Malleilactone, a polyketide synthase-derived virulence factor encoded by the cryptic secondary metabolome of Burkholderia pseudomallei group pathogens

Abstract

Sequenced bacterial genomes are routinely found to contain gene clusters that are predicted to encode metabolites not seen in fermentation-based studies. Pseudomallei group Burkholderia are emerging pathogens whose genomes are particularly rich in cryptic natural product biosynthetic gene clusters. We systematically probed the influence of the cryptic secondary metabolome on the virulence of these bacteria and found that disruption of the MAL gene cluster, which is natively silent in laboratory fermentation experiments and conserved across this group of pathogens, attenuates virulence in animal models. Using a promoter exchange strategy to activate the MAL cluster, we identified malleilactone, a polyketide synthase-derived cytotoxic siderophore encoded by this gene cluster. Small molecules targeting malleilactone biosynthesis either alone or in conjunction with antibiotics could prove useful as therapeutics to combat melioidosis and glanders.

Figure 1

(a) Venn diagram showing the relationship of number of NRPS/PKS gene clusters shared among pseudomallei group pathogens B. pseudomallei K96243, B. mallei ATCC 23344 and B. thailandensis E264 (BP, BM and BT respectively). (b) C. elegans after 24 h co-culture with: i. wild type BT (dead), ii. BT(ΔmalF), a PKS2 gene deletion mutant and iii. BT(ΔmalR), a transcription factor deletion mutant (40x magnification). (c) C. elegans survival after 24 h exposure to wild type BT, BT(ΔmalF), BT(ΔmalR) and BT(ΔmalR/malA), a malR-PKS1 double mutant. ***P<0.001, two-tailed t-test. (d) D. discoideum co-culture at 120 h with: i. wild type BT and ii. BT(ΔmalF). Arrows highlight D. discoideum aggregation and differentiation into fruiting bodies with MAL cluster disruption (white box - close-up of a fruiting body). (e) MAL clusters from BP, BM and BT with predicted functions for each MAL protein.

Figure 2

(a) HPLC traces (total diode array: 210-450 nm) of BT culture broth ethyl acetate extracts. i. wild type BT, ii. BT(ΔmalF), iii. BT(ΔmalR), iv. BT(ΔmalR/ΔmalA). (b) Promoter exchange strategy used to induce cryptic gene cluster expression. In brief, BT cells are transformed with a cassette designed to hybridize and recombine with the native promoter region resulting in the insertion of the rhamnose-inducible promoter P_RhaB directly upstream of the cryptic biosynthetic gene cluster. Small molecule production is then induced with the addition of 0.2% L-rhamnose to the culture media. (c) HPLC traces of culture broth extracts, showing that the induction of the MAL cluster leads to the production of the novel metabolite (malleilactone, 1). Cultures were grown with or without 0.2% L-rhamnose: v. wild type BT with rhamnose, vi. BT:P_RhaB-MAL, no rhamose, vii. BT:P_RhaB-MAL with rhamnose, viii. BT:P_RhaB-MAL(ΔmalF) with rhamnose.

Figure 3

(a) Key NMR arguments used to define the structure of malleilactone (1). (b) Retro-biosynthetic analysis of 1. (c) Biosynthetic proposal for 1. AT, acyltransferase; KS, ketosynthase; DH, dehydratase; KRⁱ, inactive ketoreductase; C, condensation domain; R, reductase domain; T, thiolation domain.