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

Abstract

Mycobacterium tuberculosis causes 10 million cases of active TB disease and over 1 million deaths worldwide each year. TB treatment is complex, requiring at least 6 months of therapy with a combination of antibiotics.

Functional whole genome screen of nutrient-starvedMycobacterium tuberculosisidentifies genes involved in antibiotic tolerance

“…The recent screen by Block et al evaluated antibiotic tolerance under two separate stress conditions (stationary phase and phosphate starvation), and screened ~1000 mutant subsets of a defined collection of transposon mutants (35). Their cutoffs were more stringent than ours (LFC >|2| and p<0.025 versus LFC>|0.5| and p<0.05).…”

“…We have previously performed a similar Tn-Seq screen in M. avium complex to identify genes required for antibiotic tolerance in that organism (20). Others have also utilized this methodology for investigating the intrinsic mechanisms of antibiotic susceptibility in Mtb under nutrient-rich (34) and phosphate-starvation (35) conditions. We compared the Mtb genes conferring differential susceptibility to rifampin to those previously shown to confer differential susceptibility to rifabutin in M. avium (20) (Table 1).…”

Mycobacterium tuberculosisrequires conditionally essential metabolic pathways for infection

“…We previously constructed an arrayed Tn mutant library in the M. tuberculosis Erdman strain using MtbYM rich medium, which contains many additional carbon sources, amino acids, metabolic intermediates, vitamins and co-factors that are not present in Middlebrook medium [27, 28]. Our library contains 48 Tn mutants in genes annotated as “essential” in Middlebrook medium that are non-essential in MtbYM rich medium [11, 27, 28]. We refer to these genes as M iddlebrook- Es sential (M-ES) genes.…”

“…M-ES Tn mutants were screened for secondary point mutations that disrupt PDIM production known to be present in our Tn library by PCR and Sanger sequencing. We screened for mutations in ppsD and ppsE previously identified in other Tn mutants [28] and for additional common mutations in ppsB, ppsC and ppsE identified by whole-genome sequencing of M-ES Tn mutants (Table S4). To generate a consensus sequence for each strain, paired reads were mapped to the M. tuberculosis Erdman reference genome (NC_020559.1) using the "map to reference" function in Geneious 2021 software (Biomatters, Ltd.) as previously described [28].…”

“…We screened for mutations in ppsD and ppsE previously identified in other Tn mutants [28] and for additional common mutations in ppsB, ppsC and ppsE identified by whole-genome sequencing of M-ES Tn mutants (Table S4). To generate a consensus sequence for each strain, paired reads were mapped to the M. tuberculosis Erdman reference genome (NC_020559.1) using the "map to reference" function in Geneious 2021 software (Biomatters, Ltd.) as previously described [28]. To identify single nucleotide polymorphisms (SNPs) in Tn mutant genomes, the WT Erdman and Tn mutant consensus sequences were aligned in Geneious using the "Align Whole Genomes" function with the default Mauve Genome parameters, as described [28].…”