Evolution of Mycobacterium abscessus in the human lung: Cumulative mutations and genomic rearrangement of porin genes in patient isolates

ABSTRACT

Background

Mycobacterium abscessus subspecies massiliense (M. massiliense) is increasingly recognized as an emerging bacterial pathogen, particularly in cystic fibrosis (CF) patients and CF centres’ respiratory outbreaks. We characterized genomic and phenotypic changes in 15 serial isolates from two CF patients (1S and 2B) with chronic pulmonary M. massiliense infection leading to death, as well as four isolates from a CF centre outbreak in which patient 2B was the index case.

Results

Comparative genomic analysis revealed the mutations affecting growth rate, metabolism, transport, lipids (loss of glycopeptidolipids), antibiotic susceptibility (macrolides and aminoglycosides resistance), and virulence factors. Mutations in 23S rRNA, mmpL4, porin locus and tetR genes occurred in isolates from both CF patients. Interestingly, we identified two different spontaneous mutation events at the mycobacterial porin locus: a fusion of two tandem porin paralogs in patient 1S and a partial deletion of the first porin paralog in patient 2B. These genomic changes correlated with reduced porin protein expression, diminished ¹⁴C-glucose uptake, slower bacterial growth rates, and enhanced TNF-α induction in mycobacteria-infected THP-1 human cells. Porin gene complementation of porin mutants partly restored ¹⁴C-glucose uptake, growth rate and TNF-α levels to those of intact porin strains.

Conclusions

We hypothesize that specific mutations accumulated and maintained over time in M. massiliense, including mutations shared among transmissible strains, collectively lead to more virulent, host adapted lineages in CF patients and other susceptible hosts.

KEYWORDS:

• Mycobacterium massiliense
• Mycobacterium abscessus
• cystic fibrosis
• Porin
• whole genome sequencing
• immune response

Abbreviations

ADC: albumin, dextrose and catalase, BAL: bronchoalveolar lavage, CF: cystic fibrosis, CFU: colony forming units, FEV₁: serial forced expiratory volume in 1 second, GPL: glycopeptidolipid, ICU: intensive care unit, MOI: multiplicity of infection, MTB: Mycobacterium tuberculosis, SNP: Single nucleotide polymorphisms, ORF: Open reading frame, RGM: rapidly growing mycobacteria, NTM: Nontuberculous mycobacteria.

Acknowledgements

This study was supported in part by intramural research programs of the National Institute of Allergy and Infectious Diseases (NIAID), the National Heart, Lung and Blood Institute and the NIH Clinical Center and by NIAID contract number HHSN272200900009C to the Institute for Genome Sciences and funds from the Cystic Fibrosis Foundation under grant number TETTEL13G0 to HT. We thank Dr. M. Jackson, University of Colorado for the Plasmid pCherry3 with a kanamycin drug resistance cassette.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Supplemental data

Supplemental data for this article can be accessed online at https://doi.org/10.1080/21505594.2023.2215602.

Data availability statement

The authors confirm that the data supporting the findings of this study are available within the article, its supplementary materials and NCBI https://www.ncbi.nlm.nih.gov/nuccore/ under accession numbers AKUL00000000, AKUK00000000, AKUJ00000000, AKUI00000000, AKUM00000000, AKUO00000000, JARETB000000000, AKUW00000000, AKUV00000000¸ AKUU00000000, AKUN00000000, JAOW00000000, AYTF00000000, JAOV00000000, AYTA00000000.

Additional information

Funding

The work was supported by the Cystic Fibrosis Foundation Therapeutics [TETTEL13G0]; Division of Intramural Research, National Institute of Allergy and Infectious Diseases [HHSN272200900009C]; NHLBI Division of Intramural Research [NA]; NIH Clinical Center [NA].