Culture-independent discovery of the malacidins as calcium-dependent antibiotics with activity against multidrug-resistant Gram-positive pathogens

Abstract

Despite the wide availability of antibiotics, infectious diseases remain a leading cause of death worldwide ¹ . In the absence of new therapies, mortality rates due to untreatable infections are predicted to rise more than tenfold by 2050. Natural products (NPs) made by cultured bacteria have been a major source of clinically useful antibiotics. In spite of decades of productivity, the use of bacteria in the search for new antibiotics was largely abandoned due to high rediscovery rates^2,3. As only a fraction of bacterial diversity is regularly cultivated in the laboratory and just a fraction of the chemistries encoded by cultured bacteria are detected in fermentation experiments, most bacterial NPs remain hidden in the global microbiome. In an effort to access these hidden NPs, we have developed a culture-independent NP discovery platform that involves sequencing, bioinformatic analysis and heterologous expression of biosynthetic gene clusters captured on DNA extracted from environmental samples. Here, we describe the application of this platform to the discovery of the malacidins, a distinctive class of antibiotics that are commonly encoded in soil microbiomes but have never been reported in culture-based NP discovery efforts. The malacidins are active against multidrug-resistant pathogens, sterilize methicillin-resistant Staphylococcus aureus skin infections in an animal wound model and did not select for resistance under our laboratory conditions.

Figure 1. Using a culture-independent strategy for the discovery of calcium-dependent antibiotics from the global microbiome

(a) i) Degenerate PCR primers targeting the conserved regions of adenylation domains (AD) found in nonribosomal peptide synthetase genes were used to generate amplicons from an arrayed collection of environmental DNA isolated from 2000 unique soils. The reads from these next-generation sequenced amplicons (natural product sequence tags, NPSTs) were analyzed by eSNaPD (environmental Surveyor of Natural Product Diversity). ii) A desert soil rich in AD-NPSTs from the previously unknown malacidin clade was used to build an arrayed cosmid library. Cosmids harboring all fragments of a targeted biosynthetic gene cluster were assembled and integrated into a heterologous host for production, extraction, and characterization. (b) AD-NPSTs identified by the eSNaPD analysis to be evolutionarily related to the conserved Asp4 ADs of known calcium-dependent antibiotics were used to phylogenetically map the unexplored clades of this larger family across all tested soil microbiomes. The subfamilies of calcium-dependent antibiotics and their relative abundance are illustrated on the phylogenetic tree by color and percent. Across all sampled soil metagenomes, the malacidin antibiotic-clade represents 19% of the NPSTs, and 59% of calcium-dependent antibiotic tags originate from unexplored branches. (c) Geospatial distribution of calcium-dependent antibiotics across sampled US soil metagenomes. US states containing at least one soil with AD-NPSTs from the malacidin clade are indicated in orange. States lacking malacidin tags but still containing calcium-dependent antibiotics NPSTs are indicated in blue. States with at least one sampled soil but no detected calcium-dependent antibiotics NPSTs are highlighted in dark grey.

Figure 2. Maladicin biosynthesis, heterologous expression and structure

(a) The malacidin biosynthetic gene cluster was recovered on three overlapping cosmid clones and (b) assembled from these three overlapping clones in yeast using transformation-associated recombination (TAR). The resulting bacterial artificial chromosome (BAC) was integrated into S. albus genome for heterologous expression studies. (c) A representative HPLC analysis of crude extracts derived from cultures of S. albus transformed with the malacidin biosynthetic gene cluster (BGC) shows the presence of BGC-specific small molecules. The two primary malacidin peaks are highlighted with an asterisk. (d) Unlike crude extracts of the S. albus host strain alone, only extracts from the S. albus harboring the malacidin BGC showed antibacterial activity when applied to a lawn of S. aureus USA300. Both the (c) HPLC analysis and (d) antibacterial activity are representative of 4 independent fermentations. (e) Malacidin A and B are cyclic lipopeptides containing 8 amino acid macrocycles and polyunsaturated lipids. The malacidins do not contain the conserved DXDG motif seen in all known calcium-dependent antibiotics – incorporating a rare 3-hydroxyl aspartic acid (HyAsp, highlighted in violet) and lacking the spacer residue. Biosynthetic enzymes predicted to be involved in the production of non-proteinogenic amino acid [3-methyl aspartic acid (MeAsp), 4-methyl proline (MePro), and 2,3-diamino 3-methyl propanoic acid (MeDap)] and fatty acid substrates required for the biosynthesis of the maladicins are shown and colored coded according to their activities. Stereocenters in malacidin that were predicted bioinformatically as opposed to through chemical and spectroscopic analysis are denoted with an asterisk.

Figure 3. Malacidin, a calcium-dependent antibiotic

(a) The MIC of malacidin A against MRSA was assessed at various concentrations of calcium and the antibiosis of malacidin A was found to be calcium-dependent. Error bars represent the standard deviation across two replicates over three independent experiments (n = 6). (b) Malacidin A is an effective treatment against MRSA in rat cutaneous wound infections. Error bars represent the standard deviation across replicate wounds (n = 4). (c) Unlike daptomycin, malacidin A activity against S. pneumoniae is largely unaffected by the presence of pulmonary surfactants. Error bars represent the standard deviation across two replicates over three independent experiments (n = 6). (d) After 20 days of repeated exposure to 0.5= MIC of malacidin A (Mal), we did not detect any malacidin-resistant S. aureus. Vancomycin (Van), Daptomycin (Dap), and Rifamycin (Rif) were used as controls in this assay. Error bars represent the standard deviation across three replicates for MIC determination (n = 3).

Figure 4. Malacidin mode of action

(a) Cartoon showing modes of action of daptomycin, friulimicin and malacidin. (b) In contrast to daptomycin, malacidin A does not cause MRSA membrane leakage in a SYTOX Green fluorescent assay. Error bars represent the standard deviation across three biological replicates (n = 3). (c) As seen with the cell wall biosynthesis inhibitor vancomycin, exposing MRSA to malacidin A results in the accumulation of the cell wall intermediate UDP-MurNAc-pentapeptide. The UDP-MurNAc-pentapeptide peak ([M-H]- = 1148.35) is indicated with a red asterisk on the UPLC- trace. Chromatograms are representative of at least three independent experiments. (d) Interaction of malacidin A and daptomycin with purified cell wall precursors. An interaction is indicated by a reduction of the amount of free antibiotic (visible on the thin-layer chromatography by UV light). (e) The interaction of malacidin A to cell wall precursor, lipid II, is calcium-dependent. Both TLCs in (d) and (e) are representative of at leas two replicate experiments.