NEW YORK (GenomeWeb) – A next-generation sequencing technique called genome mining could help identify new antibacterial compounds, according to researchers from the University of Illinois.
In a study published recently in the Proceedings of the National Academy of the Sciences, researchers screened 10,000 bacterial strains, sequenced the whole genomes of 278 to identify specific gene clusters, and identified 11 previously undescribed phosphonic acid natural products, including ones that they determined have antimicrobial properties.
The study has implications for drug development and demonstrates that the genome mining technique, which researchers have used for years, can be done at a larger scale, and more cost-effectively and efficiently, by implementing NGS.
William Metcalf, a microbiology professor at the University of Illinois and a senior author of the study, told GenomeWeb this week that the updated approach identified a number of natural products that hold promise for the development of new antibacterials, for which there is a "compelling need."
"There is a real health crisis with resistance to known antibiotics," Metcalf said.
According to the US Centers for Disease Control and Prevention, at least 2 million people in the US become infected with bacteria that are resistant to antibiotics every year, 23,000 of whom die as a result of those infections.
Making antibiotics from natural products is not a new concept. In fact, the vast majority of our current antibiotics were discovered from natural products produced by bacteria and fungi.
However, in the 1980s, pharmaceutical companies largely abandoned natural products as precursors to new drugs because researchers kept rediscovering the same compounds, Metcalf said. In addition, the methods for discovering new compounds involved creating bioassays for every bacteria strain thought to produce a natural product, which was costly and time-consuming.
As sequencing costs started to decline, however, it became feasible to use genomic information to winnow down the field of potential bacterial strains from which to create bioassays. Genome mining, which involves first using PCR to broadly screen thousands of bacteria, then sequencing to identify specific gene clusters, allowed researchers to focus only on those strains with the potential to make novel compounds.
The University of Illinois group was not the first to use genome mining, Metcalf said; however, it had previously been done only on a much smaller scale. Metcalf's own team previously published a study in Nature Chemical Biology using the technique to screen 800 strains, and suggested that it could be scaled up.
In the PNAS study, the team focused on a class of bacteria known as actinomycetes, which are known to be "prolific antibiotic producers," Metcalf said. In addition, many actinomycetes produce phosphonic acids, which are one of the more abundant classes of natural products, produced in about 5 percent of strains. Furthermore, a large percentage of phosphonates are active, clinically useful molecules.
The researchers used PCR as an initial screen for all 10,000 strains to look for the presence of a gene, pepM, which is involved in the production of phosphonic acid. For this step, the researchers used four pairs of degenerate primers designed to amplify a 406-bp conserved fragment within the pepM gene.
Next, they sequenced the whole genomes of the 278 strains that had the gene in order to characterize the particular gene clusters with the potential for making phosphonic acids.
Screening all 10,000 strains with PCR and then sequencing the genomes of 278 strains took the group about one year, Metcalf said. The next steps proved to be trickier.
Sequencing identified 64 distinct phosphonate biosynthetic gene clusters, of which 55 the researchers thought were likely to produce unknown compounds. Then the researchers used chemical characterization techniques such as mass spectrometry and nuclear magnetic resonance spectroscopy to figure out in which strains those clusters were active and producing compounds.
Next, they purified and determined the identities of the specific phosphonates, focusing first on strains containing one of five different gene clusters. Analysis of those five clusters enabled them to discover a new pathway for phosphonate biosynthesis; a pathway for producing H-phosphinates, a family of compounds with potential therapeutic properties; and 11 previously undescribed natural products.
Metcalf said the group estimated that 85 percent of the gene clusters were likely to produce new phosphonate natural products. One next step, he said, will be to further characterize the strains from each of the distinct gene clusters and the natural products that are produced.
The group noted that one of the novel natural products it discovered and named argolaphos has "broad-spectrum antibacterial activity." Notably, they found that it was the most effective against three pathogens responsible for illness: Salmonella typimurium, Escherichia coli, and Staphylococcus aureus.
The researchers also discovered a new class of sulfur-containing phosphonate natural products that are similar to intermediates of a compound found in broccoli, Brussels sprouts, and other plants, and potentially have anti-cancer properties. However, neither of the two strains produced sufficient amounts of the compound to test its bioactivity.
One of the strains that the researchers analyzed further due to the unusual genomic location of the pepM gene produced two novel phosphonate natural products, which the researchers said were likely to be intermediates in the formation of new phosphonate dubbed valinophos. An assessment of its activity found that it "slightly inhibited" Mycobacterium smegmatis.
Metcalf said that although his team does not focus on making drugs, some of the novel compounds they've discovered so far "are ripe and ready" for clinical testing. "What we would hope is that industrial partners interested in moving molecules from discovery to development and application stage would be interested in taking our compounds forward," he said.
In addition, he said, the 11 novel compounds the team discovered came from just seven strains. "Based on our analysis of gene clusters, there are probably at least 60 to 80 other phosphanate molecules still to be identified," which he said the group plans to follow up on.
For the time being, the group will focus on actinomycetes, Metcalf said, but noted that other types of bacteria — as well as metagenomes and uncultured bacteria — are also promising candidates for novel natural products.
There is "abundant data to suggest that we have not even come close to tapping out the potential of actinobacteria, so for now we are sticking with that group," he said.