A major catalase (KatB) that is required for resistance to H2O2 and phagocyte-mediated killing in Edwardsiella tarda

P. S. Srinivasa Rao1, Yoshiyuki Yamada1 and Ka Yin Leung1,2

1 Department of Biological Sciences, Faculty of Science, The National University of Singapore, Science Drive 4, Singapore 117543
2 Tropical Marine Science Institute, The National University of Singapore, Science Drive 4, Singapore 117543

Correspondence
Ka Yin Leung
dbslky{at}nus.edu.sg


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Edwardsiella tarda causes haemorrhagic septicaemia in fish and gastro- and extra-intestinal infections in animals including humans. Resistance to phagocyte-mediated killing is one of the virulence factors of Ed. tarda. The authors' previous studies using TnphoA transposon mutagenesis indicated that katB mutants derived from the strain PPD130/91 are at least 1·6 log higher in LD50 values than the wild-type strain. These findings suggest the involvement of catalase (KatB) in Ed. tarda pathogenesis. In this study, experiments were conducted to characterize the contribution of KatB to Ed. tarda infection. Zymographic analyses indicated that the 22 Ed. tarda strains examined expressed three different types of catalase-peroxidases (Kat1–3) based on their mobility in non-denaturing polyacrylamide gels. KatB (Kat1), the major catalase enzyme, was expressed in eight out of 22 Ed. tarda strains, and was commonly found in virulent strains except AL9379. AL9379 has a mutated katB, which has a base substitution and a deletion that translate into stop codons in the catalase gene. KatB produced by PPD130/91 was located in both periplasmic and cytoplasmic fractions and was constitutively expressed in various growth phases. Kinetics studies indicated that the catalase provided resistance to H2O2- and phagocyte-mediated killing. Infection kinetics studies of katB mutant 34 in gourami fish demonstrated its inability to survive and replicate in phagocyte-rich organs and this prevented the dissemination of infections when compared to the wild-type. Complementation of catalase mutants restored the production of catalase, and led to an increase in the resistance to H2O2- and phagocyte-mediated killing, and a decrease in LD50 values. This study has identified and characterized a major catalase gene (katB) that is required for resistance to H2O2- and phagocyte-mediated killing in Ed. tarda. The results also suggest that catalase may play a role as a virulence factor in Ed. tarda pathogenesis.


The GenBank accession numbers for the sequences determined in this work are AY178619 and AY078506.


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Edwardsiella tarda is a causative agent of edwardsiellosis in freshwater and marine fish (Thune et al., 1993). This organism is widely distributed in aquatic environments and has a wide host range, causing diseases in a variety of vertebrates including humans (Plumb, 1993). Many potential virulence factors in Ed. tarda have been reported, which include the ability to invade epithelial cells (Janda et al., 1991; Ling et al., 2000), resist serum and phagocyte-mediated killing (Ainsworth & Chen, 1990; Srinivasa Rao et al., 2001), and produce toxins such as haemolysins and dermatotoxins for disseminating infection (Ullah & Arai, 1983; Hirono et al., 1997). However, very little is known about the roles of these factors in disease occurrence.

The ‘respiratory burst’ of stimulated phagocytes is one of the major defence systems in response to bacterial infection. The bacterial killing is initiated by NADPH oxidase, which catalyses the reduction of molecular oxygen () and then superoxide dismutase converts to hydrogen peroxide (H2O2), which permeates freely through biological membranes. In the bacterial cytoplasm, H2O2 reacts with reduced iron or copper ions to generate hydroxyl radical (OH-), which causes cellular damage such as single-strand nicks in DNA leading to mutations, and oxidation of biological membranes and proteins (Storz et al., 1990). Pathogens such as Mycobacterium tuberculosis (Manca et al., 1999), Legionella pneumophila (Bandyopadhyay & Steinman, 1998) and Campylobacter jejuni (Day et al., 2000) detoxify reactive oxygen intermediates by producing enzymes such as catalase (Kat), superoxide dismutase (SOD) and alkyl hydroperoxide reductase. Catalase can specifically cleave H2O2 into water and oxygen, thereby neutralizing the bactericidal action. Once the bacteria overcome the harmful effects of the reactive oxygen species produced by phagocytes, the organism can cause systemic infection in the host. Ed. tarda is known to produce iron co-factored SOD and catalase (Yamada & Wakabayashi, 1998, 1999; Mathew et al., 2001).

