Multigenic control of tuberculosis resistance: analysis of a QTL on mouse chromosome 7 and its synergism with sst1

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Abstract

Tuberculosis remains a significant global health problem: one-third of the human population is infected with Mycobacterium tuberculosis (MTB) and 10% of those are at lifetime risk of developing tuberculosis. In the majority of individuals infected, genetic determinants of susceptibility remain largely unknown due to complex multigenic control and the influence of genes—environment interactions. Genetic variation of host resistance to MTB in animal models reflects heterogeneity among humans. Stepwise dissection of these interactions will permit the deciphering of MTB's complex virulence strategy. Previously, we have characterized a mouse supersusceptibility locus (sst1) controlling antituberculosis immunity. In this study, eight host resistance quantitative trait loci (QTLs) were mapped that counter-balance the devastating effect of sst1, among which a QTL on chromosome 7 (Chr7) was most prominent. The Chr7 and sst1 loci independently control distinct resistance mechanisms to MTB, but their effects apparently converge on macrophages in remarkable synergy. Combining these resistance alleles on a C3HeB/FeJ-susceptible background reduced the lung pathology and improved survival after MTB challenge accounting for half of the difference between susceptible and resistant parental strains. These data reveal novel gene interactions controlling MTB resistance and will enable the identification of resistance gene(s) encoded within Chr7 locus.

Introduction

Tuberculosis (TB) remains a significant global health threat; approximately one-third of the World's population is estimated to be infected with virulent Mycobacterium tuberculosis with almost 8 million new cases and nearly 2 million deaths annually.1 It is established that there is genetic heterogeneity in host population resistance to mycobacterial infection, this ranges from extreme susceptibility to avirulent mycobacteria to the effective control of fully virulent MTB. This heterogeneity is true of human populations, in which <10% of immunocompetent individuals develop clinical disease after infection with fully virulent MTB.2, 3, 4, 5 Host genetic variation is also true of experimental mouse, guinea pig and rabbit models of infection,6, 7, 8, 9 and without considering the contribution of host genetics, the development of more efficient drug and vaccine interventions will be challenging.

In most immunocompetent patients, TB is controlled systemically, but the lung is destroyed leading to propagation and transmission to new hosts through the aerosol route.3, 10 Host genetic variants that predispose immunocompetent hosts to TB by weakening their defenses enough to permit lung destruction and pathogen transmission remain to be elucidated. TB pathogenesis, a highly complex sequence of events, is multistaged with many cell types participating during chronic infection. One would expect a hierarchy of genetic factors controlling a network of biological processes. Untangling complex genetic control of TB susceptibility in humans is inherently difficult. Thus, animal models are used to identify susceptibility genes under controlled experimental conditions in which route of infection, dose, strain and virulence of M. tuberculosis as well as environmental factors, such as diet and stress, can be standardized (reviewed in Fortin et al.11 and North and Jung12). Natural variation in host genetic resistance to MTB can be addressed using forward genetic approaches, that is, from phenotype to gene(s). The strengths of this approach are the ability to identify previously unknown mechanisms of anti-TB immunity and reveal their hierarchy, interactions and individual contributions to pathogenesis during host–pathogen interactions in vivo. Mouse models of MTB infection were used in three independent studies to map resistance/susceptibility genes (reviewed in Fortin et al.11). In each study, a different combination of inbred mice was used as resistant and susceptible partners for linkage analysis.13, 14, 15, 16, 17, 18 Regardless of parental combinations, all groups reported complex multigenic control of host resistance to TB.

Previously, we investigated the genetic basis for extreme susceptibility of inbred mouse strain C3HeB/FeJ to MTB.19 These inbred mice are distinguished from most other strains, because they form large necrotic lesions in their lungs after infection with virulent MTB14 and Mycobacterium bovis (I Kramnik, unpublished observations). C3HeB/FeJ mice are striking as they lack gross immunological abnormalities in known essential mechanisms of anti-TB immunity as their (1) dendritic cells produce interleukin 12 (E Eruslanov, unpublished observations), (2) mycobacteria-specific CD4+ T cells differentiate along the Th1 pathway and (3) macrophages are capable of responding to interferon--γ.20 Nevertheless, C3HeB/FeJ mice exhibit survival time comparable to mice homozygous for knockouts in T-cell receptor α and β chains, interleukin 12 β chain, and shorter than that of the inducible nitric oxide synthase (iNOS), CD4 and MHC class II knockouts (Kramnik, in press). We used forward genetics to reveal underlying defects in host resistance to TB. The sst1 (super susceptibility to TB 1) locus on chromosome 1 was mapped19 and a candidate gene, Ipr1 (intracellular pathogen resistance 1) was identified by positional cloning.21 Two sst1 congenic strains, C3H.B6-sst1 and a reciprocal congenic strain B6.C3H-sst1 (Table 1) were generated to test the isolated effect of sst1 on C3HeB/FeJ and C57BL/6J genetic backgrounds. Survival of both sst1 congenic strains fell intermediately between the parental strains indicating that non-sst1 loci had an effect on host survival. Four of those loci on chromosomes 7, 12, 15 and 17 were mapped using a cross between sst1 resistant (sst1R) strains C3H.B6-sst1 and C57BL/6J.18 Incomplete sst1-susceptible (sst1S) allele penetrance in the original cross and higher resistance of the B6.C3H-sst1 mice compared to C3HeB/FeJ (both sst1S) indicated that host resistance mechanisms, independent of the sst1 locus and capable of compensation for the sst1 deficiency did exist.

