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Search of Soil Microbiome Turns Up New Class of Antibiotics Called Malacidins

NEW YORK (GenomeWeb) – By scouring the content of soil microbiomes, researchers have uncovered a new class of antibiotic.

Chemicals produced by microorganisms have been a key source of antibiotics.  Calcium-dependent antibiotics have been particularly helpful in treating multi-drug resistant infections, leading a Rockefeller University-led team of researchers to search for gene clusters linked to that function within a series of environmental metagenome samples.

As they reported today in Nature Microbiology, Rockefeller's Sean Brady and his colleagues uncovered and characterized a class of antibiotics they dubbed malacidins that are active against multi-drug resistant pathogens.

"While metagenome-based antibiotic discovery methods are still in their infancy, the scaling and automation of the pipeline described here should permit the systematic discovery of [natural product] antibiotics that have until now remained hidden in the global metagenome, providing a potentially powerful approach for combating antibiotic resistance," the researchers wrote in their paper.

Brady and his colleagues used a sequence-guided screen to search environmental DNA samples for biosynthetic gene clusters encoding calcium-binding motifs. They isolated DNA from 2,000 unique soil samples and used degenerate PCR primers targeting a conserved region of genes that synthesize calcium-dependent antibiotics to generate amplicons. In particular, they targeted those genes' adenylation domains.

They analyzed these amplicons using a bioinformatics platform called environmental Surveyor of Natural Products Diversity (eSNaPD). Three-quarters of the soil samples contained natural product sequence tags that mapped to at least one adenylation domain from a known calcium-dependent antibiotic biosynthetic gene cluster, the researchers reported.

Brady and his colleagues constructed a phylogenetic tree based on these adenylation domain sequences. They noted that a domain encoding a particular aspartic acid residue was linked to the functional divergence of calcium-dependent antibiotic biosynthetic gene clusters. This led them to rely on that domain to track those gene clusters.

Through that, they found an eDNA-specific clade that was present in 19 percent of metagenomes, which suggested to the researchers that it might represent a common, but uncharacterized class of antibiotics, which they dubbed malacidins.

They then extracted DNA from a soil sample rich in malacidins to piece together a complete malacidin biosynthetic gene cluster. They cloned this into a BAC, which they then grew up in yeast. They subsequently isolated the clone-specific metabolites, which they called malacidin A and B.

With a combination of mass spectrometry and NMR analyses, the researchers teased out the structure of the compounds. Malacidins are 10-membered cyclic lipoproteins that differ in the placement of a methylene on their lipid tails and have peptide cores that include for non-proteinogenic amino acid. The malacidins lack the canonical calcium binding domains of known calcium-dependent antibiotics, the researchers noted.

Still, they found through a series of assays that the function of the malacidins does rely on the presence of calcium.

In another series of assays, the researchers found that the malacidins are active against Gram-positive bacteria, including multi-drug resistant pathogens. Similarly, in a rat model, they found that topical application of malacidin A sterilized a methicillin-resistant Staphylococcus aureus-infected wound.

Brady and his colleagues also sought to induce malacidin resistance in S. aureus in the lab by exposing it to sub-lethal levels of the antibiotic. But after 20 days of such exposure, they did not observe any resistance.

Other calcium-dependent antibiotics work by affecting the lipid membrane or cell wall synthesis, but the researchers found that the malacidins don't lead to membrane leakage — rather, they appear to affect the cell wall in a manner that differs from known calcium-dependent antibiotics.

"The discovery of the malacidins supports our hypothesis that the calcium-dependent antibiotics are a larger-than-previously-thought family of [natural products] with low susceptibility to resistance and diverse modes of action," the researchers wrote.