Major Histocompatibility Complex Heterozygote Superiority during Coinfection

Genes of the major histocompatibility complex (MHC) play a criticalrole in immune recognition, and many alleles confer susceptibilityto infectious and autoimmune diseases. How these deleteriousalleles persist in populations is controversial. One hypothesispostulates that MHC heterozygote superiority emerges over multipleinfections because MHC-mediated resistance is generally dominantand many allele-specific susceptibilities to pathogens willbe masked by the resistant allele in heterozygotes. We testedthis hypothesis by using experimental coinfections with Salmonellaenterica (serovar Typhimurium C5TS) and Theiler's murine encephalomyelitisvirus (TMEV) in MHC-congenic mouse strains where one haplotypewas resistant to Salmonella and the other was resistant to TMEV.MHC heterozygotes were superior to both homozygotes in 7 outof 8 comparisons (P = 0.0024), and the mean standardized pathogenload of heterozygotes was reduced by 41% over that of homozygotes(P = 0.01). In contrast, no heterozygote superiority was observedwhen the MHC haplotype combinations had similar susceptibilityprofiles to the two pathogens. This is the first experimentalevidence for MHC heterozygote superiority against multiple pathogens,a mechanism that would contribute to the evolution of MHC diversityand explain the persistence of alleles conferring susceptibilityto disease.

INTRODUCTION

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Introduction

Major histocompatibility complex (MHC) genes are the most diversegenes known. One of the postulated mechanisms for maintainingthis diversity is MHC heterozygote superiority, or overdominance.The phrase heterozygote advantage (7, 12) is often used as asynonym for heterozygote superiority but is also used for othermeanings (36, 38). In population studies, it is used in thegeneral sense to imply that the mean fitness of heterozygotesis higher than the mean fitness of all homozygotes (8, 47).In laboratory studies and evolutionary models, it is often usedin the narrow sense to imply that heterozygotes are superiorto both parental homozygotes (46). To avoid this confusion,we have defined heterozygote advantage to mean that the heterozygoteis better than the average of both homozygotes and we have definedheterozygote superiority to mean that the heterozygote is betterthan both homozygotes (overdominance) (36). This distinctionis important because heterozygote advantage by itself cannotcontribute to MHC diversity, whereas heterozygote superioritycan (46).

For any given autoimmune or infectious disease, some MHC allelesare usually found that confer susceptibility (3, 47). It isunclear what keeps these demonstrably harmful genes from beingeliminated by natural selection. Some form of balancing anddiversifying selection must be operating consistently over longperiods of evolutionary time to explain the relatively uniqueand extreme features of MHC diversity (2). This MHC diversityis generally thought to result from pathogen-mediated selection,through either heterozygote superiority or antagonistic host-pathogencoevolution (7, 17). Both mechanisms favor rare and new alleles(selection for divergence) and also maintain large numbers ofalleles because allelic extinction rates are reduced. Moreover,both mechanisms predict the existence of some MHC alleles (orgenotypes) that confer susceptibility to certain diseases. Inthe case of heterozygote superiority, low-fitness homozygoteswill express a susceptible phenotype. In the case of antagonisticcoevolution, MHC alleles that have been the target of recentpathogen evasion (evolution) will be susceptible.

MHC heterozygotes are expected to be superior to both homozygotesbecause they can present a wider variety of peptide antigensto T lymphocytes (12), the triggering event of the adaptiveimmune response (51). Although this is the mechanistic explanationgenerally given for heterozygote superiority, single-infectionexperiments do not support this hypothesis: heterozygotes areusually similar to the resistant homozygote but are seldom better(2). This is not surprising, since most immune responses aredominated by T-cell recognition of one or a few MHC-presentedpeptides (immunodominance) (54) rather than all or most of thepresented peptides. Therefore, presentation of more peptideantigens by MHC heterozygotes may have little effect on theimmune recognition of pathogens.

Still, heterozygote superiority is expected to emerge afterserial or simultaneous coinfections (12, 24) when resistanceis dominant and the susceptibility profiles of two pathogensare opposite or reciprocal (39). We will refer to this as thedual-infection heterozygote superiority (DIHS) hypothesis. Heterozygotesshould be superior under these two conditions because, for anypathogen, the susceptibility of the worst allele would be maskedby the resistant allele (Fig. 1). In fact, the literature doesindicate that MHC-mediated resistance to infections is generallydominant (63% of 49 haplotype-pathogen combinations tested and79% of 13 pathogens tested) (Table 1). Resistance being dominantprovides a mechanistic basis for how MHC heterozygote superioritycan occur during dual infections but not during single infections.It also suggests that immunodominance generally favors T-cellresponses associated with the more-resistant allele (thoughthis mechanism is not yet characterized) (54).

