Molecular Infectious Diseases Group, Department of Paediatrics, Faculty of Medicine, Imperial College, St Marys Hospital Campus, Norfolk Place, London W2 1PG, UK1
Institute for Animal Health, Compton, Newbury, Berkshire, RG20 7NN, UK2
Author for correspondence: J. Simon Kroll. Tel: +44 20 7886 6220. Fax: +44 20 7886 6284. e-mail: s.kroll{at}ic.ac.uk
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ABSTRACT |
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Keywords: Superoxide dismutase, Salmonella, periplasm, bacterial [Cu,Zn]-SOD, bacterial virulence
Abbreviations: [Cu,Zn]-SOD, copper- and zinc-cofactored superoxide dismutase; DEDC, diethyldithiocarbamate; FCS, foetal calf serum; IFN, interferon
; i.p., intraperitoneal; ROI/RNI, reactive oxygen/nitrogen intermediate; SOD, superoxide dismutase; SPER/NO, 2,2'-hydroxy-nitrosohydrazono bis-ethanamine (spermine NONOate); X, xanthine; XO, xanthine oxidase
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INTRODUCTION |
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Superoxide dismutases (SODs) are virtually ubiquitous in bacteria, catalysing the conversion of into hydrogen peroxide and oxygen (McCord & Fridovich, 1969
) in the first of a series of reactions to remove free radicals generated during the reduction of molecular oxygen. Three types of SOD are widely found in bacteria. Two are cofactored by manganese or iron and are located in the cytoplasm where they catalyse dismutation of
generated in the course of aerobic metabolism. A third SOD is cofactored by copper and zinc ([Cu,Zn]-SOD) and has been identified in the periplasm of a wide range of Gram-negative bacteria [Photobacterium leiognathi (Steinman, 1987
); Caulobacter crescentus (Steinman, 1982
); Brucella abortus (Stabel et al., 1994
); Haemophilus species (Langford et al., 1992
; Kroll et al., 1995
); Legionella species (St John & Steinman, 1996
); Actinobacillus and Pasteurella species (Kroll et al., 1995
); Escherichia coli (Benov & Fridovich, 1994
; Imlay & Imlay, 1996
); and Salmonella typhimurium, Salmonella choleraesuis and Salmonella dublin (Canvin et al., 1996
; Farrant et al., 1997
)]. Within the periplasm [Cu,Zn]-SOD is inaccessible to cytosolic superoxide but is suitably located to protect the organism from exogenous superoxide and the toxic action of its further reaction products with hydrogen peroxide and nitric oxide. Studies have confirmed such a function both in Salmonella and Neisseria meningitidis (Farrant et al., 1997
; De Groote et al., 1997
; Wilks et al., 1998
).
The degree of dissimilarity (only 54% identity) between the translated Salmonella sodC sequence described by Farrant et al. (1997) and E. coli SodC, its aberrant map position with respect to the Salmonella and E. coli genomes, and its flanking regions encoding proteins resembling products of bacteriophage lambda, suggested that this gene was not the orthologue of the E. coli gene and that Salmonella may have acquired it through phage-mediated transfer from an unknown donor (Farrant et al., 1997
). This hypothesis has been supported by the discovery of a new Salmonella gene, sodC-2, encoding a protein 82% identical to the E. coli SodC (Fang et al., 1999
). The first sodC gene is now renamed sodC-1. Here we report an investigation of the contribution that each of these genes makes to the virulence of S. choleraesuis. Unexpectedly, we did not find the effect to be additive, but rather, that both genes were necessary for the contribution of [Cu,Zn]-SOD to be discerned.
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METHODS |
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Construction of sodC-2 mutants.
A 344 bp DNA fragment amplified by PCR from within the sodC-2 ORF (nt 508851, GenBank accession AF056931) and ligated into the suicide vector pRR10(trfA) (Fang et al., 1992
) was generously provided by Ferric Fang (University of Colorado). This plasmid was mobilized from E. coli S17-1 into S. choleraesuis A50 wild-type and sodC-1 mutant (Farrant et al., 1997
). Transconjugants were selected on LB agar containing penicillin for the sodC-2 mutants or kanamycin and penicillin for the sodC-1 sodC-2 mutants. Homologous recombination of the plasmid into the Salmonella chromosome created an interruption of sodC-2, confirmed by Southern hybridization.
Extraction of bacterial proteins and detection of SOD activity.
Periplasmic extracts were prepared from 100 ml overnight cultures of S. choleraesuis wild-type, and sodC-1, sodC-2 and sodC-1 sodC-2 mutants by the method of Higgins & Hardie (1983) . Proteins were precipitated with ammonium sulphate (650 g l-1), concentrated by centrifugation and stored at -20 °C for later analysis. To detect SOD, proteins were separated in pre-cast wide range (pH 310) IEF gels (Bio-Rad). Gels were stained to reveal SOD activity using the method of Beauchamp & Fridovich (1971)
as modified by Steinman (1985)
. [Cu,Zn]-SOD activity was identified by specific inhibition with 5 mM diethyldithiocarbamate (DEDC; Sigma), a copper chelator (Benov & Fridovich, 1994
). [Fe]-SOD was identified by inhibition of its activity with hydrogen peroxide (Crapo et al., 1978
).
