Integrated omics approach to unveil antifungal bacterial polyynes as acetyl-CoA acetyltransferase inhibitors

Abstract

Bacterial polyynes are highly active natural products with a broad spectrum of antimicrobial activities. However, their detailed mechanism of action remains unclear. By integrating comparative genomics, transcriptomics, functional genetics, and metabolomics analysis, we identified a unique polyyne resistance gene, masL (encoding acetyl-CoA acetyltransferase), in the biosynthesis gene cluster of antifungal polyynes (massilin A 1, massilin B 2, collimonin C 3, and collimonin D 4) of Massilia sp. YMA4. Crystallographic analysis indicated that bacterial polyynes serve as covalent inhibitors of acetyl-CoA acetyltransferase. Moreover, we confirmed that the bacterial polyynes disrupted cell membrane integrity and inhibited the cell viability of Candida albicans by targeting ERG10, the homolog of MasL. Thus, this study demonstrated that acetyl-CoA acetyltransferase is a potential target for developing antifungal agents.

Fig. 1. Polyynes and polyyne biosynthesis gene cluster of Massilia sp. YMA4.

a Gene expression profile around the mas BGC under polyyne production (PDA) versus non-production (YMA) medium. The expressions of additional and core biosynthesis genes of mas BGC are framed in the gray area. b Structure of polyynes collimonin C/D 1, 2 and massilin A/B 3, 4. c Extraction ion chromatography (EIC) of collimonin C/D 1, 2 (m/z 273.1132), massilin A 3 (m/z 257.1183), and massilin B 4 (m/z 275.1289) in Massilia sp. YMA4 wild type (black) and the biosynthesis null mutant strains (YMA4::masH) with a 10 ppm mass window.

Fig. 2. Heterologous co-expression of mas genes in E. coli.

a The EIC (m/z 241.1234) of engineered E. coli strain, which carried masD, masE, masF, masG, Bv4687 (homolog of masH from Burkholderia vietnamiensis LMG 10929) and masI when co-expressed with (mas–, top) or without (mas–', bottom) masJ. b The EIC (m/z 273.1132) of E. coli strain mas– and mas–' harboring additional dioxygenase (masB) were renamed as mas + and mas + '. The structures of products are illustrated on top of each chromatogram and were verified by LC-HRMS/MS analysis (Supplementary Fig. 24).

Fig. 3. Comparative analysis of polyyne biosynthetic gene clusters and the structures of the bacterial polyynes.

a Phylogenetical analysis of polyyne biosynthetic gene clusters (BGCs) in bacteria (Supplementary Figs. 23 and 29). Species in the red box have been reported to produce palmitate-derived polyynes (C16), and species in the blue box have been reported to produce stearate-derived polyynes (C18). The number of BGCs in each group is shown in parentheses. b Comparison of the polyyne BGC architectures of mas BGC in Massilia sp. YMA4 (i), ccn BGC in B. ambifaria BCC0191(ii), and col BGC in C. fungivorans Ter331(iii). Genes conserved in polyyne BGC across the phylogenetic tree are shown in blue, and those conserved in the C16 polyyne group are shown in gray. The potential protective genes in the BGC are shown in red for acetyl-CoA acetyltransferase and in yellow for MFS transporter. The corresponding homologs (over 40% identity) in BGCs between two species are shown as the light green area. Black triangles indicate the mutation sites in previous research and this study.

Fig. 4. C. albicans was rescued by heterologous overexpression of masL (OE masL) and ERG10 (OE ERG10) from the minimum inhibitory concentration of collimonin C/D 1, 2, massilin A 3, and massilin C 5.

The cell viabilities were normalized to the mock treatment. The standard deviation was calculated based on three replicates and the two-tailed Student’s t-test for statistical analysis. ***P-value < 0.000001. ns: not significant. Each experiment comprised three biological replicates (N = 3). All data values were listed in Supplementary Table 7.

Fig. 5. X-ray structures and polar interaction within the binding site of MasL-collimonin C and MasL-collimonin D complex.

Overall views of the crystal structures of MasL-collimonin C complex (a) and MasL-collimonin D (b). Four subunits per asymmetric cell are shown in red, orange, light green, and cyan, respectively. Collimonin C/D 1, 2 is shown as grey space-filling balls. In the magnified view, the residues involved in collimonin C/D 1, 2 interactions are shown as sticks with sequence identities indicated in the main chain molecule shown in gray. The R135 residue of another molecule is shown in cyan. The dotted lines indicate the hydrogen bonds and salt bridges involved in collimonin C/D 1, 2 interactions within the binding pocket.