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FIG. 1. Hypothetical MHC-associated pathogen loads for three pathogens and the predicted combined pathogen loads during different dual infections. MHC genotypes infected with single pathogens (a, b, and c) show different patterns of susceptibility. Combined pathogen loads during dual infections (d and e) are obtained by adding the single pathogen loads for each genotype and standardizing the axes. When animals are infected with two pathogens that show opposite susceptibility profiles (a and b), heterozygotes are predicted to be superior (d). When animals are infected with two pathogens that show similar susceptibility profiles (a and c), heterozygotes are predicted to be intermediate (e). MHC-mediated resistance is dominant in all cases and is required for the predicted patterns (2).

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TABLE 1. Survey of published literature to determine if MHC-dependent resistance is dominant or recessive^a

Therefore, the critical empirical test of the DIHS hypothesisrequires resistance to be dominant in experimental dual infectionswith pathogens and MHC combinations that show both opposite(Fig. 1d) and similar susceptibility profiles (Fig. 1e, negativecontrol). If MHC haplotypes confer opposite patterns of susceptibility(Fig. 1a and b), then heterozygotes should be superior wheninfected by both pathogens (Fig. 1d). However, if MHC haplotypesconfer similar patterns of susceptibility (Fig. 1a and c), thenheterozygotes should be intermediate between the two homozygotes(Fig. 1e).

In this study, we provide the first experimental test of theDIHS hypothesis by coinfection with the pathogens Salmonellaenterica (serovar Typhimurium C5TS) and Theiler's murine encephalomyelitisvirus (TMEV). Although serial infections are generally morecommon in nature, coinfections represent a potentially importantcase in which MHC-dependent escape variants emerge (causingdifferent susceptibility profiles) during the course of singleinfections, as seen for diversifying pathogens such as humanimmunodeficiency virus (HIV) (30), simian immunodeficiency virus(1), hepatitis B (5), hepatitis C (14), and human T-cell leukemiavirus (15). We demonstrate that MHC heterozygote superiorityemerges when resistance is dominant and MHC haplotypic combinationshave susceptibility profiles that are opposite. These resultsprovide a mechanism that could help explain the unprecedenteddiversity of MHC genes and the persistence of MHC alleles whichconfer disease susceptibility.

MATERIALS AND METHODS

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Materials and Methods

Mice. MHC-congenic mice (C57BL/10SnJ-H2^b, B10.D2-H2^d, B10.M-H2^f, B10.BR-H2^k,and B10.Q-H2^q) were obtained from Jackson Laboratories and bredthereafter under specific-pathogen-free conditions. In orderto randomize any genetic mutations that have become differentiallyfixed in these strains of mice (9), the F₁ heterozygotes (b/q,d/q, d/k, and f/k) were intercrossed to produce F₂ segregants,which were used for all infections. To control for potentialvariation among cages, mice were housed by sex in groups ofsix individuals representing the three genotypes to be compared(e.g., b/b, b/q, and q/q or d/d, d/q, and q/q). All q/q andk/k mice were analyzed according to their housing conditionsand thus not pooled for analysis (e.g., q/q mice housed withb/b and b/q mice were analyzed only with the b/q haplotypiccombination). All cagemates were unrelated and had not beenpreviously housed together when infected. The original coinfectionexperiment was repeated (see in Fig. 5, experiment 2) in anindependent experiment in which the F₂ mice were housed withF₁ and P₀ mice of the same genotype to evaluate any differencesbetween the generations (data not shown for F₁ and P₀). Allanimal use complied with federal regulations and the Universityof Utah's Institutional Animal Care and Use Committee guidelines.

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FIG. 5. Pooled standardized combined pathogen loads for genotypic combinations showing opposite (a and b) and similar (c) susceptibility profiles. The sexes and both experiments were pooled for the b/q and d/q combinations (a and b). All P values shown are from a Wilcoxon rank-sum test. Sample sizes are indicated above each bar.

Pathogens. Mice were anesthetized with metofane and simultaneously infectedretro-orbitally with 1 x 10⁶ CFU of the C5TS strain of S. enterica(serovar Typhimurium)/ml and intracerebrally with 2 x 10⁵ PFUof the DA strain of TMEV/ml. The C5TS strain is a temperature-sensitivemutant strain of Salmonella (22). As a control, the single infectionsalso received a sham infection of phosphate-buffered salineto control for the effects of two injections. Both agents arenatural pathogens of mice.