Table 1 Genetic composition of congenic/consomic strains used in the study

In the case of multigenic quantitative traits, identification of causal polymorphic genes encoded within each locus represents a significant challenge. Of the 2000 quantitative trait loci (QTLs) mapped in 2005, the underlying genetic polymorphisms have been identified for <1%.22 Factors that impede the progress from QTL to causal gene identification include broad candidate intervals, weak effects of individual loci on overall phenotype, unknown subphenotypes (i.e., specific functional effects) of each locus, a possibility of epistatic interactions among individual loci and the potential complexity of each candidate region that may encode several functionally related genes.11, 23, 24, 25 Thus, attempts to isolate and dissect individual loci following initial QTL mapping are often unsuccessful.

In this study, we performed genetic and functional characterization of the second most powerful TB resistance locus in our model—a quantitative trait locus on mouse chromosome 7 (Chr7) and characterized its epistatic interactions with the sst1. Our data demonstrate importance of the Chr7 QTL in control of host resistance to MTB in immunocompetent hosts and provide a basis for subsequent identification of novel TB resistance gene(s) within the Chr7 locus using positional cloning.

Results

The chromosome 7 locus is involved in the sst1-independent genetic control of host resistance to tuberculosis infection

In our previous studies, we observed a significant effect of the genetic background on phenotypic expression of the susceptible allele of the sst1 locus (sst1S) by comparing TB progression in two inbred strains homozygous for the sst1S—the C3HeB/FeJ parental inbred mice and the sst1S congenic mouse strain B6.C3H-sst1 (Table 1). The B6.C3H-sst1 congenic mice are genetically identical to the TB-resistant parental strain C57BL/6J with the exception of a 12 cM segment of chromosome 1 (47–59 cM) encompassing the sst1 locus. Even though the sst1S strains (B6.C3H-sst1 and C3HeB/FeJ) were more susceptible to MTB than their sst1R counterparts (B6 and C3H.B6-sst1, respectively), TB progressed more rapidly in C3HeB/FeJ than in B6.C3H-sst1 mice. After i.v. infection with a standard dose of MTB (Erdman strain, Trudeau Institute, Saranac Lake, NY, USA; 5 × 104 CFU (colony-forming unit) per mouse), the B6.C3H-sst1 mice survived for 9–12 weeks, whereas the C3HeB/FeJ succumbed within 3.5–5.5 weeks (Table 1). A low dose aerosol challenge with MTB resulted in a median survival time (MST) in B6.C3H-sst1 and C3HeB/FeJ mice of 225 days and 153 days, respectively.18 Using a cross of two sst1R strains C57BL/6J (B6) and C3H.B6-sst1, we have previously mapped four TB resistance loci on mouse chromosomes 7, 12, 15 and 17.18 At that time it was impossible to determine whether the same or different loci contributed to host resistance in the sst1-susceptible B6.C3H-sst1 mice, because the population of the sst1S F2 hybrids (C3HeB/FeJ × B6.C3H-sst1) was much more susceptible to i.v. infection with a standard dose of MTB, as compared to the sst1R (C3H.B6-sst1 × B6)F2 hybrids.18

In this study, we optimized the MTB infectious dose to increase phenotypic variation, used a backcross breeding strategy to reduce the genetic variation and tested larger mouse populations to make statistical analyses more powerful. The sst1S (C3HeB/FeJ × B6.C3H-sst1)F1 hybrid males were backcrossed on C3HeB/FeJ or B6.C3H-sst1 backgrounds. A total of 220 backcross progenies on B6 and 160 progenies on C3H backgrounds were analyzed for survival after infection with 2 × 104 CFU of MTB i.v. Backcrossed mice that represented 20% resistant and susceptible extremes in each backcross were selected for a whole genome scan, which was performed using a panel of 240 informative (polymorphic between the parental strains) single nucleotide polymorphism (SNP) markers. In this analysis, the Chr7 locus produced the highest linkage scores in both backcrosses on B6.C3H-sst1 and C3HeB/FeJ genetic backrounds (Z-scores 3.12 and 4.29, respectively). Mice carrying two copies of the B6-derived alleles on Chr7 were more resistant than heterozygotes in backcross on B6.C3H-sst1 background, whereas the heterozygous mice were more resistant than homozygous for the C3H-derived alleles in the backcross on the C3HeB/FeJ background. Thus, for the B6-derived resistance locus on Chr7, survival times were distributed as bb>bh>hh indicating that the B6-derived resistance allele was dominant with an additive mode of inheritance.

