Research Article | Therapeutics and Prevention

Novel Antimicrobials from Uncultured Bacteria Acting against Mycobacterium tuberculosis

Jeffrey Quigley, Aaron Peoples, Asel Sarybaeva, Dallas Hughes, Meghan Ghiglieri, Catherine Achorn, Alysha Desrosiers, Cintia Felix, Libang Liang, Stephanie Malveira, William Millett, Anthony Nitti, Baldwin Tran, Ashley Zullo, Clemens Anklin, Amy Spoering, Losee Lucy Ling, Kim Lewis
K. Heran Darwin, Editor
DOI: 10.1128/mBio.01516-20

Article Figures & Data

Figures

  • FIG 1
    FIG 1

    Structures of compounds. Shown are the chemical structures of compounds identified in screening. The genera of the producing organisms and the exact mass in Daltons of each compound are indicated.

  • FIG 2
    FIG 2

    Compound activity against exponentially growing and stationary-phase M. tuberculosis cultures. (a) M. tuberculosis was grown to mid-log phase and diluted to an OD600 of 0.003. The cultures were either left untreated (UT) or treated with each compound at 4× MIC (amycobactin) or 10× MIC (streptomycobactin, kitamycobactin, and marfomycin D). Samples were taken at the indicated time points and plated for CFU counting. (b) Stationary-phase cultures of M. tuberculosis were either left untreated or treated with each compound at 4× MIC (amycobactin) or 10× MIC (streptomycobactin, kitamycobactin, and marfomycin D). Samples were plated for CFU counting at the indicated time points. Data represent the results of two replicates and are displayed as means ± standard errors of the means.

  • FIG 3
    FIG 3

    Analysis of amycobactin mutants in M. smegmatis and M. tuberculosis. (a) Alignment of protein sequences from WT M. smegmatis SecY and amycobactin mutants N28R1 and N28R2. The deletions in each mutant are boxed in red. (b) Growth curves of WT M. smegmatis and mutants containing targeted 3- and 6-amino-acid deletions in secY conferring resistance to amycobactin. (c) Growth curves of WT M. tuberculosis and a mutant containing a targeted 3-amino-acid deletion in secY conferring resistance to amycobactin. Data in panels b and c represent the results of two independent experiments and are displayed as means ± standard errors of the means. (d and e) Side view (d) and top view (e) of the predicted crystal structure of M. tuberculosis SecY, with the amycobactin resistance-conferring mutations shown in red. For reference, helices 2 and 7, which together form the lateral gate of SecY, are shown in blue. The plug restricting secretion through the central channel of SecY is shown in orange.

  • FIG 4
    FIG 4

    Amycobactin inhibits protein secretion through the Sec translocon. (a) The M. smegmatis ′BlaTEM-1 reporter strain was used to monitor the presence of β-lactamase in the culture supernatant (CF) (red curves) and whole-cell lysate (WCL) (black curves), either untreated (UT) (circles) or after treatment with amycobactin (squares). β-Lactamase was monitored by using the cleavage of the chromogenic β-lactamase substrate nitrocefin and monitoring the absorbance at 490 nm and 390 nm every 5 min for 60 min. The data, displayed as the ratio of absorbance at 490 nm (cleaved product) to absorbance at 390 nm (uncleaved nitrocefin), represent the results of three independent experiments. (b) Culture filtrate samples were analyzed for the maximum change in absorbance at 490 nm (Vmax490) over the course of the 60-minute experiment for which results are shown in panel a. (c) Representative Western blot analysis of β-lactamase protein in the CF and WCL of the untreated or amycobactin-treated M. smegmatis ′BlaTEM-1 reporter strain. (d) Densitometry analysis, using ImageJ software, of the Western blots of WCL and CF from untreated and amycobactin-treated cultures. AUC, area under the curve. Error bars display standard errors of the means. Significance was determined by Student’s t test (b) or one-way analysis of variance (d). ***, P ≤ 0.001; ****, P ≤ 0.0001; ns, not significant. Data represent the results of three independent experiments.

Tables

  • TABLE 1

    Bioactivity of compounds

    TABLE 1

    • ↵a Values are MICs for bacteria and TC50 for mammalian cells.

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KEYWORDS

drug discovery
Mycobacterium tuberculosis
natural product discovery
nontuberculous mycobacteria
Sec translocation
antibiotic
antimicrobial

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