Functional genomics reveals strain-specific genetic requirements conferring hypoxic growth in Mycobacterium intracellulare
- Department of Bacteriology, Graduate School of Medicine and Dental Sciences, Niigata University, Japan
- Department of Microbiology, Fukushima Medical University, Japan
- Department of Mycobacterium Reference and Research, The Research Institute of Tuberculosis, Japan Anti-Tuberculosis Association, Japan
- Center for Infectious Disease Research, Fujita Health University, Japan
- Department of Microbiology and Immunology, University of Minnesota Medical School, United States
- Medical Microbiology, Faculty of Medicine, Universitas Airlangga, Indonesia
- Department of Bacteriology, Osaka Metropolitan University Graduate School of Medicine, Japan
- Division of Research Aids, Hokkaido University Institute for Vaccine Research and Development, Japan
Figures
Figure 1 with 1 supplement
Phylogenetic tree of the M. intracellulare strains and strategy of the Transposon sequencing (TnSeq) analyses 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. The detail of the phylogenetic tree is shown in Figure 1—figure supplement 1. (b) Strategy of the procedure of TnSeq analyses.
Figure 1—figure supplement 1
Subject M. intracellulare strains in this study.
The phylogenetic tree represents the global genetic diversity of the M. intracellulare strains by our previous study (Tateishi et al., 2021). The subject strains were squared in red. As for strains squared in bold line, four strains, including the type strain ATCC13950 were selected from the TMI (typical M. intracellulare) group, and five strains were selected from the MP-MIP (M. paraintracellulare-M. indicus pranii) group in this Transposon sequencing (TnSeq) study. Two strains squared in dashed lines are closely related neighbors of ATCC13950, which were additionally enrolled in the hypoxic growth assay (as explained in Supplementary file 1).
Figure 2 with 1 supplement
Identification of the essential and growth-defect-associated genes across genetically diverse nine M. intracellulare strains used in this study.
(a) Functional categories of 131 genes identified as universal essential or growth-defect-associated with an hidden Markov model (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 genes, and the number of strain-dependent essential or growth-defect-associated genes in the core genes.
Figure 2—figure supplement 1
Summary of the gene set enrichment analysis (GSEA) results.
The dataset of strain-dependent/accessory essential or growth-defect-associated genes was statistically tested to be enriched with the gene sets required for hypoxic pellicle formation observed in ATCC13950. (Top) Statistics data for the gene set enrichment analysis. (Bottom) Data of enrichment plots. On the X-axis, the left direction indicates positive correlation with the gene sets required for hypoxic pellicle formation observed in ATCC13950, and the right direction indicates negative correlation with the gene sets required for hypoxic pellicle formation observed in ATCC13950. Only 31 genes were ranked because the rest of the 144 genes were not present in the gene sets of strain-dependent or accessory essential genes. The ranking of the genes in the gene sets required for hypoxic pellicle formation observed in ATCC13950 is shown in Supplementary file 6.
Figure 3
Detection of genes showing increased or reduced genetic requirements in the clinical M. intracellulare strains.
Left panel shows the genes identified as having fewer transposon (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 ATCC13950 (Tateishi et al., 2020). *Genes identified with a combination of resampling and hidden Markov model (HMM) analyses. †Genes identified only with resampling analysis. Right panel shows the genes identified as having more Tn insertion reads than the type strain ATCC13950. NH: no homolog with ATCC13950, NS: no significant increase in Tn insertion reads by a resampling analysis (adjusted p<0.05).
Figure 4
Overview of the differences in genetic requirements between the clinical M. intracellulare strains and ATCC13950, drawn with Cytoscape (Shannon et al., 2003).
Each central node represents the functional category of genes, assigned with a Kyoto Encyclopedia of Genes and Genomes (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 transposon (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 al., 2018.
Figure 5
Preferential genetic requirements for gluconeogenesis in the clinical M. intracellulare strains inferred from the Transposon sequencing (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 genetic requirements calculated with a resampling analysis in the clinical M. intracellulare strains compared with ATCC13950. Red indicates a reduction in transposon (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 al., 2018.
Figure 6 with 4 supplements
Detection of genes required for mouse lung infection in the clinical M. intracellulare strains.
(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 genetic requirements observed in the clinical M. intracellulare strains after the infection of mouse lungs. Highlighted genes are also required for hypoxic pellicle formation by ATCC13950. Genetic requirements of the strain itself: genetic requirements compared with that of ATCC13950. Genetic requirements in mouse lungs: changes in the genetic 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’.
