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Journal of Bacteriology, July 2007, p. 4708-4717, Vol. 189, No. 13
0021-9193/07/$08.00+0     doi:10.1128/JB.00299-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

In Vivo-Selected Mutations in Methyl-Directed Mismatch Repair Suppress the Virulence Attenuation of Salmonella dam Mutant Strains following Intraperitoneal, but Not Oral, Infection of Naïve Mice{triangledown}

Douglas M. Heithoff,1 Golnaz Badie,1 Steven M. Julio,1,{dagger} Elena Y. Enioutina,2 Raymond A. Daynes,2 Robert L. Sinsheimer,1 and Michael J. Mahan1*

Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, California 93106,1 Department of Pathology, University of Utah School of Medicine, Salt Lake City, Utah 841322

Received 27 February 2007/ Accepted 18 April 2007


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ABSTRACT
 
Salmonella enterica serovar Typhimurium that lacks the DNA adenine methylase (Dam) ectopically expresses multiple genes that are preferentially expressed during infection, is attenuated for virulence, and confers heightened immunity in vaccinated hosts. The safety of dam mutant Salmonella vaccines was evaluated by screening within infected mice for isolates that have an increased capacity to cause disease relative to the attenuated parental strain. Since dam mutant strains are sensitive to the DNA base analog 2-aminopurine (2-AP), we screened for 2-AP-resistant (2-APr) isolates in systemic tissues of mice infected with dam mutant Salmonella. Such 2-APr derivatives were isolated following intraperitoneal but not oral administration and were shown to be competent for infectivity via intraperitoneal but not oral infection of naïve mice. These 2-APr derivatives were deficient in methyl-directed mismatch repair and were resistant to nitric oxide, yet they retained the bile-sensitive phenotype of the parental dam mutant strain. Additionally, introduction of a mutH null mutation into dam mutant cells suppressed the inherent defects in intraperitoneal infectivity and nitric oxide resistance, as well as overexpression of SpvB, an actin cytotoxin required for Salmonella systemic survival. These data suggest that restoration of intraperitoneal virulence of dam mutant strains is associated with deficiencies in methyl-directed mismatch repair that correlate with the production of systemically related virulence functions.


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INTRODUCTION
 
Salmonella enterica is an important pathogen of reptiles, birds, and mammals and is a significant food-borne pathogen of humans, where clinical manifestations range from gastroenteritis to bacteremia and typhoid fever, depending on the bacterial strain and host immune and nutritional status (38). Such infections are often caused by consumption of contaminated meat and animal products (38). Since multiple Salmonella serovars are endemic to many intensive livestock farms, it is important to design and implement broad prophylactic strategies that are efficacious for many salmonellae. While the use of Salmonella vaccines is common (62), most commercial products are bacterins of limited efficacy, presumably since on-farm exposure often occurs during the first few hours after birth, limiting the prospect of stimulating an acquired immune response with a bacterin (33).

Modified live Salmonella vaccines have the capacity to infect and proliferate within the gut-associated lymphoid tissue, often resulting in the elicitation of strong innate and adaptive mucosal responses, as well as cell-mediated immunity (20, 32-34, 61), and a limited number of the vaccines are currently available commercially (12). Oral administration of modified live Salmonella vaccines has been shown to reduce fecal shedding, decrease severity of disease, and/or reduce mortality following experimental Salmonella challenge in sheep (48) and calves (4, 6, 36, 46, 56, 60, 63, 64, 69, 70). Vaccine constructs have included aromatic amino acid-dependent (aro), streptomycin-dependent, and galE mutants (13, 17, 27, 63, 69). These vaccines are generally effective at inducing protective acquired immunity against homologous Salmonella challenge but have limited protective efficacy against heterologous Salmonella serovars. Thus, the ideal Salmonella vaccine should confer both early- and late-onset immunity against a wide spectrum of Salmonella serovars.

Modified live vaccines of S. enterica serovar Typhimurium that lack the DNA adenine methylase (dam) gene have been developed that are avirulent yet confer cross-protective immunity to multiple Salmonella strains in murine (21, 29, 30), avian (18, 19), and bovine (17, 47) models of typhoid fever. Such vaccines were shown to confer early-onset immunity in livestock, which is critical to commercial applications where neonatal exposure to Salmonella is prevalent (17). Here we examined the safety of dam mutant Salmonella vaccines by screening within infected mice (in vivo) for isolates that were competent for infectivity. Derivatives were isolated that exhibited a heightened capacity to cause disease via the intraperitoneal but not oral route of infection. The molecular basis of enhanced intraperitoneal virulence was associated with deficiencies in methyl-directed mismatch repair that correlated with restored production of systemically related virulence functions.


