Département de Génétique et Microbiologie, Faculté de Médecine, Université de Genève, 1 Rue Michel Servet, 1211 Genève, Switzerland1
Author for correspondence: Menno Kok. Tel: +41 22 7025652. Fax: +41 22 7025702. e-mail: menno.kok{at}medecine.unige.ch
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ABSTRACT |
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Keywords: Salmonella, virulence, thymidylate synthase
Abbreviations: i.p., intraperitoneal; HBSS, Hanks basic salt solution; TMP, thymidine monophosphate
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INTRODUCTION |
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The capacity of S. typhimurium to survive and grow in the endosomal compartment of macrophages in vitro correlates strongly with mouse virulence (Fields et al., 1986 ; Bäumler et al., 1994
; Uchiya et al., 1999
), underscoring the important role of these cells in host defence against bacterial infection. Foreign antigens expressed in attenuated salmonellae may be delivered efficiently to the host immune system. A number of promising attempts have been reported to exploit the lifestyle of this bacterium for vaccination purposes (Coulson et al., 1994
; Verma et al., 1995
; Hopkins et al., 1996
; Nayak et al., 1998
). Salmonella thyA mutants have also been tested in this context and were found not to be fully attenuated (Smith & Tucker, 1976
; Nnalue & Stocker, 1987
; Curtiss et al., 1988
).
We further examined the behaviour of S. typhimurium thyA mutants, which lack the enzyme thymidylate synthase (EC 2 . 1 . 1 . 45) and are incapable of synthesizing DNA in the absence of precursors of thymidine monophosphate (TMP) in the growth medium (Neuhard & Kelln, 1996 ). We anticipated that these mutants would be incapable of normal DNA synthesis during intracellular growth, due to the absence of TMP and TMP precursors in the endosomal compartment, and that the bacteria would thus suffer thymidineless death (Medoff, 1972
; El-Hajj et al., 1992
). If this assumption was confirmed, it would suggest that multiplication of the S. typhimurium thyA mutants in the mouse would be limited to the extracellular space. In this report we analyse the persistence of thymidine-requiring mutants of the highly mouse-virulent strain S. typhimurium ATCC14028 in cultured epithelial and macrophage-like cells and evaluate their virulence in BALB/c mice.
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METHODS |
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Bacterial growth was followed by measuring the OD450 using a 10 mm quartz cuvette in a Uvikon 810 spectrometer (Kontron instruments). The growth rates and generation times of Salmonella mutants in thymidine-free media were followed by taking samples every 15 min during the first 90 min following dilution and plotting the optical density versus time. The thymidine requirements of S. typhimurium mutants were determined by growing bacteria overnight in complete NCE medium with 0·4% D-glucose and various amounts of thymidine. The final cell density and efficiency of plating were determined for each growth condition. Under non-limiting conditions, wild-type and thyA mutants grew to a cell density of approximately 3x109 ml-1. The thymidine requirement was defined as the lowest concentration that did not reduce the viable cell count.
Nucleotide sequence determination of the S. typhimurium thyA gene.
The S. typhimurium thyA coding sequence was amplified with 20-mer oligonucleotides complementary to the 5' and 3' ends of the gene (Washington University Genome Sequence Center) in a PCR reaction using the MWG Biotech Primus 96 plus thermocycler. The 30 cycle reaction was carried out with 1 U DyNAzyme (Finnzymes), 0·2 µg genomic DNA, 200 µM each dNTP, 0·5 µM each primer and 5 µl 10-fold concentrated DyNAzyme buffer supplied by the manufacturer, in a 50 µl reaction. The reaction products were analysed on an ABI Prism 377 DNA sequencer.
Growth and maintenance of S. typhimurium in cultured cells.
The macrophage-like cell line P388D1 and human epithelial cell line Hep-2 were obtained from the American Type Culture Collection. P388D1 cultures were maintained on RPMI-1640 supplemented with 10% heat-treated foetal calf serum (FCS; Gibco-BRL), 100 mg streptomycin l-1 and 100 U penicillin ml-1, at 37 °C under 5% carbon dioxide. Hep-2 cells were grown in Dulbeccos modified Eagle medium (DMEM; Gibco-BRL) supplemented with 0·03% L-glutamine, 10% FCS, 100 mg streptomycin l-1 and 100 U penicillin ml-1. Salmonella infection experiments were done in 24-well cell culture dishes (Costar) containing approximately 2x105 cells per well. Before infection, the cell cultures were washed with Hanks basic salt solution (HBSS; Gibco-BRL) and supplemented with 1 ml fresh growth medium without antibiotics.
