 1988), PhlB from P. luminescens (59 %) (Brillard et al., 2002) and a putative haemolysin activation protein from Yersinia pestis CO92 (51 %) (CAC93188). According to the SignalP program, the protein YhlB carries a signal peptide of 18 amino acids. In addition, the analysis of its amino acid sequence with the program PSLPred, which predicts the subcellular location of proteins, suggested that YhlB would be probably located in the outer membrane. This is in agreement with the secretory function of these proteins, being necessary for the transport of the respective toxins through the outer membrane as well as for their activation during this process (Schönherr et al., 1993).
Another ORF, of 4893 bp, designated yhlA, was found 69 bp downstream of yhlB. The product of this gene has high sequence identity with haemolysins of the Serratia-type pore-forming toxins. The identity was 52, 48 and 45 % with the haemolysins ShlA from S. marcescens (Poole et al., 1988) and PhlA from P. luminescens (Brillard et al., 2002), and a hypothetical protein from Y. pestis CO92 (NP_407172), respectively. These toxins typically have a large molecular size and are secreted to the extracellular milieu by means of a type V or two-partner secretion system (TPSS). The deduced amino acid sequence has a putative signal peptide of 30 amino acid residues and contains the conserved motifs which are characteristic of this kind of protein: QLAG (92 to 95), ILNEV (111 to 115), NPNG (140 to 143), CGFIN (149 to 153), LWGNP (159 to 164), WGGIGG (553 to 558) and LQGT (1259 to 1262) (Hirono et al., 1997). According to Schönherr et al. (1993), the most relevant motif is NPNG, which seems to be involved in the secretion of the haemolysin as well as in the exertion of the haemolytic activity. These toxins bind to the membrane of erythrocytes and other cell types and are able to create pores. In addition, they have been related to invasive properties (Hertle et al., 1999). Interestingly, these haemolysins have also been found in genomic analyses of other Yersinia species, though their function is still to be determined (Deng et al., 2002; Chain et al., 2004). Therefore, as far as we know, this is the first study on the role of this group of toxins in the genus Yersinia.
Transcriptional analysis
RT-PCRs were carried out to confirm the prediction made by sequence analysis that genes yhlB and yhlA form an operon. The results obtained with this analysis are represented in Fig. 2 and show that a region overlapping the two genes can be amplified when using RNA from the parental strain and, therefore, that yhlB and yhlA are co-transcribed (Fig. 2b). On the other hand, the fact that no mRNA corresponding to yhlA or to the overlapping region is present in the mutant Y. ruckeri 150RyhlB reveals that this mutation has a polar effect (Fig. 2c). However, the gene located downstream of yhlA, orf1, is expressed in both the parental and the mutant strains, which demonstrates that it is not affected by the interruption of yhlB (Fig. 2b, c). This operon structure also occurs for genes encoding other Serratia-type haemolysins such as PhlA from P. luminescens (Brillard et al., 2002), although it is not a general rule. For example, the genes responsible for the production of the EthA haemolysin from Edwardsiella tarda are transcribed independently (Hirono et al., 1997).
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), which was used to study the regulation of the expression of this gene in response to two important environmental signals, namely iron and temperature. These factors are well studied, given the significance they have for the pathogen to sense the transition into the host, which generally involves a change in temperature and a limitation in the amount of accessible iron (Mekalanos, 1992). Studies on the regulation of Serratia-type haemolysins have demonstrated the role of iron availability in regulation of expression (Hirono et al., 1997; Brillard et al., 2002). The influence of temperature, however, is still to be determined.
The results obtained by the determination of -galactosidase activity in cultures grown in different conditions showed that both iron and temperature exert an important influence on the transcription levels of yhlB, which is repressed by iron and induced by temperature downshift. Thus, the levels of -galactosidase activity (expressed as A420 ml–1 min–1 per OD600 unit) were 0.47±0.03, 2.83±0.21 and 4.9±0.20 when the cells were incubated in M9 supplemented with FeCl3, M9 and M9 supplemented with 2,2'-dipyridyl, respectively (P=0.002). This induction under iron-restricted conditions also occurred with the haemolysins produced by S. marcescens (Schiebel et al., 1989), P. luminescens (Brillard et al., 2002) and Ed. tarda (Hirono et al., 1997). These results agree with the hypothesis that haemolysin production might be a way of releasing iron from erythrocytes so that it can be used by the bacterial cell (Litwin & Calderwood, 1993). In addition, the expression from the yhlB promoter was approximately sevenfold higher at 18 °C (2.83±0.21 A420 ml–1 min–1 per OD600 unit), the infection temperature, than at 28 °C (0.42±0.11 A420 ml–1 min–1 per OD600 unit), the optimal growth temperature of this micro-organism (P=0.003). This matches the results obtained for gene regulation of the protease Yrp1 and ruckerbactin production (Fernández et al., 2003, 2004) and reinforces the importance of temperature as an environmental signal regulating the virulence of Y. ruckeri. The temperature-dependent modulation of virulence genes tends to trigger the expression of these in conditions mimicking those encountered in the host. This is not, however, a general rule and each case must be analysed independently. In the case of the genus Yersinia, the regulation of virulence genes by temperature is a well-characterized phenomenon (Straley & Perry, 1995; Konkel & Tilly, 2000).