Figure 6—figure supplement 1
Mouse infection experiment for transposon sequencing (TnSeq).
Mouse infection experiment for Transposon sequencing (TnSeq) (a) Schematic representation of mouse infection experiment. #Three mice were excluded from TnSeq because the number of harvested colonies was <1×104 /mouse. § Three samples of M.i.198Tn at week 12 were grouped into M.i.198Tn at week 16 to represent the chronic phase of infection. $ Three mice were excluded from TnSeq because the number of harvested colonies was only 103–104 /mouse. (b) Time-course changes in colony-forming units (CFUs) in mouse lungs after infection with M.i.198Tn or M.i.27Tn. Data on CFUs are from one biological experiment (N=1). Each dot represents an individual mouse. (c) Resampling assay to detect significantly reduced Tn insertion reads in infected mouse lungs compared with before infection. The data on time-course changes in CFUs in mouse lungs after infection with wild-type strains of M.i.198 or M.i.27 were published previously (Tateishi et al., 2023). (d) Number of genes showing significantly reduced transposon (Tn) insertion reads in the infected mouse lungs compared with before infection according to a resampling analysis. As described above, the samples of M.i.198Tn in week 16 were derived from two mice killed in week 16 and one mouse killed in week 12.
Figure 6—figure supplement 2
Summary of the gene set enrichment analysis (GSEA) results.
The dataset of fitness costs for infection in mouse lungs in transposon (Tn) mutant libraries of M.i.27 and M.i.198 from day 1 to week 16 of infection was statistically tested to be enriched with the gene sets required for hypoxic pellicle formation observed in ATCC13950 and with those showing increased genetic requirements observed in the Mycobacterium avium–intracellulare complex pulmonary disease (MAC-PD) clinical strains. (Top) Statistics data for the gene set enrichment analysis. (Bottom) Data of enrichment plots. On the X-axis, the left direction indicates positive correlation with the conditions after infection in mouse lungs, and the right direction indicates positive correlation with the conditions outside of mouse lungs (i.e. negative correlation with the conditions after infection in mouse lungs). The ranking of the genes in the gene sets required for hypoxic pellicle formation observed in ATCC13950 and those showing increased genetic requirements observed in the MAC-PD clinical strains is shown in Supplementary file 12.
Figure 6—figure supplement 3
Summary of the gene set enrichment analysis (GSEA) results.
The dataset of fitness costs for infection in mouse lungs in transposon (Tn) mutant libraries of M.i.27 and M.i.198 was statistically tested to be enriched with the gene sets required for hypoxic pellicle formation observed in ATCC13950. (Top) Statistics data for the gene set enrichment analysis. (Bottom) Data of enrichment plots. On the X-axis, the left direction indicates positive correlation with the conditions after infection in mouse lungs, and the right direction indicates positive correlation with the conditions outside of mouse lungs (i.e. negative correlation with the conditions after infection in mouse lungs). The ranking of the genes in the gene sets required for hypoxic pellicle formation observed in ATCC13950 is shown in Supplementary file 16.
Figure 6—figure supplement 4
Summary of the gene set enrichment analysis (GSEA) results.
The dataset of fitness costs for infection in mouse lungs in transposon (Tn) mutant libraries of M.i.27 and M.i.198 was statistically tested to be enriched with those showing increased genetic requirements observed in the Mycobacterium avium–intracellulare complex pulmonary disease (MAC-PD) clinical strains. (Top) Statistics data for the gene set enrichment analysis. (Bottom) Data of enrichment plots. On the X-axis, the left direction indicates positive correlation with the conditions after infection in mouse lungs, and the right direction indicates positive correlation with the conditions outside of mouse lungs (i.e. negative correlation with the conditions after infection in mouse lungs). The ranking of the genes showing increased genetic requirements observed in the MAC-PD clinical strains is shown in Supplementary file 17.
Figure 7 with 2 supplements
Evaluation of the effect of the suppression of Transposon sequencing (TnSeq)-hit genes on the bacterial growth 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.
Figure 7—figure supplement 1
Comparative growth rates of the knockdown strains relative to those of the vector control strains in several accessory and strain-dependent essential or growth-defect-associated genes.
Data are shown as the means ± SD of triplicate experiments. Data from one experiment representative of two independent experiments (N=2) are shown.
Figure 7—figure supplement 2
Quantitative reverse transcription PCR (qRT-PCR) validation of the suppression of gene expression in knockdown strains of universal and strain-dependent/ accessory essential or growth-defect-associated genes.
