Research ArticleMICROBIOLOGY

Posttranslational modification of a histone-like protein regulates phenotypic resistance to isoniazid in mycobacteria

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Science Advances  02 May 2018:
Vol. 4, no. 5, eaao1478
DOI: 10.1126/sciadv.aao1478
  • Fig. 1 Identification of mycobacterial subpopulations that grow in the presence of drug.

    (A, B, D, and E) M. smegmatis. (C) M. tuberculosis. (A) Colonies formed on INH plates with indicated concentrations as a percentage of colonies formed on plates without drug. (B) Time of initial detection of colonies, as determined by CellProfiler, in the presence and absence of INH (4 μg/ml). (C) Distribution of M. tuberculosis colony sizes after 22 days of growth on INH. (D) Example series of one plate tracked over time with CellProfiler (INH, 4 μg/ml). Plates were run through custom program, and colored circles indicate the actual size of the detected colonies. (E) Histogram of major axis length of colonies for all colonies plated over time (INH, 4 μg/ml).

  • Fig. 2 Phenotypic drug resistance is heritable and semi-stable.

    (A) Small and large colonies were picked, resuspended in liquid media, and plated immediately (t = 0) and then passaged through liquid media without drug and plated at indicated time points. Ratio of colonies on INH plates/plates without INH was measured. SP, starting population. Significance was determined by unpaired t test. (B) LCVs and SCVs were picked from INH plates, resuspended in 7H9 media, and replated onto plates without drug. The histograms of the resulting colony sizes from each colony type after immediate replating are shown. (C) Large and small colonies were picked from INH plates, and cells were loaded into a microfluidic device and imaged for ~48 hours in media with INH (4 μg/ml). (D) Quantification of cell size at birth. Significance was determined by unpaired t test. (E) Quantification of time from birth to division for individual cells. Significance was determined by unpaired Student’s t test. For all panels, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

  • Fig. 3 Variants have distinct transcriptional signatures.

    Pools of LCVs and SCVs were assayed in duplicate. (A) Hierarchical clustering analysis of genes differentially expressed more than threefold. (B) Volcano plot analysis of RNA-seq data comparing gene expression profiles of SCVs and LCVs. Dots indicated in red and blue are genes that are down-regulated and up-regulated, respectively, in the SCVs more than twofold with a P value of <0.01. (C) Gene categories of genes differentially expressed >10-fold in LCVs versus SCVs.

  • Fig. 4 Loss of HupB results in significant reduction of colony formation in the presence of INH and alters gene expression.

    (A) Percent survivors were calculated from the number of colonies counted on the plates with drug as compared to the number of colonies on the plates without drug. (B) Representative images of ΔHupB_pjeb:WT and ΔHupB_pjeb:empty vector plated on INH (4 μg/ml). (C) RNA-seq analysis comparing gene expression patterns in ΔHupB_pjeb:WT and ΔHupB_pjeb:empty. Dots indicated in green are up-regulated in the ΔHupB_pjeb:empty strain. Strains were collected at late stationary phase. (D) GSEA of genes differentially expressed in ΔHupB_pjeb:empty strain versus ΔHupB_pjeb:WT as compared to the genes identified as down-regulated more than fourfold with a P value of <0.01 using DESeq analysis in comparison to SCV versus LCV. ES, enrichment score.

  • Fig. 5 Posttranslational modification sites on HupB.

    (A) Modified amino acids and posttranslational modification sites on HupB identified by whole-cell proteomics. (B) Predicted structure of M. tuberculosis HupB reprinted with permission from Bhowmick et al. (30). Modified amino acid residues identified in this study are circled. The K86 residue, the spectrum of which is depicted in (C), is circled in black. (C) MS/MS of tryptic peptide identified after a Mascot database search, where methylation on Lys86 can be inferred from the peptide mass/charge ratio (m/z) and the ion fragmentation pattern.

  • Fig. 6 K86R mutation results in the preferential loss of small colonies.

    (A) Total colony formation in the presence of INH (4 μg/ml) as a percentage of colonies formed on plates without drug. Colonies were counted after 7 days. (B) Representative plates of indicated strains plated on INH (4 μg/ml) at day 7. (C) Representative plates tracked over time using CellProfiler. (D) Histograms of colony sizes on INH plates (4 μg/ml) at day 7. Frequency distribution between ΔHupB_pjeb:WT and ΔHupB_pjeb:K86R is significantly different as determined by the Kolmogorov-Smirnov (KS) test. (E) Number of SCVs and LCVs at day 7 on INH plates (4 μg/ml). In (A) and (E), significance is determined by the Mann-Whitney test and is indicative of over 10 independent biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. NS, not significant.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/5/eaao1478/DC1

    table S1. Bacterial strains used in this study.

    table S2. Genes down-regulated >10-fold in small versus large colonies.

    table S3. Genes up-regulated more than fivefold in HupBΔ versus WT.

    fig. S1. M. tuberculosis colony sizes and numbers plated on increasing concentrations of INH.

    fig. S2. M. smegmatis strains expressing WT and mutant HupB alleles grown at WT rates.

    fig. S3. Modifications identified in previous study and current work.

    fig. S4. His-tagged HupB was purified from M. smegmatis, and modifications were identified by MS.

    fig. S5. Effects of disrupting HupB modification sites on HupB protein abundance.

  • Supplementary Materials

    This PDF file includes:

    • table S1. Bacterial strains used in this study.
    • table S2. Genes down-regulated >10-fold in small versus large colonies.
    • table S3. Genes up-regulated more than fivefold in HupBΔ versus WT.
    • fig. S1. M. tuberculosis colony sizes and numbers plated on increasing concentrations of INH.
    • fig. S2. M. smegmatis strains expressing WT and mutant HupB alleles grown at WT rates.
    • fig. S3. Modifications identified in previous study and current work.
    • fig. S4. His-tagged HupB was purified from M. smegmatis, and modifications were identified by MS.
    • fig. S5. Effects of disrupting HupB modification sites on HupB protein abundance.

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