Experiment
Infection with Mycobacterium tuberculosis (Mtb) produces heterogeneous outcomes that are influenced by genetic and phenotypic variation in both the host and the pathogen. The outcome of an encounter with Mtb depends on the pathogen's ability to adapt to the variable immune pressures exerted by the host. The authors leveraged the genetically diverse Collaborative Cross (CC) mouse panel in conjunction with a library of Mtb mutants to create a resource for associating bacterial genetic requirements with host genetics and immunity.
To characterize the diversity of disease states that are possible in a genetically diverse mouse population, the authors infected a panel of 52 CC lines and the eight founder strains with Mtb. To enable bacterial transposon sequencing (TnSeq) studies downstream, the animals were infected via the intravenous (IV) route with a saturated library of Mtb transposon mutants (infectious dose of 105 CFU), which in sum produce an infection that is similar to the wild-type parental strain (Bellerose et al., 2020; Sassetti and Rubin, 2003). Groups of three to six male mice per genotype were infected and TB disease-related traits were quantified at one-month post-infection.
Male mice from 52 CC strains were purchased from the UNC Systems Genetics Core Facility (SGCF) between July 2013 and August 2014. The 52 CC strains used in this study include: CC001/Unc, CC002/Unc, CC003/Unc, CC004/TauUnc, CC005/TauUnc, CC006/TauUnc, CC007/Unc, CC008/GeniUnc, CC009/Unc, CC010/GeniUnc, CC011/Unc, CC012/GeniUnc, CC013/GeniUnc, CC015/Unc, CC016/GeniUnc, CC017/Unc, CC018/Unc, CC019/TauUnc, CC021/Unc, CC022/GeniUnc, CC023/GeniUnc, CC024/GeniUnc, CC025/GeniUnc, CC027/GeniUnc, CC028/GeniUnc, CC029/Unc, CC030/GeniUnc, CC031/GeniUnc, CC032/GeniUnc, CC033/GeniUnc, CC034/Unc, CC035/Unc, CC037/TauUnc, CC038/GeniUnc, CC039/Unc, CC040/TauUnc, CC041/TauUnc, CC042/GeniUnc, CC043/GeniUnc, CC044/Unc, CC045/GeniUnc, CC046/Unc, CC047/Unc, CC051/TauUnc, CC055/TauUnc, CC056/GeniUnc, CC057/Unc, CC059/TauUnc, CC060/Unc, CC061/GeniUnc, CC065/Unc, CC068/TauUnc.
The bacterial burden after 4 weeks of infection was assessed by plating (colony-forming units, CFU) and quantifying the number of bacterial chromosomes in the tissue (chromosome equivalents, CEQ). These two metrics were highly correlated (r = 0.88) and revealed a wide variation in bacterial burden across the panel. The authors report that CC strains vary dramatically in their susceptibility to infection and produce qualitatively distinct immune states. Global analysis of Mtb transposon mutant fitness (TnSeq) across the CC panel revealed that many virulence pathways are only required in specific host microenvironments, identifying a large fraction of the pathogen's genome that has been maintained to ensure fitness in a diverse population.
A subset of the inbred CC mice used in the analysis were genotyped on the GigaMUGA array (Morgan et al., 2015) available from Neogen Inc. The inbred parents, F1s and F2 mice from the (CC030 x CC029) cross (see below) were genotyped on the MiniMUGA array (Sigmon et al., 2020) at Neogen Inc. For QTL mapping in the CC panel, the Most Recent Common Ancestor (Srivastava et al., 2017) 36-state haplotypes were downloaded from the UNC Systems Genetics Core Facility and simplified to 8-state haplotype probabilities (for the 8 CC founder strains), which is appropriate for additive genetic mapping. The authors generated 36-state haplotype probabilities from the individual CC mice genotyped on GigaMUGA and combined these data with the MRCA data to obtain a common genome cache. For clinical trait scans, batch was included as an additive covariate. Significance thresholds for QTL were estimated using 10,000 permutations. LOD profiles and effect plots were generated using the plotting functions of the R/qtl2 package. Multiple QTL at similar genetic locations were assessed for independence using qtl2pleio with 400 bootstrap samples.
