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The Polyene Natural Product Thailandamide A Inhibits Fatty Acid Biosynthesis in Gram-Positive and Gram-Negative Bacteria Yihan Wu, and Mohammad R. Seyedsayamdost Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00678 • Publication Date (Web): 05 Jul 2018 Downloaded from http://pubs.acs.org on July 6, 2018
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Biochemistry
The Polyene Natural Product Thailandamide A Inhibits Fatty Acid Biosynthesis in Gram-Positive and Gram-Negative Bacteria Yihan Wu† and Mohammad R. Seyedsayamdost†,‡,* Department of Chemistry, Princeton University, Princeton, NJ 08544, United States Department of Molecular Biology, Princeton University, Princeton, NJ 08544, United States KEYWORDS. Natural Product, Burkholderia thailandensis, Thailandamide, Fatty Acid Biosynthesis Supporting Information Placeholder ABSTRACT: Burkholderia thailandensis produces an impressive array of secondary metabolites, most with yet unknown targets. One of these metabolites is thailandamide, a linear polyene natural product that is constitutively synthesized by the corresponding tha gene cluster. Using broad bioactivity screens, we observed strong yet selective antibacterial activity by thailandamide against Grampositive and cell wall-weakened Gram-negative bacteria. Bacterial cytological profiling and comparison with ten antibiotics with known modes of action revealed a unique profile for thailandamide, suggesting a distinct mechanism of inhibition. To address the target of the drug, we obtained resistant mutants of Bacillus subtilis and mapped the resistant phenotype to accA, the product of which catalyzes the first committed step in fatty acid biosynthesis. Interestingly, the tha gene cluster encodes an accA homolog with a similar amino acid substitution. Heterologous expression showed that it confers resistance to otherwise susceptible Escherichia coli cultures, indicating that it provides immunity to thailandamide-producing B. thailandensis cells. Aside from moiramide B and andrimid, thailandamide represents only the second class of natural products that inhibits bacterial growth by targeting AccA.
thailandamide derivatives have been structurally elucidated and regulators that control their production have been investigated as well.17-19 The MoA of thailandamide, however, remains unknown. Given that many polyene natural products exhibit antimicrobial activities, we examined the effect of thailandamide on the growth of a battery of diverse, pathogenic and non-pathogenic bacteria. The compound exhibited strong inhibitory activity against some strains tested, notably Bacillus subtilis and Staphylococcus aureus with a minimal inhibitory concentration (MIC) of ~10 μM (Table 1). Apart from Neisseria gonorrhoeae, which thailandamide kills with a MIC of 1 μM, the drug was largely ineffective against Gram-negative bacteria. However, growth of cell wall-weakened E. coli, carrying a mutation in the lipopolysaccharide assembly protein LptD (ΔlptD), was efficiently inhibited by thailandamide, indicating that it was likely not taken up by the Gram-negative bacteria tested (Table 1).20 Thus, thailandamide is a potent, selective antimicrobial agent, with its spectrum of activity likely limited by poor uptake. O
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Burkholderia thailandensis has emerged as a model system for investigating the biosynthesis, function, and regulation of diverse natural products.1 Its repertoire includes the broad-spectrum ribosomal inhibitor bactobolin,2-5 the synergistically acting hydroxyalkylquinolines (HAQs) that inhibit pyrimidine biosynthesis and dissipate the proton motive force,6,7 the RNA polymerase-targeting capistruin A,8,9 as well as the antiparasitic malleilactones.10,11 Burkholdac and terphenyl, inhibitors of histone deacetylases and