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Functional genomics reveals strain-specific genetic requirements conferring hypoxic growth in
https://doi.org/10.7554/eLife.99426.5
contribution by elucidating the genetic determinants of growth and fitness across multiple clinical strains of Mycobacterium intracellulare, an understudied non-tuberculous mycobacterium. Using transposon sequencing (Tn-seq), the authors identify a core set of 131 genes essential for bacterial adaptation to hypoxia, providing a
foundation for anti-mycobacterial drug discovery.
https://doi.org/10.7554/eLife.99426.5.sa0
: Findings that have theoretical or practical implications for a subfield
: Appropriate and validated methodology in line with current state-of-the-art
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is a major etiological agent of the recently expanding
Mycobacterium avium–intracellulare
complex pulmonary disease (MAC-PD). Therapeutic regimens that include a combination of macrolides and antituberculous drugs have been implemented with limited success. To identify novel targets for drug development that accommodate the genomic diversity of
, we subjected eight clinical MAC-PD isolates and the type strain ATCC13950 to genome-wide profiling to comprehensively identify universally essential functions by transposon sequencing (TnSeq). Among these strains, we identified 131 shared essential or growth-defect-associated genes by TnSeq. Unlike the type strain, the clinical strains showed increased requirements for genes involved in gluconeogenesis and the type VII secretion system under standard growth conditions, the same genes required for hypoxic pellicle-type biofilm formation in ATCC13950. Consistent with the central role of hypoxia in the evolution of
, the clinical MAC-PD strains showed more rapid adaptation to hypoxic growth than the type strain. Importantly, the increased requirements of hypoxic fitness genes were confirmed in a mouse lung infection model. These findings confirm the concordant genetic requirements under hypoxic conditions in vitro and hypoxia-related conditions in vivo and highlight the importance of using clinical strains and host-relevant growth conditions to identify high-value targets for drug development.
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 (
). 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 (
). 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).
has been divided into four subspecies, including subspecies
has been divided into two subgroups, typical
M. paraintracellulare–M. indicus pranii
(MP-MIP), with currently two additional distinguished subspecies,
). 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 (
). 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 (
). 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
). 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 (
). 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
). They reported the diversity of genetic requirements among strains, including the type strain, such as
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
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.
To obtain comprehensive information on the genome-wide gene essentiality of
, we included nine representative strains of
with diverse genotypes for TnSeq in vitro in this study (
) to obtain 2–6 million reads of Tn insertions. The average number of Tn insertion reads was >50 at Tn-inserted TA sites, which confirmed the TnSeq data (
). The numbers of essential genes in the clinical MAC-PD strains, calculated with the hidden Markov model (HMM; a transition probability model), tended to be smaller than that in ATCC13950 (
). In total, 131 genes were identified as essential or growth-defect-associated with the HMM analysis across all
strains, including the eight MAC-PD clinical strains and ATCC13950 (
). The genes identified as universal essential or growth-defect-associated were involved in fundamental functions, such as glyoxylate metabolism, purine/pyrimidine metabolism, amino acid synthesis, lipid biosynthesis, arabinogalactan/peptidoglycan biosynthesis, porphyrin metabolism, DNA replication, transcription, amino acid tRNA ligases, ribosomal proteins, general secretion proteins, components of iron–sulfur cluster assembly, some ABC transporters, and the type VII secretion system. The genes identified corresponded to the genes that encode conventional and under-development