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Biotin-dependent cell envelope remodelling is required for Mycobacterium abscessus survival in lung infection

Nature Microbiology volume 8, pages 481–497 (2023)Cite this article

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Abstract

Mycobacterium abscessus is an emerging pathogen causing lung infection predominantly in patients with underlying structural abnormalities or lung disease and is resistant to most frontline antibiotics. As the pathogenic mechanisms of M. abscessus in the context of the lung are not well-understood, we developed an infection model using air–liquid interface culture and performed a transposon mutagenesis and sequencing screen to identify genes differentially required for bacterial survival in the lung. Biotin cofactor synthesis was required for M. abscessus growth due to increased intracellular biotin demand, while pharmacological inhibition of biotin synthesis prevented bacterial proliferation. Biotin was required for fatty acid remodelling, which increased cell envelope fluidity and promoted M. abscessus survival in the alkaline lung environment. Together, these results indicate that biotin-dependent fatty acid remodelling plays a critical role in pathogenic adaptation to the lung niche, suggesting that biotin synthesis and fatty acid metabolism might provide therapeutic targets for treatment of M. abscessus infection.

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Fig. 1: Air–liquid interface culture model for M. abscessus lung infection.
Fig. 2: Biotin synthesis is required in culture medium and lung infection model despite presence of biotin.
Fig. 3: Physiological environments impose demand for biotin synthesis.
Fig. 4: Biotin is required to support fatty acid remodelling that sustains envelope fluidity.
Fig. 5: Physiological pH alters fatty acid profile and imposes increased demand for biotin.

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Materials availability

All reagents generated in this study are available upon request from the corresponding author.

Data availability

All relevant data generated in this study are present within the manuscript and its Supplementary Information, with the following exceptions. Whole genome sequencing data for strain T35 are available on SRA (https://www.ncbi.nlm.nih.gov/sra) under project number PRJNA840944, accession number SAMN28571509. Raw TnSeq data are available on SRA under project number PRJNA902827. Source data are provided with this paper.

Code availability

R scripts used for this study are included as Supplementary File 4, and all files called by that R script are included as Supplementary Files 5–8.

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Acknowledgements

We thank all members of the Rubin and Fortune labs for input and advice on the manuscript; J.-A. Park and C. Mwase for assistance with air–liquid interface cultures; the Biopolymers Facility at Harvard Medical School for sequencing; and the Microscopy Resources on the North Quad (MicRoN) core at Harvard Medical School for assistance with microscopy. Electron microscopy imaging was performed in the HMS Electron Microscopy Facility. M.R.S. is a Merck Fellow of the Damon Runyon Cancer Research Foundation, DRG-2415-20. E.J.R. was supported by a Dean’s Innovation Award from Harvard Medical School, and by NIH/NIAID under award number R21AI156772. A.M. acknowledges support from the Ludwig Center for Metastasis. D.B.M acknowledges NIH R01 AI049313 and NIH U19 AI162584.

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Authors and Affiliations

  1. Department of Immunology and Infectious Disease, Harvard T.H. Chan School of Public Health, Boston, MA, USA

    Mark R. Sullivan, Kerry McGowen, Chidiebere Akusobi, Ian D. Wolf & Eric J. Rubin

  2. Department of Medicinal Chemistry, University of Minnesota College of Pharmacy, Minneapolis, MN, USA

    Qiang Liu & Courtney C. Aldrich

  3. Division of Rheumatology, Immunity and Inflammation, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA

    David C. Young, Jacob A. Mayfield, Sahadevan Raman & D. Branch Moody

  4. Ben May Department for Cancer Research, University of Chicago, Chicago, IL, USA

    Alexander Muir

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M.R.S. and E.J.R. conceptualized the project. M.R.S. and C.A. developed the methodology. M.R.S., D.C.Y. and J.A.M. conducted formal analysis. M.R.S., K.M., I.D.W., D.C.Y., S.R. and A.M. conducted the investigations. J.A.M. developed software. Q.L. and C.C.A. procured resources. M.R.S., D.C.Y. and J.A.M. performed visualization. M.R.S. wrote the original draft. M.R.S., K.M., C.A., D.C.Y., J.A.M., A.M., D.B.M. and E.J.R. reviewed and edited the second draft. M.R.S. and E.J.R. acquired funding. E.J.R., C.C.A. and D.B.M supervised the project.