Several attenuated mutants of Ed. tarda PPD130/91 have been identified by TnphoA transposon mutagenesis (Mathew et al., 2001; Srinivasa Rao et al., 2003). Two of them had insertions in the katB gene, and this indicated the possibility of direct involvement of catalase in pathogenesis. In this study, we describe the identification and characterization of an Ed. tarda catalase (KatB), and examine its role in resisting H2O2- and phagocyte-mediated killing and in disseminating infection.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Bacterial strains and media.
The bacteria and plasmids used in this study are listed in Table 1. Ed. tarda strains were routinely cultured at 25 °C on tryptic soy agar (TSA, Difco) or in tryptic soy broth (TSB, Difco) without shaking. Escherichia coli strains were maintained in Luria broth (LB, Difco) or on LB agar at 37 °C. When required, media were supplemented with antibiotics such as ampicillin and neomycin at 100 and 50 µg ml-1, respectively.


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Table 1. Bacteria and vectors used in this study

 
Bacterial lysate preparation and catalase-peroxidase activity analyses.
Forty-eight-hour cultures of Ed. tarda cells were washed, resuspended in 50 mM phosphate buffer (pH 7·4), and sonicated at 4 °C for 2 min (Misonix). The lysate was then centrifuged at 20 000 g for 10 min at 4 °C and the supernatant was collected as the whole-cell lysate. Periplasmic proteins were prepared by a combined lysozyme/osmotic shock treatment as described by French et al. (1996). After the periplasmic protein extraction, the residual cell pellets were sonicated to obtain cytoplasmic proteins. Protein concentrations in various fractions were determined by the DC protein kit (Bio-Rad) with bovine serum albumin as the standard. Fifty micrograms of each lysate or protein fraction was electrophoresed through an 8 % non-denaturing polyacrylamide gel and stained for catalase or peroxidase activity as described by Clare et al. (1984) and Heym & Cole (1992), respectively.

Catalase assay was performed as described by Beers & Sizer (1952). Briefly, each dilution of bacterial cell lysate was added to 3 ml 10 mM H2O2 in 50 mM phosphate buffer (pH 7·0). The decrease in absorbance at 240 nm was monitored for 2 min, and the linear part of the curve was used to quantitate the rate of decrease by using an absorption coefficient of H2O2 at 240 nm of 0·0435 mM-1 cm-1. One unit (U) is defined as the amount of catalase that degrades 1 µmol H2O2 min-1 at 25 °C. Results are expressed as the means ± SEM of experiments carried out in triplicate.

DNA manipulation, PCR and Southern hybridization.
Bacterial genomic DNA and plasmid DNA were extracted using the Genome DNA kit (Bio 101) and the QIAprep miniprep kit (Qiagen), respectively. All the primers used in the present study are listed in Table 2. Primers specific for katB (catB and catC) and ankB (catE and catF) were designed from the known DNA sequences of PPD130/91. PCR was performed by using Advantage 2 polymerase mix (Clontech) using the following protocol: 94 °C for 25 s; 30 cycles of 15 s at 94 °C, 30 s at 62 °C, 3 min at 72 °C; and a final extension for 3 min at 72 °C. Southern blotting was performed using the BluGene Non-Radioactive Nucleic Acid Detection System (Gibco-BRL). Transfers of the DNA to nylon membranes (GeneScreen, NEM Research Products), hybridization conditions, and visualization with streptavidin-alkaline phosphatase conjugates were carried out as recommended by the manufacturer's protocol.