phosphodiesterases, respectively, further bolster the arsenal of bioactive metabolites.12-15 Thailandamide, a linear polyene polyketide was among the first natural products identified from B. thailandensis, but its bioactivity and mode of action (MoA) have remained elusive.16 We have herein addressed the function and target of thailandamide in both Gram-positive and Gram-negative bacteria. Using a variety of methods, including bacterial cytological profiling, generation of spontaneous resistant mutants, and genetic assays, we identified acetyl-CoA carboxylase, which catalyzes the first step in fatty acid biosynthesis, as the target of thailandamide. The intrinsic resistance mechanism to the drug in B. thailandensis was also investigated, leading to the discovery of a thailandamideresistant form of AccA encoded in the tha biosynthetic gene cluster. Thailandamide A (hereafter thailandamide) was first identified in an effort to better understand trans-acting acyltransferase domains in polyketide biosynthetic pathways (Fig. 1).16 Several
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n=1, Moiramide B n=2, Andrimid
Figure 1. Structures of thailandamide A, moiramide B, and andrimid. Table 1. Antimicrobial activity of thailandamide A against select agents. Strain Bacillus subtilis 3610 Staphylococcus aureus MSSA Staphylococcus aureus MRSA Neisseria gonorrhoeae Escherichia coli ΔlptD Escherichia coli Vibrio parahaemolyticus Klebsiella pneumoniae Acinetobacter baumannii Saccharomyces cerevisiae
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To interrogate the MoA of thailandamide, we first conducted bacterial cytological profiling, a quantitative imaging technique that has emerged as an effective MoA-determining tool.21,22 Specifically, we exposed ΔlptD E. coli to ten antibiotics with known MoAs as well as to thailandamide. Subsequently, the cells were treated with three different dyes that stain the cell membrane (FM464), chromosomal DNA (DAPI), or report on cell wall permeability (Sytox green) (Fig. 2A). The cells were then imaged, which enabled extraction of fourteen features. Multi-variate analysis of these features allowed us to group the antibiotics by their cytological profiles, a proxy for MoA. Somewhat surprisingly, thailandamide exhibited a unique profile, indicating that its MoA is likely different from those of the ten known antibiotics tested (Fig. 2B). This conclusion was also reached by visual inspection of drug-treated cells, which revealed a unique phenotype after exposure to thailandamide (Fig. 2A).
Figure 2. Bacterial cytological profiles of thailandamide A. (A) Representative fluorescence microscopy images of E. coli cells after treatment with the antibiotic indicated. White scale bar, 1 μm; red scale bar, 3 μm. (B) Principal component analysis to cluster antibiotics by MoA. Thailandamide A treatment results in a unique profile. The relative contribution of each principal component (PC) is indicated by the color key. We next decided to generate spontaneous thailandamide-resistant mutants to define the mutation(s) that contribute to resistance. B. subtilis was selected for this analysis, as it is highly sensitive to thailandamide, and because it has been suggested that B. subtilis is less likely to generate permeability-mutants, thus favoring mutations in the target site.4 After prolonged exposure of B. subtilis to 10-fold of the MIC of thailandamide, two spontaneous resistant mutants emerged on agar plates. In comparison with wt B. subtilis, the two mutants were much less susceptible to thailandamide, exhibiting a 10-fold increased half-maximal inhibitory concentration (IC50) (Fig. 3A). The genomes of both resistant
