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

In contrast to the ongoing decline in the rate of tuberculosis, Mycobacterium avium–intracellulare complex pulmonary disease (MAC-PD) is increasing in many parts of the world (Adjemian et al., 2012; Namkoong et al., 2016). The standard therapy for MAC-PD involves multidrug chemotherapy that includes clarithromycin and several antituberculous drugs. Unfortunately, treatment failure is common, resulting in relapse, disease progression, and death (Daley et al., 2020). Therefore, it is critical to investigate the bacterial physiological characteristics that underlie the ability of MAC-PD strains to establish and maintain chronic drug-recalcitrant infections.

Recent advances in comparative genomics have revealed major differences in the genomic features of reference type strains and circulating pathogenic clinical isolates. Extensive genomic diversity has been recognized across clinical isolates of nontuberculous mycobacteria (NTM). Mycobacterium avium has been divided into four subspecies, including subspecies avium, hominissuis, silvaticum, and paratuberculosis (Uchiya et al., 2017). Whereas M. intracellulare has been divided into two subgroups, typical M. intracellulare (TMI) and M. paraintracellulare–M. indicus pranii (MP-MIP), with currently two additional distinguished subspecies, yongonense and chimaera (Tateishi et al., 2021; Tortoli et al., 2019). Despite the increasing number of sequenced MAC-PD strains, the molecular genetic mechanisms that underlie their virulence and pathogenesis are still poorly understood.

The burgeoning field of functional genomics has allowed the rapid assessment of molecular aspects of organisms on a genome-wide scale. Transposon (Tn) sequencing (TnSeq) is a functional genomic approach that combines saturation-level transposon-insertion mutagenesis with next-generation sequencing (NGS) to comprehensively evaluate the fitness costs associated with gene inactivation in bacteria. Over the last decade, TnSeq has been applied to numerous bacterial species using various approaches (van Opijnen and Camilli, 2013). Whereas initial studies applied TnSeq to reference strains, recent approaches have included the comparative assessment of representative clinical strains to better understand the fundamental aspects of pathogen biology (Carey et al., 2018; Akusobi et al., 2025). We have previously used TnSeq to globally characterize the genes that are essential for growth versus those that are essential for hypoxic pellicle formation in the M. intracellulare type strain ATCC13950 (Tateishi et al., 2020). However, given the recent expansion of MAC-PD and the well-recognized high levels of genomic diversity in this complex of bacteria, data obtained from the study of a decades-old reference strain is not expected to represent the diversity of extant clinical isolates. ATCC13950 was originally isolated from the abdominal lymph node of the 34-month-old female (Cuttino and McCABE, 1949). Even though the etiological bacterial species are the same, the clinical manifestation of infant lymphadenitis differs markedly from MAC-PD, which often occurs in middle-aged and elderly female patients with no predisposing immunological disorder. So far, there are a total of two reports on TnSeq that compare genetic requirements between clinical mycobacterial strains and the type strains in M. tuberculosis (Carey et al., 2018) and M. abscessus (Akusobi et al., 2025). They reported the diversity of genetic requirements among strains, including the type strain, such as M. tuberculosis H37Rv and M. abscessus ATCC19977. Therefore, it is essential to investigate a collection of strains that captures the diversity of recent clinical MAC-PD strains in order to define key molecular aspects of their pathogenesis and virulence.

In this study, we used TnSeq to analyze a set of recently isolated clinical M. intracellulare strains with diverse genotypes. We integrated the gene essentiality data for these strains and the type strain to identify the common essential and growth-defect-associated genes that represent the genomic diversity of this group of pathogens. We also identified the greater requirements of genes involved in gluconeogenesis, the type VII secretion system, and cysteine desulfurase in the clinical MAC-PD strains than in the type strain. Furthermore, the requirement for these genes was confirmed in a mouse lung infection model. The profiles of genetic requirements in clinical MAC-PD strains suggest the mechanism of hypoxic adaptation in NTM in terms of promising drug targets, especially for strains that cause clinical MAC-PD.