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Table 2. Primers used in this study

 
Construction of complement mutants.
Catalase (katB) was PCR amplified using catA and catD primers, and katB–ankB operon was amplified using catA and catG primers (Table 2, Fig. 2). The PCR-amplified products were later cloned into pGEM-T Easy vector (Promega), and then transformed into E. coli Top 10 F' cells. Plasmids having katB and katB–ankB were transformed into mutants 34 and 309 for complementation. These mutants with their respective plasmids were used for carrying out the various experiments described below.



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Fig. 2. Comparison of the genetic organization of katB–ankB operon in P. aeruginosa and the wild-type strains of Ed. tarda. ORFs within the katB–ankB operon, with the direction of transcription, are indicated by solid arrows. The small arrows above the PPD130/91 diagram indicate the forward and reverse primers for the PCR analyses.

 
Sequencing of the katB–ankB operon in Ed. tarda PPD130/91 and AL9379.
DNA sequencing was carried out on a PRISM 377 automated DNA sequencer by the dye terminator method (Applied Biosystems). Sequence assembly and further editing were carried out with DNASIS DNA analysis software (Hitachi Software). BLASTN, BLASTX, BLASTP sequence homologies and protein conserved domain analyses (cd-search) were performed by using the national centre for biotechnology information BLAST network service. The katB–ankB sequences of AL9379 and PPD130/91 have been deposited in GenBank under accession numbers AY178619 and AY078506, respectively.

Determination of H2O2 sensitivity.
Strains of Ed. tarda examined for H2O2 sensitivity were cultured in TSB or TSA for 48 h. Cells were harvested and diluted to OD540 1·0. An equal volume of H2O2 in phosphate-buffered saline (PBS; 137 mM NaCl, 2·7 mM KCl, 1·5 mM KH2PO4 and 8 mM Na2HPO4, pH 7·4) was added to the bacteria to a final concentration of 60 mM, and incubated for 1 h at 25 °C. The numbers of viable bacteria in the cell preparation before and after the treatment were determined via dilution plating on TSA. The data were obtained from three independent experiments.

Intracellular replication in fish phagocytes and LD50 determinations.
Healthy blue gourami (Trichogaster trichopterus Pallas) were obtained from a commercial fish farm, maintained in well-aerated de-chlorinated water at 25±2 °C, and acclimatized to the laboratory conditions for at least 15 days. Phagocytes were isolated from the head kidney of naive gourami and purified following the procedure of Secombs (1990). Purified phagocytic cells (1x106 to 2x106 cells per well) were allowed to adhere to 48-well tissue culture plates (Falcon) in fetal calf serum-supplemented L-15 medium (Sigma). After 2 h incubation at 25 °C in a 5 % (v/v) CO2 atmosphere, the cells were washed twice using Hanks' balanced salt solution (HBSS) (Sigma) to remove unattached cells. Intracellular replication inside the phagocytes was assayed by the method of Leung & Finlay (1991) with the following modifications. Thirty minutes after infection, phagocytes were washed once with HBSS and then incubated for 1·5 h in 5 % fetal calf serum-supplemented fresh L-15 medium with 100 µg gentamicin ml-1. Gentamicin-treated phagocytes were then washed three times in HBSS and incubated with antibiotic-free L-15 medium. After 5 h incubation, phagocytes were lysed by treatment with 1 % (v/v) Triton X-100 solution, and the viable bacterial cells were counted by plating on TSA with appropriate antibiotics.

LD50 determination was conducted to assess the virulence of the Ed. tarda strains. Three groups of 10 gourami fish were injected intramuscularly with 0·1 ml PBS-washed bacterial cells adjusted to the required concentrations. The fish were monitored for mortalities for 7 days and LD50 values were calculated by the method of Reed & Muench (1938).

