TUN Analogues Show Potent Antimicrobial Activity against a Range of Bacteria
We evaluated the analogues (TUN-7,7, -8,8, -9,9, -10,10, -11,11, -12,12) for potency against a range of Gram-negative and Gram-positive bacteria that cause infections in hospitals (Staphylococcus, Pseudomonas, and Escherichia), as well as reference bacteria (Bacillus, Staphylococcus, and Micrococcus) used in previous tunicamycin bioactivity studies (Ward, 1977). Kirby-Bauer disc diffusion susceptibility tests (Figures S4A–S4E), revealed potent activity against Bacillus subtilis (EC1524) and opportunistic pathogen Bacillus cereus (ATCC 11778). There was a weaker but significant effect on the pathogenic bacteria Staphylococcus aureus (ATCC 29219) and Pseudomonas aeruginosa (ATCC 27853); the latter is a strain resistant to natural tunicamycin. No activity was seen against Micrococcus luteus, a bacterium noted to have some resistance to tunicamycin (Ward, 1977). Consistent with the critical role of the lipid, none of the non-lipidated analogues (e.g., TUN or TUN-Ac,Ac) or synthetic intermediates showed any activity. Lipid-length (X = 7, 8…12) in the TUN-X,X analogues systematically modulated activity; the most potent analogues TUN-8,8 and TUN-9,9 were those with C8 and C9 chain lengths.
Figure S4 Kirby-Bauer Disc Diffusion Tests and Dose Response Curves Used for MIC, MBC, and IC50 Determination for Tunicamycin and the TUN-X,X Analogues against Several Bacterial Strains, Related to Figure 5
(A–E) Kirby-Bauer disc diffusion tests for the TUN analogues against bacterial strains (A) B. subtilis EC 1524, (B) M. luteus, (C) B. cereus ATCC 11778, (D) S. aureus ATCC 29219 and (E) P. aeruginosa ATCC 27853. Discs impregnated with 5 μg, unless otherwise indicated, of the compound were laid onto plates with lawns of bacteria. The compounds are labelled: TC: commercial tunicamycin, TS: tunicamycin from S. chartreusis, 2: (2) N-acetyl tunicamine, Ac: TUN-Ac,Ac, Boc: TUN-Boc,Boc, 7: TUN-7,7, 8: TUN-8,8, 9: TUN-9,9, 10: TUN-10,10, 11: TUN-11,11, 12: TUN-12,12, Cit: TUN-Cit,Cit, Amp: ampicillin, Gen: gentamycin.
(F–J) Dose response curves for tunicamycin and the analogues with (F) B. subtilis EC1524. (G) B. cereus ATCC11778. (H) S. aureus ATCC29219. (I) P. aeruginosa ATCC27853 and (J) E. coli ATCC25922. These dose-response curves were generated by Prism 6.0 software by plotting percent growth (normalized OD600 values) vs. logarithmic scale of the concentrations. The data shown are mean ± SEM errors of three independent experiments. See Table S3 for the MIC, MBC and IC50 values.
The minimal and half maximal inhibitory concentrations (MIC and IC50) and minimal bactericidal concentrations (MBC) were determined by both a micro-broth dilution test and drop plate test, respectively (Figures 5B and 5C, Figures S5F–S5J, Table S3). Only lipidated variants (tunicamycin and TUN-X,X) displayed antibacterial activity, with MICs down to 0.02 ± 0.01 μg/ml for TUN-9,9 against B. subtilis and 0.33 ± 0.11 μg/ml against B. cereus, with TUN-10,10.
Figure S5 On-Target Effects Demonstrated for the TUN-8,8 Analogue, Related to Figure 5
(A) Glucosamine incorporation within 48 h was reduced with 10x TUN-8,8. Positive controls were tunicamycin, meropenem/clavulanate (MCA) and D-cycloserine (DCS). All compounds tested at 1- and 10-fold MIC concentrations (n=2).
(B) GFP release assay. Mtb expressing GFP was treated with 1- and 10-fold MIC concentrations of TUN-8,8 or tunicamycin. Lysis was monitored by measurement of fluorescence in cell-free supernatants daily over a week of exposure as a measure of release of cytosolic protein (GFP) (n=2).
(C) Confocal microscopy of BODIPY-vancomycin used to track synthesis of PG, showing abnormal PG formation with tunicamycin and TUN-8,8 treated Mtb, both differing from the effects of pentapeptide labelling by the BODIPY-vancomycin caused by meropenem/clavulanate treatment.
Data presented in (A) and (B) are means ± SD.
TB, through its etiological agent Mycobacterium tuberculosis (Mtb) is a global concern. Testing of the lipid-altered analogues (TUN-7,7 to -12,12) against pathogenic Mtb strain H37Rv revealed striking MIC values (0.03 ± 0.001 μg/ml in minimal growth medium and 0.22 ± 0.02 μg/ml in rich 7H9-based growth medium) for TUN-9,9: some 5-fold more potent than even tunicamycin itself (Figure 5D, Table S4).
The on-target effect of tunicamycin analogues was explored by multiple approaches. Firstly, TUN-8,8-resistant Mtb mutants (Methods S1) carried mutations in Rv0751c (mmsB, 3-hydroxyisobutyrate dehydrogenase), an enzyme in fatty acid metabolism, suggesting possible inactivation of TUN-8,8 by destruction of fatty acid/lipid or an intergenic mutation between Rv2980 and Rv2918c (the central D-alanine-D-alanine ligase ddlA involved in peptidoglycan (PG) synthesis), suggesting possible regulation of this key PG biosynthetic enzyme (perhaps via small regulatory noncoding RNA). Secondly, macromolecular incorporation assay using 14C-glucosamine as radiolabeled precursor (Figure S5A) confirmed that TUN-8,8 and tunicamycin have similar effects on PG biosynthesis, suggesting that they act on the same target. Thirdly, extracellular release of green fluorescent protein (GFP) from Mtb expressing GFP showed lytic effects of TUN-8,8, even outstripping those of tunicamycin (Figure S5B), consistent with on-target PG inhibition activity. Finally, fluorescently-labeled vancomycin (Daniel and Errington, 2003, Tiyanont et al., 2006) revealed similar phenotypes (Figure S5C) following treatment with TUN-8,8 or tunicamycin with disrupted, non-uniform cell-wall, consistent with targeting of the PG pathway. Together these data suggested that TUN-8,8 is inhibiting the same target as tunicamycin in Mtb.