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MATERIALS AND METHODS
 
Bacterial strains, phage, and media. The Salmonella pathogenic strains used in this study (Table 1) were derived from S. enterica serovar Typhimurium strain ATCC 14028 (CDC 6516-60). dam mutant derivatives contained a dam-102::Mud-Cm insertion (MT2116) or {Delta}dam-232 (MT2188, a nonpolar in-frame deletion strain) (30). The {Delta}mutH358::Km mutant (MT2934) was constructed using internal oligonucleotides that served as PCR primers designed to construct a 589-bp deletion of defined mutH sequence marked at the joint point with a kanamycin resistance cassette (37); the deletion retains 24 and 81 bp of the 5' and 3' ends of the mutH coding sequence, respectively. The nonpolar in-frame {Delta}mutH359 deletion strain (MT2994) was constructed in serovar Typhimurium by using internal oligonucleotides that served as PCR primers designed to construct a 591-bp deletion of internal mutH sequence (30); the deletion retains 105 nucleotides of coding sequence. S. enterica serovar Dublin strains MT2999 ({Delta}mutH359) and MT3000 ({Delta}dam-232 {Delta}mutH359) were constructed by standard allelic replacement. The {Delta}spvB121 strain (MT2891) was constructed using internal oligonucleotides that served as PCR primers designed to construct a 1,752-bp nonpolar deletion of internal spvB sequence; the deletion retains 24 nucleotides of coding sequence.


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  		TABLE 1. Bacterial strains used in this study

The high-frequency generalized transducing bacteriophage P22 mutant HT105/1 int-201 was used for all transductional crosses (57), and phage-free, phage-sensitive transductants were isolated as previously described (7). Unless otherwise specified, Luria-Bertani (LB) broth (14) was the laboratory medium used in these studies. Final concentrations of antibiotics (Sigma) were as follows: ampicillin, 100 µg/ml; chloramphenicol, 20 µg/ml; kanamycin, 50 µg/ml; streptomycin, 100 µg/ml; and 2-aminopurine (2-AP), 0.6 mg/ml.

Virulence assays. (i) CI. Mutant and wild-type Salmonella strains were grown overnight in LB broth with shaking at 37°C. Six- to 8-week-old BALB/c mice were infected intraperitoneally with a 1:1 ratio of mutant to wild type at a dose of ~500 cells each. At 5 days postinfection, the bacterial cells were recovered from the spleen. The competitive infectivity index (CI) is the ratio of mutant to wild-type bacteria recovered from target tissues (spleen and liver) divided by the ratio of the input inoculum; bacterial cell number was enumerated by direct colony count. The wild-type (dam+) strain (MT2057) contains a Lac+ MudJ transcriptional fusion which is used to discern it from other Salmonella strains which are inherently Lac– (11, 28).

(ii) LD50. An oral virulence 50% lethal dose (LD50) assay was used to determine the dose required to kill 50% of the animals. Briefly, mutant and wild-type Salmonella strains were grown overnight in LB medium with shaking at 37°C. Six- to 8-week-old BALB/c mice were perorally infected by gastrointubation in 0.2 ml of 0.2 M phosphate buffer (pH 8.0). The intraperitoneal LD50 was determined by infecting five BALB/c mice per challenge dose in 0.2 ml of 0.15 M NaCl solution. Mice were examined daily following challenge for morbidity and mortality. To determine the number of bacteria in host tissues, moribund mice were sacrificed and bacteria were recovered from host tissues and plated for colony counts. Host tissues assayed were Peyer's patches (the Peyer's patches proximal to the ileal-cecal junction), liver, and spleen.

In vivo selection for 2-APr derivatives of dam mutant Salmonella. BALB/c mice were infected with dam mutant cells (MT2188) (intraperitoneally at 105 cells per dose or orally at 109 cells per dose) as described above for virulence assays. To determine the number of bacteria in the spleen and liver at various time points, mice were sacrificed and bacteria were recovered from host tissues and plated for colony counts on LB medium incubated for 16 to 20 h at 37°C. The resultant colonies were either patched or replica plated to LB plates containing 0.6 mg/ml 2-AP, and 2-APr derivatives of dam mutant Salmonella were isolated after incubation for 16 to 20 h at 37°C.

Nitric oxide sensitivity assay. Salmonella cultures containing dam+, dam mutant, mutH mutant, and dam mutH mutant derivatives of strain 14028 were grown for 16 h in LB medium (pH 5.0) buffered with MES [2-(N-morpholino)ethanesulfonic acid] (Sigma). Approximately 103 cells of the overnight culture were added to individual microtiter wells containing LB medium (pH 5.0) buffered with MES and supplemented with the indicated final concentration of the nitric oxide source, sodium nitrate (Sigma) (5). Cells were incubated for an additional 20 h without shaking at 37°C in the absence of sodium nitrate supplementation; all strains tested grew to similar cell densities (optical density at 600 nm [OD600]) under these conditions. Values given are averages of OD600 measurements from a triplicate of a representative experiment; standard deviations were <20% of the means.