Next, we performed a high-density genotyping using SNP markers that covered the entire Chr7 at 1–2 cM intervals in all mice that have been tested in four independent experiments, which included the (C3H.B6-sst1 × C57BL/6J)F2 and (C3HeB/FeJ × B6.C3H-sst1)F2 intercross mice reported previously18 and the backcross mice described above. As shown in Table 2, the Chr7 locus effect was prominent in the sst1R intercross progeny C3H-sst1RB6F2, but was of borderline significance in the sst1S progeny C3FeB6-sst1F2 after infection with the same dose of MTB. When a lower infectious dose of MTB was used to test the sst1S backcross progeny, the Chr7 locus effect was greater. Of note, the QTL candidate regions on Chr7 identified in each of four independent experiments were large, spanning 50–70 Mb and only partially overlapping between 30 and 66 Mb of Chr7.

Table 2 Summary of independent replication of the tuberculosis resistance QTL on chromosome 7

Thus, the TB resistance QTL on Chr7 (Chr7 QTL) was replicated in a total of four independent crosses (Table 2). However, additional experiments neither allowed clearer delineation of the candidate region, nor excluded a presence of several resistance/susceptibility loci on this chromosome. Therefore, for further functional and genetic dissection of the Chr7 QTL, we generated a Chr7 consomic strain C3H-Chr7B6, which carried two copies of the entire B6-derived Chr7 on the C3HeB/FeJ genetic background.

Individual effect of the Chr7 locus on host resistance to TB infection and its synergy with the sst1 locus

An isolated effect of the Chr7 QTL on host TB resistance was tested by comparing the survival of the parental C3HeB/FeJ and C3H-Chr7B6 consomic mouse strains after i.v. and aerosol infections with MTB. The Chr7 locus-resistant allele produced a small increment in survival after i.v. infection with a standard dose of MTB—the C3HeB/FeJ mice had succumbed to infection by 42 days after i.v. challenge, whereas the C3H-Chr7B6 died within the next week (Figure 1a, left panel). After a low-dose aerosol infection, however, the susceptible parental strain C3HeB/FeJ mice died much earlier with an MST of 75 days, whereas the C3H-Chr7B6 mice survived for 120–150 days post infection (pi; Figure 1a, right panel). Thus, the Chr7 locus significantly affected TB progression only during chronic infection caused by a low-dose aerosol infection with MTB.

Figure 1
figure1

Effect of the Chr7 locus on the course of tuberculosis in C3HeB/FeJ genetic background mice. (a) Survival of the parental C3HeB/FeJ and Chr7 consomic strain (C3H-Chr7B6) following systemic i.v. challenge with 4 × 104 CFU of M. tuberculosis (Erdman; left panel). Survival of parental C3HeB/FeJ (C3H) and C3H-Chr7B6 consomic mice following aerosol challenge with 30–50 CFU of M. tuberculosis Erdman (right panel). Six to eight mice per group were used in each experiment. (b) Interactions of the Chr7 and sst1 loci during the course of tuberculosis following i.v. challenge with M. tuberculosis. MTB burden (CFU) in the organs of the C3H–Chr7B6 (sst1S, S) and C3H.B6-sst1, Chr7B6 (sst1R, R) at 45 days post i.v. challenge with 5 × 104 CFU of MTB (four mice per group). (c) Histopathology of representative tuberculosis lung lesions of the C3H-Chr7B6 (left and middle panels) and C3H.B6-sst1, Chr7B6 (right panel) double congenic mice 45 days post i.v. challenge. H&E, original magnification × 40; inset acid-fast fluorescent staining of MTB, original magnification × 400. CFU, colony-forming unit; MTB, Mycobacterium tuberculosis; Chr7, chromosome 7; H&E, hematoxylin and eosin stain.

We hypothesized that the individual effect of the Chr7 resistance allele was suppressed due to the absence of other B6-derived resistance loci after infection with higher dose of MTB i.v. To test interactions of the Chr7 and sst1 loci, the C3H-Chr7B6 consomic mice were crossed with the sst1R congenic strain C3H.B6-sst1 to produce F1 hybrids heterozygous at both loci. The (C3H.B6-sst1 × C3H-Chr7B6)F1 hybrids were backcrossed on C3H-Chr7B6 consomic strain to produce progeny that were homozygous for the Chr7-resistant allele, but segregated at the sst1 locus. The backcross mice were infected with a standard dose (5 × 104 CFU) of MTB i.v. and killed 45 days pi to assess TB progression. Bacterial loads in the organs of mice grouped by their sst1 allele are presented in Figure 1b. Mice with the susceptible allele of the sst1 locus (S) developed necrotic lung lesions despite the presence of the resistant allele at the Chr7 locus (Figure 1c, left and middle panels). The backcross progeny carrying the sst1-resistant allele (R) had significantly fewer bacteria in their organs (Figure 1b), and did not develop lung necrosis (Figure 1c, right panel). Therefore, the resistant allele of the Chr7 locus failed to prevent the development of lung necrosis, which was controlled by the sst1 locus. However, the Chr7-resistant allele limited the extent of necrosis associated with TB lung lesions in the sst1-susceptible mice: in the Chr7-resistant C3H-Chr7B6 mice, the areas of necrosis were found in the center of well-organized tubercles (Figure 1c, left panel), and advanced necrotic areas were separated from normal lung tissue by a wall of inflammatory cells (Figure 1c, middle panel). This is in contrast with the Chr7-susceptible parental C3HeB/FeJ mice (also sst1S), in which extensive inflammation resembling caseous pneumonia occurred within 25–35 days of infection and resulted in earlier death of these mice.