Nine Tuberculosis ImmunoPhenotype QTL (TipQTL) were identfied in the CC population (genome coordinates relative to GRCm38/mm10):
Tip5 (tuberculosis immunophenotype 5, spleen CEQ) maps to Chr 2: 174.29 - 178.25 Mb with a peak LOD score of 9.14 at 178.25 Mb. The NOD/ShiLtJ haplotype was associated with high values at Tip5.
Tip6 (tuberculosis immunophenotype 6, spleen CFU) maps to Chr 2: 73.98 - 180.10 Mb with a peak LOD score of 7.04 at 174.29 Mb. The NOD/ShiLtJ haplotype was associated with high values at Tip6.
Tip7 (tuberculosis immunophenotype 7, IL-9) maps to Chr 2: 33.43 - 41.48 Mb with a peak LOD score of 8.61 at 41.4 Mb.
Tip8 (tuberculosis immunophenotype 8, IL-9) maps to Chr 2: 22.77 - 25.65 Mb with a peak LOD score of 7.85 at 24.62 Mb.
Tip9 (tuberculosis immunophenotype 9, IL-17) maps to Chr 15: 67.98 - 82.11 Mb with a peak LOD score of 7.84 at 74.14 Mb.
Tip10 (tuberculosis immunophenotype 10, CXCL1) maps to Chr 7: 30.43 - 46.72 Mb with a peak LOD score of 7.57 at 45.22 Mb. The CAST/EiJ haplotype was associated with both low bacterial burden and CXCL1 at Tip10.
Tip11 (tuberculosis immunophenotype 11, lung CFU) maps to Chr 7: 31.06 - 45.22 Mb with a peak LOD score of 7.47 at 37.78 Mb. The CAST/EiJ haplotype was associated with both low bacterial burden and CXCL1 at Tip11.
Tip12 (tuberculosis immunophenotype 12, IL-10) maps to Chr 17: 80.98 - 83.55 Mb with a peak LOD score of 7.16 at 82.47 Mb.
Tip13 (tuberculosis immunophenotype 13, lung CFU) maps to Chr 15: 77.00 - 78.70 Mb with a peak LOD score of 7.13 at 78.16 Mb.
The authors took an F2 intercross approach to independently assess the importance of the lung CFU QTL on chromosomes 7 and 15 (Tip10, Tip11, Tip13). Given that the associations at all QTL were driven by the CAST/EiJ haplotype, the authors generated an F2 population based on two CC strains, CC029/Unc and CC030/GeniUnc, that contained CAST/EiJ sequence at the Tip10, Tip11, and Tip13 loci. The F2 validation cohort (n = 251 mice) were genotyped (Sigmon et al., 2020) and infected with the Mtb strain H37Rv (IV route with infectious dose of 105 CFU, as per the original CC screen). At 1 month post infection, lung CFU was quantified, and the authors conducted QTL mapping in R/qtl2 (Broman et al., 2019) to identify host loci underlying bacterial burden in the lung.
Markers were filtered to 2499 markers that differentiated between CC029 and CC030 haplotypes. Sex and infection batch were also considered as covariates for mapping.The authors identified a QTL significantly associated with lung CFU that overlapped with Tip10 and Tip11:
Tip14 (tuberculosis immunophenotype 14, lung CFU) maps to Chr 7 with a peak LOD score of 6.81 at 28.6 Mb (p < 0.05; 10,000 permutations).
Additionally in the mapping validation study, the authors identified a new resistance (low lung CFU) locus:
Tip15 (tuberculosis immunophenotype 15, lung CFU) maps to Chr 8 with a peak LOD score of 4.08 at 116.1 Mb. Tip15 is driven by the CC029/Unc cross partner with the CAST/EiJ haplotype.
To investigate the host genetic determinants of the bacterial microenvironment, the authors leveraged TnSeq as a high-resolution phenotyping platform to associate Mtb mutant fitness profiles with variants in the mouse genome. When the relative abundance of each Mtb mutant phenotype was considered individually, the corresponding 'Host Interacting with Pathogen QTL' (HipQTL) were distributed across the mouse genome. Forty-seven HipQTL were identified:
Hiwp1 (host interacting with pathogen QTL 1, rv0770) maps to Chr 1: 40.43 - 43.32 Mb with a peak LOD score of 9.81 at 42.73 Mb.
Hiwp2 (host interacting with pathogen QTL 2, rv0309) maps to Chr 1: 57.99 - 62.79 Mb with a peak LOD score of 7.95 at 58.18 Mb.