Bile sensitivity assay. Salmonella cultures containing dam+, dam mutant, mutH mutant, and dam mutH mutant derivatives of strain 14028 were grown for 16 h in LB medium. Approximately 5 x 102 of the overnight grown cells were added to individual microtiter wells containing LB supplemented with the listed final concentration of Ox-bile (sodium choleate; Sigma) as described previously (3). Cells were incubated for an additional 16 h without shaking at 37°C without bile supplementation, all strains tested grew to similar cell densities (OD600) under these conditions. Values given are averages of OD600 measurements from at least two experiments in triplicate; standard deviations were <20% of the means. Values of <0.02 represent no detectable growth under the conditions tested.

Western blot analysis. Whole-cell protein extracts prepared from ~107 cells were processed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (~20 µg of protein/well), transferred to polyvinylidene difluoride membranes (Pierce), and probed with SpvB primary antibody (a gift from Don Guiney, UCSD) which was purified by passage over a Sepharose (Sigma) column to which was bound protein derived from a crude lysate of the serovar Typhimurium spvB deletion strain MT2891. Peroxidase-conjugated donkey anti-rabbit immunoglobulin G (Amersham Biosciences) was used as secondary antibody. Whole-cell protein extracts were serially diluted twofold for SpvB quantitation relative to wild-type cells. Signal was detected by chemiluminescence using Supersignal West Femto Maximum Sensitivity Substrate (Pierce) followed by exposure to film. MT2891 was used as an SpvB– control (10).

Spontaneous mutation frequency assay. Salmonella cells were grown overnight in LB medium with shaking at 37°C, serially diluted, and plated on solid LB medium containing 100 µg/ml streptomycin. The streptomycin-resistant cells were enumerated after 20 h of growth at 37°C. The mutation frequency was calculated relative to that exhibited by the wild type (dam+). Values given are averages from at least one triplicate; standard deviations were <10% of the means.

Sequencing of genomic regions adjacent to Tn10d-Tc insertions by inverse PCR. Inverse PCR was performed by a modification of methods described previously (49). Genomic DNA prepared from Salmonella cells harboring a Tn10d-Tc insertion linked to the desired mutation was digested to completion with HhaI or AluI. The DNA fragments were circularized by large-volume overnight ligation (50 µl) at 16°C, and sequences adjacent to the Tn10d-Tc were amplified by inverse PCR, using outward-reading oligonucleotide primers that are complementary to the Tn10d-Tc sequence (5'-CGCGGATCCTGTTGACAAAGGGAATC-3' and 5'-CGGAATTCCGGGAATTGACGTTCCTTC-3'). PCR products were run on a 1.2% agarose gel, and amplified DNA fragments (~400 to 600 bp) were gel extracted and sequenced using the Tn10d-Tc complementary primers (University of Utah sequencing center).

Mapping 2-APr isolates of dam mutant Salmonella to mutHLS loci. Phage P22 lysates were grown on Salmonella strains containing transposon insertions adjacent to mutH, mutL, or mutS (ygdR::Tn10dTc, 90% linked to mutH [MT3023]; purA3131::Tn10dTc, 50% linked to mutL [MT1057] [28]; and invF::Tn5 lacZY-12-5, 50% linked to mutS [EE639] [obtained from C. A. Lee], respectively). Such lysates were used to transduce 2-APr mutants of Salmonella dam strains to the appropriate antibiotic resistance corresponding to the donor insertion, and genetic linkage was determined by the frequency of 2-APs transductants among the total CFU enumerated.

Sequencing of mutHLS alleles. Taq high-fidelity DNA polymerase (New England Biolabs) was used to amplify mutHLS regions, using overlapping oligonucleotide primers sufficient to generate double-stranded sequence of the cognate mutHLS regions. Products from two independent PCRs were run on a 0.8% agarose gel, and amplified DNA fragments were gel extracted and sequenced using mutHLS complementary primers (University of Utah sequencing center).