In this study, using TnSeq of nine genetically diverse M. intracellulare strains, we have identified 131 shared essential and growth-defect-associated genes with an HMM analysis. The genes identified are promising drug targets, representing the genomic diversity of the clinical MAC-PD strains and reflecting the actual clinical situation. The strategy of narrowing the essential genes of bacteria is a useful approach for refining the database of drug targets. We have also demonstrated an overview of the differences in genetic requirements for hypoxic growth between the ATCC type strain and the clinical M. intracellulare strains responsible for MAC-PD, with the evidence of earlier entry to log phase followed by slower growth rate observed in most of these clinical strains compared to ATCC13950. This is the first study to demonstrate the positive relationship between the profiles of increased genetic requirements for hypoxic metabolic remodeling and the phenotypes advantageous for bacterial survival under hypoxic conditions.

In this study, 121 genes were detected as significantly reduced Tn insertion in the clinical MAC-PD strains compared to ATCC13950, which was higher than the case of Mtb showing 10–50 genes with altered Tn insertion reads detected by a resampling analysis (Carey et al., 2018). This may reflect the genomic diversity of M. intracellulare compared with that of Mtb. The proportion of accessory genes is larger in NTM than in Mtb (Yang et al., 2018). In a clinical trial of phage therapy for M. abscessus infection, the number of accessory genes was shown to decrease with the loss of virulence in M. abscessus during the course of therapy (Nick et al., 2022), suggesting some role for accessory genes in its clinical pathogenesis. Our data on the overlap of the accessory essential or growth-defect-associated genes, such as pckA and mycP5 (Supplementary file 7) with genes required for mouse lung infection (Figure 6b, Figure 6—figure supplement 1) supports the idea that accessory genes (non-core genes) may encode functions required for bacterial survival, especially under non-optimal conditions, such as hypoxia and during the infection of human and animal bodies.

Our TnSeq data are consistent with metabolomic data that have shown the importance of the gluconeogenic carbon flow of TCA cycle intermediates for bacterial growth under hypoxic conditions in terms of the essentiality of PckA and the reverse TCA cycle (Eoh et al., 2017; Lim et al., 2021; Marrero et al., 2010; Watanabe et al., 2011). Gluconeogenesis is essential for bacterial growth when fatty acids are used as a major nutrient (Marrero et al., 2010). PckA catalyzes the conversion of the metabolites derived from the TCA cycle to the metabolites of gluconeogenesis (Lim et al., 2021). The reaction catalyzed by PckA is essential for bacterial growth when the available nutrients are predominantly fatty acids (Gouzy et al., 2021; Quinonez et al., 2022). The main nutrients for mycobacteria inside hypoxic granulomas in an infected human or mouse body are lipids, such as fatty acids and cholesterol, rather than carbohydrates (Marrero et al., 2010; Pandey and Sassetti, 2008). In the present study, M. intracellulare grew under 5% oxygen, whereas Mtb stops growing under 1% oxygen and enters a dormant state (Lim et al., 2021). The depletion of phosphoenolpyruvate in the pckA deletion mutant of Mtb reduced the phosphoenolpyruvate–carbon flux essential for bacterial growth and drug sensitivity (Lim et al., 2021). The present study supports the idea that PckA is essential for the microaerobic growth of mycobacteria. Our TnSeq data imply the preferential adaptation of the clinical MAC-PD strains for circumstances in vivo, which are characterized by hypoxia.

On the other hand, such metabolic shift towards gluconeogenesis and reverse TCA cycle can also be observed under reactive nitrogen species and in vivo infection as well. This suggests that the metabolic shift observed under hypoxia is a general stress response rather than being specific to hypoxia due to the nature of the provided/available carbon source (Prosser et al., 2017). Several factors other than hypoxia can be estimated to be related to the change of the genetic requirements in the clinical MAC-PD strains, such as host-related factors and general stress conditions (i.e. nutrition of abundant fatty acids and less carbohydrates, low pH circumstances) (Marrero et al., 2010; Gouzy et al., 2021; Quinonez et al., 2022; Pandey and Sassetti, 2008). Except for our data of the profile of genes required for hypoxic pellicle formation in M. intracellulare (Tateishi et al., 2020), no fundamental genome-wide data are available on the change of the genetic requirements in response to each stress factor in non-tuberculous mycobacteria. Further studies are needed to discriminate which factors are specifically involved in the increased requirements of each gene. Although of interest, it is beyond the scope of the current study.