Corresponding author

Correspondence to Eric J. Rubin.

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Competing interests

D.B.M consults with Pfizer and EnaraBio. The other authors declare no competing interests.

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Nature Microbiology thanks Laurent Kremer, Thomas Dick and Luiz Pedro de Carvalho for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Air-liquid interface culture model system.

(A) Scanning electron microscope images of apical surface of lung epithelial cells at 0 and 14 days after initiation of air-liquid interface. Images were obtained at 2000X magnification. Scale bar = 10 µm. Images representative of 3 biological replicates for day 0, 4 biological replicates for day 14. (B) Scanning electron microscope image of apical surface of lung epithelial cells at 14 days after initiation of air-liquid interface. Image was obtained at 10,000X magnification. Scale bar, 1 µm. Image representative of 4 biological replicates. (C) Widefield microscope images of NuLi-1 lung epithelial cells stained for F-actin, MUC5AC, and with DAPI to highlight nuclei. Images were obtained at 40X magnification. Scale bar, 20 µm. Images representative of 3 biological replicates. (D) Monolayer permeability as measured by amount of sodium fluorescein that penetrated through the epithelial layer at successive days after initiation of air-liquid interface. Data are normalized to empty, collagen-coated transwells and are presented as mean +/− SD. n = 3 biological replicates. (E) Fraction of M. abscessus remaining after aspiration of excess liquid to re-generate air-liquid interface for the M. abscessus type strain ATCC19977 and clinical isolate T35. Data are presented as individual values along with mean +/− SD. n = 6 biological replicates. (F) Colony forming units of M. abscessus ATCC19977 infected at a multiplicity of infection = 1 on the apical surface of lung epithelial cells. n = 3 biological replicates. Data are presented as mean +/− SD. (G) Luminescence emitted by M. abscessus ATCC1997 or clinical isolate T35 in lung infection model infected at the indicated multiplicity of infection (MOI) over 48 hr of infection. Data are presented as mean +/− SD. n = 3 biological replicates per condition. (H) Lactate dehydrogenase (LDH) release from lung epithelial cells at 0 and 24 hr post-infection. LDH release is normalized to uninfected control cells that were lysed to release maximal LDH. Data are presented as individual values along with mean +/− SD. n = 3 biological replicates per condition. (I) Scanning electron microscope image of apical surface of lung infection model at 48 hr post-infection. Image was obtained at 3000X magnification. Scale bar, 5 µm. (J) Number of M. abscessus colony forming units that were detached from the apical surface by vigorous washing after 48 hours in the lung infection model (Extracellular) and those that were attached to or inside the lung epithelial layer (Intracellular). Data are presented as individual values along with mean +/− SD. n = 3 biological replicates.

Extended Data Fig. 2 Development of biotin synthesis pathway knockouts.

(A) Proliferation rate of M. abscessus ATCC19977 grown either in tissue culture medium or on the apical surface of air-liquid interface lung cultures. Data are presented as individual values along with mean +/− SD. n = 3 biological replicates. p-value derived from unpaired, two-tailed t-test. (B) Schematic of biotin biosynthesis pathway. ACP: acyl carrier protein. CoA: coenzyme A. SAM: S-adenosyl methionine. ATP: adenosine triphosphate. AMP: adenosine monophosphate. PPi: inorganic phosphate. (C) Schematic of recombineering knockouts of bioA. zeoR: zeocin resistance cassette (D) Agarose gel electrophoresis of PCR products demonstrating insertion of zeoR into bioA. Expected PCR product sizes are indicated in (C). (E) Agarose gel electrophoresis of PCR products demonstrating excision of zeoR from bioA::zeoR. Expected PCR product sizes are indicated in (C). (D) and (E) are representative of 2 independent experiments.

