Mycobacterium tuberculosis Requires the Outer Membrane Lipid Phthiocerol Dimycocerosate for Starvation-Induced Antibiotic Tolerance

Alisha M. Block, Sarah B. Namugenyi, Nagendra Prasad Palani, Alyssa M. Brokaw, Leanne Zhang, Kenny B Beckman, Anna D. Tischler

Research output: Contribution to journalArticlepeer-review


Tolerance of Mycobacterium tuberculosis to antibiotics contributes to the long duration of tuberculosis (TB) treatment and the emergence of drug-resistant strains. M. tuberculosis drug tolerance is induced by nutrient restriction, but the genetic determinants that promote antibiotic tolerance triggered by nutrient limitation have not been comprehensively identified. Here, we show that M. tuberculosis requires production of the outer membrane lipid phthiocerol dimycocerosate (PDIM) to tolerate antibiotics under nutrient-limited conditions. We developed an arrayed transposon (Tn) mutant library in M. tuberculosis Erdman and used orthogonal pooling and transposon sequencing (Tn-seq) to map the locations of individual mutants in the library. We screened a subset of the library (;1,000 mutants) by Tn-seq and identified 32 and 102 Tn mutants with altered tolerance to antibiotics under stationary-phase and phosphate-starved conditions, respectively. Two mutants recovered from the arrayed library, ppgK::Tn and clpS::Tn, showed increased susceptibility to two different drug combinations under both nutrient-limited conditions, but their phenotypes were not complemented by the Tn-disrupted gene. Whole-genome sequencing revealed single nucleotide polymorphisms in both the ppgK::Tn and clpS::Tn mutants that prevented PDIM production. Complementation of the clpS::Tn ppsD Q291* mutant with ppsD restored PDIM production and antibiotic tolerance, demonstrating that loss of PDIM sensitized M. tuberculosis to antibiotics. Our data suggest that drugs targeting production of PDIM, a critical M. tuberculosis virulence determinant, have the potential to enhance the efficacy of existing antibiotics, thereby shortening TB treatment and limiting development of drug resistance.

Original languageEnglish (US)
Issue number1
StatePublished - Jan 2023

Bibliographical note

Funding Information:
This work was supported by a National Institutes of Health Director’s New Innovator Award DP2AI112245 from NIAID (A. D. Tischler), T90 DE02273 from NIDCR (A. M. Block), and a University of Minnesota Doctoral Dissertation Fellowship (S. B. Namugenyi). The content is solely the responsibility of the authors and does not reflect the official views of the National Institute of Dental and Craniofacial Research, the National Institute of Allergy and Infectious Diseases, or the National Institutes of Health.

Publisher Copyright:
Copyright © 2023 Block et al.


  • antibiotic resistance
  • drug tolerance
  • membrane permeability
  • Mycobacterium tuberculosis
  • nutrient limitation
  • PDIM
  • Tn-seq

PubMed: MeSH publication types

  • Journal Article
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't


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