As in the glpX-knockdown strains, csd knockdown caused growth suppression in several MAC-PD strains. This type II Csd is involved in the biosynthesis of the iron–sulfur clusters of the SUF machinery. Compared with the type I cysteine desulfurase IscS, the SUF machinery is active under oxidative-stress and iron-limiting conditions (Takahashi and Tokumoto, 2002). Representatives of iron–sulfur cluster proteins include components of the electron-transport system, such as NADH dehydrogenase I (Nuo) and succinate dehydrogenase (Sdh), which are components of respiratory complexes I and II, respectively. Under hypoxic conditions, the non-proton-translocating type II NADH dehydrogenase Ndh is upregulated, and cytochrome bd oxidase (CydAB) and the proton-pumping type I NADH dehydrogenase (Nuo) are downregulated (Prosser et al., 2017). In the reverse TCA cycle on the same reaction pathway as Sdh, fumarate reductase (Fnr) is also an iron–sulfur cluster protein that produces succinate and is a proposed alternative electron acceptor under hypoxic conditions (Watanabe et al., 2011; Cecchini et al., 2002). The increased essentiality of the csd gene observed in the clinical MAC-PD strains seems to reflect the switching of the electron-transport system to less-energy-efficient respiration to support preferential hypoxic survival, including in vivo.

About 10% of the genes required for mouse infection are also required for hypoxic pellicle formation. The genes showing increased genetic requirements in the clinical MAC-PD strains, such as pckA and genes encoding the ESX-5 type VII secretion system, including eccC5 and mycP5, also showed increased genetic requirements in mouse infections. In a TnSeq study of mice infected intravenously with M. avium, the genes of the ESX-5 type VII secretion system were required for infection of the liver and spleen (Dragset et al., 2019). The ESX-5 type VII secretion system is involved in the transport of fatty acids and the virulence of mycobacteria (Ates et al., 2015; Ates et al., 2016). These similarities in the profiles of genetic requirements prompt us to propose some role of biofilm formation in mycobacterial pathogenesis in animals and humans. In the present study, we shed light on the impact of hypoxia on biofilm formation, the acquisition of virulence, and the in vivo pathogenesis of NTM in terms of global genetic requirements. Although the morphologies of the pellicle and macroscopic lesions (including cavitation and nodular bronchiectasis) are not similar, monitoring the expression of the genes identified in this study may provide clues to the mechanism of biofilm formation in vivo.