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RESULTS
 
Isolation of 2-AP-resistant derivatives of dam mutant Salmonella from murine liver and spleen after intraperitoneal but not oral infection. dam mutants are sensitive to the DNA base analog 2-AP due to their inherent inability to discriminate DNA strands for the efficient excision of the defective base during methyl-directed mismatch repair (22, 23, 42). We have utilized resistance to 2-AP as a screen for isolates of dam mutant Salmonella recovered from infected murine tissues that have reverted to the wild-type (Dam+) 2-APr phenotype. Five independent colonies of dam mutant Salmonella ({Delta}dam-232) were grown overnight in LB and used to intraperitoneally infect five BALB/c mice at a dose of 105 bacteria. (The intraperitoneal LD50s of Salmonella dam+ and dam mutant 14028 cells are <10 and >104 organisms, respectively [30]). At day 5 postinfection, the mice were sacrificed and the bacteria present in the spleen and liver were enumerated and tested for their 2-APr phenotype. Typically, 5 x 104 CFU per gram of tissue were recovered from the livers and spleens of surviving animals (Fig. 1), from which 0.025 to 4% of the cells were resistant to 2-AP, the phenotype of the wild type (dam+) (Fig. 1A). This resulted in the isolation of five independent 2-APr derivatives of dam mutant Salmonella (MT3008 to MT3012); additionally, two other independent 2-APr mutants (MT2334 and MT3007) were isolated from systemic tissues from other intraperitoneally infected mice (Table 2). PCR analysis of the dam locus showed that all (seven of seven) 2-APr isolates retained the dam deletion ({Delta}dam-232), indicating that the 2-APr phenotype was not attributable to a repair of the parental dam mutation. Additionally, pulsed-field gel electrophoresis analysis revealed that all seven 2-APr isolates exhibited a dam mutant methylation pattern as assessed by complete MboI digestion of chromosomal DNA, where MboI digests only nonmethylated Dam target GATC sequences (42) (data not shown).


Figure 1
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  		FIG. 1. 2-AP-resistant derivatives of dam mutant Salmonella were isolated from murine systemic tissues upon intraperitoneal, but not oral, infection. (A) BALB/c mice were intraperitoneally infected at a dose of 105 dam mutant serovar Typhimurium cells. The number of 2-APs (open boxes) or 2-APr (closed boxes) Salmonella organisms in the spleen or liver was enumerated at day 5 postinfection. (B) BALB/c mice were infected perorally via gastrointubation at a dose of 109 dam mutant serovar Typhimurium cells. The number of 2-APs (open boxes) or 2-APr (closed boxes) Salmonella organisms from the spleen or liver were enumerated at 2, 3, and 4 weeks postinfection.


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  		TABLE 2. In vivo-selected 2-APr derivatives of dam mutant Salmonella are virulent upon intraperitoneal but not oral infection of naïve mice

Next, we determined whether the route of administration affected the ability to isolate 2-APr derivatives of dam mutant Salmonella. Five independent colonies of the dam mutant Salmonella were grown overnight in LB and used to orally infect five individual groups of BALB/c mice (two to five per group) by gastrointubation (dose, 109). (The oral LD50s of Salmonella dam+ and dam mutant 14028 cells are 105 and >109 organisms, respectively [30]). Since complete clearance of dam mutant cells occurs at 5 to 6 weeks after oral infection (29), bacteria present in the spleen and liver were enumerated and tested for their 2-AP phenotype at 2 to 4 weeks postinfection. As expected, bacterial counts in the target tissues declined steadily over this time period (Fig. 1). In contrast to the case for intraperitoneal infection, none (0 of 15) of the orally infected mice harbored 2-APr organisms in the liver or spleen (Fig. 1B). Additionally, dam mutant Salmonella present in the target tissues after oral infection was not capable of causing overt disease as evidenced by visual inspection of vaccinated animals up to 4 weeks postinfection. These data indicate that 2-APr derivatives of dam mutant Salmonella can be readily isolated from target tissues after intraperitoneal, but not oral, infection of naïve mice.

In vivo-selected 2-APr derivatives of dam mutant Salmonella are virulent upon intraperitoneal but not oral infection of naïve mice. LD50 and CI virulence assays were utilized to determine whether the 2-APr organisms recovered from systemic tissues after intraperitoneal administration were fully virulent upon infection of naïve mice. The LD50 is the dose required to kill 50% of infected animals; the CI is the ratio of mutant to wild-type cells recovered from the spleen after infection with equal numbers of mutant (2-APr) and wild-type (dam+) cells divided by the ratio of the input inoculum. The parental dam mutant strain exhibits a >104-fold defect in virulence by the intraperitoneal and oral routes of administration (21, 30).

After intraperitoneal infection of naïve BALB/c mice, LD50 and CI virulence assays revealed that all (seven of seven) 2-APr derivatives of dam mutant Salmonella (MT2334 and MT3007 to MT3012) had regained partial virulence relative to the parental dam mutant strain (Table 2); four of these were within 10-fold of the virulence exhibited by the wild type (dam+) as assessed by the intraperitoneal CI assay. In contrast, all 2-APr isolates remained highly attenuated following an oral infection of naïve mice; six of seven 2-APr isolates were attenuated >1,000-fold as assessed by the oral LD50 assay via gastrointubation. These data indicate that the in vivo-selected 2-APr derivatives of dam mutant Salmonella had gained virulence attributes pertaining to intraperitoneal but not oral infection of naïve mice.

