The chemical arsenal of Burkholderia pseudomallei is essential for pathogenicity

Abstract

Increasing evidence has shown that small-molecule chemistry in microbes (i.e., secondary metabolism) can modulate the microbe-host response in infection and pathogenicity. The bacterial disease melioidosis is conferred by the highly virulent, antibiotic-resistant pathogen Burkholderia pseudomallei (BP). Whereas some macromolecular structures have been shown to influence BP virulence (e.g., secretion systems, cellular capsule, pili), the role of the large cryptic secondary metabolome encoded within its genome has been largely unexplored for its importance to virulence. Herein we demonstrate that BP-encoded small-molecule biosynthesis is indispensible for in vivo BP pathogenicity. Promoter exchange experiments were used to induce high-level molecule production from two gene clusters (MPN and SYR) found to be essential for in vivo virulence. NMR structural characterization of these metabolites identified a new class of lipopeptide biosurfactants/biofilm modulators (the malleipeptins) and syrbactin-type proteasome inhibitors, both of which represent overlooked small-molecule virulence factors for BP. Disruption of Burkholderia virulence by inhibiting the biosynthesis of these small-molecule biosynthetic pathways may prove to be an effective strategy for developing novel melioidosis-specific therapeutics.

Figure 1

Examining the influence of secondary metabolism on bacterial pathogenicity. (A) Bacterial pathogens typically produce a limited number of metabolites under laboratory fermentation conditions, (B) while having the capacity to produce multiple metabolites within the context of an infection, which can function as virulence factors. (C) Cryptic/silent gene clusters can be identified bioinformatically in sequenced pathogen genomes. Through genome engineering, individual biosynthetic clusters can be both activated for accessing the encoded molecules and disrupted for determining their influence on bacterial pathogenicity. (D) Virulence factor biosynthesis can be targeted for the development of new disease-specific therapeutics.

Figure 2

Comparison of the secondary metabolomes encoded by B. pseudomallei1026b (BP), B. mallei ATCC 23344 (BM), and B. thailandensis E264 (BT). Positioning of individual NRP- and PK-encoding gene clusters within each genome is displayed. Clusters shared among the three species are indicated by the connective lines between genomes (detailed in Table S1). Genomes are linearized and arranged for clarity, with the purple arrowhead designating direction from the first gene in each chromosome (i.e., nucleotide + 1). Clusters 2, 14, and 15 (red) are unique to BP. Cluster 11 (yellow) is shared by BP and BM but absent in BT. Genes dedicated to NRP/PK-based secondary metabolism encompass approximately 6% of the BP genome.

Figure 3

21-day murine intranasal infection challenge of wild-type BP and the disruption mutants of biosynthetic clusters 2, 11, 14, and 15. The number of surviving mice is charted over time. Inoculum: 10⁵ (10⁶ for cluster 15) colony-forming units.

Figure 4

Characterization of the MPN cluster. (A) Biosynthetic gene cluster encoding the malleipeptins (genes BP1026B_II1742-1746, Table S2), with P_RhaB promoter exchange (red). (B) Predicted NRPS domain architecture and adenylation domain selectivity for MpnBCD. Parentheses indicate amino acids observed in the NMR-determined final malleipeptin structure that differ from the bioinformatics prediction. Abbreviations: C, condensation domain; C_S, starter condensation domain; C_d, dual condensation/epimerization domain; A, adenylation domain; T, peptidyl carrier domain; TE, thioesterase; Dhb, 2,3-dehydrobutyric acid; Dab, 2,4-diaminobutyric acid; hGlu, 4-hydroxyglutamic acid. (C) HPLC traces (diode array: 254 nm) of culture broth extracts from (a) Bp82, (b) Bp82:P_RhaB-MPN (no rhamnose), and (c) Bp82:P_RhaB-MPN (rhamnose induced). (D) Structure of malleipeptins A (1) and B (2) with key HMBC/ROESY correlations. Predicted tailoring enzyme functionalities (MpnA and MpnE) are highlighted. Comprehensive NMR assignments are detailed in Figure S3.

Figure 5

Characterization of SYR cluster. (A) Biosynthetic gene cluster encoding for the syrbactins (genes BP1026B_II1345-1353; Table S2) with P_RhaB promoter exchange (red). (B) HPLC traces (diode array: 254 nm) of culture broth extracts from (a) Bp82, (b) Bp82:P_RhaB-SYR (no rhamnose), and (c) Bp82:P_RhaB-SYR (rhamnose induced). (C) Structures of glidobactin C (3) and deoxyglidobactin C (4).

Figure 6

Malleipeptin activity. (A) Emulsification activity of cell culture supernatants. Equal parts toluene and supernatant were combined, vortexed, and then let stand for 2 h. (a) Bp82, no rhamnose; (b) Bp82, plus rhamnose; (c) Bp82:P_RhaB-MPN, no rhamnose; (d) Bp82:P_RhaB-MPN, plus rhamnose; (e) LB medium (blank). (B) Microtiter plate assay showing increasing disruption of Bp82 top biofilm with increasing concentrations of 2 (200 μL cultures, 30 °C, 48 h; 20× magnification).

Figure 7

Putative metabolomics basis for bacterial virulence. Small-molecule biosynthesis is predicted to influence BP virulence through diverse mechanisms: (A) bacterial invasion, (B) systemic toxicity, and (C) acquisition of nutrients. The inhibition of these overlooked components of BP virulence should provide novel avenues for the development of BP-specific anti-infective agents.