Bacterial sensitivity to superoxide and nitric oxide generated in vitro.
Sensitivity to increased cytosolic flux induced by paraquat was assessed by the method of De Groote et al. (1995)
. Sensitivity to exogenous
and NO was assessed by exposure of suspensions (105 c.f.u. ml-1) of stationary-phase organisms in phosphate-buffered saline (pH 7·4) (PBS) to these radicals, generated in solution respectively from reactions between xanthine (X) and xanthine oxidase (XO) (Fridovich, 1970
) and on acidification of ice cold alkaline 2,2'-hydroxy-nitrosohydrazono bis-ethanamine (spermine NONOate, SPER/NO) (De Groote et al., 1997
). Bacterial suspensions in these solutions were incubated with shaking at 37 °C and aliquots were removed periodically and assayed for viable counts.
Preparation of macrophages.
Porcine alveolar macrophages were isolated as previously described (Farrant et al., 1997 ). Macrophages were suspended in Iscoves modified Dulbeccos medium containing 10% (v/v) foetal calf serum (FCS), 100 mg gentamicin l-1 and 100 U ml-1 each of penicillin and streptomycin, and distributed into 1 ml flat-bottomed wells of tissue culture plates to give 5x105 cells per well. Wells were incubated overnight at 37 °C in an atmosphere of 5% CO2 to allow cells to adhere as monolayers. Two hours before infection monolayers were washed and covered with fresh antibiotic-free medium. Murine peritoneal macrophages were isolated from BALB/c mice at 4 days after intraperitoneal (i.p.) injection of 5 mM sodium periodate by peritoneal lavage. Macrophages were suspended in RPMI 1640 medium containing 10% FCS and 20 U murine interferon
(IFN
) ml-1, and distributed into 0·1 ml flat-bottomed wells at 5x104 cells per well. Cells were left to adhere as monolayers as before, and washed and covered with fresh medium at the start of experiments.
Infection of monolayers.
Bacteria from overnight cultures were washed and resuspended in tissue culture medium at a concentration of 5x106 c.f.u. ml-1. For opsonization, bacteria were incubated with 10% normal porcine or murine serum at 37 °C for 30 min on a rolling platform at 120 r.p.m. Bacteria were added to monolayers to give a verified multiplicity of infection of 510 bacteria per macrophage. In experiments with IFN-stimulated macrophages, the infected monolayers were gently centrifuged (150 g for 10 min at 4 °C). Experiments were performed in triplicate.
Bacterial uptake and killing by macrophages.
Bacterial uptake and killing were assessed by a modification of the gentamicin-protection assay of Buchmeier & Heffron (1989) . Infected monolayers of IFN
-stimulated macrophages were incubated for 15 min, followed by a wash and further incubation with Iscoves modified Dulbeccos Medium or RPMI 1640 containing 10% v/v FCS and 100 mg gentamicin l-1. After 124 h incubation, monolayers were washed twice with PBS to remove any remaining superficially adherent viable bacteria and lysed with 0·1% (w/v) sodium deoxycholate in PBS. To assess viable counts of phagocytosed bacteria, lysates were plated on to MacConkey agar containing 30 mg nalidixic acid l-1. For experiments with unstimulated macrophages, the initial incubation was extended to 1 h.
Experimental infection of mice.
Mice were purchased from Charles River Laboratories. In a first experiment, groups of five 810 week-old BALB/c female mice were injected i.p. with 103 c.f.u., and five C3H/HeN mice with 5x104 c.f.u. A group of sham-inoculated mice received saline only. At 4 and 5 days post-infection, respectively, when animals infected with the wild-type S. choleraesuis began to exhibit symptoms of systemic disease (ruffled fur, reduced activity), all the mice were killed by CO2 asphyxiation followed by cervical dislocation. Spleens and livers were removed, weighed and homogenized in 0·9% saline containing 1% Triton X-100. Viable counts were determined by plating serial dilutions on MacConkey agar containing nalidixic acid. Replica plating on appropriate selective media was carried out to exclude the theoretical possibility that mutant strains might have reverted to wild-type.
In a second experiment, groups of three 810 week-old BALB/c female mice were infected i.p. with 100 c.f.u. Salmonella suspended in 0·1 ml 0·9% saline or saline only (sham infection). At 4 days post-infection the mice began to exhibit symptoms of systemic disease and were killed by CO2 asphyxiation. Spleens and livers were removed and number of viable organisms in the organs was determined as above.
Groups of three 810 week-old BALB/c female mice were infected by oral administration of 106 c.f.u. Salmonella suspended in 0·1 ml 0·9% saline. Sham-infected mice received saline only. At 5 days post-infection, as mice began to exhibit symptoms of systemic disease, they were killed by CO2 asphyxiation. Spleens and livers were removed and the number of viable organisms in the organs was determined as above.
