Microbiology (2003), 149, 2635–2644
DOI 10.1099/mic.0.26478-0
A major catalase (KatB) that is required for resistance to H2O2 and phagocyte-mediated killing in Edwardsiella tarda P. S. Srinivasa Rao,1 Yoshiyuki Yamada1 and Ka Yin Leung1,2 Correspondence Ka Yin Leung
[email protected]
Received 10 May 2003 Revised
16 June 2003
Accepted 16 June 2003
Department of Biological Sciences, Faculty of Science1 and Tropical Marine Science Institute2, The National University of Singapore, Science Drive 4, Singapore 117543
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.
INTRODUCTION 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 phagocytemediated 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 GenBank accession numbers for the sequences determined in this work are AY178619 and AY078506.
0002-6478 G 2003 SGM
Printed in Great Britain
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 (O2) to superoxide (O{ 2 ) and then superoxide dismutase converts to hydrogen peroxide (H2O2), which permeates freely O{ 2 through biological membranes. In the bacterial cytoplasm, H2O2 reacts with reduced iron or copper ions to generate hydroxyl radical (OH2), 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 2635
P. S. S. Rao, Y. Yamada and K. Y. Leung
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 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 uC on tryptic soy agar (TSA, Difco) or in tryptic soy broth (TSB, Difco) without shaking. Escherichia coli strains were
Table 1. Bacteria and vectors used in this study Bacteria and plasmids Pathogenic Ed. tarda PPD130/91 AL9379 E22 E381 NE8003 NuF84 NuF251 Su35 Su226 Non-pathogenic Ed. tarda AL92448 ATCC15947T NuF194 PPD499/84 PPD453/86 PPD76/87 PPD125/87 PPD200/87 PPD129/91 Su20 Su100 Su119 Su138 Mutants 34 309 34C1 34C2 309C1 309C2
Source* and characteristics
Reference
Serpae tetra, AVA; Neos Colr Amps Channel catfish, AU Japanese eel, TU Tilapia, TU Japanese flounder, TU Japanese flounder, TU Japanese flounder, TU Japanese eel, TU Eel pond water, TU
Ling et al. Ling et al. Yamada & Yamada & Yamada & Yamada & Yamada & Yamada & Yamada &
(2000) (2000) Wakabayashi Wakabayashi Wakabayashi Wakabayashi Wakabayashi Wakabayashi Wakabayashi
Unknown, AU Human faeces, ATCC Eel pond water, TU Diseased fish, AVA Arowana, AVA Sword tail, AVA Guppy, AVA Discus, AVA Tilapia, AVA Eel pond water, TU Eel pond water, TU Eel pond sediment, TU Intestine of healthy eel, TU
Ling et al. Ling et al. Yamada & Ling et al. Ling et al. Ling et al. Ling et al. Ling et al. Ling et al. Yamada & Yamada & Yamada & Yamada &
(2000) (2000) Wakabayashi (2000) (2000) (2000) (2000) (2000) (2000) Wakabayashi Wakabayashi Wakabayashi Wakabayashi
(katB : : TnphoA) in PPD130/91, Neor Colr (katB : : TnphoA; ssrB : : TnphoA) in PPD130/91, Neor Colr 34 (pkatB), Ampr, Neor Colr 34 (pkatBankB), Ampr Neor Colr 309 (pkatB), Ampr Neor Colr 309 (pkatBankB), Ampr Neor Colr
Mathew et al. (2001) This study This study This study This study This study
E. coli Top 10 F9
Cols Amps Neos
Promega
Plasmids pGEM-Teasy pkatB pkatBankB
Ampr pGEM-Teasy+katB, Ampr pGEM-Teasy+katB and ankB, Ampr
Promega This study This study
(1999) (1999) (1999) (1999) (1999) (1999) (1999)
(1999)
(1999) (1999) (1999) (1999)
*ATCC, American Type Culture Collection; AVA, Agri-Food Veterinary Authority of Singapore, Singapore; AU, Department of Fishery, Auburn University, USA; TU, Department of Aquatic Bioscience, University of Tokyo, Japan. 2636
Microbiology 149
Characterization of KatB in Ed. tarda maintained in Luria broth (LB, Difco) or on LB agar at 37 uC. When required, media were supplemented with antibiotics such as ampicillin and neomycin at 100 and 50 mg ml21, respectively. 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 uC for 2 min (Misonix). The lysate was then centrifuged at 20 000 g for 10 min at 4 uC 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 mM21 cm21. One unit (U) is defined as the amount of catalase that degrades 1 mmol H2O2 min21 at 25 uC. 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 uC for 25 s; 30 cycles of 15 s at 94 uC, 30 s at 62 uC, 3 min at 72 uC; and a final extension for 3 min at 72 uC. Southern blotting was performed using the BluGene NonRadioactive Nucleic Acid Detection System (Gibco-BRL). Transfers of the DNA to nylon membranes (GeneScreen, NEM Research Products), hybridization conditions, and visualization with streptavidinalkaline phosphatase conjugates were carried out as recommended by the manufacturer’s protocol. 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 PCRamplified products were later cloned into pGEM-T Easy vector
(Promega), and then transformed into E. coli Top 10 F9 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. 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 uC. 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 wellaerated de-chlorinated water at 25±2 uC, 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 (16106 to 26106 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 uC 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 mg gentamicin ml21. 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.
Table 2. Primers used in this study Primer catA catB catC catD catE catF catG
Nucleotide sequence 59-CAG TAC GTA TGT CTC GC-39 59-ATG CCA CAA CAA CCG AG-39 59-TGC CCA TCC TGG TTG CCA-39 59-ACT ATG GCC TTA TCA GGT GA-39 59-TTA TCA AGC CTA ATG GCG-39 59-AAC GCT GTT GCC GTG AG-39 59-CGA GAT ACC GTG ATG CAG A-39
Description* Nucleotides Nucleotides Nucleotides Nucleotides Nucleotides Nucleotides Nucleotides
2526 to 2519, used for amplifying full-length katB and katB–ankB 85 to 102, used for PCR amplification of katB 1291 to 1274, used for PCR amplification of katB 1721 to 1703, used for amplifying full-length katB 1749 to 1766, used for amplifying ankB 2234 to 2218, used for amplifying ankB 2295 to 2277, used for amplifying full-length katB–ankB
*Nucleotide numbers are relative to the katB–ankB DNA sequence of the Ed. tarda PPD130/91 strain, wherein start codon ATG is taken as nucleotides 1 to 3. http://mic.sgmjournals.org
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P. S. S. Rao, Y. Yamada and K. Y. Leung 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 intra-
muscular route of administration was used to study the infection kinetics of Ed. tarda in vivo. Briefly, the fish were injected with 1?06105 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 161 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 LabBlender, 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 uC 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 P108?0 107?7 107?5
Catalase activity [U (mg protein)”1] 138?9±5?3 12?1±0?5 205?5±10?1 321?7±21?7 5?5±0?2 150?0±4?4 195?8±5?0
*The values represent the ratio of opsonized Ed. tarda obtained by dividing the total count of bacteria at 5 h by an initial count at 2 h after infection (n=3). Within the column, values followed by different superscript letters (a to e) differ significantly (P