Salmonella loads. Mice were sacrificed 4 weeks postinfection. Salmonella loadswere assayed from platings of homogenized spleens. Briefly,spleens were collected under antiseptic conditions, immediatelyhomogenized, and plated on Shigella-Salmonella agar, which wasthen incubated overnight at 30°C. When this strain was createdin the early 1970s (22) with a UV screen, the authors foundthat a small (unspecified) percentage reverted to the wild-typeC5 virulent strain. This revertant phenotype was seen in 8.7%of the mice infected in this study. To check for possible wild-typerevertants, the spleen homogenate was also plated on Shigella-Salmonellaagar and incubated overnight at 37°C. If more than 50% ofthe colonies of an infected mouse also grew at 37°C, thenthe infection was deemed to be dominated by a revertant andthe data point was excluded on the basis that the revertantwas more lethal (i.e., faster replication rate than C5TS). Thus,comparing mortality or pathogen loads between animals that didor did not contain a revertant would be inappropriate. A totalof 205 (out of 1,061) animals were excluded either because theydied or were sacrificed prior to day 28 postinfection or becausethey contained Salmonella revertants. Of the excluded mice,78 died early, 94 were sacrificed prior to day 28 postinfectiondue to severe illness, 31 contained revertants, and 2 were excludeddue to unknown genotype. Of the 94 mice (8.9%) sacrificed early,61 contained revertants. The other 33 mice that were sacrificedearly were ill due to high TMEV loads or high C5TS loads. Forthe 78 that died early (7.4%), the cause of death was probablydue to a reversion in C5TS or high TMEV or C5TS loads. Therewas no association with MHC genotype in the excluded animalsthat died (or were sacrificed) early ( ${chi}$ ² = 4.914, P = 0.77).However, there was a significant association with MHC genotypein animals that contained revertants versus those that did not( ${chi}$ ² =20.515, P = 0.0086). The genotype with the highest numberof revertants was the most susceptible (b/b) while the genotypewith the lowest number of revertants was the most resistant(q/q), suggesting that the pattern may simply be due to mutationrate (i.e., more revertants would occur in hosts with fasterpathogen replication). We believe this makes our results moreconservative because the animals with the high pathogen loadswere disproportionately thrown out, making differences smaller.

TMEV loads. TMEV loads were assayed by conducting viral plaque assays onbrain and spinal cord homogenates (26). Briefly, brain and spinalcords were collected and stored in 0.5 ml of TMEV diluent (phosphate-bufferedsaline, 1% antibiotics, 1% fetal calf serum) at -70°C untilassayed. Brain and spinal cords were homogenized and freeze-thawedtwice. Samples were then centrifuged at 450 x g for 10 min,and the supernatant was collected. Supernatant fluid was thenadded to baby hamster kidney-21 (BHK) cells and incubated for1 h at 37°C, with hand rocking every 10 to 15 min. The fluidwas then aspirated off the BHK cells, and a 1:1 mixture of 1%agarose-2x 199 medium was added to the wells. Plates were incubatedat 37°C for 4 days before fixation with 2.5% formalin followedby staining with 0.1% crystal violet and enumeration of plaques.

Standardized combined pathogen loads. Because the actual pathogen loads of Salmonella and TMEV differedby at least 1 order of magnitude, we standardized the loadsto make them comparable and to obtain an overall pathogen loadfor every mouse. Thus, the standardized combined pathogen loadwas calculated as follows. The mean Salmonella and TMEV loadswere determined for each MHC genotype. For example, the Salmonellaloads of each b/b, b/q, and q/q mouse in the group were dividedby the mean of the worst genotype and multiplied by 100. Allother genotypic combinations were calculated in the same way.This was repeated for the TMEV loads, and then the Salmonellaand TMEV percents were averaged for every mouse. Thus, the overallpathogen load for every mouse was standardized by sex and experiment,allowing all of the data to be pooled. This standardized combinedpathogen load gives equal weight to both pathogens. This maynot be true in relation to actual fitness impacts on the host,but at this point we have no way to test for possible fitnessdifferences.

