Mode of Action and Heterologous Expression of the Natural Product Antibiotic Vancoresmycin

Abstract

Antibiotics that interfere with the bacterial cytoplasmic membrane have long-term potential for the treatment of infectious diseases as this mode of action is anticipated to result in low resistance frequency. Vancoresmycin is an understudied natural product antibiotic consisting of a terminal tetramic acid moiety fused to a linear, highly oxygenated, stereochemically complex polyketide chain. Vancoresmycin shows minimum inhibitory concentrations (MICs) from 0.125 to 2 μg/mL against a range of clinically relevant, antibiotic-resistant Gram-positive bacteria. Through a comprehensive mode-of-action study, utilizing Bacillus subtilis reporter strains, DiSC(5) depolarization assays, and fluorescence microscopy, we have shown that vancoresmycin selectively targets the cytoplasmic membrane of Gram-positive bacteria via a non-pore-forming, concentration-dependent depolarization mechanism. Whole genome sequencing of the producing strain allowed identification of the 141 kbp gene cluster encoding for vancoresmycin biosynthesis and a preliminary model for its biosynthesis. The size and complex structure of vancoresmycin could confound attempts to generate synthetic analogues. To overcome this problem and facilitate future studies, we identified, cloned, and expressed the 141 kbp biosynthetic gene cluster in Streptomyces coelicolor M1152. Elucidation of the mode-of-action of vancoresmycin, together with the heterologous expression system, will greatly facilitate further studies of this and related molecules.

A membrane-depolarizing toxin substrate of theStaphylococcus aureusType VII secretion system mediates intra-species competition

“…Pore-forming proteins are widely used as toxins to target either prokaryotic or eukaryotic cells 33, 34 . To determine whether TspA forms pores we used single-cell microscopy that combines the voltage-sensitive dye DiSC 3 (5) with the membrane-impermeable nucleic acid stain Sytox Green 35 . E. coli cells incubated with Polymyxin B, which produces large ion-permeable pores in the E. coli cell envelope 36 , showed strong labelling with Sytox Green, indicative of permeabilisation, coupled with very low DiSC 3 fluorescence (Fig 3E-G).…”

Streptomycetes: Surrogate hosts for the genetic manipulation of biosynthetic gene clusters and production of natural products

“…The 141-kb gene cluster of vancoresmycin (Figure 2), a potent antibiotic active against vancomycin-resistant Enterococcus spp. (VRE) and methicillin-resistant Staphylococcus aureus (MRSA), was expressed in S. coelicolor M1152 yielding vancoresmycin at 2.2 mg/L (Kepplinger et al, 2018). …”

The “Three Cs” of Novel Antibiotic Discovery and Production through Synthetic Biology: Biosynthetic Gene Clusters, Heterologous Chassis, and Synthetic Microbial Consortia

“…The large BGC (141kb) of the antibiotic vancoresmycin was also recently conjugated into S. coelicolor to elucidate its mode of action and biosynthetic pathway. This BGC is hypothesized to be the largest heterologously expressed gene cluster at present …”