Extended Data Fig. 3 Characterization of BioA inhibitor compound 36 in M. abscessus.

(A) Luminescence of M. abscessus ATCC19977 grown 48 hr with either vehicle or 16 µM compound 36 treatment. Media were either tissue culture medium or basal medium sampled from infected or mock infected air-liquid interface lung cultures dialyzed against tissue culture medium to replenish small molecules while retaining protein factors. Values are normalized within each condition to vehicle-treated. (B) Luminescence of the indicated M. abscessus clinical isolates grown in tissue culture medium for 48 hr with the specified final concentrations of the BioA inhibitor compound 36 in the medium. Values are normalized within each medium to the vehicle treated condition. (C) Luminescence of M. abscessus ATCC19977 grown in the lung infection model for 48 hr in the presence or absence of 16 µM compound 36 and/or 2 µM biotin added to the basal medium. Values are normalized to the vehicle treated condition. (D) Trypan blue measurement of viability of lung epithelial cells after 48 hr treatment with the indicated concentrations of compound 36. Values are normalized to vehicle treated condition. (E) Uncropped western blot (corresponding to Fig. 3E) for total biotinylated protein in M. abscessus ATCC19977 grown in tissue culture medium with either vehicle or 16 µM compound 36 along with the indicated supplementation of propionate. SYPRO Ruby panel depicts total protein. Blot is representative of 3 independent experiments. (F) Luminescence of M. abscessus ATCC19977 grown in tissue culture medium for 48 hr with either vehicle or 64 µM compound 36 along with the indicated supplementation of cholesterol. Values are normalized within each condition to vehicle-treated. (G) Luminescence of M. abscessus ATCC19977 grown in tissue culture medium for 48 hr with either vehicle or 16 µM compound 36 along with the indicated supplementation of sodium pyruvate. Values are normalized within each condition to vehicle-treated. For all graphs, data are presented as mean +/− SD. n = 3 biological replicates. All p-values derived from unpaired, two-tailed t-tests.

Extended Data Fig. 4 Altered biotin metabolism induces M. abscessus envelope remodeling.

(A) Fluorescence intensity scan from 440 nm to 490 nm from either laurdan stained (+cells, stained) or unstained (+cells, unstained) M. abscessus ATCC19977 samples or samples containing laurdan but no cells (-cells, stained). One representative sample is depicted for each condition. (B) Difference in laurdan generalized polarization (GP) between the same sample measured at 23 °C, then rapidly shifted to 37 °C and re-measured. n = 65 biological replicates. (C) Heatmap depicting relative abundance of 24 fatty acid species measured by GC/MS in M. abscessus ATCC19977 grown 48 hr in tissue culture medium treated either with vehicle or 16 µM compound 36. Samples and fatty acid species are both hierarchically clustered. n = 3 biological replicates. (D) Heatmap depicting relative abundance of 24 fatty acid species measured by GC/MS in M. abscessus ATCC19977 grown 48 hr in tissue culture medium treated with 16 µM compound 36 along with either vehicle or 1 mM sodium propionate. Samples and fatty acid species are both hierarchically clustered. n = 3 biological replicates. (E) Schematic of propionate utilization. CoA: coenzyme A. TCA: tricarboxylic acid. (F) Volcano plots depicting log2-fold change in abundance versus significance for ‘molecular events’ with linked retention time, mass, and intensity representing potential lipid species detected by HPLC/MS. Molecular events detected in M. abscessus ATCC19977 grown 48 hr in tissue culture medium containing vehicle are contrasted against those detected in cells treated with 16 μM compound 36 (left) or with 16 μM compound 36 and 1 mM propionate (right). Peaks significantly changed (p < 0.05 based on two-tailed moderated t-test using linear model and Bayesian shrinkage of variance methods with multiple hypothesis adjustment by the Benjamini-Hochberg method) in both contrasts (red circles) and a peak with the mass of compound 36 (blue outline) are indicated. Peak that is significantly depleted upon compound 36 treatment is depicted as an asterisk in both volcano plots. (G) Plot of retention time versus mass to charge ratio for all significantly changed peaks depicted in (F), which clusters peaks by shared chemical properties. Peaks significant in both contrasts (red), a peak with the mass of compound 36 (blue, [M + H]+) and select alternate compound 36 adducts (blue) are indicated.