mutants were analyzed by whole-genome sequencing. After mapping the Illumina sequencing reads to a B. subtilis reference genome, we identified a single shared mutation in both mutants: A Gto-A alteration at position 679 in the accA gene that codes for acetyl-CoA carboxylase A. AccA is a subunit of the ACC complex that catalyzes the first committed step in fatty acid biosynthesis, the formation of malonyl-CoA from acetyl-CoA (Fig. 3B).23 The 679-Gto-A alteration leads to a E227K mutation in AccA. We verified that the mutant accA was sufficient for conferring thailandamide resistance by expressing it from a neutral chromosomal site. Specifically, the accAG679A gene, under the control of a lac promoter, was inserted into the neutral amylase E (amyE) site in the B. subtilis chromosome. As control, we generated a strain carrying the wt accA allele, controlled by a lac promoter, at the neutral amyE locus. Both constructs were grown in the presence and absence of thailandamide in liquid and solid agar cultures. In the presence of IPTG, liquid B. subtilis cultures overexpressing AccAE227K were unsusceptible to thailandamide, while those expressing wt accA showed a similar IC50 as determined above (Fig. 3C). A similar result was observed in disc diffusion assays, where expression of AccAE227K abolished a zone of growth inhibition, while expression of wt AccA did not (Fig. 3D). The finding that overexpression of a mutant AccA protein alone can confer resistance to thailandamide, suggests that the AccA protein or the AccA/AccD complex is the cellular target of thailandamide. With the molecular target identified, we next examined the mechanism of immunity or intrinsic resistance of B. thailandensis to thailandamide. We reasoned that the resistance gene must be coregulated with the biosynthetic ones and may therefore be found in the tha gene cluster. Intriguingly, we noticed a gene, annotated as thaC, with high homology to accA. ThaC is a duplicate copy, as B. thailandensis encodes another accA gene (BTH_I1943) elsewhere in its genome. The thaC gene carries a substitution at residue 228, adjacent to the site where we observed the resistance-conferring Eto-K mutation. Because of this unusual extra copy of accA (thaC) and the substitution within the same motif as the resistant B. subtilis isoform, we hypothesized that thaC provides immunity to thailandamide. To evaluate this hypothesis, we expressed thaC ectopically in susceptible ΔlptD E. coli cells, as previous work has shown that exogenous ACC enzymes from Gram-negative bacteria can efficiently form an active ACC complex with E. coli-endogenous subunits.24 B. thailandensis accA or thaC were placed under the control of the araBAD promoter, inducible by L-arabinose (L-ara), and subsequently expressed from a low-copy plasmid. Cells expressing thaC were significantly less susceptible to thailandamide than those expressing accA (Fig. 3E) A similar result was observed in disk diffusion assays, with thaC-expressing cells exhibiting a significantly reduced zone of inhibition (Fig. 3F). These resistant phenotypes were dependent on the presence of the inducer L-ara. Together, these results demonstrate that thaC is resistant or less-susceptible to inhibition by thailandamide and thereby provides immunity to cells producing the metabolite. Its coregulation with the biosynthetic enzymes ensures that the resistance gene is produced upon synthesis of the antibiotic. Interestingly, a bioinformatic search for the thaC allele reveals that several Burkholderia spp. encode a homolog without the corresponding tha gene cluster (and in the presence of a second copy of accA, Fig. S1). This finding suggests that some Burkholderia strains produce other, yet-unknown AccA-targeting antibiotics, or alternatively, that the acquisition of thaC provides a competitive advantage by allowing these bacteria to persist in the presence of thailandamide-producing relatives. Accumulation of antibiotic-resistance genes in bacteria that do not produce the corresponding toxin is well-documented, and the prevalence of thaC is another demonstration of this phenomenon.