2-APr derivatives of dam mutant Salmonella are present within the inocula and proliferate within systemic tissues following intraperitoneal but not oral infection. To discern whether the 2-APr derivatives were present in the inocula and/or arose or proliferated within the infected animals, the frequency of reversion from 2-AP sensitivity to resistance was determined in vitro. Ten different colonies of dam mutant Salmonella were inoculated in LB, grown overnight, and plated on solid LB medium containing 2-AP. The frequency of 2-APr cells was ~1/104 for all 10 colonies tested, indicating that ~10 2-APr cells were present in the 105 cells of the intraperitoneal inoculum. However, the total numbers of 2-APr cells recovered from the spleens and livers of five mice at day 5 after intraperitoneal infection were 10, 30, 91, 360, and 1,780 cells, respectively (Fig. 1A). Thus, the number of 2-APr cells was either maintained or increased 3- to 178-fold in the spleen and liver from that present in the inoculum. In contrast, although 105 2-APr cells were present in the oral inoculum (109 cells), no 2-APr isolates were recovered from the spleen or liver after infection by the oral route (0 of 15 mice) (Fig. 1B), nor did such mice exhibit morbidity or mortality up to 4 weeks postinfection. These data indicate that 2-APr derivatives are present within the intraperitoneal and oral inocula and propagate within systemic tissues following administration by the intraperitoneal, but not oral, route.

In vivo-selected 2-APr derivatives of dam mutant Salmonella harbor mutations in methyl-directed mismatch repair. To determine the molecular basis of the reversion of Salmonella dam mutant cells to virulence after intraperitoneal but not oral infection, the 2-APr phenotype was used as a genetic marker to map the loci resulting in enhanced intraperitoneal infectivity. Briefly, a pool of 30,000 to 40,000 Tn10d-Tc transposon insertions was used to transduce two parental 2-AP-resistant mutants to sensitivity, the phenotype of dam mutant cells. By backcrossing the corresponding Tn10d-Tc insertions into the two parental 2-APr mutants and scoring 2-APs, the Tn10d-Tc insertions were shown to be genetically linked to the 2-APr phenotype.

The chromosomal regions adjacent to the Tn10d-Tc insertions were identified by inverse PCR (49) using outward-reading oligonucleotide primers that are complementary to sequences within the Tn10d-Tc. Sequence analysis of the PCR products revealed that the two classes of Tn10d-Tc insertions mapped near mutH and mutS, respectively. Transductional mapping revealed that additional Tn10d-Tc insertions linked to the 2-APr phenotype of the other five in vivo-selected derivatives also mapped near mut loci. To determine whether the 2-APr isolates harbored a mut mutation(s), the relevant mut loci of these isolates were cloned and sequenced. Briefly, oligonucleotide primers that flank the mutH, mutL, and mutS genes were used to amplify the cognate mut loci by PCR. Sequence analysis of the PCR products revealed that all (seven of seven) 2-APr isolates contained a mutation(s) in mutH, mutL, or mutS relative to the corresponding wild-type (mut+) sequence (Table 3).


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  		TABLE 3. In vivo-selected 2-APr derivatives of dam mutant Salmonella contain mutations in mutHLS loci

Since all in vivo-selected 2-APr isolates contained mutations in mut loci, we tested the hypothesis that defects in methyl-directed mismatch repair suppressed the intraperitoneal virulence attenuation inherent to dam mutant Salmonella. A mutH deletion ({Delta}mutH358::Km) was constructed by standard allelic replacement and introduced into a dam mutant strain, and the resultant dam mutH strain (MT2937) was assessed for virulence. LD50 and CI virulence assays revealed that MT2937 (dam-102::Mud-Cm {Delta}mutH358::Km) was partially restored for virulence via the intraperitoneal but not the oral route of infection relative to the dam mutant parental strain, similar to what was exhibited by the in vivo-selected 2-APr isolates (MT2334 and MT3007 to MT3012) (Table 2). These results are consistent with a previous report that showed that mutHLS mutations partially suppressed the virulence defect of dam mutant cells (52).

To determine whether allelic differences occur in 2-APr mutants that arise after in vitro compared to in vivo selection, 10 different colonies of 2-APs dam mutant Salmonella were inoculated in LB, grown overnight, and plated on solid LB medium containing 2-AP. Ten independent 2-APr mutants were isolated, and the 2-APr phenotype was mapped relative to known transposon insertions linked to mut loci. All (10 of 10) in vitro-selected 2-APr mutants mapped to mutH, mutL, or mutS, suggesting that in vivo- and in vitro-selected 2-APr derivatives have mutations in similar mut loci.