In vivo characterization of attenuated mutants.
An intramuscular route of administration was used to study the infection kinetics of Ed. tarda in vivo. Briefly, the fish were injected with 1·0x105 c.f.u. of Ed. tarda PPD130/91 (Ling et al., 2001) or mutant 34. A control group of fish was injected with 0·1 ml PBS. Four fish from each group were sampled on days 1, 3, 5 and 7 post-infection. The gall bladder, spleen, kidney, intestine, liver and heart were aseptically removed. Blood was aseptically collected from the caudal vein. A piece of body muscle from the site of injection, measuring approximately 1x1 cm, was also taken. Samples from each treatment were pooled based on organ types and put into sterile sample bags (Whirl-Pak). One millilitre of PBS was added to all the sample bags and the samples were homogenized with a Stomacher Lab-Blender, model 80 (Seward Medical). The homogenized samples were serially diluted and plated in triplicate onto either TSA with ampicillin or TSA with neomycin, and incubated at 25 °C for 48 h.

Statistical analysis.
All data were expressed as means±SEM. The data were analysed using one-way analysis of variance (ANOVA) and a Duncan multiple range test (SAS software). Values of P<0·05 were considered significant.


   RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Zymographic analyses of Ed. tarda catalase activity
The zymograms for catalase detected three types of catalase activity in the whole-cell lysates of Ed. tarda (Fig. 1a, Table 3). These enzymes were named Kat1, Kat2 and Kat3 based on their migration rates in the non-denaturing polyacrylamide gels (Fig. 1a). Kat1 showed the highest catalase activity but with the slowest mobility. Kat2 and Kat3 had lower catalase activities and Kat3 demonstrated the fastest mobility. According to the distribution of these catalase enzymes, 22 strains of Ed. tarda were divided into three groups: group 1 expressed both Kat1 and Kat2, as represented by PPD130/91 (eight strains); group 2 expressed only Kat2 (one strain: AL9379); group 3 expressed only Kat3, as represented by PPD125/87 (13 strains) (Table 3). Similar to Ed. tarda, other pathogenic bacteria such as E. coli, Legionella pneumophila and Pseudomonas aeruginosa also have multiple catalase enzymes (Loewen et al., 1985; Bandyopadhyay & Steinman, 1998, 2000; Howell et al., 2000). Some of these enzymes are bifunctional catalase peroxidases, such as KatG and KatE of E. coli (Loewen et al., 1985), KatG of Mycobacterium tuberculosis (Manca et al., 1999), and KatA and KatB of L. pneumophila (Bandyopadhyay & Steinman, 1998, 2000). We therefore examined whether these Ed. tarda catalases also had peroxidase activity. A zymographic analysis showed that all the three catalases (Kat1, 2 and 3) were bifunctional catalase-peroxidases (data not shown).



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Fig. 1. (a) Zymographic analyses of Ed. tarda catalase activity in wild-type strains. Lanes 1 to 10 are PPD130/91, NE8003, NuF251, E381, AL9379, ATCC 15947T, PPD125/87, AL92448, Su100 and NuF194, respectively. (b) Catalase activity in mutants and complemented mutants. Lanes 1 to 7 are PPD130/91, 34, 34C1, 34C2, 309, 309C1 and 309C2, respectively.

 

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Table 3. Distribution of catalase enzymes and katB and ankB genes in various strains of Ed. tarda

 
KatB-deficient mutants 34 and 309 were previously obtained by TnphoA transposon mutagenesis from strain PPD130/91 (Mathew et al., 2001; Srinivasa Rao et al., 2003). The zymographic analyses of catalase activities revealed that Kat1 was missing in these KatB-deficient mutants (Fig. 1b). Kat1 was therefore renamed KatB (Fig. 1, Table 3). All the virulent strains except AL9379 produced KatB and it was not found in any avirulent strains. Since KatB is the major catalase enzyme produced by our pathogenic Ed. tarda isolates, we focus on this protein in the present study.