Statistics. Male and female animals were analyzed separately because theyhad significantly different pathogen loads (Fig. 2 and 3). Becausesex, genotype, and experiment were independent (Fig. 4), a binomialdistribution was used to test whether the results of these independentexperiments showed a significant pattern of heterozygote superiority.Since there were three possible patterns, (i) overdominance(the heterozygote was better than both homozygotes), (ii) underdominance(the heterozygote was worse than both homozygotes), or (iii)codominance (the heterozygote was intermediate between the twohomozygotes), a value of 1/3 was used for P (the probabilityof obtaining any one of the three patterns). All other comparisonswere analyzed with the Wilcoxon rank-sum test, unless otherwisestated. All pathogen loads were determined without knowledgeof the MHC genotype.

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FIG. 2. Salmonella and TMEV loads for single infections. Pathogen loads of female (a) and male (c) animals of different MHC heterozygote combinations showing opposite susceptibility profiles. Pathogen loads of female (b) and male (d) animals of different MHC heterozygote combinations showing similar susceptibility profiles. In the case of opposite susceptibility profiles (a and c), all homozygotes were significantly different (P < 0.05 by t test), except the following: Salmonella loads for the d/d and q/q genotypic combinations in females (P = 0.08), TMEV loads for the d/d and k/k genotypic combinations in females (P = 0.17), and TMEV loads for the d/d and k/k genotypic combinations in males (P = 0.08). Sample sizes are indicated above each bar. spcd, spinal cord.

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FIG. 4. Standardized combined pathogen loads for animals showing opposite susceptibility profiles. We calculated the combined pathogen load from the individual Salmonella and TMEV loads for the two different genotypic combinations showing opposite susceptibility profiles, b/q (a, c, e, and g) and d/q (b, d, f, and h). This was done for two independent experiments and for each sex. Sample sizes are indicated above each bar.

RESULTS

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Results

To verify that the MHC haplotypic combinations that we chosefrom the literature indeed showed opposite (or similar) susceptibilityprofiles, we evaluated these profiles in both single infectionsand coinfections. The results of single-infection experimentswith the pathogen Salmonella or TMEV are indicated in Fig. 2.MHC had a significant effect on resistance, as predicted fromthe published literature (34, 43). MHC haplotypic combinations(b with q, d with q, and d with k) showed opposite susceptibilityprofiles, whereas the MHC haplotypic combination (f with k)showed similar susceptibility profiles when comparing thesetwo pathogens. Resistance was usually dominant (11 out of 16comparisons), with some exceptions. These haplotypic combinationswere then coinfected, and pathogen loads were determined 4 weekspostinfection.

Figure 3 illustrates the pathogen loads for MHC genotypes ofcoinfected mice showing either opposite (Fig. 3a and c) or similar(Fig. 3b and d) patterns of susceptibility. Female data aredepicted in Fig. 3a and b, and male data are shown in Fig. 3c and d.As expected, the MHC b/b and q/q genotypes show oppositepatterns of susceptibility to Salmonella and TMEV, as do theMHC d/d and q/q genotypes (Fig. 3a and c). Also as expected,the MHC f/f and k/k genotypes show similar patterns of susceptibilityto these two pathogens. However, the MHC genotypes d/d and k/k(Fig. 3b and d) showed similar patterns of susceptibility toSalmonella and TMEV, the reverse of what we found in the single-infectionexperiments (Fig. 2).

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FIG. 3. Salmonella and TMEV loads for coinfected mice of either opposite or similar susceptibility profiles. Pathogen loads are given for coinfected female (a) and male (c) animals of different MHC heterozygote combinations showing opposite susceptibility profiles. Pathogen loads are given for coinfected female (b) and male (d) animals of different MHC heterozygote combinations showing similar susceptibility profiles. In the case of opposite susceptibility profiles (a and c), all homozygotes were significantly different (P < 0.05 by t test), except TMEV loads for the b/b and q/q genotypic combinations in males (P = 0.07), and resistance was dominant (pathogen loads of heterozygotes were more like the resistant than the susceptible homozygote). In the case of similar susceptibility profiles, all homozygotes were significantly different (P < 0.05 by t test) for the Salmonella infection but not for the TMEV infection (P > 0.60). Sample sizes are indicated above each bar.

This apparent reversal of susceptibility patterns between singleinfections and coinfections suggests there is an interactionbetween these two pathogens during coinfection and these specificMHC genotypes, as seen in other coinfections such as HIV andmany parasitic diseases (19). When we tested this hypothesis,there was not a significant difference in the overall patternof susceptibility (opposite or similar) between single infectionsand coinfections (data not shown). However, there was no significantdifference between the d/d and k/k homozygotes for either thesingle infections (Fig. 2) or coinfections (Fig. 3). Thus, forthis genotypic combination, we cannot determine whether theTMEV loads show an opposite or similar pattern of susceptibility,and so we excluded them from further analysis. All patternsseen in single infections and coinfections (Fig. 2 and 3) aresimilar to those seen in previous studies (34, 43), except thed/k genotypic combination.