The data on the validation of the universal essential or growth-defect associated genes by the CRISPR-i system was overall acceptable in the clinically MAC-PD strains whose knockdown strains could be constructed (Figure 7a). However, we have encountered the difficulties of validating the TnSeq-hit genes by CRISPR-i experiment, especially accessory and strain-dependent essential or growth-defect-associated genes in the MAC-PD strains other than M.i.198 or M003 (Figure 7b, Figure 7—figure supplement 1). We found the lower efficiency of the suppression of gene expression by CRISPR-i in M. intracellulare than in Mtb that is reported to be suppressed to 6% under 10 ng/ml anhydrotetracycline (aTc) and by <1% under 100 ng/ml aTc in the knockdown strains, compared to strains expressing scrambled single guide RNAs (sgRNA) (McNeil and Cook, 2019). However, because the levels of knockdown efficiency were comparable between strain-dependent/accessory essential genes and universally essential genes (Figure 7—figure supplement 2), the low efficiency of knockdown does not seem to explain the reason for the discrepancy between TnSeq and CRISPR-i results. Although it is difficult to find the clear definitive reason for this phenomenon only from the current study, the possible reason may be speculated by the bypass mechanism of gene essentiality which is characteristically observed in strain-dependent/accessory essential or growth-defect-associated genes. According to the publication by Rosconi et al., 2022 reporting the ‘forced-evolution experiments’ of 36 clinical Streptococcus pneumoniae strains, gene essentiality can be bypassed by several mechanisms, including the composition of the accessory genome and pathway rewiring. They successfully recovered knockout mutants from transformation experiments in strain-dependent/accessory essential genes, such as cytidine monophosphate kinase, a folate pathway enzyme formate tetrahydrofolate ligase and an undecaprenyl phosphate-biosynthesis pathway enzyme farnesyl-diphosphate synthase. The bypassing of gene essentiality could be suggested by observing suppressor mutations and synthetic lethality in knockout strains. By contrast, universal essential genes were reported to fulfill the three categories, including high levels of conservation within and often across species, limited genetic diversity, and high and stable expression levels. Consequently, universal essential genes were estimated to be rigid, largely immutable key components to an organism’s survival. The knockout recovery of the universal essential genes was reported to fail, as shown by no colonies or only the appearance of merodiploids. Taking into consideration such bypass mechanism of gene essentiality, the inconsistent effect of gene silencing of strain-dependent/accessory essential genes on bacterial growth may reflect some kinds of pathway rewiring that helps the bacteria grow under suppression of the target gene expression. Nevertheless, our study has opened the avenue for functional analysis of NTM that leads to the development of novel drugs targeting universal and accessory/strain-dependent essential metabolic pathways, especially by making use of M.i.198 and M003 as optimal NTM strains for gene manipulation.

In this study, we revealed the pattern of hypoxic adaptation characteristic to the clinical MAC-PD strains by the growth curve analysis: an early entry to log phase followed by a reduced growth rate (Figure 8). This pattern implicates the continuous impact on the infected hosts during logarithmic bacterial growth, which may be involved in the persistent and steadily progressive illness for years in humans (Daley et al., 2020). The advantage of slow growth in mycobacteria may be inferred by the role of a histone-like nucleoid protein, mycobacterial DNA-binding protein 1 (MDP1), also designated HupB (Savitskaya et al., 2018; Enany et al., 2017; Shaban et al., 2023). The expression of MDP1 is enhanced in the stationary phase, resulting in reduced growth speed and long-term survival with increased resistance to reactive oxygen species (ROS) (Savitskaya et al., 2018; Enany et al., 2017; Shaban et al., 2023). The reduced growth rate after entry to the log phase may allow long-term survival of mycobacteria under ROS-rich circumstances inside host cells. The pattern of hypoxic adaptation as observed in the clinical MAC-PD strains may enhance pathogenesis by extending the period of the repeated process of bacterial invasion and replication in infected macrophages.

Contrary to our expectation, not all clinical strains showed rapid adaptation from aerobic to hypoxic conditions relative to ATCC13950. The two strains belonging to the MP-MIP subgroup, MOTT64 and M001, showed similar time at midpoint under aerobic conditions. However, the time at midpoint was significantly different between MOTT64 and M001 under hypoxia, the latter showing great delay of timing of entry to logarithmic phase. In contrast to the majority of the clinical strains that showed slow growth at midpoint under hypoxia, neither strain showed such a phenomenon. These data suggest that some, but not a major proportion of, clinical M. intracellulare strains do not have an advantageous phenotype for continuous logarithmic growth under hypoxia. In Mtb, the adaptation to hypoxia is considered to be necessary for clinical pathogenesis because the conditions in vivo, such as inside macrophages or granulomas, are hypoxic (Prosser et al., 2017; Rustad et al., 2009). Our inability to construct knockdown strains in M001 and MOTT64 prevented us from clarifying the factors that discriminate against the pattern of hypoxic adaptation. Although the implication in clinical situations has not been proven, strains without slow growth under hypoxia may have different (possibly strain-specific) mechanisms of hypoxic adaptation corresponding to their growth phenotypes under hypoxia. Nevertheless, because we have shown that most of the M. intracellulare strains with different genotypes showed preferential adaptation to hypoxia in this study, our results largely reflect the clinical situation of the etiological agents of MAC-PD. A possible strategy for identifying factors that confer clinical pathogenesis will be to focus on the major clinical strains that show preferential hypoxic adaptation.