Sequencing and genetic organization of the katB–ankB operon in Ed. tarda
The genetic organization of the katB–ankB operon of Ed. tarda is shown in Fig. 2. The katB operon of Ed. tarda PPD130/91 is composed of two ORFs, catalase precursor (katB) and ankyrin B (ankB). KatB and AnkB comprise 552 and 183 aa, with a predicted molecular mass of 61·7 kDa and 19·6 kDa, respectively (accession no. AY078506). The katB and ankB of Ed. tarda PPD130/91 show the highest similarities with the corresponding genes in P. aeruginosa (accession no. Q59635), with amino acid identities of 77 % and 59 %, respectively. In P. aeruginosa, KatB expression is one of the oxidative stress responses and is regulated by the transcriptional activator OxyR (Howell et al., 2000; Ochsner et al., 2000). The OxyR-binding site is a motif consisting of two ATAG and two CTAT elements spaced at 10 bp intervals and is located upstream of katB. Optimal H2O2 detoxification was facilitated by AnkB, which formed an antioxidant scaffolding with KatB in the periplasm at the cytoplasmic membrane. In the case of Ed. tarda PPD130/91, ankB was also found downstream of katB; however no OxyR-binding site was found in the upstream sequence.

Distribution of katB and ankB in various strains of Ed. tarda
PCR and Southern analyses using specific primers and probes for Ed. tarda katB and ankB were performed to investigate the distribution of the katB–ankB operon among the 22 strains (Table 3). katB and ankB were found in all the virulent strains, but not in avirulent strains. These results agree with the detection of KatB in a zymographic analysis, except for AL9379, which has katB and ankB genes but does not produce KatB protein. The distribution of the katB gene and the KatB protein presented here indicates that KatB is a specific enzyme for virulent strains. KatB shows the highest catalase activity among the three catalases (Fig. 1a) and, hence, the catalase activity of the pathogenic Ed. tarda group is very high compared to the non-pathogenic Ed. tarda group, except for AL9379 (Table 3). This strongly suggests that KatB may be an important virulent factor in Ed. tarda pathogenesis.

Further sequence analysis revealed that the katB of AL9379 has a nucleotide substitution from C to T (position 64 of PPD130/91) and a base deletion (C, position at 234), which may turn off the translation of KatB at the same positions (position 1 or 112) as that of KatB of PPD130/91 (Fig. 3). Biocomputational analysis using DNASIS software showed that the AL9379 sequence may have a short ORF starting from the 619th nucleotide position compared to PPD130/91 (Fig. 3). This can be translated into a truncated protein of 346 aa with a molecular mass of 39·3 kDa. The KatB of AL9379 was further compared with the catalase consensus sequence (pfam00199.5) and other catalase sequences (KatB of P. aeruginosa and Ed. tarda PPD130/91) by multiple sequence alignment analysis using DNASIS (Fig. 4). The results clearly indicate that the KatB of AL9379 lacks the first 136 aa when compared to pfam00199.5 and other catalases. KatB of P. aeruginosa and Ed. tarda PPD130/91 have PROSITE (PDOC00395) catalase signatures which include proximal active sites (PS00438) and proximal haem-ligands (PS00437) (Fig. 4). KatB of AL9379 lacks the first of these signatures, which is important for catalase activity. Welinger (1991, 1992) also reported that catalase and peroxidase activities are associated with amino-terminal domains. The loss of the amino-terminal domain (PS00438) would have made the KatB of AL9379 non-functional. This is supported by the lack of a catalase activity band for AL9379 in the zymographic analysis (Fig. 1a). The LD50 value of AL9379 was 105·9, which is higher than that of other virulent strains (Ling et al., 2000). A naturally occurring mutation on the katB of AL9379 may be related to the decrease of its pathogenicity.