In vivo-selected 2-APr derivatives are less mutagenic than Salmonella strains containing defined null mutations in dam and mutH. Since dam mutants are mutagenic (43) and the elevated mutation rate is exacerbated in combination with other mutations in methyl-directed mismatch repair (23), we compared the mutability of the in vivo-selected 2-APr mutants, which contain mutations in dam and mut loci, to that exhibited by strains containing defined null mutations in dam and mutH, alone and in combination. As expected, the frequency of spontaneous streptomycin-resistant mutants increased in a dam mutant (35-fold), a Dam-overproducing strain (196-fold), a mutH mutant (456-fold), and a dam mutH mutant (1,508-fold) relative to the dam+ parent strain (Table 4). Although the in vivo-selected 2-APr mutants exhibited an increase in mutation frequency compared to the parental dam mutant strain, the mutability of the seven 2-APr mutants (69- to 296-fold relative to the wild type) was lower than that exhibited by a strain containing null mutations in dam and mutH (1,508-fold; MT2937). These data suggest that in vivo-selected 2-APr isolates contain mut alleles that do not cause a complete loss of function and/or that additional mutations have been selected that reduce the high mutability inherent to dam mutH mutant strains constructed in vitro.


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  		TABLE 4. In vivo-selected 2-APr derivatives of dam mutant Salmonella exhibit a reduced mutation frequency compared to that of a defined dam mutH mutant strain

To discern between these two possibilities, methyl-directed mismatch repair was abrogated in all in vivo-selected 2-APr mutants by introduction of a defined mutH null allele ({Delta}mutH358::Kmr) by standard allelic replacement (15). All (seven of seven) 2-APr mutant derivatives harboring the null mutation in mutH exhibited the reduced mutation rate similar to that of the in vivo-selected 2-APr parent, indicating that an extragenic suppressor mutation(s) accounts for the reduced mutation rate (data not shown).

In vivo-selected 2-APr derivatives exhibit elevated resistance to nitric oxide while retaining the bile-sensitive phenotype of the dam mutant parental strain. The induced production of nitric oxide is an important immune defense mechanism for inhibiting the intracellular growth of Salmonella residing within infected macrophages (1, 58, 59). To understand the molecular basis by which 2-APr derivatives of dam mutant Salmonella are selected for and/or are maintained systemically within infected animals and to understand why such mutants regained virulence following intraperitoneal but not oral infection of naïve mice, we assessed the relative sensitivity of such 2-APr derivatives to growth under conditions containing the nitric oxide source acidified sodium nitrate (ASN) (5). The parental dam mutant strain (MT2188) was threefold more sensitive than the wild type (14028) to growth with 1.5 mM ASN (Table 5). Introduction of a defined mutH mutation suppressed the ASN sensitivity of dam strains: the dam mutH strain (MT2937) was ninefold more resistant than dam mutant cells and fourfold more resistant than the wild type (dam+) to growth with 3.0 mM ASN. Similarly, the in vivo-selected 2-APr derivatives, which contain both dam and mutHLS mutations (Table 3), exhibited a 3- to 11-fold-elevated ASN resistance relative to the dam mutant parental strain (1.5 mM and 3.0 mM ASN). These data indicate that mutHLS mutations suppress the nitric oxide sensitivity inherent to dam mutant cells.


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  		TABLE 5. In vivo-selected 2-APr derivatives of dam mutant Salmonella exhibited elevated resistance to nitric oxide and retained the bile-sensitive phenotype of the dam mutant parental strain

Pathogenic strains of Salmonella are resistant to bile and exploit bile concentrations as a signal for the temporal and spatial production of virulence factors required for intestinal survival and for the induction of other adaptive mechanisms (26, 51). Mutants that lack or overproduce dam are highly sensitive to bile (3, 29, 53). To examine the failure of 2-APr derivatives of dam mutant Salmonella to be recovered from systemic tissues after oral infection and to understand why they are restored for virulence after intraperitoneal but not oral infection of naïve mice, we assessed whether such mutants retained the inherent bile sensitivity phenotype of the parental dam mutant strain (MT2188). MT2188 is >7-fold more sensitive than the wild type to growth with 5% bile (Table 5). The in vivo-selected 2-APr derivatives as well as the dam mutH strain (MT2937) retained much of the enhanced bile sensitivity of the parental dam mutant strain. Such a bile-sensitive phenotype may contribute to their attenuated virulence by the oral route of administration.