(a) List of target universal essential or growth-defect-associated genes analyzed with qRT-PCR. (b) Levels of gene expression of the target universal essential or growth-defect-associated genes in knockdown strains compared with those in control strains not expressing guide RNA. (c) List of target strain-dependent/ accessory essential or growth-defect-associated genes analyzed with qRT-PCR. (d) Levels of gene expression of the target strain-dependent/ accessory essential or growth-defect-associated genes in knockdown strains compared with those in control strains not expressing guide RNA. Aliquots (10 mL) of log-phase bacterial cultures were prepared and the expression of guide RNA was induced by the addition of aTc on day 0 and day 2 (final concentrations of 50 ng/ml for ATCC13950 and M.i.27; 200 ng/ml for M.i.198, M018, M019, M003, and M021). The bacteria were harvested, RNA extracted, and qRT-PCR performed to evaluate the gene expression levels. Gene expression data are the means ± SD of triplicate experiments. Data from one experiment representative of two independent experiments (N=2) are shown.
Figure 8 with 1 supplement
Comparison of the timing of entry into logarithmic growth and logarithmic growth rate by the clinical M. intracellulare strains and ATCC13950.
(a) Representative data on the growth curves of each strain under aerobic and 5% oxygen conditions. The assay was performed three times in 96-well plates containing 250 μl of broth medium per well, in triplicate. Data are represented as colony-forming units (CFUs) in 4 μl samples at each timepoint. Data on the growth curves are the means of three biological replicates from one experiment. Data from one experiment representative of three independent experiments (N=3) are shown. (b) 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. (c) 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.
Figure 8—figure supplement 1
Data on the growth curve in ATCC13950 and its neighbor clinical M. intracellulare strains M005 and M016.
(a) Representative data on the growth curves of ATCC13950, M005, and M016 under aerobic and 5% oxygen conditions. The assay was performed three times in 96-well plates containing 250 μl of broth medium per well, in triplicate. Data are represented as colony-forming units (CFUs) in 4 μl samples at each timepoint. Data from one experiment representative of three independent experiments (N=3) are shown. (b) Comparison of the time at the inflection point (midpoint) on the sigmoid growth curve between the clinical M. intracellulare strains M005 and M016 and the type strain 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. (c) Comparison of the logarithmic growth rate at midpoint between the clinical M. intracellulare strains M005 and M016 and the type strain ATCC13950. Open bars: aerobic; closed bars: 5% O2. Data are shown as the means ± SD of triplicate experiments.
Additional files
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Supplementary file 1
Genotypes of study strains and the strains used for each experiment.
- https://cdn.elifesciences.org/articles/99426/elife-99426-supp1-v1.docx
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Supplementary file 2
Number of reads obtained and TA coverage in each replicate of transposon (Tn) mutant library strains.
Data for ATCC13950 are from our previous study (Tateishi et al., 2020).
- https://cdn.elifesciences.org/articles/99426/elife-99426-supp2-v1.xlsx
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Supplementary file 3
Number of essential, growth-defect-associated, nonessential, and growth-advantage-associated genes detected with an hidden Markov model (HMM) analysis in each M. intracellulare strain.
Data for ATCC13950 are from our previous study (Tateishi et al., 2020).
- https://cdn.elifesciences.org/articles/99426/elife-99426-supp3-v1.xlsx
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Supplementary file 4
List of genes identified as universal essential or growth-defect-associated among the nine M. intracellulare strains analyzed in this study.
ES: essential, GD: growth-defect-associated.
- https://cdn.elifesciences.org/articles/99426/elife-99426-supp4-v1.xlsx
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Supplementary file 5
List of genes identified as accessory essential or growth-defect-associated in the accessory genome and strain-dependent essential or growth-defect-associated in the core genes of M. intracellulare.
‘Hit’ represents the genes identified as essential or growth-defect-associated in each strain. The data of the pan-genome were referred to our previous study (Tateishi et al., 2021).
- https://cdn.elifesciences.org/articles/99426/elife-99426-supp5-v1.xlsx
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Supplementary file 6
Result of the gene set enrichment analysis (GSEA) in strain-dependent/accessory essential or growth-defect-associated genes.
Genes required for hypoxic pellicle formation observed in ATCC13950 were ordered by their position in the ranked list of genes. Only 31 genes were ranked because the rest of the 144 genes were not present in the gene set of strain-dependent or accessory essential genes.