We tried to use inhibitors of phosphoenolpyruvate carboxykinase to confirm the increased essentiality of the pckA gene. However, we detected no positive effect because millimolar 3-mercaptopicolinic acid is required to show the effect of inhibition, even in eukaryotic cells, which have no cell wall (Brearley et al., 2020). Moreover, 3-alkyl-1,8-dibenzylxanthine (Montal et al., 2019) also only effectively inhibits PckA in eukaryotic cells. However, PckA has been proposed as a drug target for Mtb (Mcleod et al., 2019), so our TnSeq data seem to be consistent with studies directed towards antimycobacterial drug discovery.

We found the difference in capacity of transformation between strains belonging to the same genomic M. paraintracellulare-M. indicus pranii (MP-MIP) subgroup. Although the direct mechanism determining the competency for foreign DNA has not been elucidated in M. intracellulare and other NTM species, several studies on general bacteria suggest the difficulties of introducing foreign DNA into clinical strains compared to the laboratory strains. As suggested in Staphylococcus aureus (Corvaglia et al., 2010), some clinical strains develop elimination systems of foreign nucleic acids, such as a type III-like restriction endonuclease. As suggested in gram-negative bacteria (Qin et al., 2022), there may be some difference in cell surface structures between strains, resulting in the necessity of polymyxin B nonapeptide targeting cell membrane for transforming clinical strains. The efficiency of eliminating foreign DNA may be attributed to various kinds of strain-specific factors, including restriction endonuclease, natural CRISPR-interference system, and cell wall structures rather than a simple genotype factor.

There may be a claim that the saturation of Tn insertion in most of each replicate were 50–60% in our libraries. However, our method of constructing Tn mutant libraries is in accordance with the previous studies of ‘high-density’ transposon libraries (Akusobi et al., 2025) because the saturation of the Tn mutant libraries by combining replicates are 62–79% as follows: ATCC13950: 67.6%, M001: 72.9%, M003: 63.0%, M018: 62.4%, M019: 74.5%, M.i.27: 76.6%, M.i.198: 68.0%, MOTT64: 77.6%, M021: 79.9%. That is, we predicted gene essentiality from the Tn mutant libraries with 62–79% saturation in each strain. However, there is a non-permissive sequence pattern in mariner transposon mutagenesis and using more than 10 replicates of Tn mutant libraries is reported to be a more accurate method (DeJesus et al., 2017; Rifat et al., 2021). The issue of high probability of judging nonessential regions as mistakenly as ‘essential’ are mainly found in small ORFs (2–9 TA sites) rather than larger ORFs (TA sites >= 10) (DeJesus et al., 2017). There are 4136 (6.43% of total ORFs) nonpermissive TA sites in ATCC13950, which is less than in M. tuberculosis H37Rv (9% of total ORFs) DeJesus et al., 2017 and in M. abscessus ATCC19977 (8.1% of total ORFs) (Rifat et al., 2021). As for small ORFs (2–9 TA sites), there are non-permissive TA sites in 41 genes (ORFs) of common essential or growth-defect-associated (1.35% of a total of 3021 larger ORFs in ATCC13950). With respect to the covering of non-permissive sites, our TnSeq data may need to be improved to classify quite accurately the gene essentiality, particularly on the nonstructural small regulatory genes, including small RNAs, although the study by DeJesus shows the comparable number of essential genes in large genes possessing more than 10 TA sites between 2 and 14 TnSeq datasets, most of which seem to be structural genes (DeJesus et al., 2017).