These data demonstrate that an individual effect of the resistant allele of the Chr7 QTL on TB progression can be detected in the susceptible C3HeB/FeJ genetic background. However, an overall increase in host resistance due to the Chr7 QTL does not prevent the formation of necrosis within TB lung lesions. The latter is specifically controlled by the sst1 locus. Therefore, mechanistically, the Chr7 QTL phenotypic expression is sst1-independent, but its impact on TB progression at the whole organism level is limited in the presence of the sst1S allele.

The Chr7 locus mediates systemic control of TB at later stages of infection

To examine the effect of the Chr7 locus on TB resistance in the presence of the sst1R allele, the B6-derived Chr7 allele was introduced on the C3H.B6-sst1 background. Progeny, homozygous for the sst1R allele and segregated at the Chr7 locus, was produced by backcross–intercross breeding, and infected with MTB i.v. as described above. Survival curves of the intercross mice, grouped by their Chr7 allele, are presented in Figure 2a. The Chr7 locus exhibited a strong effect on survival in the presence of the sst1R allele: the MST for mice homozygous for the B6-derived resistant Chr7 allele (bb) was 131 days; the MST for C3H homozygotes (hh) was 56 days. Chr7 heterozygotes (bh) had an intermediate MST, which agrees with additive effects of this locus determined by linkage analyses (Table 2). Subsequently, detailed Chr7 locus studies were performed in the sst1R background using a double consomic/congenic strain C3H.B6-sst1, Chr7B6 carrying homozygous B6-derived Chr7 and sst1 alleles on the C3HeB/FeJ background (Table 1).

Figure 2
figure2

The Chr7 effect on the course of tuberculosis in the sst1R genetic background following i.v. challenge with M. tuberculosis (Erdman). (a) Dose-dependent effect of the B6-derived resistance allele of the Chr7 locus on survival of the sst1R mice after systemic i.v. infection with 7 × 104 CFU of MTB. The C3H.B6-sst1, Chr7bh and C3H.B6-sst1, Chr7bb carry one or two B6-derived resistant alleles of Chr7, respectively. Seven to twelve mice per group were used in the experiment. (b) Tuberculosis lung lesion histopathology in the sst1R congenic mouse strains C3H.B6-sst1 and double congenic C3H.B6-sst1, Chr7B6 56 days postinfection with MTB (H&E, original magnification × 40). (c) Kinetic analysis of mycobacterial loads in spleens, lungs and livers of the parental C57BL/6 (B6), C3H.B6-sst1 and C3H.B6-sst1, Chr7B6 mice 18, 38 and 56 days post i.v. challenge with 5 × 104 CFU of MTB (y axis—CFU per organ). CFU, colony-forming unit; MTB, Mycobacterium tuberculosis; Chr7, chromosome 7; H&E, hematoxylin and eosin stain.

TB progression in two Chr7 disparate sst1R strains—C3H.B6-sst1 and C3H.B6-sst1, Chr7B6 and the resistant parental strain B6—were compared after i.v. infection with a standard dose of MTB. The initial growth of MTB up to 38 days pi was similar in C3H.B6-sst1 and C3H.B6-sst1, Chr7B6 mice (Figure 2c), whereas a strong effect of the Chr7 locus occurred at 8 weeks pi: a 1.5–2 log increase in MTB burden was observed in all organs of the C3H.B6-sst1 mice carrying the Chr7-susceptible allele (Figure 2c). The systemic effect of the Chr7-resistant allele was strongly protective: the bacterial load stabilized in the lungs and decreased significantly in spleens and livers of the C3H.B6-sst1, Chr7B6 congenic mouse (Figure 2c). At 8 weeks pi histopathology revealed greater lung inflammation, a decrease in airspaces and consolidation of the lung lesions in the Chr7-susceptible mice C3H.B6-sst1 (Figure 2b, left panel). Meanwhile, the Chr7-resistant C3H.B6-sst1, Chr7B6 mice displayed moderate localized interstitial lung inflammation (Figure 2b, right panel), which at that time was similar to the parental resistant B6 mice (not shown).