mutH mutation restores the overexpression defect in SpvB production associated with dam mutant cells. dam mutants express in vitro a number of genes that are normally preferentially expressed during bacterial infection (29, 30). For example, dam mutants overexpress spvB, encoding the SpvB actin cytoxin (40), which is required for Salmonella survival in systemic tissues (24, 25). To understand the molecular basis of the capacity of mismatch repair deficiencies to restore intraperitoneal virulence to dam mutant cells, we assessed the effect of defined null alleles in mutH and dam, alone and in combination, on the production of SpvB. Since SpvB expression under laboratory growth conditions is relatively low in serovar Typhimurium strains compared to the closely related S. enterica serovar Dublin (67), we used serovar Dublin for the SpvB expression analysis. As expected, the lack of dam resulted in an eightfold induction of SpvB relative to wild-type cells grown under laboratory conditions (Fig. 2). In contrast, introduction of a mutH mutation into dam mutant cells resulted in only a threefold induction of SpvB relative to the wild type, resulting in levels exhibited by mutH mutant cells. This is consistent with microarray analysis in which the global gene expression pattern of a number of genes in dam mutS mutant cells of Escherichia coli was intermediate relative to that observed in wild-type and dam mutant cells (55). Taken together, these data suggest that mismatch repair deficiencies are correlated with the restoration of defects in systemically related virulence functions, such as nitric oxide resistance and SpvB production, which are inherent to dam mutant cells.


Figure 2
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  		FIG. 2. mutH mutation suppresses the elevated SpvB production inherent to dam mutant cells grown in vitro. Whole-cell protein extracts corresponding to ~107 cells from the wild type as well as dam (MT2255), mutH (MT2999), and dam mutH (MT3000) derivatives of S. enterica serovar Dublin, Lane strain (8), were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. Membranes were probed with SpvB primary antiserum that was purified by passage through a Sepharose column bound to proteins derived from SpvB– serovar Typhimurium ({Delta}spvB121; MT2891). Whole-cell protein extracts were serially diluted twofold for SpvB quantitation relative to that in wild-type cells. Signal was detected as described in Materials and Methods.


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DISCUSSION
 
The commercial success of any vaccine is dependent on the therapeutic index, i.e., the ratio of the efficacy of the therapy to the toxicity, and the principal safety concern of modified live vaccines is reversion to virulence. Here we examined the safety of a dam mutant Salmonella vaccine by screening within infected mice for derivatives that have an increased capacity to cause disease relative to the virulence-attenuated parental dam mutant strain. Mutants that were productive for the infective process following the intraperitoneal but not the oral route of infection were isolated. The molecular basis of intraperitoneal virulence reversion correlated with deficiencies in methyl-directed mismatch repair that may suppress defects in the production of systemically related virulence functions.

Elucidation of the molecular mechanisms underlying microbial virulence provides key insights into the requisite parameters that dictate the elicitation of effective innate and adaptive immune responses. Salmonella is cytotoxic to M cells, antigen-sampling cells that reside in the follicle-associated epithelium above the Peyer's patch (35). This is followed shortly thereafter with the infection of neighboring enterocytes and additional bacterial spread into the surrounding lamina propria (34). Salmonella also infects cell types residing in the lymphoid organ itself, such as macrophages, dendritic cells, and T and B cells, all of which represent known cellular targets (9, 35, 68). These early events, coexisting in time with the mounting of the host inflammatory innate defenses, cause severe damage to the affected lymphoid organ (24). Such a generalized toxicity to Peyer's patch cellularity might functionally retard the initiation of adaptive immune responses to Salmonella antigens, and the sheer magnitude of the inflammatory process caused by infection might have a modifying influence on new and preexistent immune responses. In contrast to the wild-type infective process, dam mutant Salmonella is attenuated for virulence (21, 30) and elicits potent states of cross-protective immunity against heterologous serovars (18, 29, 47). The molecular basis of attenuation and protection appears to be the result of a number of changes in the physiology of dam mutant bacteria, including changes in the levels of bacterium-associated and secreted proteins, a reduction in the ability of the bacteria to invade nonphagocytic cell types, and a minimal cytotoxicity to M cells in vivo (21, 29, 53). Consequently, pathways involved in antigen processing and presentation by dendritic cells and the ability to effectively initiate antigen-specific T- and B-cell responses within the Peyer's patch may be better preserved in animals exposed to dam mutant than in animals exposed to dam+ Salmonella.

Safety is the primary concern for any vaccine, particularly in the case of modified live attenuated vaccines that have the capacity to revert to full virulence. The safety of dam mutant vaccines was examined by screening within infected mice for 2-AP-resistant organisms that have an enhanced capacity to cause disease. Such 2-APr cells were recovered from infected tissues following intraperitoneal but not oral infection and were shown to be competent for the infective process via intraperitoneal but not oral infection. The molecular mechanism of reversion of dam mutant cells to intraperitoneal virulence was shown to be associated with deficiencies in methyl-directed mismatch repair that were correlated with the restoration of virulence functions required for systemic survival: mut mutations suppressed defects in nitric oxide sensitivity and SpvB actin cytotoxin overproduction. This may reflect an enhanced capacity to survive within infected macrophages and splenocytes, contributing to enhanced intraperitoneal virulence. Such restoration of SpvB expression is consistent with E. coli microarray analysis in which the overexpression of many genes in dam mutant cells (41, 50) is suppressed in mismatch repair-deficient cells (55). However, the molecular mechanism by which gene expression is restored in mismatch repair-deficient dam mutant cells is poorly understood (55).