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Fig. 3. Variation in the nucleotide sequences of the katB gene in Ed. tarda AL9379 and PPD130/91. Bold letters indicate nucleotide substitutions (S) and deletions (D) that affected the ORF in Ed. tarda AL9379. Letters in normal print indicate other nucleotide substitutions (S), deletions (D) and insertions (I). The numbers in parentheses indicate the relative nucleotide position based on the PPD130/91 katB start codon. The three start codons at positions 1, 112 and 619 indicate the possible start codons for PPD130/91 and AL9379. The stop codons at positions 64 and 349 may disrupt the translation of AL9379.

 


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Fig. 4. Multiple alignment of amino acid sequences of various Ed. tarda KatB (PPD130/91 and AL9379) with homologous catalase sequence from P. aeruginosa (PAKATB) and catalase consensus sequence (pfam00199.5) from the conserved domain database (CDD) using DNASIS. Biocomputational analysis showed the presence of two catalase signatures, shown in bold letters [between 108–124 aa (proximal active site, PS00438) and 395–403 aa (proximal haem-ligand, PS00437) of KatB of PPD130/91]. Amino acid residues involved in catalase activity are marked by inverted triangles and the ligand for haem binding is denoted by #.

 
Characteristics of KatB
A zymographic analysis using periplasmic and cytoplasmic fractions of Ed. tarda PPD130/91 revealed that KatB was located in both fractions (Fig. 5a). Mutant 34 had a single TnphoA insertion in katB of PPD130/91 and was alkaline phosphatase positive. This indicates that KatB of PPD130/91 may be secreted into the periplasmic space. Further experiments are required to confirm the presence of catalase in the periplasmic fraction. KatB of PPD130/91 was produced constitutively irrespective of the presence or absence of oxidative stress agents such as H2O2 and paraquat. A slight increase in catalase activity was observed when Ed. tarda PPD130/91 was grown with the addition of 1 mM H2O2 once every 20 min over a period of 2 h, while no significant variation in the activity was observed when the strain was grown with the addition of 120 µM paraquat (Fig. 5b). KatB of P. aeruginosa, which has a high sequence similarity to KatB of Ed. tarda, was induced by paraquat and H2O2, but not induced throughout the aerobic growth phases (Brown et al., 1995). KatB of Ed. tarda was constitutively expressed regardless of its growth phase (Fig. 5c). On the other hand, KatG of E. coli and KatB of L. pneumophila showed an increased catalase activity during exponential growth (Gonzalez-Flencha & Demple, 1997; Bandyopadhyay & Steinman, 1998).



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Fig. 5. Catalase activity of Ed. tarda PPD130/91. (a) Lane 1, periplasmic fraction; lane 2, cytoplasmic fraction. (b) Catalase activity of total cell extract. Lane 1, control; lane 2, six 1 mM H2O2 treatments for every 20 min for 2 h; lane 3, 120 µM paraquat. (c) Catalase activity of PPD130/91 grown in TSB over a period of 48 h. Lanes 1–4 are samples taken at 6 h, 12 h, 24 h and 48 h respectively.

 
Complementation of catalase mutants
Mutants 34 and 309 were complemented using plasmids carrying katB alone and katB–ankB. A zymographic analysis indicated that both mutants lacked KatB expression while the complemented mutants (34C1, 34C2, 309C1 and 309C2) regained KatB expression (Fig. 1b). Higher catalase activity was found in complemented mutants of 34 than of 309 (Table 4). All the complemented mutants produced higher catalase activities than the wild-type; this might be due to the high copy number of the pGEM-T easy vector. Mutations in the ankB gene of P. aeruginosa resulted in a fourfold reduction in KatB activity and rendered the mutant sensitive to H2O2 (Howell et al., 2000). We also found a slight increase in catalase activities in Ed. tarda mutants when they were complemented with katB–ankB compared to katB alone (Table 4). Zymographic analyses showed that the catalases of 34C1 and 309C1 had lower mobility in non-denaturing PAGE compared to the wild-type, whereas 34C2 and 309C2 showed similar mobility to that of the wild-type strain PPD130/91 (Fig. 1b). This observation suggests that AnkB may play a role in post-translational modifications or the assembly of the catalase protein. Further studies are required to validate the above hypothesis.