We have provided the data suggesting the preferential hypoxic adaptation in clinical strains compared to the ATCC type strain by the growth assay of individual strains. To strengthen our claim, several experiments are suggested, including mixed culture experiments of clinical and reference strains under hypoxia. However, co-culture conditions introduce additional variables, including inter-strain competition or synergy, which can obscure the specific contributions of hypoxic adaptation in each strain. Therefore, we took the current approach using monoculture growth curves under defined oxygen conditions, which offers a clearer interpretation of strain-specific hypoxic responses. Furthermore, one of the limitations of this study is the lack of validation of TnSeq results with individual gene knockouts. Contrary to the case of Mtb, the technique of constructing knockout mutants of slow-growing NTM, including M. intracellulare has not been established for a long time. We have just recently succeeded in constructing the vector plasmids for making knockout mutants of M. intracellulare (Tateishi et al., 2024). A growth assay of individual knockout strains of genes showing increased genetic requirements, such as pckA, glpX, csd, eccC5, and mycP5 in the clinical strains is suggested to provide the direct involvement of these genes on the preferential hypoxic adaptation in clinical strains. We have a future plan to construct knockout mutants of these genes to confirm the involvement of these genes in preferential hypoxic adaptation.

In summary, we have identified 131 universal essential and growth-defect-associated genes that cover the genomic diversity of M. intracellulare strains responsible for MAC-PD, which narrowed down the drug targets against MAC-PD. Furthermore, we have demonstrated preferential adaptation to hypoxia in clinical NTM strains compared with the type strain in terms of the increased genetic requirements for hypoxic growth, such as those encoding phosphoenolpyruvate carboxykinase, the ESX-5 type VII secretion system, and cysteine desulfurase. These genes were shown to have a significant impact on infection in vivo by increasing their genetic requirements. Our data highlight the importance of using clinical strains and hypoxic conditions in experimental research and provide fundamental resources for developing novel drugs directed towards nontuberculous mycobacterial strains that cause severe clinical diseases.

The TnSeq sequencing datasets are openly available in the DDBJ Sequence Read Archive database with the primary accession numbers DRA017301. The authors confirm that the data supporting the findings of this study other than the TnSeq sequencing datasets are available within the article.

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Author details

1. Yoshitaka Tateishi

   1. Department of Bacteriology, Graduate School of Medicine and Dental Sciences, Niigata University, Niigata, Japan
   2. Department of Microbiology, Fukushima Medical University, Fukushima, Japan

   Contribution

   Conceptualization, Resources, Data curation, Software, Formal analysis, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing – original draft, Project administration, Writing – review and editing

   For correspondence

   y-tateishi@med.niigata-u.ac.jp

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-1200-0000

2. Yuriko Ozeki

   Department of Bacteriology, Graduate School of Medicine and Dental Sciences, Niigata University, Niigata, Japan

   Contribution

   Resources

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-0672-1039

3. Akihito Nishiyama

   Department of Bacteriology, Graduate School of Medicine and Dental Sciences, Niigata University, Niigata, Japan

   Contribution

   Resources

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-4416-7710

4. Yuta Morishige

   Department of Mycobacterium Reference and Research, The Research Institute of Tuberculosis, Japan Anti-Tuberculosis Association, Kiyose, Japan

   Contribution

   Resources, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-3818-5288

5. Yusuke Minato

   Center for Infectious Disease Research, Fujita Health University, Toyoake, Japan

   Contribution

   Resources, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-0888-8564

6. Anthony David Baughn

   Department of Microbiology and Immunology, University of Minnesota Medical School, Minneapolis, United States

   Contribution

   Resources, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-1188-4238

7. Sohkichi Matsumoto

   1. Department of Bacteriology, Graduate School of Medicine and Dental Sciences, Niigata University, Niigata, Japan
   2. Medical Microbiology, Faculty of Medicine, Universitas Airlangga, Surabaya, Indonesia
   3. Department of Bacteriology, Osaka Metropolitan University Graduate School of Medicine, Osaka, Japan
   4. Division of Research Aids, Hokkaido University Institute for Vaccine Research and Development, Sapporo, Japan

   Contribution

   Resources, Supervision, Funding acquisition, Project administration, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-6106-7538

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