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RESULTS |
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DISCUSSION |
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While a [Cu,Zn]-SOD gene has by now been found in many bacterial species, the discovery of two different, functional, versions of the gene and very recently a third, sodC-3 (Figueroa-Bossi et al., 2001 ) in the one pathogen is without precedent. The gene sodC-2 in Salmonella is the true orthologue of sodC in E. coli, established by extended sequencing of the chromosomal locus in each organism. In E. coli a 7 kb domain contains consecutive ORFs: slyAB1643B1644B1645sodCB1647B1648B1649nemA. A search with the translation of each of these genes/putative genes against the S. typhimurium genome database (at http://genome.wustl.edu/gsc/Projects/S.typhimurium/) demonstrated in each case high identity (>80%) for corresponding ORFs flanking sodC-2. In contrast, sodC-1 and sodC-3appear to have been acquired on lysogenic bacteriophages (Gifsy-2 and Fels-1 respectively) (Figueroa-Bossi & Bossi, 1999
; Figueroa-Bossi et al., 2001
). A likely origin of sodC-1 from outside Salmonella is revealed by all-against-all protein sequence comparison of a set of SodC sequences. SodC-1 is as divergent in sequence from Salmonella SodC-2 and E. coli SodC as the examples of the sodC from such upper respiratory tract pathogens as H. influenzae. While an anomalously high mutation rate could theoretically be the explanation for the SodC-1 sequence divergence, the association with phage genes strongly supports the hypothesis that this gene has an exogenous origin: sodC-1 joins a growing list of virulence genes likely to have been acquired through horizontal transfer. Carriage of sodC-1 on lysogenized Gifsy-2 is a characteristic of the more virulent Salmonella serotypes, such as S. typhimurium, S. choleraesuis, S. dublin and Salmonella enteritidis. The less virulent serotypes have only sodC-2 (Fang et al., 1999
). Taken with the data presented here for S. choleraesuis, this suggests that selection pressure may have favoured acquisition of one or even two extra [Cu,Zn]-SOD genes by Salmonella serovars adapted to survive within macrophages. A mechanism to promote sodC-1 acquisition may be proposed. Gifsy-2 elements are released upon exposure to H2O2, raising the intriguing possibility that infection of macrophages and exposure to reactive oxygen species generated in the respiratory burst may, through induction of a lytic cycle for Gifsy-2, potentiate transmission of the genes it carries, among them protective sodC-1, to other salmonellae (Figueroa-Bossi et al., 1999
).
The proposition that horizontally acquired [Cu,Zn]-SOD genes may make a special contribution to bacterial virulence, not conferred by endogenous sodC, gains support from observations in the genus Neisseria. Commensal neisserial species, common colonists of the human upper respiratory and genital mucosa, do not in general have sodC genes (our unpublished results) but N. meningitidis, the only neisserial species regularly causing invasive, life threatening, infection, is the exception and in this pathogen [Cu,Zn]-SOD unequivocally contributes to virulence (Wilks et al., 1998 ). As seen for sodC-1 in Salmonella, meningococcal sodC appears to have been acquired by horizontal transfer, in this case probably from co-commensal Haemophilus species (Kroll et al., 1998
). In contrast, studies of other pathogens with only a single sodC gene and in which there is no evidence, for example from sequence anomaly at the locus, of horizontal acquisition of the gene, have generally yielded equivocal or negative evidence for the involvement of [Cu,Zn]-SOD in virulence [B. abortus (Tatum et al., 1992
; Latimer et al., 1992
), A. pleuropneumoniae (Sheehan et al., 2000
) and M. tuberculosis (Dussurget et al., 2001
; Piddington et al., 2001
)].
Fang et al. (1999) have studied the contribution that SodC-1 and SodC-2 make to the virulence of S. typhimurium and reached a different conclusion to ours. It is not, however, possible directly to compare our findings as there are significant differences in experimental design. In a different experimental model much more prolonged murine infection to a fatal end point they observed that only the sodC-1 sodC-2 strain was attenuated in lethality for Itys mice (a different strain to that used here). In an Ityr strain (the same as that used here), all sodC mutants, single and double, were slower to kill mice than the wild-type. Although the result reported for the double mutant suggested greater attenuation, the reproducibility of this single observation cannot be assessed with the data available. It may be that the contrast reflects differences in the pathogenesis of S. typhimurium and S. choleraesuis in mice. Despite being highly virulent for pigs (Watson et al., 2000
), S. choleraesuis A50 is significantly less virulent for mice than S. typhimurium and S. dublin (Farrant et al., 1997
).
In conclusion, in S. choleraesuis it seems clear that the acquisition of an additional sodC gene on a lysogenic bacteriophage has effectively led to a gain of function (plausibly through increased total activity) for [Cu,Zn]-SOD, contributing to virulence. Until now the contribution that sodC makes to bacterial virulence has seemed wholly capricious clearly contributing in some organisms, but not at all in others. From this work we would tentatively suggest that it is in particular in those organisms in which there is evidence of horizontal acquisition of sodC by bacteriophage or plasmid transfer, or through transformation with chromosomal DNA that evidence may be found of a virulence-associated gain of function, the capacity to neutralize host defences that depend on the generation of superoxide and its further reaction products.
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ACKNOWLEDGEMENTS |
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Received 9 August 2001;
revised 22 October 2001;
accepted 15 November 2001.