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Table 4. Intracellular replication in blue gourami phagocytes, LD50 values and catalase activity of Ed. tarda PPD130/91 and mutants

 
Catalase is essential for Ed. tarda to resist H2O2- and phagocyte-mediated killing
Catalase is an important enzyme required for overcoming the harmful effects of H2O2, one of the reactive oxygen intermediates produced by phagocytes. We therefore studied the effect of H2O2 and phagocyte-mediated killing in vitro on katB-deficient (34 and 309) and complemented (34C1, 34C2, 309C1 and 309C2) mutants. Sixty millimolar H2O2 (60 mM) caused significant killing of catalase mutants 34 and 309 within 1 h of incubation compared to the wild-type PPD130/91 (Fig. 6). The complemented mutants showed a complete restoration of survivability when incubated under similar conditions. The results clearly demonstrate the importance of KatB protein in overcoming H2O2-mediated killing. A resistance to H2O2-mediated killing by catalase has been demonstrated in other pathogens such as Campylobacter jejuni (Day et al., 2000) and M. tuberculosis (Manca et al., 1999).



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Fig. 6. Resistance to H2O2-mediated killing by Ed. tarda PPD130/91, katB-deficient and complemented mutants. Bacterial strains were incubated with phosphate buffer solution (white bars) and 60 mM H2O2 (shaded bars) for 1 h at 25 °C and viable bacteria enumerated. The data presented are means±SEM from three independent trials. The asterisks indicate statistically significant differences between the phosphate buffer and H2O2-treated strains.

 
Tests on the ability of the wild-type, KatB-deficient and complemented mutants to replicate within blue gourami phagocytes were also carried out (Table 4). The wild-type had a replication rate of 4·5±0·5 at 5 h post-incubation within phagocytes, while the mutants had a significantly lower replication rate, indicating their inability to survive and replicate efficiently in phagocytes. Complemented mutants regained some degree of ability to replicate inside phagocytes as indicated by the increase in the replication rate (Table 4). Mutant 309 had the lowest replication ability followed by mutant 34; when complemented, 34C2 had a higher replication rate than 309C2. Like KatB of Ed. tarda, KatG of M. tuberculosis and KatA of C. jejuni also contributed to intramacrophage survival (Manca et al., 1999; Day et al., 2000).

LD50 assays were carried out using the wild-type, mutants and complemented mutants (Table 4). Both the mutants had a significantly higher LD50 than the wild-type, with 309 being highly attenuated compared to 34. Complementation restored virulence to a greater extent for mutant 34 than for mutant 309, but no significant difference was noticed between 34C1 and 34C2, and 309C1 and 309C2.

The results obtained show that there is full recovery of resistance to H2O2-mediated killing when mutants are complemented. Since catalase is the enzyme which is directly involved in breaking down H2O2, it is reasonable that mutants that have single (34) and double (309) transposon insertion are susceptible to H2O2-mediated killing. On the other hand, only partial restoration was seen in LD50 values and intracellular replication assays in both mutants. This is to be expected, since processes like resistance to phagocyte-mediated killing and virulence in hosts are governed by many genes. Complementation of mutant 34 showed a higher restoration in both intracellular replication and LD50 assays compared to mutant 309. This may be due to the double transposon insertions, one in katB and the other in ssrB in the case of mutant 309 (Srinivasa Rao et al., 2003). SsrB is homologous to a secretory system regulatory gene that regulates the type III secretion system (TTSS), a contact dependent secretory system of Salmonella pathogenicity island 2. The TTSS is required for replication inside phagocytes and for systemic infection (Hensel, 2000). SsrB is also known to activate a global regulon of horizontally acquired genes (Worley et al., 2000). All this indicates that TTSS genes may be more important than KatB for intramacrophage survival and replication.

