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. 2022 Feb 3;11:e74419. doi: 10.7554/eLife.74419

Figure 4. Mtb genetic requirements vary across diverse hosts.

(A) The number of Mtb genes required for growth or survival in each diverse mouse strain across the panel (Qval ≤0.05). Orange indicates the mutants required for each strain; turquoise shows the cumulative requirement as each new host strain is added. (B) Venn diagram showing the composition of Mtb gene sets required in each category of host (white, largest circle), only required in the CC panel (gray), required in specific immunological KO mice (blue) and genes required in B6 mice (red). Note, 1* is required in B6 and KO. In order to be called ‘essential’ in each mouse strain, Mtb genes had to be significantly over or underrepresented in at least two genotypes. (C) Each box shows log2 fold change (LFC) of individual mutants from the TnSeq screen relative to the input pool in indicated mouse strains (top); log2 fold change of the indicated deletion mutants relative to WT from a pooled mutant validation infection (middle panel); relative fitness calculated from (middle panel) to account for generation differences in each host due to differential growth rate. Bars are the average of 3–6 mice per mutant/genotype ± SD. Statistical differences between mini-pool validation groups was assessed by Welch’s t-test. (D) Lung CFU and spleen CFU from single strain low-dose aerosol infections of ∆bioA mutant or WT H37Rv strain in B6 and CAST mice at 2- or 5 weeks post-infection. Dashed line indicates the limit of detection. Each point indicates the average CFU ± SD of 4–5 mice per group. Statistical differences between groups were assessed by mixed effects models (Tukey’s test). (E) Log2 fold change of selected mutants from the TnSeq screen across the CC panel and immunological KO mice. Each dot represents the average LFC per mouse genotype; KO mouse strains (on a B6 background) dots are shown larger for clarity. All mice in the large CC TnSeq screen were male; mice in the ∆bioA aerosol validation were female; mice in the mini-pool validation studies were male and female with no significant differences detected. Source file of the TnSeq screen is available in Figure 4—source data 1; source count data of the TnSeq validation experiment is available in Figure 4—source data 2.

Figure 4—source data 1. TnSeq summary table.
LFC values represent the log2 fold change (LFC) between input and mouse-selected pools. ‘NA’ indicates genes with fewer than three occupied TA transposon insertion sites for the indicated comparison. Qvals represent adjusted p-values comparing mutant abundance in input and selected pools. ‘NA’ indicates genes with fewer than three occupied TA transposon insertion sites for the indicated comparison. Required in vivo: ‘TRUE’ indicates the mutant is significantly underrepresented (Qval <0.05) after in mouse-selection in at least two mouse strains. Required in B6: ‘TRUE’ indicates the mutant is significantly underrepresented (Qval <0.05) after in selection in B6 mice. Required in KO mice: "TRUE" indicates the mutant is significantly underrepresented (Qval <0.05) after in selection in Rag-/-, Nos2-/-, Cybb-/-, or Ifnγ-/- mice. Core gene set: ‘TRUE’ indicates the mutant is significantly underrepresented (Qval <0.05) in 30 mouse strains. U = uninformative; fewer than three occupied TA transposon insertion sites in all strains in panel. F = filtered; essential in only a single strain. ‘Module’ corresponds to WGCNA module number as illustrated in Figure 5A. Mouse strains are listed in the same order as Figure 5B, with the corresponding cluster designation.
Figure 4—source data 2. Validation counts table.
CFU and normalized barcode counts from Mtb mutant mini-pool infection in individual mice, including mbtA, glpK, pstC2, eccB1, RNaseJ, and WT (H37Rv).

Figure 4.

Figure 4—figure supplement 1. Sampling additional B6 libraries does not appreciably increase the estimate of genes necessary for growth.

Figure 4—figure supplement 1.

To approximate the experimental design of Figure 4A but for B6 only, six biological replicate TnSeq libraries from 4-week post-infection B6 mice were randomly paired, and genes required for growth or survival in each of the three runs or cumulatively were identified and counted exactly as for Figure 4A. The process was repeated ten times with the results shown. Error bars indicate mean ± SD for the 10 runs. Cumulative gene count stabilizes after two runs.