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Salmonella serovars are closely related at the DNA level, and yet show substantial variations in the hosts they are able to infect and the types of diseases they elicit within each host. Certain strains of Salmonella typhimurium have been extensively studied, providing well-defined genetic and physical maps, a wide collection of mutant strains and selectable genetic markers, efficient mechanisms for gene transfer, and an excellent animal model for virulence (14, 23, 24). In contrast, other Salmonella serovars are less amenable to experimental analysis. For example, Salmonella typhi causes typhoid fever in humans but does not infect other animals. The lack of a simple in vivo assay for virulence has hampered the genetic analysis of S. typhi. Instead, most research has focused on the virulence of S. typhimurium in mice as a model for typhoid fever in humans. Overall S. typhimurium and S. typhi are nearly identical at the nucleotide level and cause similar diseases in the appropriate hosts. Thus, it is not surprising that many of the virulence factors identified in S. typhimurium are also present in S. typhi (1). However, there are clearly some important differences between virulence factors in the two serovars. For example, some virulence factors in S. typhimurium are located on a large plasmid which is absent from S. typhi (11). In addition, the genetic determinants specifying host range must differ between these Salmonella serovars (6).

Although these two serovars are closely related, regions of DNA from the S. typhimurium chromosome cannot be freely replaced with the corresponding chromosomal region from S. typhi due to strong barriers to genetic recombination. The recombination of cognate DNA sequences that are similar but not identical is termed "homeologous" recombination (17, 18). Although S. typhimurium and S. typhi are 98 to 99% identical at the nucleotide level, they undergo homeologous recombination at frequencies which are often undetectable (<10⁹). The barriers to homeologous recombination include restriction systems, components of the mismatch repair system (mutSL), and the exonuclease activity of the RecBCD complex (recD). Restriction barriers can be overcome by brief exposure to high temperature, which temporarily inactivates restriction endonucleases (7). However, overcoming the other two barriers requires inactivation of the mutS and recD genes (29). Inactivation of either the mutS or recD gene increases the homeologous recombination frequency about 10³-fold. Inactivation of both the mutS and recD genes increases the recombination frequency to levels normally observed during homologous recombination (about 10⁶-fold) and increases the length of DNA which is exchanged.

We reasoned that the inactivation of barriers to recombination would allow the construction of genetic hybrids between S. typhi and S. typhimurium, allowing potential virulence genes from S. typhi to be directly studied in vivo in a surrogate S. typhimurium host (15). However, certain genes involved in DNA repair and recombination are required for growth or survival of Salmonella in animals. For example, the recA and recBC gene products are required for homologous recombination, and mutations in these genes attenuate the virulence of S. typhimurium both in mice and in murine macrophages (3, 4). The recD gene product is not required for homologous recombination, but it is induced during growth of S. typhimurium in murine macrophages (12). To determine whether hybrids constructed in a mutS recD host can be used to study Salmonella pathogenesis in an animal model, we assayed the effect of mutS and recD mutations on virulence of S. typhimurium 14028s in mice.

Effect of mutS and recD mutations on virulence of S. typhimurium in mice. In vivo competition assays were initially used to compare the virulence of mutS, recD, and mutS recD mutant strains to that of wild-type S. typhimurium 14028s (Table 1). Mice which were infected with the mutS or recD derivatives had similar ratios of wild-type to mutant bacteria in both the spleen and liver compared to the ratio of bacteria in the initial inoculum (Fig. 1). In contrast, the ratio of mutS recD to wild-type bacteria recovered from the spleen or liver was diminished 10³- to 10⁴-fold relative to the initial infecting ratio (Fig. 1). These results indicate that 4 to 5 days after infection, the interval required for mice to succumb following inoculation with wild-type S. typhimurium 14028s, the mutS recD mutant was unable to establish a lethal infection.