Hiwp3 (host interacting with pathogen QTL 3, rv3657c) maps to Chr 1: 136.39 - 143.60 Mb with a peak LOD score of 7.90 at 138.24 Mb.
Hipw4 (host interacting with pathogen QTL 4, rv0110) maps to Chr 2: 170.67 - 178.84 Mb with a peak LOD score of 7.79 at 174.00 Mb.
Hiwp5 (host interacting with pathogen QTL 5, rv3577) maps to Chr 3: 3.32 - 14.67 Mb with a peak LOD score of 9.23 at 10.03 Mb.
Hiwp6 (host interacting with pathogen QTL 6, rv3005c) maps to Chr 3: 20.31 - 26.12 Mb with a peak LOD score of 8.03 at 26.12 Mb.
Hiwp7 (host interacting with pathogen QTL 7, dinX) maps to Chr 3: 26.99 - 33.85 Mb with a peak LOD score of 9.97 at 30.29 Mb.
Hiwp8 (host interacting with pathogen QTL 8, fadA6) maps to Chr 3: 29.23 - 37.11 Mb with a peak LOD score of 8.74 at 35.22 Mb.
Hiwp9 (host interacting with pathogen QTL 9, dinX) maps to Chr 3: 36.22 - 38.27 Mb with a peak LOD score of 9.22 at 36.83 Mb.
Hiwp10 (host interacting with pathogen QTL 10, rv2707) maps to Chr 3: 100.90 - 115.82 Mb with a peak LOD score of 8.17 at 103.23 Mb.
Hiwp11 (host interacting with pathogen QTL 11, rv3701c) maps to Chr 4: 74.00 - 87.00 Mb with a peak LOD score of 7.90 at 78.25 Mb.
Hiwp12 (host interacting with pathogen QTL 12, ahpC) maps to Chr 6: 19.75 - 23.31 Mb with a peak LOD score of 8.12 at 22.21 Mb.
Hiwp13 (host interacting with pathogen QTL 13, umaA) maps to Chr 7: 117.87 - 120.15 Mb with a peak LOD score of 8.32 at 118.41 Mb.
Hiwp14 (host interacting with pathogen QTL 14, rv2566) maps to Chr 7: 123.21 - 126.67 Mb with a peak LOD score of 7.86 at 126.67 Mb.
Hiwp15 (host interacting with pathogen QTL 15, rv3173c) maps to Chr 7: 137.41 - 138.36 Mb with a peak LOD score of 8.17 at 138.36 Mb.
Hiwp16 (host interacting with pathogen QTL 16, rv3173c) maps to Chr 7: 139.15 - 141.88 Mb with a peak LOD score of 8.12 at 140.76 Mb.
Hiwp17 (host interacting with pathogen QTL 17, rv3502c) maps to Chr 9: 15.91 - 18.72 Mb with a peak LOD score of 8.16 at 16.33 Mb.
Hiwp18 (host interacting with pathogen QTL 18, mycP1) maps to Chr 9: 28.47 - 31.10 Mb with a peak LOD score of 9.09 at 29.45 Mb.
Hiwp19 (host interacting with pathogen QTL 19, rv0057) maps to Chr 9: 36.78 - 40.36 Mb with a peak LOD score of 8.39 at 40.07 Mb.
Hiwp20 (host interacting with pathogen QTL 20, hycE) maps to Chr 9: 47.40 - 51.80 Mb with a peak LOD score of 8.21 at 47.93 Mb.
Hiwp21 (host interacting with pathogen QTL 21, mbtA) maps to Chr 10: 64.48 - 75.42 Mb with a peak LOD score of 8.30 at 68.09 Mb.
Hiwp22 (host interacting with pathogen QTL 22, eccD1) maps to Chr 10: 64.56 - 71.04 Mb with a peak LOD score of 8.08 at 68.12 Mb.
Hiwp23 (host interacting with pathogen QTL 23, rv2989) maps to Chr 10: 74.30 - 81.03 Mb with a peak LOD score of 9.16 at 77.63 Mb.
Hiwp24 (host interacting with pathogen QTL 24, mce4A) maps to Chr 10: 78.88 - 88.25 Mb with a peak LOD score of 7.91 at 81.36 Mb.
Hiwp25 (host interacting with pathogen QTL 25, treS) maps to Chr 11: 20.80 - 44.06 Mb with a peak LOD score of 7.94 at 36.14 Mb.
