Phylogenetic tree of the M. intracellulare strains and strategy of the experiment in this study.

a Phylogenetic tree of the M. intracellulare strains used in this study. The tree was generated based on average nucleotide identity (ANI) with the neighbor-joining method. TMI: typical M. intracellulare; MP-MIP: M. paraintracellulare–M. indicus pranii. b Strategy of the experimental procedure.

Identification of the essential and growth-defect-associated genes across all nine M. intracellulare strains used in this study.

a Functional categories of 131 genes identified as universal essential or growth-defect-associated with an HMM analysis. The genes were categorized according to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. b Universal essential or growth-defect-associated genes corresponding to the genes of existing antituberculous drug targets. c The number of the essential or growth-defect-associated genes in the accessory genome, and the number of strain-dependent essential or growth-defect-associated genes in the core genome.

Detection of genes showing increased or reduced gene essentiality in the clinical M. intracellulare strains.

Left panel shows the genes identified as having fewer Tn insertion reads than the type strain ATCC13950. The fold changes in the number of Tn insertion reads calculated by a resampling analysis are represented by the color scale. Red squares indicate genes required for hypoxic pellicle formation in ATCC139509. *Genes identified with a combination of resampling and HMM analyses. †Genes identified only with resampling analysis. Right panel shows the genes identified as having more Tn insertion reads than type strain ATCC13950. NH: no homolog with ATCC13950, NS: no significant increase in Tn insertion reads by a resampling analysis (adjusted P < 0.05).

Overview of the differences in gene essentiality between the clinical M. intracellulare strains and ATCC13950, drawn with Cytoscape55.

Each central node represents the functional category of genes, assigned with a KEGG pathway analysis. Each peripheral node represents the genes showing significant changes in the number of Tn insertion reads. The size of each node represents the number of strains identified. Each edge represents a strain identified as showing significant changes in the number of Tn insertion reads. The thickness of each edge represents the adjusted P value. The color scale of each edge represents the fold change in the number of Tn insertion reads calculated by a resampling analysis. The graphical organization was referenced to the previous publication by Carey, et al8.

Preferential gene requirements for gluconeogenesis in the clinical M. intracellulare strains inferred from the TnSeq results.

a Carbohydrate pathway showing the changes in Tn insertion reads. Genes identified with the resampling analysis are framed by squares. The essentiality of gluconeogenesis-related genes (pckA, glpX) was higher and that of glycolysis-related genes (aceE, lpdC) was lower in the clinical M. intracellulare strains compared with those in ATCC13950. b Data on gene essentiality calculated with a resampling analysis in the clinical M. intracellulare strains compared with ATCC13950. Red indicates a reduction in Tn insertion reads in the clinical M. intracellulare strains. Blue indicates an increase of Tn insertion reads in the clinical M. intracellulare strains. Asterisks indicate statistical significance of log2 fold changes (log2FCs). The graphical organization was referenced to the previous publication by Carey, et al8.

Significance of increased gene essentiality profiles in vitro on the pathogenesis of mouse lung infection.

a Consistency between the genes required for infection in mouse lungs and the 175 genes required for hypoxic pellicle formation in ATCC13950. b Changes in the requirements for genes with increased essentiality observed in the clinical M. intracellulare strains after the infection of mouse lungs. Highlighted genes are also required for hypoxic pellicle formation by ATCC13950. Gene essentiality of the strain itself: gene essentiality compared with that of ATCC13950. Gene requirements in mouse lungs: changes in the gene requirements in the infected mouse lungs compared with those before infection. Gene set enrichment analysis: The genes listed as core enrichment are shown as “Yes” and the genes not listed as core enrichment are shown as “No.”

Comparison of the timing of entry into logarithmic growth and logarithmic growth rate by the clinical M. intracellulare strains and ATCC13950.

a Time at the inflection point (midpoint) on the sigmoid growth curve. *significantly earlier than an aerobic culture of ATCC13950; #significantly earlier than a hypoxic culture of ATCC13950; †significantly later than an aerobic culture of ATCC13950; ‡significantly later than a hypoxic culture of ATCC13950. b Growth rate at midpoint of the growth curve in each strain. #significantly slower than a hypoxic culture of ATCC13950; †significantly slower than an aerobic culture of the corresponding strain; ‡significantly faster than an aerobic culture of ATCC13950. Open bars: aerobic; closed bars: 5% O2. Data are shown as the means ± SD of triplicate experiments. Data from one experiment representative of three independent experiments (N = 3) are shown.

Validation of the inhibition of bacterial growth caused by the suppression of the expression of TnSeq-hit genes using the CRISPR-i system.

Open bar: day 3; closed bar: day 7. a Comparative growth rates of the knockdown strains relative to those of the vector control strains in the representative universal essential or growth-defect-associated genes: glcB, inhA, gyrB and embB. b Comparative growth rates of the knockdown strains relative to those of the vector control strains in the representative accessory and strain-dependent essential or growth-defect-associated genes: pckA, glpX, csd and ESX-5 type VII secretion components. Data are shown as the means ± SD of triplicate experiments. Data from one experiment representative of two independent experiments (N = 2) are shown.