View larger version (50K): [in this window] [in a new window]   FIG. 1.   Competition between mutant and wild-type strains in infected mice. Strains were grown overnight in rich medium prior to inoculation. Wild-type and mutant bacteria were administered intraperitoneally at 0.2 ml with 100 to 500 CFU of bacteria to groups of five 6- to 8-week-old female BALB/c mice (Harlan Sprague, Indianapolis, Ind.). The ratios of mutant to wild-type bacteria used ranged from 0.9:1 to 5:1. Mice were sacrificed 4 to 5 days after infection. Bacteria were recovered from the spleen and liver by homogenizing the tissues in 0.85% NaCl and plating serial dilutions onto rich medium. The number of mutant bacteria recovered was determined by replica plating onto rich medium supplemented with the appropriate antibiotic. The competition index is expressed as the (CFU of mutant recovered/CFU of wild type recovered)/(CFU of mutant inoculated/CFU of wild type inoculated). The value reported is the median competition index for five separate experiments. The error bars represent the maximal and minimal competition indices for each mutant versus wild-type bacteria in individual mice.

Survival of mutS recD mutants in murine peritoneal macrophages. Survival of Salmonella within macrophages is essential for virulence (9) and involves a dynamic equilibrium between bacterial growth and death (2). To determine whether the mutS recD phenotype was due to diminished survival in macrophages, we compared the survival of wild-type and mutant strains of S. typhimurium in murine peritoneal macrophages in vitro (Fig. 2). As expected, the number of wild-type bacteria recovered from infected macrophages increased with time after infection. The number of recA bacteria recovered from infected macrophages decreased about 10-fold by 6 h after infection, reflecting the increased sensitivity of recA mutants to the initial oxidative attack by macrophages. In contrast, the number of mutS recD bacteria recovered from macrophages remained nearly constant for the first 6 h after infection, suggesting that these mutants fail to proliferate in macrophages but are not rapidly killed by the oxidative burst (Fig. 2). Furthermore, although mutants defective for DNA repair and recombination are often sensitive to oxidative DNA damage (3, 4), wild-type and mutS recD strains showed similar sensitivities to H₂O₂ in vitro (data not shown). Thus, the delay in virulence observed in vivo is probably due to an inability of mutS recD mutants to multiply within the host macrophages.

View larger version (13K): [in this window] [in a new window]   FIG. 2.   Survival of S. typhimurium derivatives in murine peritoneal macrophages. Macrophages were harvested from the peritoneal cavity of 6- to 8-week-old BALB/c mice 3 days after intraperitoneal injection with 1.5 ml of 10% sterilized proteose peptone (Difco). Macrophages removed from the peritoneal cavity with a 23-gauge needle and 30-ml syringe were resuspended in endotoxin-free RPMI medium containing 5% fetal calf serum and 1 U of heparin per ml. Contaminating erythrocytes were removed by centrifugation, and the number of viable macrophages was quantitated on a hemocytometer following the addition of trypan blue. Approximately 105 peritoneal macrophages were seeded per well in 48-well tissue culture plates. Macrophages were allowed to adhere for 1 h in a 37°C incubator containing 5% CO2. Bacteria were diluted in 0.85% NaCl, centrifuged, and resuspended in 25% mouse serum. After opsonization in mouse serum at 37°C for 15 min, RPMI medium was added to give a final multiplicity of infection of bacteria to macrophages of between 1 and 5. Immediately after the addition of bacteria, tissue culture plates were centrifuged to promote bacterial adherence. Bacterial internalization was allowed to proceed for approximately 30 min, and then each reaction mixture was divided into two aliquots. In one aliquot, extracellular bacteria were killed by the addition of RPMI buffer containing 12.5 µg of gentamicin per ml. Macrophages were lysed with 0.5% deoxycholate, and the number of bacteria in each aliquot was determined by spotting serial dilutions onto rich medium plates. The number of internalized bacteria was initially determined 1.5 h after gentamicin treatment. Survival in macrophages was determined after 6 and 19 h of incubation. The number of surviving bacteria at these times is expressed as a percentage of the number of bacteria internalized. Values represent the mean from experiments performed in triplicate. The standard error was <2.0% of the mean for all values. Strains were wild-type (), mutS recD (), or recA () derivatives of S. typhimurium 14028s.