Hiwp26 (host interacting with pathogen QTL 26, pckA) maps to Chr 11: 85.95 - 91.75 Mb with a peak LOD score of 7.67 at 89.78 Mb.
Hiwp27 (host interacting with pathogen QTL 27, aspB) maps to Chr 11: 114.69 - 117.08 Mb with a peak LOD score of 8.32 at 116.99 Mb.
Hiwp28 (host interacting with pathogen QTL 28, rv1227c) maps to Chr 12: 25.23 - 28.54 Mb with a peak LOD score of 9.16 at 25.23 Mb.
Hiwp29 (host interacting with pathogen QTL 29, rv0219) maps to Chr 12: 40.65 - 47.22 Mb with a peak LOD score of 7.94 at 42.65 Mb.
Hiwp30 (host interacting with pathogen QTL 30, rv3643) maps to Chr 13: 95.43 - 97.79 Mb with a peak LOD score of 8.89 at 97.08 Mb.
Hiwp31 (host interacting with pathogen QTL 31, ansA) maps to Chr 13: 96.82 - 99.09 Mb with a peak LOD score of 8.28 at 97.79 Mb.
Hiwp32 (host interacting with pathogen QTL 32, echA19) maps to Chr 13: 113.20 - 117.64 Mb with a peak LOD score of 9.68 at 114.59 Mb.
Hiwp33 (host interacting with pathogen QTL 33, rv1836c) maps to Chr 14: 74.94 - 76.43 Mb with a peak LOD score of 9.19 at 76.40 Mb.
Hiwp34 (host interacting with pathogen QTL 34, rv2183c) maps to Chr 16: 12.18 - 17.92 Mb with a peak LOD score of 7.75 at 14.06 Mb.
Hiwp35 (host interacting with pathogen QTL 35, rv1178) maps to Chr 17: 80.92 - 83.23 Mb with a peak LOD score of 8.19 at 80.92 Mb.
Hiwp36 (host interacting with pathogen QTL 36, rv0492c) maps to Chr 18: 5.85 - 12.40 Mb with a peak LOD score of 8.90 at 5.85 Mb.
Hiwp37 (host interacting with pathogen QTL 37, cysM) maps to Chr 19: 4.20 - 6.46 Mb with a peak LOD score of 8.47 at 6.46 Mb.
Hiwp38 (host interacting with pathogen QTL 38, atsA) maps to Chr 19: 31.21 - 37.93 Mb with a peak LOD score of 8.58 at 37.86 Mb.
Hiwp39 (host interacting with pathogen QTL 39, galE2) maps to Chr X: 6.01 - 9.12 Mb with a peak LOD score of 8.10 at 6.01 Mb.
Hiwp40 (host interacting with pathogen QTL 40, pks11) maps to Chr X: 50.43 - 52.29 Mb with a peak LOD score of 8.25 at 51.75 Mb.
Hiwp41 (host interacting with pathogen QTL 41, pknK) maps to Chr X: 95.01 - 130.04 Mb with a peak LOD score of 8.73 at 102.02 Mb.
Hiwp42 (host interacting with pathogen QTL 42, module 3) maps to Chr 10: 64.7 - 77.07 Mb with a peak LOD score of 7.80 at 68.27 Mb. NOD/ShiLtJ and WSB/EiJ haplotypes were associated with higher bacterial numbers at Hiwp42.
Hiwp43 (host interacting with pathogen QTL 43, module 4) maps to Chr 10: 65.23 - 74.30 Mb with a peak LOD score of 7.79 at 69.94 Mb.
Hiwp44 (host interacting with pathogen QTL 44, module 16) maps to Chr 10: 74.30 - 87.61 Mb with a peak LOD score of 7.53 at 81.36 Mb.
Hiwp45 (host interacting with pathogen QTL 45, module 19) maps to Chr 11: 60.87 - 63.26 Mb with a peak LOD score of 7.64 at 62.20 Mb.
Hiwp46 (host interacting with pathogen QTL 46, module 10) maps to Chr 15: 100.39 - 103.36 Mb with a peak LOD score of 6.95 at 102.25 Mb.
Hiwp47 (host interacting with pathogen QTL 47, module 10) maps to Chr 19: 32.74 - 37.48 Mb with a peak LOD score of 6.32 at 32.87 Mb.
Potential candidate genes are discussed in the manuscript.
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