Figure 4 compares the results of eight coinfection groups ofMHC genotypic combinations showing opposite (reciprocal) patternsof susceptibility. These eight experiments represent comparisonsof two haplotype combinations (b with q plus d with q), bothsexes, and two independent experiments. The pattern of heterozygotesuperiority was observed for standardized combined pathogenloads in seven of the eight groups (P = 0.0024 by binomial distribution,where P = 1/3). Even if we conservatively excluded the two groupswhere the heterozygote load was similar to the best homozygote(Fig. 4d and h), the pattern of heterozygote superiority isstill significant (five out of six groups, P = 0.01 by binomialdistribution, where P = 1/3). Analyzing all eight groups, thestandardized combined pathogen load in heterozygotes was 41%lower than the load in homozygotes (P = 0.01, by the Wilcoxonrank-sum test).

To estimate the relative strength that each genotypic combinationcontributed to the overall significant pattern of heterozygotesuperiority (Fig. 4), we pooled the standardized data acrosssex and experiments for each genotype. Figure 5 illustratesthe standardized combined pathogen loads for the genotypic combinationsshowing opposite (Fig. 5a and b) and similar (Fig. 5c) susceptibilityprofiles. For opposite susceptibility profiles, the b with qhaplotypic combinations showed the largest effect, with pathogenloads of the b/q heterozygotes approximately 71% lower thanthose of both the b/b and q/q homozygotes (P < 0.0001 andP = 0.04, respectively, by the Wilcoxon rank-sum test) (Fig.5a). The d and q genotypic combination (Fig. 5b) showed a nonsignificantpattern of heterozygote superiority, and the relative strengthwas smaller than that of b and q, with pathogen loads of thed/q heterozygote being 9% lower than those of d/d homozygotesand 20% lower than those of q/q homozygotes. When the MHC genotypiccombination showing similar patterns of susceptibility was examined,MHC f/k heterozygotes were worse than both homozygotes (Fig.5c). This nonsignificant pattern may be due to the susceptibilitybeing dominant for the Salmonella infection (Fig. 3b and d).

We used pathogen load as a measure of host immunocompetencedue to its strong inverse correlation with overall host fitness(e.g., HIV) (41). We tested the effects of MHC genotype on deathand weight, but no significant differences were found (datanot shown). These infections were generally benign, with theseyoung, growing animals gaining weight during the infection,although at a slower rate than their uninfected siblings (datanot shown).

DISCUSSION

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Discussion

This is the first experimental evidence for MHC heterozygotesuperiority, a pathogen-mediated mechanism that could contributeto the evolution of MHC diversity. A pattern of MHC heterozygotesuperiority was seen in 7 out of 8 comparisons (P = 0.0024)(Fig. 4), and the mean standardized load of the heterozygoteswas 41% lower than the mean of the homozygotes (P = 0.01). Thepooled data also showed a significant pattern of heterozygotesuperiority for the b/q genotypic combination (Fig. 5). Thenegative-control combination of f and k haplotypes did not showa heterozygote superiority pattern. This control excludes thealternative hypothesis that MHC heterozygotes have a generaladvantage under all coinfections, as would be the case if thebasis of MHC heterozygote superiority were the presentationof more peptide antigens.

For the DIHS hypothesis to be a general explanation (at leastin part) for MHC diversity, the infections experienced overthe lifetimes of individuals must in general lead to heterozygotesuperiority. This requires two relationships. First, MHC heterozygotesmust be superior in dealing with infections separated in time,as this will be more common than coinfections. While our experimentsused simultaneous dual infections, there are no a priori reasonsthat multiple infections separated in time would not also leadto heterozygote superiority. The combined pathogen load, althoughoccurring over a longer time period, would still be expectedto be lower for heterozygotes. Second, the susceptibility andresistance patterns of host MHC alleles to natural pathogensmust be somewhat balanced or the worst alleles will become extinct(28). A recent survey of MHC-dependent susceptibility and resistancepatterns across a number of pathogens shows that any given haplotypetends to be resistant to some infectious agents and susceptibleto others (37). Whether these two relationships are generallytrue is an empirical question and remains to be tested.