To further examine the Chr7 effect in a physiologically more relevant TB model, the C3H.B6-sst1 and C3H.B6-sst1, Chr7B6 mice were infected with a low dose of MTB (30–50 CFU per animal) through aerosol. Four mice of each strain were killed at 2, 6, 12 and 20 weeks pi to monitor TB progression (Figure 3a). Similar kinetics of the bacterial growth was observed until the 12th week pi. However, there was a 1.5–2 log increase in the MTB burden in the organs of C3H.B6-sst1 mice between the 12th and the 20th week pi, whereas bacterial loads remained stable in the Chr7-resistant consomic mice C3H.B6-sst1, Chr7B6.

Figure 3
figure3

Effect of the Chr7 on the course of tuberculosis following the low-dose aerosol challenge with M. tuberculosis (Erdman). (a) Bacterial loads in organs of C3H.B6-sst1 and C3H.B6-sst1, Chr7B6 mice at 2, 6, 12 and 20 weeks after aerosol challenge with 30–50 CFU of MTB strain (Erdman). Four mice per strain were killed at each time point. (b) Tuberculosis lung lesion histopathology of the C3H.B6-sst1 and C3H.B6-sst1, Chr7B6 mice 20 weeks following the low-dose aerosol infection with MTB. Upper panels—H&E, original magnification × 200, lower panels—auramine-rhodamine staining of acid fast mycobacteria, original magnification × 400. CFU, colony-forming unit; MTB, Mycobacterium tuberculosis; Chr7, chromosome 7; H&E, hematoxylin and eosin stain.

Histopathological examination at 20 weeks pi revealed that the Chr7-susceptible mouse lungs contained larger areas of inflammation and less airspace, as compared to those of the C3H.B6-sst1, Chr7B6 consomic mice (Figure 3b, upper left and right panels, respectively). No necrosis in lung lesions of either strain was found consistent with their sst1R genotype. In agreement with higher bacterial loads determined by plating organ homogenates, acid fast fluorescent staining of the lung sections revealed greater numbers of mycobacteria within the lung lesions of the Chr7-susceptible mice (Figure 3b, lower panels). The bacteria were intracellular in both strains. However, macrophages in the lung lesions of C3H.B6-sst1, Chr7B6 consomic mice contained 1–3 bacteria per cell, whereas those in Chr7-susceptible C3H.B6-sst1 mice were loaded with 10–20 or more MTB per cell (Figure 3b, right and left lower panels, respectively). These data suggest that the Chr7 locus is involved in control of intracellular multiplication of MTB in vivo. Whether this genetic locus influences macrophage function directly or this is a secondary effect due to insufficient activation of macrophages by exogenous stimuli produced within TB lesions by other cell types remains to be determined.

To summarize, the Chr7 locus mediates the control of intracellular multiplication of MTB in macrophages within TB granulomas in vivo, and its effect is displayed systemically at a later stage of the disease. The phenotypic expression of the Chr7 locus is significantly more pronounced in the presence of the sst1R allele, which prevents necrosis in TB lung lesions, extracellular multiplication of the pathogen and early lethality.

Fine mapping of the Chr7 locus using sst1-resistant subcongenic strains

As the difference in TB susceptibility between mice that carried the Chr7-resistant or susceptible alleles was maximal in the presence of the sst1R allele, for fine mapping of the Chr7 locus, we produced a set of subcongenic strains that carried recombinant Chr7 on the C3H.B6-sst1 genetic background. Being derived from the same C3H.B6-sst1, Chr7B6 grandparent male after a second backcross on C3H.B6-sst1 female, each subcongenic strain was of C3HeB/FeJ genetic background, homozygous for the B6-derived sst1-resistant allele and carried a homozygous recombinant Chr7 with a single recombination event, such that a B6-derived segment in each subcongenic strain partially overlapped the candidate QTL region, as shown in Figure 4a.

Figure 4
figure4

The Chr7 locus mapping using Chr7 interval-specific subcongenic mouse strains. (a) Schematic diagram of the Chr7 interval-specific subcongenic mouse strains. Black and white boxes represent homozygous C57BL/6J and C3HeB/FeJ-derived alleles, respectively. The Chr7 subcongenic mouse strains (A–D) are sst1R. The Chr7 allele is determined based on survival (B and C) and organ bacterial loads (D) after i.v. infection with MTB. (b) Survival of four chromosome 7 subcongenic mouse strains (A–D) and the parental C3H.B6-sst1 and C3H.B6−sst1, Chr7B6 mice after i.v. infection with a standard dose of MTB (Erdman). (c) Pairwise comparisons of survival curves of each subcongenic strain with the parental strains to determine allele of the Chr7 locus. (d) Bacterial burdens in the organs of the Chr7 subcongenic and parental mouse strains 10 weeks postinfection with 5 × 104 CFU of MTB i.v. CFU, colony-forming unit; MTB, Mycobacterium tuberculosis; Chr7, chromosome 7.

