Baulamycins A and B, broad-spectrum antibiotics identified as inhibitors of siderophore biosynthesis in Staphylococcus aureus and Bacillus anthracis

Abstract

Siderophores are high-affinity iron chelators produced by microorganisms and frequently contribute to the virulence of human pathogens. Targeted inhibition of the biosynthesis of siderophores staphyloferrin B of Staphylococcus aureus and petrobactin of Bacillus anthracis hold considerable potential as a single or combined treatment for methicillin-resistant S. aureus (MRSA) and anthrax infection, respectively. The biosynthetic pathways for both siderophores involve a nonribosomal peptide synthetase independent siderophore (NIS) synthetase, including SbnE in staphyloferrin B and AsbA in petrobactin. In this study, we developed a biochemical assay specific for NIS synthetases to screen for inhibitors of SbnE and AsbA against a library of marine microbial-derived natural product extracts (NPEs). Analysis of the NPE derived from Streptomyces tempisquensis led to the isolation of the novel antibiotics baulamycins A (BmcA, 6) and B (BmcB, 7). BmcA and BmcB displayed in vitro activity with IC50 values of 4.8 μM and 19 μM against SbnE and 180 μM and 200 μM against AsbA, respectively. Kinetic analysis showed that the compounds function as reversible competitive enzyme inhibitors. Liquid culture studies with S. aureus , B. anthracis , E. coli , and several other bacterial pathogens demonstrated the capacity of these natural products to penetrate bacterial barriers and inhibit growth of both Gram-positive and Gram-negative species. These studies provide proof-of-concept that natural product inhibitors targeting siderophore virulence factors can provide access to novel broad-spectrum antibiotics, which may serve as important leads for the development of potent anti-infective agents.

Figure 1

Biosynthesis of the virulence-associated siderophore (A) staphyloferrin B in S. aureus (B) petrobactin in B. anthracis. Target biosynthetic enzymes SbnE and AsbA are highlighted by the yellow circles.

Figure 2

Triage of hits derived during high throughput screening of SbnE and AsbA against the full microbial NPE library.

Figure 3

Planar structure of baulamycins A (6) and B (7) showing COSY correlation with bold bonds and HMBC correlations with arrow.

Figure 4

H-H, C-H coupling values and relative configuration determined for C1’-C11. Arrows showing ROESY correlations.

Figure 5

H-H, C-H coupling values and relative configuration determined for C10-C4. Arrows showing ROESY correlations.

Figure 6

(A–C) In vitro bioactivities against purified enzyme recorded for BmcA and BmcB against the NIS synthetase (A) SbnE, (B) AsbA, or (C) AsbB. (D–H) BmcA inhibition against live cultures of microbial strains (D) S. aureus (Newman), (E) B. anthracis (Sterne 34F2), F) MRSA (USA 300), G) S. flexneri (BS103), or (H) E. coli (MC 1061) in iron-depleted (IDM) or iron-rich (IRM) media. The y-axis represents a comparison of the optical densities of inhibitor- (OD_I) and DMSO control-treated (OD_C) cultures. Assays were conducted in duplicate due to the current limited availability of the baulamycins.