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Biochemistry
Figure 3. Target of and resistance to thailandamide. (A) Growth of wt and thailandamide-resistant B. subtilis mutants in the presence of varying concentrations of the drug. Both mutants exhibit ~10-fold lower susceptibility to thailandamide and carry an E227K mutation in the AccA protein. The average of three biological replicates is shown. Error bars represent standard deviation (SD). (B) Reaction catalyzed by AccA. AccA forms a complex with AccD and catalyzes the biotin-dependent carboxylation of acetyl-CoA to malonyl-CoA, the first committed step in fatty acid biosynthesis. Biotin is carboxylated by AccC. (C, D) Growth of B. subtilis expressing a wt or E227K-substituted AccA from a neutral chromosomal site as function of varying thailandamide concentrations in liquid (C) or agar (D) cultures. The strain expressing E227K-AccA exhibits significantly lowered susceptibility to the drug. (E, F) ΔlptD E. coli expressing a plasmid-encoded wt B. thailandensis accA or thaC grown in liquid (E) or on agar (F). Cells expressing thaC exhibit lowered susceptibility to thailandamide. In (E), both cultures contained L-ara. In (F), the presence of L-ara, essential for reduced susceptibility, is indicated in each of the three cultures. Studies addressing the MoAs of linear polyene natural products have been surprisingly limited, especially compared to the polyene macrolides, represented by nystatin.25,26 We have herein investigated the function of the large polyene thailandamide and found it to inhibit bacterial growth, especially that of Gram-positive bacteria, by targeting AccA, the enzyme that catalyzes the first step in fatty acid biosynthesis. In light of the reaction carried out by AccA (Fig. 3B), it is tempting to speculate that the long linear structure of thailandamide mimics the similarly lengthy linear backbones of acetyl-CoA and carboxy-biotin, perhaps facilitating competitive inhibition with respect to both substrates. Thailandamide is only the second class of secondary metabolites known to inhibit AccA, with the other class comprised of andrimid and moiramide B (Fig. 1), both hybrid non-ribosomal peptides containing a pyrrolidinedione moiety, which is essential for binding to AccA.24,27,28 Structures of AccA bound to moiramide B have defined a binding pocket of the inhibitor that is distant from E227,27 which when mutated to Lys confers resistance to thailandamide (Fig. 4). Like tha, the andrimid biosynthetic gene cluster also encodes a resistance gene, in this case an alternative AccD, rather than AccA.24 The requirement of the pyrrolidinedione moiety, the reported crystal structure of moiramide B in complex with AccA/AccD, the resistance mechanism against andrimid/moiramide B, and our analysis of thailandamideresistant mutants all suggest that the binding pocket of thailandamide is likely to be different from that of moiramide B (Fig. 4). Our proposed MoA of thailandamide A is in agreement with very recent results by Wozniak et al.29 who found a similar MoA for the less abundant analog thailandamide B against Salmonella typhimurium. B. thailandensis boasts an imposing collection of small molecules that are constitutively produced. Under normal growth conditions, it generates the ribosome-inhibiting broad-spectrum bactobolin antibiotics, the HAQs, which interfere with energy production and pyrimidine biogenesis, as well as thailandamide, which inhibits fatty acid biosynthesis. All three metabolites accumulate to
significant levels, thus precluding microbial competitors of B. thailandensis access to fatty acids, pyrimidine nucleotides, and proteins. Moreover, the bacterium boasts an inducible metabolome that includes capistruin, terphenyl, burkholdac, and acybolin.15 This prolific capacity to produce structurally and functionally diverse secondary metabolites in part explains the ubiquitous distribution of B. thailandensis and its survival in diverse environments.30,31
Figure 4. Crystal structure of S. aureus AccA (brown)/AccD (blue) in complex with moiramide B (green).27 Only a magnified view of the moiramide B binding pocket is depicted. Residues interacting with the pyrrolidinedione and Val moieties of moiramide B are rendered in magenta and yellow, respectively. Residue E224 (E227 in B. subtilis AccA) and G225 (G228 in B. subtilis), which are altered in resistant AccA isoforms or in ThaC are shown in red and white, respectively.
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ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Detailed Materials and Methods, Supplemental Table and Figure (PDF).
AUTHOR INFORMATION Corresponding Author E-mail:
[email protected]. Telephone: (609) 258 5941.
Author Contributions Y.W. and M.R.S. conceived the project, carried out and analyzed experiments, and wrote the manuscript.
Funding Sources This work was supported by the National Institutes of Health (ODAI-124786 to M.R.S.) and the Pew Biomedical Scholars Program.
Notes The authors declare no competing financial interests.
ACKNOWLEDGMENT We thank Lance Parsons and Robert Leach at the Lewis Sigler Institute Genomics Core Facility for assistance with genome sequencing and analysis, and Chen Zhang, Étienne Gallant, Dr. Marcus Gibson, and Dr. Fei Xu for helpful discussions.
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