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
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ABSTRACT |
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INTRODUCTION |
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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.
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METHODS |
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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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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.
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RESULTS AND DISCUSSION |
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Sequencing and genetic organization of the katBankB operon in Ed. tarda
The genetic organization of the katBankB 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 katBankB 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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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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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 10 May 2003;
revised 16 June 2003;
accepted 16 June 2003.
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