Analysis of a set of recombinant congenic strains

Our preliminary data thus far suggest that susceptibility to tuberculosis in our strain combination is a multigenic trait. Major advances in the analysis of the multigenic traits were made possible recently with the development of the high density genome maps of humans and mice using microsatellite markers (reviewed by Lander & Schork 1994). A different strategy is based on the premise that when the trait of interest is controlled by a limited number of genes they could be separated by random breeding, and thus can be studied either individually or in a setting in which the complexity of interactions is significantly reduced. The concept and production of the unique set of inbred recombinant congenic strains (RCS) is described in detail by Demant and colleagues (Demant & Hart 1986, Groot et al 1992). Briefly, an initial cross was made between two distinct inbred strains C3H/HeSnDem (which is susceptible to M. tuberculosis) and C57Bl/B10. The next two generations were produced by backcrossing, without selection, to one of the parental strains (recipient, C3H). This was followed by strict brother—sister mating for more than 14 generations in order to produce a set ofhomozygous inbred strains (HcB/Dem series), which have mosaic genomes. On average each strain carries a random 12.5% fraction of the donor genome (B10 in our case) and the rest of the genome is of recipient origin (C3H). The RCS analysis offers two unique advantages over classical genetic analysis: (i) epistatic effects of other genes that can confound analysis of multiple genetic pathways that affect the resistant or susceptible phenotype can be eliminated by separation of the parts of the donor genome (Frankel & Schork 1996, Van Wezel et al 1996, Fijneman et al 1996); and (ii) the RCS are homozygous inbred strains, which can be studied for functional activities long before the classical approach would permit meaningful immunological and physiological analysis.

The HcB/Dem series consists of 42 strains, of which 28 strains were obtained by inbreeding after two backcrosses to C3H background and thus theoretically contain about 12.5% of the B10 genome, on average. Another 14 strains are the progeny of N4 (three backcrosses to C3H) and thus contain half the amount of B10 genome (6.25% on average). All 42 strains were tested for their susceptibility to infection with 106 CFU of M. tuberculosis at least twice in three independent experiments

TABLE 2 Distribution of survival phenotypes of recombinant congenic strains of HcB/Dem series

Phenotjpe

Mean survival time (days)

No. of recombinant congenic strains

As resistant as BIO <150

More resistant than C3H 150-100 Intermediate susceptibility 100-50

Supersusceptible < 50

4 19

(Table 2). The 21 strains that were the most resistant or susceptible were genotyped using microsatellite markers in order to obtain genetic maps with the marker intervals of 5-10 cM. According to computational analysis, more than 90% of donor chromosomal segments should be detected at that density of the markers. From the maps it is clear that the strains differ in the proportion of donor genome: the genotyped strains have between four and 10 chromosomes containing donor segments of varying sizes. It was clear that no B10 chromosomal segment is common to all resistant or all susceptible mice, and that no identical or overlapping patterns for RCS, either resistant or susceptible to tuberculosis, were observed.

The results of these experiments confirm our previous finding in (C3HXB6)F2 of a multigenic control of resistance/susceptibility to tuberculosis in our strain combination. Were there single-gene control, all strains would be segregated into two groups with polar phenotypes representing the phenotypes of the parental strains. In reality, survival time forms a continuous distribution pattern that is the hallmark of multigenic control. Also, it was clear that a number of strains are significantly more susceptible than our susceptible parental strain: strains 10,15,18, 22 and 31 died within 31-47 days after infection in two independent experiments, whereas the MST for the parental C3H/HeSnDem substrain in our experiments was 65-75 days. Importantly, the survival times of these supersusceptible strains in our experiments are not significantly different from the survival times of mice rendered immunodeficient by y-interferon or inducible nitric oxide synthase (iNOS) gene knockouts (Flynn et al 1993, MacMicking et al 1997). This observation suggests that some segments of B10 chromosomes introduced into those particular strains contain gene(s) which dramatically exacerbate susceptibility to M. tuberculosis upon interaction with the C3H background. Therefore, the F2 hybrids between the supersusceptible strains HcB-22 and C3H are being used for the mapping of that gene(s).

In order to correlate the survival of RCS with other parameters of the disease progression, we compared the bacterial growth in the lungs and spleens of two resistant and two supersusceptible RCS. The virulent M. tuberculosis grew progressively in the spleens and livers of all strains of mice for the initial seven days of infection. Few bacteria were detected in the lungs on the seventh day of infection. This early seeding of the lungs was detected in both resistant and susceptible mice and was confirmed by acid-fast fluorescent staining of the lung tissue. Once the bacteria seeded the lungs, they grew progressively in all strains of mice including the F1 hybrids (most resistant), although the growth rates, being the best correlate of the survival times, differed significantly among resistant and susceptible mice. Two strains of supersusceptible mice showed indistinguishable kinetics of the CFU in their lungs, albeit the growth in the spleens was different. The bacterial loads in the spleens and livers of HcB-18 were higher than the number of CFU per lung. This strain is characterized by the

FIG. 4. Lungs of two mice of supersusceptible recombinant congenic strains at a time of death. Left, HcB-22; right, HcB-18. Bar = 2 cm.

generalized progressive infection. On the contrary, in HcB-22 the number of viable bacteria per organ did not increase in the spleens and livers between Day 7 and 25 of infection, whereas M. tuberculosis grew progressively only in the lungs of mice that developed dramatic macroscopical lesions (Fig. 4). Microscopic analysis revealed large areas of necrosis in the lungs of that strain, with tissue debris loaded with M. tuberculosis in the lumen of the airways, which is unusual for murine tuberculosis. Therefore, we assume that this strain (HcB-22) could be particularly useful for studying the mechanisms and underlying genetic control of the higher vulnerability of the lung tissue to M. tuberculosis.

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