Microbiology and Infectious Disease

Functional genomics reveals strain-specific genetic requirements conferring hypoxic growth in Mycobacterium intracellulare

Yoshitaka Tateishi author has email address
Yuriko Ozeki
Akihito Nishiyama
Yuta Morishige
Yusuke Minato
Anthony D Baughn
Sohkichi Matsumoto

Department of Bacteriology, Graduate School of Medicine and Dental Sciences, Niigata University, Niigata, Japan
Department of Microbiology, Fukushima Medical University, Fukushima, Japan
Department of Mycobacterium Reference and Research, Research Institute of Tuberculosis, Japan Anti-Tuberculosis Association, Kiyose, Tokyo, Japan
Department of Microbiology, Fujita Health University School of Medicine, Toyoake, Japan
Department of Microbiology and Immunology, University of Minnesota Medical School, Minneapolis, United States
Laboratory of Tuberculosis, Institute of Tropical Disease, Universitas Airlangga, Surabaya, Indonesia
Department of Bacteriology, Osaka Metropolitan University Graduate School of Medicine, Osaka, Japan
Division of Research Aids, Hokkaido University Institute for Vaccine Research & Development, Sapporo, Japan

https://doi.org/10.7554/eLife.99426.3

Open access
Copyright information

Figures and data

Phylogenetic tree of the M. intracellulare strains and strategy of the 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 phylogenetic tree is shown in Supplementary Fig. 1. b Strategy of the procedure of TnSeq analyses.

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 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.

Detection of genes showing increased or reduced genetic requirements 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 ATCC13950¹⁰. *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 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).

Overview of the differences in genetic requirements between the clinical M. intracellulare strains and ATCC13950, drawn with Cytoscape⁶³.
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 genetic 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 genetic requirements 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.

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.”

Evaluation of the effect of the suppression of 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.

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 CFUs in 4 μl sample 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.

Sign up for email alerts