Subcongenic strains A, B, C and D, and the corresponding parental strains were tested for survival after i.v. challenge with a standard dose of MTB. MST of the subcongenic strain A (101 days) was significantly shorter than in subcongenic strains B, C and D (MST=147, 151 and 142.5, respectively, Figure 4b). Pairwise comparisons of Kaplan–Mayer survival curves using log-rank test demonstrated that the survival of the subcongenic strain A was statistically indistinguishable from that of the Chr7-susceptible parental strain C3H.B6-sst1 and significantly shorter than that of the resistant consomic strain C3H.B6-sst1, Chr7B6 (Figure 4c). Survival curves of subcongenic strains B, C and D were significantly different from those of C3H.B6-sst1 and subcongenic strain A, and these subcongenic strains were as resistant as the consomic parental strain C3H.B6-sst1, Chr7B6. These data indicate that the subcongenic strains B, C and D carried the resistant and strain A, the susceptible alleles of the Chr7 QTL, and therefore the Chr7 critical interval is delimited by microsatellite markers D7Mit230 and D7Mit350 (Figure 4a).

As, in addition to its effect on mouse survival, the Chr7 QTL had a clear systemic effect on the bacterial loads at late stages of the disease progression, in a second experiment, we used both the survival and the bacterial burdens of spleen, liver and lung at 10 weeks post i.v. infection to differentiate the Chr7-resistant and susceptible alleles. As shown in Figure 4d, the bacterial burden in the lungs, spleens and livers of strains A and C3H.B6-sst1 were similar and significantly higher (P<0.05) than in strains B, C and C3H.B6-sst1, Chr7B6. In this experiment, the MST of the susceptible strains A and C3H.B6-sst1 equaled 103 and 104 days, respectively, whereas the MST of strains B, C and C3H.B6-sst1, Chr7B6 equaled 150, 163 and 143 days, respectively. Thus according to both criteria, the Chr7 subcongenic strains could be divided into two groups: resistant (strains B and C) and susceptible (strain A) indicating that the same Chr7 interval delimited by microsatellite markers D7Mit230 and D7Mit350 controls both mouse survival and the MTB organ burden. As there were no statistically significant differences between the resistant subcongenic strains B, C and the resistant parental strain C3H.B6-sst1, Chr7B6 (P>0.3), according to both criteria, we concluded that the entire effect of the Chr7 QTL on TB resistance was due to this 8.7-cM interval and confidently excluded the irf3 and irf7 genes from the Chr7 locus.

Discussion

Unusually severe lung pathology combined with early lethality that develops after infection with virulent MTB is the characteristic for C3HeB/FeJ inbred mice. Because these mice fail to control TB progression in the lungs despite a functional immune system,20 a situation also typical in humans, a better understanding of pulmonary MTB pathogenesis may emerge by identifying causal genetic defects in these mice. Using a cross of these mice with a relatively TB resistant inbred mouse strain B6, we have determined that the genetic control of TB resistance in our model is multigenic and mapped several TB resistance loci on mouse chromosomes 1 (sst1), 7, 12, 15 and 17.

In our previous mapping study, the effect of the Chr7 locus was the strongest in a cross of two inbred strains that carried the sst1-resistant allele—B6 and the sst1 congenic C3H.B6-sst1. However, in a cross of two sst1-susceptible mouse strains, C3HeB/FeJ and the B6-derived sst1-susceptible congenic mouse strain B6.C3H-sst1, the Chr7 effect fell below the statistical threshold. This raised a possibility that the phenotypic expression of the Chr7 QTL as well as of other non-sst1 loci identified previously were dependent on the presence of the sst1-resistant allele. Several observations, however, suggested the existence of sst1-independent genetic factors capable of counteracting the devastating effect of the sst1S allele on TB progression. We repeated our attempt to map novel TB resistance loci in a cross of two sst1-susceptible parental strains using optimized infectious dose and backcross breeding strategy. Surprisingly, the highest linkage scores were obtained for loci on chromosomes 7, 15 and 17, which overlap with QTLs identified in our previous cross of the sst1R parents. These data demonstrate that, in principle, similar host resistance mechanisms may operate in the presence and in the absence of the sst1-dependent mechanism, although their effects are less pronounced in the sst1S genetic background.

Because of ‘genetic noise’ produced by multiple unlinked loci in QTL analysis, candidate intervals are difficult to determine precisely. In each of our crosses, the location of the Chr7 QTL peak varied. Therefore, a so-called consomic (chromosome substitution) mouse strain C3H-Chr7B6 was constructed by transferring the whole B6-derived Chr7 on the C3HeB/FeJ genetic background using a marker-assisted breeding strategy.25 Initially, the individual effect of the Chr7 locus on mouse survival was tested after i.v. infection. Although statistically significant, it was weak and hardly amenable to further dissection and gene identification. In isolation, the Chr7 locus-resistant allele was unable to prevent necrosis within TB lung lesions in C3HeB/FeJ mice. On the sst1R genetic background, however, the B6-derived Chr7 locus produced much more pronounced chromosome dose-dependent phenotypic effect consistent with predicted dominant/additive pattern of inheritance.