The virulence of the in vivo-selected 2-APr derivatives of dam mutant Salmonella following intraperitoneal challenge remains a valid safety concern for dam mutant vaccines. However, the relative safety of the vaccine by the natural oral route of administration was supported by the failure to recover 2-APr mutants from systemic tissues after oral infection and the avirulent phenotype of 2-APr mutants following an oral infection of naïve mice. These data suggest that 2-APr mutants are defective for survival within the gastrointestinal tract and/or lack the capacity to disseminate and/or survive within systemic tissues after introduction by the oral route. Consistent with this hypothesis, in vivo-selected 2-APr isolates retained the bile-sensitive phenotype of the parental dam mutant vaccine strain. This may contribute to the safety of the vaccine when orally administered, since bile is utilized by Salmonella as a signal for the temporal and spatial production of virulence factors required for intestinal survival and for the induction of other adaptive mechanisms such as antimicrobial resistance (26, 51). These results are consistent with a previous reports indicating that mismatch repair deficiencies suppress the virulence defect of dam mutant cells (52), although the 2-APr derivatives retained the bile-sensitive phenotype at the relatively high bile concentrations used in these studies. Additionally, a mutation in a Salmonella outer membrane component (enterobacterial common antigen) conferred a bile-sensitive and an attenuated-virulence phenotype following an oral but not intraperitoneal infection, similar to the phenotype exhibited by the 2-APr derivatives reported here (54).

dam mutants exhibit a mutator phenotype due to the lack of strand specificity in methyl-directed mismatch repair (43). Overexpression of dam also leads to a mutator phenotype, which may reflect the inability of the methyl-directed mismatch repair machinery to act on fully methylated DNA trailing the replication fork (31, 71). Mismatch repair-deficient cells exhibit a mutator phenotype that is exacerbated when in combination with a dam deficiency (23). The physiological relevance of microbial mutator genotypes is demonstrated by the fact that they are prevalent in natural populations of pathogenic (39) and commensal (45) bacteria, as well as in bacteria grown under defined laboratory conditions (65). Presumably, mutator genotypes are prevalent since rare beneficial mutations may arise more frequently and undergo more rapid selection relative to that exhibited by the wild type (66). Thus, mutator cell populations may respond more effectively to the dynamic challenges encountered during the microbial life cycle. However, high mutation rates can be deleterious, as it is likely that most mutations would not enhance cell fitness (16); additionally, high mutation rates may not be advantageous to distinct populations of beneficial variants, as a superior strain will eventually drive all other variants to extinction (2). Consistent with this notion, although the mutation frequency of 2-APr isolates was elevated with respect to that of the parental dam mutant strain, they exhibited a reduced mutation frequency (via extragenic suppression) relative to a defined dam mutH null mutant constructed in vitro. However, the infectivity of 2-APr isolates was not enhanced relative to that of the defined dam mutH mutant strain as evidenced by similar virulence capacities in LD50 and CI assays.

Safety issues, cost, ease of administration, and the ability to confer strong and long-lasting protection against disease represent parameters associated with vaccine development that are always open to further improvement. This is especially the case as we learn more about the mechanisms underlying microbial virulence and the subtle nuances associated with the innate and adaptive immune mechanisms possessed by susceptible hosts. Although the relative safety of modified live dam mutant Salmonella vaccines was established via the oral route of administration, safety remains a valid concern since derivatives that were competent via the intraperitoneal route have been isolated. Commercial success of this vaccine can be further improved by the introduction of additional attenuating and/or epistatic mutations that do not compromise the cross-protective capacity or the early- and late-onset immunity conferred by the current formulation.


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ACKNOWLEDGMENTS
 
We are grateful to Don Guiney (UCSD) for the gift of SpvB antibody and to Bill Shimp (UCSB) and Diana Bareyan (Utah) for technical assistance in the construction of a mutH null mutant used in these studies and in the nitric oxide assay, respectively.

This work was supported by G. Harold & Leila Y. Mathers Foundation, National Institutes of Health (NIH) grants AI 61399-01 and AI 59242-04A1, and National Research Initiative of the USDA Cooperative State Research, Education and Extension Service grant 2004-04574 (to M.J.M.).


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, CA 93106. Phone: (805) 893-7160. Fax: (805) 893-4724. E-mail: mahan{at}lifesci.lscf.ucsb.edu Back

{triangledown} Published ahead of print on 27 April 2007. Back

{dagger} Present address: Department of Biology, Westmont College, 955 La Paz Rd., Santa Barbara, CA 93108. Back


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Journal of Bacteriology, July 2007, p. 4708-4717, Vol. 189, No. 13
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