Abstract
Isonitrile natural products exhibit promising antibacterial activities. However, their mechanism of action (MoA) remains largely unknown. Based on the nanomolar potency of xanthocillin X (Xan) against diverse difficult-to-treat Gram-negative bacteria, including the critical priority pathogen Acinetobacter baumannii, we performed in-depth studies to decipher its MoA. While neither metal binding nor cellular protein targets were detected as relevant for Xan’s antibiotic effects, sequencing of resistant strains revealed a conserved mutation in the heme biosynthesis enzyme porphobilinogen synthase (PbgS). This mutation caused impaired enzymatic efficiency indicative of reduced heme production. This discovery led to the validation of an untapped mechanism, by which direct heme sequestration of Xan prevents its binding into cognate enzyme pockets resulting in uncontrolled cofactor biosynthesis, accumulation of porphyrins, and corresponding stress with deleterious effects for bacterial viability. Thus, Xan represents a promising antibiotic displaying activity even against multidrug resistant strains, while exhibiting low toxicity to human cells.
Highlights
Natural product antibiotics are evolutionarily optimized to hit the soft spots of bacterial viability
Synthesis of Xan commenced according to published procedures and subsequent screening revealed a broad spectrum bioactivity of Xan against most strains tested with the notable exception of enterococcal species (Table 1).[29]
We investigated the direct effects of Xan on heme biosynthesis by comparing the full proteomes of treated and untreated A. baumannii
Summary
Natural product antibiotics are evolutionarily optimized to hit the soft spots of bacterial viability. The slower resistance formation compared to standard antibiotics is in accordance with a previous study that treated staphylococci with sub-MICs of Xan for 240 days, which failed to produce any resistant strains.[9] Whole-genome sequencing of three mutants each from three independent experiments and three colonies of the negative control unveiled that only the gene hemB encoding the enzyme porphobilinogen synthase (PbgS) was consistently mutated in response to Xan but not upon DMSO treatment (Figure 3A). Uncontrolled heme biosynthesis induced by impaired feedback inhibition would lead to enhanced tetrapyrrole levels To validate this hypothesis, we fluorescently quantified porphyrin intermediates using an established protocol.[58−60] A. baumannii was incubated with either Xan or DMSO for 1 h, porphyrins were extracted from cell lysate and fluorescence spectra subsequently measured (λex = 406 nm) (Supplementary Figure 13A). In line with published data, Xan did not show any significant toxicity in human cells up to its solubility limit of 10 μM highlighting a suitable therapeutic window for single or combination treatment (Supplementary Figure 14).[9,18,28]
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