At the whole organism level, the combined effect of the two loci, sst1 and Chr7, transformed an extremely TB-susceptible parental mouse C3HeB/FeJ that succumbs in 3.5–5 weeks to systemic MTB infection into a mouse with a relatively resistant phenotype that survives for 18–24 weeks, approximately a half of the difference between parental strain survival times. Each locus had a specific effect in vivo: the sst1R worked at an earlier time point and was responsible for preventing necrosis within forming TB granulomas specifically in the lungs. In the sst1 resistant setting, the Chr7 locus controlled intracellular MTB multiplication within TB inflammatory lesions. The effect of this locus, observed in lungs, spleens and livers, was systemic and manifested at later time after infection. Differences in phenotypic expression of the sst1 and Chr7 loci suggest that each locus mediates a distinct molecular pathway. We propose the following explanation of the synergistic effect of the two loci: within the lung granulomas the sst1R locus ensures survival of the MTB-infected macrophages, whereas the resistant allele of the Chr7 locus limits a rate of the intracellular MTB multiplication in these cells. Because the sst1S macrophages are sensitized to necrotic cell death induced by virulent MTB,21 formation of necrotic microfoci within the lung lesions of the sst1S mice creates a favorable environment for rampant extracellular growth of the bacteria in the lungs20 and, thus limits the effect of the Chr7 locus explaining a hierarchical relationship between the two loci. Although the activities of the two TB resistance loci seemingly converge on macrophages, our ongoing studies will establish whether the effect of the Chr7 locus is macrophage cell autonomous, or it is dependent on interactions of macrophages with other cell types within TB granulomas.

To date, linkage of TB resistance to Chr7 was identified in two independent studies. Mitsos et al.16, 17 mapped the TB resistance locus on Chr7 (trl3) in a cross of B6 and DBA/2 mouse strains. Peak location of the trl3 on proximal Chr7 overlaps with the Chr7 QTL described here. As C3H inbred mouse strain was derived from a cross of DBA and Bagg albino mice,26 the two susceptible strains are related and the Chr7 locus may represent a common ancestral genetic polymorphism. On the contrary, the susceptible allele of the sst1 locus represents a recent mutation, which occurred in the C3HeB/FeJ, the only substrain of C3H that carries the sst1-susceptible allele. The interplay of the Chr7 and sst1 loci illustrates how the effects of common ancestral polymorphisms may be modified, or even obscured, by less frequent recent mutations. Multigenic hierarchical genetic control of TB resistance presented in this study explains why previous attempts to attribute strain differences in TB susceptibility to a single known polymorphic candidate locus9, 27 were unsuccessful.28 Instead, identification of causal polymorphisms and physiological pathways whose interactions determine the outcomes of TB infection is possible through systematic forward genetic analysis.

The reduced Chr7 region mapped here contains many attractive candidate genes. However, commenting on the role of individual genes would be highly speculative without further narrowing the critical interval. For example, analysis of interval-specific subcongenic strains in our studies excluded irf3 and irf7, which previously were considered top candidate genes, from the candidate region. It is important to note that the reduced candidate region is partially homologous to human chromosome 15q11–13, which has been previously identified as a TB susceptibility locus with suggestive evidence of linkage using genome-wide linkage analysis in humans by Cervino et al.29 In a follow-up study, these authors found significant association of TB susceptibility with a 7-bp deletion in UBE3A gene encoded within the 15q11–13 region and concluded that UBE3A or a closely flanking gene may be a TB susceptibility locus. The reduced candidate region on mouse Chr7 also encompasses the UBE3A gene. UBE3A is a ubiquitin ligase known to function in the uniquitination and degradation of several proteins including p53.30 The role of UBE3A in response to human papilloma virus has been studied, and this protein was shown to mediate the association of the E6 protein of human papilloma virus with p53.30 However, the function of this gene in control of bacterial infections remains unknown.

Further work to identify the candidate genes and understand their function may reveal novel mechanisms of TB resistance common in mice and humans. Together with the analysis of the pathogen itself, this approach will permit more complete understanding of the pathogenesis of TB infection as well as the evolutionary successful and sophisticated virulence strategy of MTB.

Materials and methods

Animals

C3HeB/FeJ and C57BL/6J mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). C3H.B6-sst1 mice were generated by introgression of a B6-derived interval of chromosome 1 (49–60 cM) encompassing the mouse TB-susceptibility sst1 locus on the susceptible genetic background, C3HeB/FeJ, using marker-assisted breeding and 10 backcrosses.21 The chromosome 7 consomic mice, C3H-Chr7B6, were generated using a microsatellite marker-assisted speed congenic breeding strategy.25 The entire B6-derived chromosome 7, which encompasses the Chr7 QTL peak region identified by linkage analysis, was transferred to the C3HeB/FeJ parental strain yielding the C3H-chr7B6 consomic strain or to the C3H.B6-sst1 congenic strain yielding the C3H.B6-sst1, Chr7B6 double congenic/consomic strain (Table 1). Chr7 subconsomic strains A–D were derived from the C3H.B6-sst1, Chr7B6 strain after backcross on to C3H.B6-sst1 background. Mice used in this study are presented in Table 1.