S. typhimurium was grown overnight under aeration at 37 °C in LB, supplemented, if required, with 300 mg thymidine l-1. The culture was diluted to 2x107 c.f.u. ml-1 in fresh LB or LBT medium containing 250 mM KCl and 0·5% KNO3, and incubated at 37 °C without aeration until a cell density of approximately 5x108 c.f.u. ml-1 was reached. Bacteria were collected by centrifugation (1500 g, 2 min), and serial dilutions were prepared in the appropriate growth medium (RPMI or DMEM) prewarmed to 37 °C. For infection, 0·1 ml bacterial suspension was added to each well and the plates were briefly centrifuged (3 min at 250 g) to enhance bacterial adsorption to the cell monolayer. After an incubation of 15 min at 37 °C, the cells were washed with 1x HBSS and supplemented with 1 ml medium with 10% FCS containing 100 mg gentamicin l-1 to eliminate extracellular bacteria. The gentamicin concentration was reduced to 20 mg l-1 2 h after infection.
Viable intracellular bacteria, expressed as c.f.u., were quantified by lysing the infected cells with 0·05% Triton X-100 in HBSS and plating serial dilutions (in 15 mM MgCl2) on LBT agar. The total number of cells per well was determined after trypsin treatment using a haemocytometer. The number of infected cells was determined by serial dilution in RPMI or DMEM medium of trypsinized cells, stored on ice for 15 min prior to dilution. The cells were lysed on LBT agar with 0·05 mM EDTA. The number of c.f.u. thus obtained was arbitrarily considered to be equal to the number of infected cells.
Infection of mice.
Five-week-old female BALB/c mice (IFFA CREDO) were used for virulence tests. Bacteria were grown to mid-exponential phase (3x108 c.f.u. ml-1) on LB or LBT, collected by centrifugation (1500 g; 3 min at 4 °C) and resuspended in 1 vol. PBS. Appropriate dilutions in PBS were used to verify the viable cell count (c.f.u.). Groups of five mice were injected intraperitoneally (i.p.) with 0·5 ml bacterial suspension and kept under daily surveillance for 3 weeks.
Mice immunized with live or heat-killed (5 min at 95 °C) S. typhimurium thyA were challenged (i.p.) 28 or 95 d after the first immunization with 5x103 wild-type S. typhimurium ATCC14028 in PBS and their survival was monitored.
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RESULTS |
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The nature of the mutation was verified by genetic complementation with a plasmid-encoded E. coli thymidylate synthase gene (Kok, 1995 ). All 40 mutants were fully complemented for growth on LB and simple mineral media, and were thus denoted thyA mutants. Six of the mutants spontaneously reverted to auxotrophy at frequencies between 7x10-5 and 2x10-7; the remaining 34 mutants were used for further experiments.
All mutants displayed an unstable phenotype with respect to their thymidine requirements. Following their isolation on LBT/trimethoprim plates, at least 5 mM thymine or 1 mM thymidine had to be added to the growth media to maintain viability. However, partial suppression of the thymine/thymidine dependence occurred spontaneously during maintenance of the mutants on LBT plates; the thymidine requirement in NCE medium of each of four independent thyA mutants going down to approximately 200 µM. We decided to use the partially suppressed mutants, which we will refer to as low-thymidine-requiring thyA mutants, for further experiments.
Genetic defect in thyA mutants
The thyA coding sequences of wild-type S. typhimurium ATCC14028 and of several high- and low-thymidine-requiring mutants were amplified by PCR for DNA sequencing. The oligonucleotides used for amplification were complementary to the extremities of the S. typhimurium TR7095 thyA gene, according to the preliminary genome sequence information available from the Washington University of St. Louis Genome Sequence Center (http://www.genome.wustl.edu/gsc/bacterial/salmonella.shtml). The sequence of the 792 bp thyA gene from the virulent parent strain used in this study was found to be identical to the sequence of strain TR7095 used in the Washington University genome-sequencing project. Two high-thymidine-requiring mutants, thyA11 and thyA12, were found to have single-nucleotide mutations in the coding sequences at codon positions Lys140 (GAAAA) and Trp80 (TGG
TAG), resulting in premature termination of the ORFs. No additional mutations in the respective coding sequences were detected in the two corresponding low-thymidine-requiring mutants thyA111 and thyA121.