- https://cdn.elifesciences.org/articles/99426/elife-99426-supp6-v1.xlsx
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Supplementary file 7
Classification of the genes showing increased genetic requirements in the clinical Mycobacterium avium–intracellulare complex pulmonary disease (MAC-PD) strains compared to ATCC13950, and the genes of gluconeogenesis, fructose-1,6-bisphosphatase glpX, with respect to the core and accessory genomes.
- https://cdn.elifesciences.org/articles/99426/elife-99426-supp7-v1.xlsx
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Supplementary file 8
List of genes showing significantly increased or reduced transposon (Tn) insertion reads in the clinical M. intracellulare strains compared with ATCC13950 by a resampling analysis, with information of the genetic requirements detected by an hidden Markov model (HMM) analysis.
- https://cdn.elifesciences.org/articles/99426/elife-99426-supp8-v1.xlsx
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Supplementary file 9
Number of transposon (Tn) insertion reads in samples from infected mouse lungs.
Individual mice are represented as A–E after the sampling time points.
- https://cdn.elifesciences.org/articles/99426/elife-99426-supp9-v1.xlsx
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Supplementary file 10
List of genes identified with a resampling analysis in mouse lungs infected with M.i.198 transposon (Tn) mutant library strains.
- https://cdn.elifesciences.org/articles/99426/elife-99426-supp10-v1.xlsx
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Supplementary file 11
List of genes identified with a resampling analysis in mouse lungs infected with M.i.27 transposon (Tn) mutant library strains.
- https://cdn.elifesciences.org/articles/99426/elife-99426-supp11-v1.xlsx
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Supplementary file 12
List of genes required for mouse lung infection that are also required for hypoxic pellicle formation by ATCC13950.
- https://cdn.elifesciences.org/articles/99426/elife-99426-supp12-v1.xlsx
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Supplementary file 13
List of genes required for mouse lung infection at all time points from Day 1 to Week 16, and from Week 4 to Week 16.
- https://cdn.elifesciences.org/articles/99426/elife-99426-supp13-v1.xlsx
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Supplementary file 14
List of genes required for mouse lung infection at all time points from Day 1 to Week 16, and from Week 4 to Week 16, as well as required for hypoxic pellicle formation in ATCC13950.
- https://cdn.elifesciences.org/articles/99426/elife-99426-supp14-v1.xlsx
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Supplementary file 15
Result of the gene set enrichment analysis (GSEA).
Genes in the gene sets required for hypoxic pellicle formation observed in ATCC13950 and those showing increased genetic requirements observed in the clinical Mycobacterium avium–intracellulare complex pulmonary disease (MAC-PD) strains were ordered by their position in the ranked list of genes.
- https://cdn.elifesciences.org/articles/99426/elife-99426-supp15-v1.xlsx
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Supplementary file 16
Result of the gene set enrichment analysis (GSEA) in mouse Transposon sequencing (TnSeq) datasets.
Genes required for hypoxic pellicle formation observed in ATCC13950 were ordered by their position in the ranked list of genes.
- https://cdn.elifesciences.org/articles/99426/elife-99426-supp16-v1.xlsx
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Supplementary file 17
Result of the gene set enrichment analysis (GSEA) in mouse Transposon sequencing (TnSeq) datasets.
Genes showing increased genetic requirements observed in the clinical Mycobacterium avium–intracellulare complex pulmonary disease (MAC-PD) strains were ordered by their position in the ranked list of genes.
- https://cdn.elifesciences.org/articles/99426/elife-99426-supp17-v1.xlsx
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Supplementary file 18
Correlation analysis between the fitness costs (log2FC), efficiency of knockdown (quantitative reverse transcription PCR, qRT-PCR) and growth suppression in the knockdown strains of strain-dependent/accessory essential or growth-defect-associated genes.
- https://cdn.elifesciences.org/articles/99426/elife-99426-supp18-v1.xlsx
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Supplementary file 19
The raw data of colony-forming units (CFUs) used for drawing growth curves in Figure 8a.
- https://cdn.elifesciences.org/articles/99426/elife-99426-supp19-v1.xlsx
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Supplementary file 20
Oligonucleotides and primers used in this study.
- https://cdn.elifesciences.org/articles/99426/elife-99426-supp20-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/99426/elife-99426-mdarchecklist1-v1.docx
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Functional genomics reveals strain-specific genetic requirements conferring hypoxic growth in Mycobacterium intracellulare
eLife 13:RP99426.
https://doi.org/10.7554/eLife.99426.5