All sst1R strains carried the C3H-derived resistant allele of the Slc11a1 gene (formerly known as Nramp1).21 Mice were housed under pathogen-free conditions in barrier animal facilities at the Harvard Medical School and were provided autoclaved chow and water ad libitum. Moribund mice were humanely killed. Time to death experiments were performed with the full knowledge and approval of the standing committee on animals at Harvard Medical School (protocol #03000).

Bacteria

Frozen stocks of M. tuberculosis (Erdman strain)) were prepared from the lungs of B6 mice infected i.v. at least 6 months before harvest. Lung tissues were ground in a stomacher in 10 ml 1 × PBS (phosphate-buffered saline). To culture mycobacteria, 500 μl from neat and 10 × diluted samples were plated on oleic albumin complex-enriched 7H10 Middlebrook (Difco, MI, USA) agar plates. After 10–12 days of growth on solid media at 37 °C, mycobacteria were collected by scraping the plates that produced visible growth of the bacteria, washed in PBS containing 0.05% Tween-80 and resuspended in 50 ml of oleic acid/albumin/dextrose/catalase-enriched Middlebrook 7H9 medium. Clumps were removed by sedimentation at 1 g and liquid cultures were grown in roller bottles to an optical density (OD) of 0.4–0.6. Aliquots in 500-μl volumes were frozen and stored at −80 °C. The viability of cultures was tested after 1 week by plating dilutions from a vial to determine colony forming units. The virulence of prepared stocks was tested by infecting susceptible C3H and resistant B6 female mice i.v.

Infection

An aliquot of M. tuberculosis was thawed, diluted 10 × in PBS (1 × ) containing 0.05% Tween-80 and sonicated for 10 min in a cup horn sonicator. For i.v. infection, bacteria were diluted with PBS 1 × containing 0.05% Tween-80. Mice were injected i.v. in the tail vein with 2–10 × 104 CFU live bacilli in 100 μl. Aerosol infections were performed using a Madison aerosol chamber.21 Aliquots of the bacterial suspension used for each infection were plated to determine the actual infectious dose.

To enumerate bacterial loads, organs were harvested under aseptic conditions at the stated times, and were homogenized individually in PBS 1 × containing 0.05% Tween-80 and were plated on Middlebrook 7H10 agar enriched with 10% oleic acid/albumin/dextrose/catalase (Difco) after the serial 10-fold dilutions, and CFU were counted 21–28 days of incubation at 37 °C. Four to five animals per group were tested at each time point. Mice were killed using isoflurane anesthesia.

Histopathology

Organs were fixed in 10% formalin for >24 h, embedded in paraffin, sectioned (5 μm thickness) and stained with hematoxylin and eosin stain by standard procedure at the Harvard Medical School Rodent Histopathology Core Facility.

Auramine/rhodamine staining

Acid-fast bacilli were identified in tissue sections using auramine/rhodamine dye (0.1% auramine O, 0.01% rhodamine B in H2O; Sigma, MO, USA). Sections were stained for 20 min at RT in the dark, destaining with 3% HCl in 70% EtOH (5 min, RT) and counterstained with Mayer's hematoxylin (VWR, West Chester, PA, USA).

Linkage analysis

Survival phenotype linkage analyses to autosomal loci were performed using the Mapmaker/SURVIVOR extension of MAPMAKER.31 This software computes logarithm of odds scores for the Cox proportional hazards model using a variant of the expectation maximization algorithm with Monte Carlo simulation. Mapmaker/SURVIVOR is available from MJD at mjdaly@genome.wi.mit.edu.

Statistical analysis

Mycobacterium tuberculosis loads were compared using a t-test (GraphPad Prizm, Version 4.0) and are presented as means±s.d. Kaplan–Meier survival curves of mouse populations stratified by genotype were plotted using GraphPad Prizm, and the log-rank tests were used to identify statistical differences between them.

Abbreviations

Chr7:

chromosome 7 locus

Ipr1:

intracellular pathogen resistance protein 1

MTB:

Mycobacterium tuberculosis

QTL:

quantitative trait locus

sst1 :

super susceptibility to tuberculosis 1

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Acknowledgements

We thank Drs Edward Jarroll and Angele Nalbandian for critical reading of this paper.

Author information

Correspondence to I Kramnik.

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Keywords

  • Mycobacterium tuberculosis (MTB)
  • sst1
  • Ipr1
  • chromosome 7
  • mouse infection model

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