Role of catalase in Ed. tarda systemic infection
Infection kinetics experiments were carried out to investigate the ability of the mutants to proliferate and cause infection in vivo. An intramuscular infection model was used to simulate infection by physical injury under normal conditions. The wild-type and mutant 34 were injected separately into gourami fish and the infection kinetics was studied over a period of 7 days. The fish injected with PPD130/91 showed a high number of bacteria in all the different organs (Fig. 7a). By day 1, the wild-type PPD130/91 had survived, colonized and proliferated to reach very high numbers in all the organs sampled, thereby causing mortality of fish within 3 and 5 days. In the case of catalase-deficient mutant 34, the bacterial numbers were significantly lower than that of the wild-type in all the organs except the muscle (Fig. 7b). Mutant 34 survived and multiplied in the muscle and reached levels comparable to that of the wild-type, but still could not cause fish mortality. Fish injected with the wild-type and mutant 34 showed haemorrhages around the site of injection, which persisted and progressed in the case of the wild-type but decreased and healed in the fish injected with mutant 34. The significant reduction in bacterial numbers in all the organs except the muscle in fish injected with mutant 34 indicates that the bacteria might have been killed by the host-defence response. Organs such as the kidney, blood, heart and spleen are rich in phagocytes, which help in killing the bacteria. Phagocytes often use oxygen-dependent mechanisms to defend bacterial infection; O- and H2O2 are some of the weapons used by phagocytes to kill bacteria. Since mutant 34 is deficient in catalase production, phagocytes may bring the bacterial numbers to lower levels and abort the infections.



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Fig. 7. Infection kinetics of Ed. tarda strains in blue gourami. Fish were injected with 1·0x105 c.f.u. of (a) PPD130/91 and (b) mutant 34. Four fish each were sampled per datum point and the error bars represent the mean±SEM of Ed. tarda per sample in triplicates. For PPD130/91 sampling was done till day 5, when all the fish had died due to severe infection. Fish from other groups were sampled till day 7. The blood (—{bullet}—), body muscle (---{blacktriangleup}---), liver (---{blacktriangledown}---), kidney (...{blacklozenge}...), gall bladder (—{blacksquare}—), spleen (.—{diamondsuit}—.), heart (..—{bullet}—..) and intestine (--{blacksquare}--) were dissected and homogenized and the bacterial enumeration was done by plating on TSA, supplemented with appropriate antibiotics.

 
Conclusions
We believe that our studies are the first molecular genetic investigations of catalase-peroxidase in Ed. tarda. We have successfully cloned and sequenced the katB–ankB operon from two Ed. tarda strains. We have shown that KatB of Ed. tarda PPD130/91 is constitutive in production irrespective of the presence or absence of H2O2 or paraquat, and of growth phase. We have also demonstrated that the expression of KatB is required for optimal resistance to H2O2- and phagocyte-mediated killing. Mutations in katB can decrease the virulence of Ed. tarda, suggesting that catalase is an important virulence factor. Further studies on the identification and characterization of the kat2 and kat3 genes are in progress. Studies of these three catalases will help us in understanding their roles in Ed. tarda pathogenesis.


   ACKNOWLEDGEMENTS
 
The authors are grateful to The National University of Singapore for providing the research grant for this work. We are grateful to Dr John Grizzle (Auburn University, USA) Drs T. Ngiam and H. Loh (Agri-Food Veterinary Authority of Singapore) and T. Nakai (University of Hiroshima, Japan), who kindly provided the isolates of Ed. tarda.


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ABSTRACT
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Received 10 May 2003; revised 16 June 2003; accepted 16 June 2003.



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