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Antimicrobial Peptides (AMPs) Mined from Human Gut Metagenome Are Effective Against Antibiotic Resistant Pathogens

The collective genomes of the human gut microbiome, known as the metagenome, encode peptides that are effective against antibiotic-resistant bacteria.

Problem:

Antimicrobial resistance poses a major threat to public health. The development of drug-resistance outpaces traditional drug discovery approaches. As a result, the supply of drugs available to treat multi-drug resistant microbes, a leading cause of nosocomial infections, is shrinking. There is a need for new antimicrobial drugs and new methods of their discovery for more rapid drug development. Thus far, no significant technological advance has capitalized on this market, which is set to grow at a compound annual growth rate (CAGR) of 5.27% through 2030.

Solution:

The human gut is lined with a diverse population of microbes competing for limited resources. Thus, these microbes have evolved strategies to survive including antimicrobial peptides (AMPs), which can be leveraged to treat infections in humans. These peptides kill pathogens by puncturing their outer membrane and, importantly, this mechanism of action is less likely to cause antimicrobial resistance.

Technology:

The inventors mined the human gut metagenome by analyzing publicly available metatranscriptomic data for small open reading frames (smORFs). Candidate AMPs were synthesized and analyzed for membrane permeabilization and depolarization, propensity toward alpha helix formation, and functional antimicrobial activity. The inventors used two separate mouse models of infection to test their candidate AMPs. In the deep thigh infection model, mice were pre-treated with immunosuppressant drugs (cyclophosphamide) immediately preceding infection to simulate nosocomial infection of a deep tissue. The authors identified five lead peptides, of which one has antimicrobial activity comparable to that of polymyxin B (an approved antibiotic).

Advantages:

Development of resistance toward AMPs, which have non-specific mechanisms of action, is rare.
Five lead compounds showed antimicrobial activity that reduced infection by as many as four orders of magnitude, which is comparable to the approved antimicrobial drug polymyxin B.
Many of the discovered peptides have antimicrobial activity and synergize with each other.
These AMPs modulate commensal bacteria, which may be useful to engineer the gut microbiome.

Figure 1. Schematic of the workflow employed to generate lead AMPs and the subsequent screening conducted to confirm their functional efficacy. From bottom left, publicly available metatranscriptomic data was annotated with MetaProdigal to identify smORFs, which were clustered with CD-Hit into 444,054 families. These families were then analyzed further by SmORFinder to generate 4,539 High Confidence Families likely to code for peptides and compared against peptides with purported antimicrobial activity generated by AmPEP to produce 323 high confidence families with predicted antimicrobial activity. 79 were synthesized and screened for bacterial growth inhibition of pathogenic and commensal bacteria and the top candidates subjected to functional membrane permeabilization assays, circular dichroism and structure prediction, and in vivo activity.