Extended Data Fig. 5 pH is a determinant of biotin demand and fatty acid composition.

(A) Correlation between medium pH and sensitivity to biotin synthesis inhibition as measured by ratio of luminescence of M. abscessus ATCC19977 in tissue culture medium treated with 32 µM compound 36 compared to vehicle-treated after 48 hr. Medium pH changes are a secondary effect of adding pools of metabolites from mycobacterial medium to tissue culture medium (see Materials and Methods for composition of pools), and pH was measured by potentiometric pH meter. Data are presented as mean +/− SD. R2 and p-value derived from two-tailed Pearson correlation. Line of best fit derived from simple linear regression. (B) Uncropped western blot (corresponding to Fig. 5B) for total biotinylated protein in M. abscessus ATCC19977 grown in tissue culture medium adjusted to the indicated pH and treated with either vehicle or 16 µM compound 36. SYPRO Ruby panel depicts total protein. Blot is representative of 3 independent experiments. (C) Principal component analysis of M. abscessus ATCC19977 grown in tissue culture medium adjusted to the indicated pH based on GC/MS measurement of 24 fatty acid species. (D) Loading plot depicting individual fatty acid contributions to the principal components displayed in (C). (E) Ratio of colony forming units of M. abscessus ATCC19977 in air-liquid interface lung cultures treated with 128 µM compound 36 compared to vehicle-treated after 48 hr infection. Initial basal pH was adjusted to either 7.6 or 6.8, and final apical pH in each condition was determined to be 7.8 and 7.1, respectively. Data are presented as individual values along with mean +/− SD. n = 3 biological replicates. p-value derived from unpaired, two-tailed t-test. (F) Scanning electron microscope images of apical surface of lung infection model at 48 hr post-infection treated with 128 μM compound 36 with medium pH adjusted to 7.8 or 7.1. Images were obtained at 10,000X magnification. Scale bar = 1 µm. (G) pH of liquid sampled from the basal and apical surfaces of infected air-liquid interface lung cultures treated with 128 µM compound 36 as measured by phenol red absorbance. Data are presented as individual values along with mean +/− SD. n = 3 biological replicates.

Supplementary information

Reporting Summary

Supplementary Table

Table 1. Library statistics for TnSeq. Table 2. Relative requirement for genes responsible for recycling propionate. Table 3. Oligonucleotides used in this study. File 1. Formulation of tissue culture and mycobacterial media. File 2. Resampling analysis of relative gene requirements in lung infection model and tissue culture medium versus input library. File 3. Fatty acid quantitation in various media conditions.

Supplementary File 4

R markdown file with code for LC/MS lipidomic analysis.

Supplementary Data

Sample identification used by code in Supplementary File 4 for negative mode analysis.

Supplementary Data

Sample identification used by code in Supplementary File 4 for positive mode analysis.

Supplementary Data

LC/MS data used by code in Supplementary File 4 for negative mode analysis.

Supplementary Data

LC/MS data used by code in Supplementary File 4 for positive mode analysis.

Source data

Source Data Fig. 1

Uncropped scans of streptavidin-HRP blot.

Source Data Fig. 2

Visible light image to demonstrate protein size markers for streptavidin-HRP blot.

Source Data Fig. 3

SYPRO Ruby staining.

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Sullivan, M.R., McGowen, K., Liu, Q. et al. Biotin-dependent cell envelope remodelling is required for Mycobacterium abscessus survival in lung infection. Nat Microbiol 8, 481–497 (2023). https://doi.org/10.1038/s41564-022-01307-5

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