Ability of mutS recD mutants to adapt to nutrient-limiting conditions. To determine if the differential survival of wild-type bacteria relative to mutS recD mutants was due to differences in bacterial growth, we compared the growth characteristics of wild-type, mutS, recD, and mutS recD strains in vitro (Fig. 3). The wild-type and mutant strains showed similar growth rates when continuously cultured in rich medium (Fig. 3A) or when continuously cultured in minimal medium (Fig. 3B). The wild-type, mutS, and recD strains also exhibited similar lag times and growth rates when grown in rich medium and subcultured into minimal medium (Fig. 3C). However, mutS recD strains exhibited a much longer lag time when grown initially in rich medium and subcultured into minimal medium (Fig. 3C). This inability to adapt was not simply the result of stationary-phase growth, because mutS recD derivatives immediately resumed growth and behaved similarly to the wild-type strain when strains were continuously subcultured in the same type of medium (Fig. 3A and B). Thus, mutS recD mutants appear unable to readily adapt to nutrient-limiting conditions in vitro.

View larger version (19K): [in this window] [in a new window]   FIG. 3.   Growth of S. typhimurium 14028s derivatives in vitro. The growth rate was determined by measuring the optical density at 600 nm (OD600) as a function of time. Wild-type (), mutS (), recD (), or mutS recD () strains were either grown in rich medium and subcultured into rich medium (A), grown in minimal medium and subcultured into minimal medium (B), or grown in rich medium and subcultured into minimal medium (C). All assays were performed in 96-well microtiter dishes with a BioTek Instruments ELx808 Automated Microdish Reader. Strains were routinely grown in 200-µl volumes and incubated at 37°C with shaking. Growth was monitored over 36 h by measuring the OD600 at 15-min intervals. The values shown represent the mean optical densities from cultures grown in triplicate. The standard error was <5% of the mean for all time points measured.

Purine supplementation alleviates the mutS recD-dependent growth phenotype. Growth of Salmonella in macrophages requires the ability to synthesize certain metabolites that are limiting in the host (27). To determine if a common metabolite could restore the ability of mutS recD strains to adapt to nutrient downshift, we analyzed the lag time required to resume growth in minimal medium supplemented with various pools of amino acids, nucleotides, cofactors, or vitamins (13). When subcultured from rich medium into minimal medium supplemented with pools of nutrients that included purines or minimal medium supplemented with only adenine, guanine, or adenine plus guanine, the growth lag for mutS recD strains was eliminated (Table 2). Thus, mutations in mutS and recD affect the ability of S. typhimurium to grow under conditions in which purines are limiting.

Conclusions. The results of the in vivo competition and LD₅₀ assays indicate that the development of a systemic infection is slower for the mutS recD strain than for the isogenic wild-type strain. The phenotype of mutS recD mutants observed in vivo was not simply the result of the disruption of either the mutS or recD genes, because derivatives of S. typhimurium carrying mutations in either one of these genes behaved similarly to the wild-type strain (Fig. 1). Nor was the alteration in virulence simply the result of a silent secondary mutation, because the same phenotype was also observed after these mutations were backcrossed into a wild-type S. typhimurium 14028s background (data not shown). Rather, the alteration in virulence was the direct result of the inactivation of both the mutS and recD gene products. The results indicate that mutS recD double mutants experience a general downshift in purine metabolism. The reasons for the purine requirement in the double mutants are not yet clear. However, these results explain the slower progression of the double mutants observed in vivo, because the environment which S. typhimurium encounters during an infection, probably within the resident macrophages, is nutrient limiting for purines (2, 27).