Growth of S. typhimurium thyA
When deprived of TMP precursors, E. coli thyA mutants undergo thymidineless death (El-Hajj et al., 1992 ; Medoff, 1972
). We evaluated the behaviour of low-thymidine-requiring S. typhimurium mutants thyA111 and thyA121 when deprived of TMP precursors in vitro. First, we established that starved bacteria, washed and resuspended in 15 mM MgSO4, remained viable (plating efficiency >0·9) for at least 24 h. Next, bacteria starved for 1 h in MgSO4 at 37 °C were diluted in NCE medium containing glucose or glycerol as the carbon source. Bacterial growth resumed virtually without lag after dilution in fresh medium, and for 35 h samples were taken to determine the optical density of the culture and the plating efficiency. The correlation between cell growth on thymidine-free medium and viability is shown in Fig. 1
. We observed a simple relationship between the mean number of cell divisions (generations) in thymidineless medium and the efficiency of plating. Apparently, cell death increases exponentially with time, after approximately one cell division of thymidineless growth.
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Virulence of thyA mutants
In view of the very limited life span of low-thymidine-requiring S. typhimurium thyA mutants in the endosomal compartments of cultured cells, we anticipated that these mutants would be strongly attenuated in mice. This assumption was tested in BALB/c mice, which were infected by i.p. injection. The low-thymidine-requiring mutants thyA111 (LD50=8x104 c.f.u.) and thyA121 (LD50=1·1x105 c.f.u.) were found to be attenuated, whereas both wild-type S. typhimurium ATCC14028 and the S. typhimurium thyA111 and thyA121 mutants complemented in trans with the E. coli thyA gene efficiently infected and killed BALB/c mice at very low doses (LD50<10 c.f.u.). A single immunization with a sublethal dose of the low-thymidine-requiring thyA121 mutant provided only partial protection against a subsequent challenge, 4 weeks later, with virulent S. typhimurium (Table 2). When boosted with a second dose, protection was improved. We could not induce similar protection by i.p. injection of equivalent amounts of heat-killed bacteria, suggesting that live bacteria are required to induce protective immunity.
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DISCUSSION |
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We have shown that low-thymidine-requiring S. typhimurium thyA mutants cannot survive intracellularly. Interestingly, these mutants are readily eliminated from both phagocytic and non-phagocytic cells. This rapid, complete and dose-independent elimination from cultured cells strongly suggests that intracellular bacteria suffer thymidineless death, a direct consequence of growth in an environment that lacks TMP precursors. Moreover, intracellular survival is stimulated under conditions that appear to make at least some thymidine available to the bacterial cells: bacterial endocytosis in the presence of high concentrations of thymidine and evasion of Salmonella into the nutrient-rich cytoplasm.
We found, in agreement with previous reports (Smith & Tucker, 1976 ; Nnalue & Stocker, 1987
; Curtiss et al., 1988
), the impact of the thyA mutation on virulence of Salmonella in the mouse to be moderate. This apparent discrepancy between the extremely rapid elimination of intracellular bacteria in vitro and the moderate attenuation of Salmonella thyA mutants in vivo, has also been reported by Watson et al. (2000)
when comparing bacterial survival in alveolar macrophages with virulence in the pig. We hypothesize that extracellular multiplication in vivo may be responsible for the observed residual virulence of S. typhimurium thyA mutants, with infected organs such as the spleen providing extracellular sources of TMP precursors at concentrations sufficient to sustain bacterial multiplication.
Whereas the S. typhimurium thyA mutant provides a useful tool to study the intracellular behaviour of Salmonella, its utility in the development of live-vaccination vectors remains to be established. Although low-thymidine-requiring thyA mutants offer some protection of mice against a challenge with the virulent parent strain, at least one additional attenuating mutation would have to be introduced into the Salmonella genome to render the strain safe for use as a vaccination vector. However, under these conditions the immunogenicity of the Salmonella vector may be seriously compromised. An alternative application of Salmonella thyA mutants in vaccine development indirectly depends on its virulence effects. Morona et al. (1994) have constructed plasmid vectors encoding E. coli thyA as a selectable marker for antigen production in a Salmonella thyA background, exploiting the selective advantage of thymidine independence in vivo to achieve stable plasmid inheritance.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 9 August 2000;
revised 6 November 2000;
accepted 22 November 2000.
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