Simulation models demonstrate that symmetrical heterozygotesuperiority (where fitness of heterozygotes is greater thanthat of homozygotes and all homozygotes and heterozygotes haveequal lifetime fitnesses within these two genotypic classes)is a powerful diversity-maintaining force that can account forMHC diversity (46). The degree of asymmetry that can be toleratedand still maintain MHC diversity is more controversial (19,28). This study provides evidence for the hypothesis that heterozygotesuperiority can emerge under conditions where resistance isdominant and susceptibility profiles are opposite, which arecommonly observed (37) (Table 1). However, determining whetherheterozygote superiority emerges under the variety of pathogensexperienced in the wild will be difficult, as it will requiremeasuring lifetime fitness.

Observational evidence for heterozygote advantage (which mayor may not be due to heterozygote superiority) (38) has beenreported for HIV (8) and hepatitis B virus (47) in humans. Althoughthese studies found heterozygote advantage for single infections,the specific pathogens involved can emulate a multiple infection.Both HIV (40, 42) and hepatitis B virus (23) are polymorphicinfections due to mutations that accumulate rapidly during chronicinfection. Also, both HIV (30) and hepatitis B virus (5) havebeen shown to escape MHC-dependent immune recognition, so itis possible that some of these polymorphisms could contain viralvariants that would create opposite susceptibility profilesin MHC-heterozygous hosts. Both studies propose that MHC heterozygoteshad an advantage because they present more viral epitopes, oras found in the HIV study, that MHC heterozygotes may preventthe escape of MHC-dependent immune recognition. The DIHS hypothesisprovides a new way to explain both the HIV and hepatitis B results.However, since both studies only compared heterozygotes to themeans of homozygotes, their results may simply be heterozygoteadvantage and not heterozygote superiority (36, 38), a resultexpected simply by MHC resistance being dominant over susceptibility(Table 1).

Simulation models demonstrate that small levels of (symmetrical)heterozygote superiority (approximately 1%) are sufficient toexplain the observed levels of MHC genetic diversity (44, 46).It is generally impossible to obtain sample sizes with vertebratesto statistically demonstrate a 1% effect, which may explainwhy MHC heterozygote superiority has been so hard to find inprevious studies (21). The 9 to 20% effect found for the d andq genotypic combination was not significant by itself (Fig.5), but when combined with the b and q genotypic combination,it contributed to the significance of overall heterozygote superiority(Fig. 4). The fact that both genotypic combinations showingopposite susceptibility profiles in this study were considerablylarger than 1% is not too surprising because the 1% requirementapplies to the combined effects of all pathogens over a lifetime.Any pathogen pair and genotypic combination that might showa large effect will be countered by other pathogen or genotypiccombinations showing small or opposite effects.

This is the first experimental evidence for MHC heterozygotesuperiority under coinfection, a mechanism that could explain(at least in part) the divergence of MHC genes and the maintenanceof high levels of allelic diversity found in nature. The excessof MHC heterozygotes found in some natural populations is consistentwith such a mechanism (20). However, these results do not excludeother proposed mechanisms of selection for MHC diversity suchas pathogen evasion, autoimmunity, mating preferences or otherreproductive mechanisms (2, 19). Any or all of them may be moreimportant than heterozygote superiority. Future studies willbe required to evaluate the relative importance of these proposedevolutionary forces for diversifying MHC genes and the functionalsignificance these polymorphisms have on vertebrate immunity.

ACKNOWLEDGMENTS

We thank Linda Morrison, Jane Libbey, and Elena Enioutina forexcellent technical assistance; Lara Carroll, Andrew Pacejka,Bradley Demarest, Sarah Zala, Patricia Slev, David Witherspoon,and Robert Fujinami for helpful comments; Carlos Hormaeche fordonation of the Salmonella strain C5TS; Jay Renolds, MaureenWilkinson, and numerous undergraduates for help with mouse care;and Fred Adler, Jon Seger, Pat Corneli, and Paul Bernhardt forstatistical advice.

This work was supported by an NIH grant to W.K.P. (GM39578)and partially by an NIH grant to Robert S. Fujinami (NS34497).

FOOTNOTES

* Corresponding author. Mailing address: Biology Department, University of Utah, 257 South 1400 East, Salt Lake City, UT 84112. Phone: (801) 585-9678. Fax: (801) 581-4668. E-mail: Potts{at}biology.utah.edu.

Editor: A. D. O'Brien

Present address: Konrad Lorenz Institute, Austrian Academy ofSciences, A-1160 Vienna, Austria.

REFERENCES

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