Phenotypic characterization and virulence determination of strains with mutations in yhlB and yhlA
Independent mutations in yhlB and yhlA were generated (as described in Methods) to allow studies on the function and importance of the haemolysin, YhlA, in Y. ruckeri.
The haemolytic activity of YhlA was barely detectable in blood agar plates, as with other haemolysins of this type (Braun et al., 1985; Brillard et al., 2002). This phenomenon could be a consequence of the low diffusion of these proteins into the culture medium due to their high molecular mass and/or to the adherence of the protein to the cell surface. For this reason, haemolytic activity was measured using liquid cultures corresponding to the parental (150R) and the mutant strains (150RyhlA and 150RyhlB) (Table 1). Two independent experiments showed that, under the assayed conditions, the percentage of lysed erythrocytes was 41.5±6 %, 21.7±4 % and 6.6±1 % for Y. ruckeri 150R, Y. ruckeri 150RyhlA and Y. ruckeri 150RyhlB supernatants, respectively.
The growth of the yhlA and yhlB mutant strains was not retarded relative to that of the parental strain, in either iron-rich or iron-depleted conditions (data not shown). This indicates that the mutations do not cause any defect in the growth ability and, therefore, that the differences in virulence could be due to the specific involvement of these proteins in the in vivo conditions. In some cases, the production of haemolysins has been related to the metabolism of iron in the cell, which implies that their main function would be the release of the iron bound to the haem group of erythrocytes (Poole et al., 1988).
Once the main characteristics of the mutant strains in vitro had been analysed, their behaviour in a fish infection model and in cell cultures was studied. LD50 experiments indicated that the mutant strains are attenuated in their virulence, because the values obtained with Y. ruckeri 150RyhlA and Y. ruckeri 150RyhlB were approximately 10- and 100-fold higher, respectively, than those of the parental strain. Thus, the means of the LD50 values obtained for the parental strain, mutant yhlA and mutant yhlB were 2.7x104, 3.9x105 and 3x106 c.f.u. per fish, respectively. This relationship between haemolytic activity and virulence was not found in other fish pathogens such as Edwardsiella ictaluri (Williams & Lawrence, 2005). Cytotoxicity assays demonstrated that the deficient production of the haemolysin YhlA, caused by the mutations, led to a significant reduction of the cytopathic effects produced by this Y. ruckeri. Thus, microscopic analysis of BF-2 cell cultures infected with the mutant and parental strains revealed that only the latter was able to lyse the eukaryotic cells after 2 days incubation, whereas the tissue organization remained unaltered not only in the control wells inoculated with PBS, but also in the wells infected with the mutant strains (data not shown). This cytotoxic effect has already been demonstrated for other haemolysins of this type produced by micro-organisms such as Haemophilus ducreyi (Palmer et al., 1996), Ed. tarda (Strauss et al., 1997) and S. marcescens (Hertle et al., 1999).
Presence of yhlB and yhlA in Y. ruckeri strains of different origins
By using the liquid culture assay, previously described for the analysis of mutant strains, the supernatants of 12 Y. ruckeri strains from different sources were tested for the production of haemolytic activity. All of them showed the ability to lyse erythrocytes, with percentages of lysis ranging from 40 to 75 %. Likewise, all the strains gave a positive result in the PCR detection analysis of genes yhlB and yhlA (Fig. 3), in which the amplification of two bands of 735 and 938 bp, respectively, indicated the presence of these two genes. This result confirms that Y. ruckeri is a highly homogeneous species at the genetic level, as several authors have pointed out before on the basis of fingerprinting (Romalde et al., 1993) and multilocus sequence typing (Kotetishvili et al., 2005) analyses. The results obtained by Fernández et al. (2003, 2004) also showed that all the strains tested harbour the genes necessary for the production of the protease Yrp1 and the siderophore ruckerbactin.
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ACKNOWLEDGEMENTS |
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We thank J. Méndez for providing the blood for the haemolysin assays, and P. Solano for contributing to the experiments on cytotoxicity. We thank A. F. Braña and T. Fabek for critical reading of the manuscript.
Edited by: S. C. Andrews
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Received 6 July 2006;
revised 27 September 2006;
accepted 25 October 2006.
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