High Persister Mutants in <i>Mycobacterium tuberculosis</i>

<div><p><i>Mycobacterium tuberculosis</i> forms drug-tolerant persister cells that are the probable cause of its recalcitrance to antibiotic therapy. While genetically identical to the rest of the population, persisters are dormant, which protects them from killing by bactericidal antibiotics. The mechanism of persister formation in <i>M</i>. <i>tuberculosis</i> is not well understood. In this study, we selected for high persister (<i>hip</i>) mutants and characterized them by whole genome sequencing and transcriptome analysis. In parallel, we identified and characterized clinical isolates that naturally produce high levels of persisters. We compared the <i>hip</i> mutants obtained <i>in vitro</i> with clinical isolates to identify candidate persister genes. Genes involved in lipid biosynthesis, carbon metabolism, toxin-antitoxin systems, and transcriptional regulators were among those identified. We also found that clinical <i>hip</i> isolates exhibited greater <i>ex vivo</i> survival than the low persister isolates. Our data suggest that <i>M</i>. <i>tuberculosis</i> persister formation involves multiple pathways, and <i>hip</i> mutants may contribute to the recalcitrance of the infection.</p></div

Distinct dynamics of cytokine mRNA expression in the lungs of MTb-infected NFATp^−/− and WT mice.

<p>NFATp^−/− and WT mice were infected with MTb and total RNA was purified from the lungs at 2, 4, 6, 8 (8.4), and 10 weeks post-infection. (<b>A</b>) IFN-γ, (<b>B</b>) TNF, and (<b>C</b>) IL-4 mRNA was measured by real-time PCR and values were normalized to ubiquitin mRNA. Error bars indicate mean of 3 samples ±SD.</p

Characterization of <i>hip</i> mutants obtained <i>in vitro</i>.

<p>Persister assays, performed by antibiotic treatment with streptomycin (10 μg/ml) and rifampicin (1 μg/ml) for 14 days, reveal the number of drug tolerant persister cells based on CFU counts. Exponential (A) and stationary phase (B) treatment of mutagenized strain mc²6020 at each stage of the <i>hip</i> mutant selection process. Time-dependent persister assays in exponential (C) and stationary phase (D) with independent mutants KL2801, KL2825, KL2849, and wild type strain (mc²6020). Late exponential phase cultures were treated with various concentrations of streptomycin (E) or rifampicin (F) or with antibiotics not used in the selection process, kanamycin (50 μg/ml) or ofloxacin (10 μg/ml) (G). Cultures grown in minimal media with glycerol, butyrate, or propionate as the sole carbon source were treated in exponential phase (H). Data represent the average of three biological replicates and the error bars represent standard deviation.</p

Summary of the study of <i>hip</i> mutants in <i>M</i>. <i>tuberculosis</i>.

<p>Schematic depicting the comparative analysis of <i>hip</i> mutants, generated <i>in vitro</i> and identified in clinical isolates, by whole genome sequencing and transcriptome analysis to identify candidate persister genes.</p

TNF, IFN-γ, and IL-4 production by CD4⁺ Th1 and Th2 cells from NFATp-deficient and WT mice.

<p>Primary CD4⁺ T cells from NFATp^−/− or WT mice were polarized into Th1 and Th2 populations for six days and subsequently stimulated with anti-CD3 plus anti-CD28 for 4 h before intracellular cytokine staining (ICS) for IL-4 and IFN-γ (<b>A</b>) or TNF and IFN-γ (<b>B</b>). NFATp-dependent production of IFN-γ protein by Th1 cells and IL-4 protein by Th2 cells is evident, while both Th1 and Th2 cells produce TNF protein with a more marked decrease in TNF expression in Th2 cells from NFATp^−/− mice.</p

Correlation of serum TNF protein levels with MTb disease progression in WT and NFATp^−/− mice.

<p>WT and NFATp^−/− mice were infected with the aerosolized clinical MTb strain HN878 at ∼100 CFU/mouse as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041427#pone.0041427-Barczak1" target="_blank">[50]</a>. Four random mice were bled at four-week intervals after initial infection and serum levels of TNF were measured by ELISA.</p

Varying NFATp requirement for cytokine expression in different primary cell populations.

<p><b>A and B.</b> TNF expression in BM-derived dendritic cells is not NFATp-dependent. Primary BM-DC were derived from WT and NFATp^−/− mice. After 7 days cells were stimulated with MTb lysates. At indicated time points the cells were lysed to prepare total RNA. (<b>A</b>) TNF mRNA was measured by real-time PCR. (<b>B</b>) TNF protein levels in the supernatants were measured by ELISA. <b>C and D.</b> TNF and IFN-γ expression in murine primary T cells is NFATp-dependent. Purified primary total CD4⁺ and naïve (CD4⁺CD62L⁺CD45RB^high) T cells from uninfected WT and NFATp^−/− mice were stimulated immediately with anti-CD3/CD28 antibody and then left in culture in neutral conditions for another six days and re-stimulated with anti-CD3/CD28 antibodies. Aliquots of cells were harvested at different time points post-induction as indicated. Purified total RNA was analyzed by real-time PCR with TNF and IFN-γ-specific primers, and cellular supernatants were used to measure TNF and IFN-γ protein levels by ELISA.</p

Increased susceptibility of NFATp^−/− mice to aerosolized <i>M. tuberculosis</i>.

<p><b>A.</b> Survival of BALB/c (WT) vs. NFATp^−/− mice after infection with MTb. 20 WT and 16 NFATp^−/− mice were infected with the aerosolized clinical MTb strain HN878 (∼149 CFU) and monitored for survival. <b>B</b> and <b>C.</b> Lung pathology. (<b>B</b>) NFATp^−/− and (<b>C</b>) WT mice were infected with MTb, and lungs taken from premortal NFATp^−/− mice and from the WT mice sacrificed at the same time were fixed in 10% neutral buffered Formalin. Fixed tissues were embedded in paraffin, and 6–10 µm sections were H&E or Ziehl-Neelsen acid-fast stained as indicated.</p

Genetic analysis of <i>hip</i> mutants obtained <i>in vitro</i>.

<p>Representative antibiotic survival plots of 18 <i>hip</i> mutant strains, obtained from 12 independent mutageneses, are presented along with lists of genes containing non-synonymous mutations within each strain.</p

Non-synonymous mutations identified in individual <i>in vitro hip</i> mutant strains by whole genome sequencing.

<p>Non-synonymous mutations identified in individual <i>in vitro hip</i> mutant strains by whole genome sequencing.</p