Novel Leech Antimicrobial Peptides — Hirunipins

Antimicrobial resistance (AMR) is among the top global health threats identified by the WHO. The clinical antibiotic pipeline has stagnated, with few novel mechanism classes entering development. Antimicrobial peptides (AMPs) represent a promising alternative: they employ membrane-disruption mechanisms fundamentally different from conventional antibiotics, making cross-resistance less likely.

Leech saliva is a rich protein source — 189 proteins were identified in the salivary cell secretion of H. medicinalis (Babenko et al. 2020). Kumar et al. (2025) screened a transcriptome of the related East Asian leech Hirudo nipponia for antimicrobial peptides and identified the hirunipins.[R1]

The researchers assembled a transcriptome from the head, salivary gland and teeth of the East Asian medicinal leech Hirudo nipponia, predicted 19 candidate antimicrobial peptides in silico and synthesized them chemically; only three (named hirunipins 1, 2 and 3) had antibacterial activity (MIC 32–128 µg/mL). The peptides were not isolated from leech saliva, and no proteomic confirmation of their expression is reported. The hirunipins showed structural features including amphipathic alpha-helical topology, cationic charge, and membrane-active conformation (random coil in aqueous buffer, α-helical in 50% TFE or 30 mM SDS).[R1]

Hirunipins kill bacteria by disrupting their membranes. For hirunipin 2 this was shown with membrane-depolarization, outer/inner-membrane permeability, propidium-iodide influx, scanning electron microscopy and optical diffraction tomography (ODT) assays:

A particularly significant finding: hirunipin 2 inhibited biofilm formation by multidrug-resistant A. baumannii (3D-ODT time-lapse) and eradicated about 50% of a preformed biofilm at 32 µg/mL (hirunipins 1 and 3: 64 and 256 µg/mL); the authors describe its antibiofilm activity as nearly equivalent to LL-37. Biofilms are a major clinical challenge — they protect bacteria from both antibiotics and immune cells, contributing to chronic and device-associated infections.

Checkerboard testing against multidrug-resistant Acinetobacter baumannii (strain 329-53) showed synergy (FICI ≤ 0.5) between hirunipin 2 and all four antibiotics tested (chloramphenicol, ciprofloxacin, tetracycline, rifampicin):

Beyond direct antimicrobial activity, hirunipin 2 showed anti-inflammatory properties in cell-based assays. At 16 µg/mL it reduced TNF-α, IL-6 and MCP-1 production in LPS-stimulated RAW 264.7 murine macrophages by about 40%, 70% and 90%, respectively:

The hirunipins study demonstrates in vitro potential of leech transcriptomes (here Hirudo nipponia) as a bioprospecting source of novel antimicrobial peptides. With the WHO reporting that only 13 new antibiotics were approved between 2017 and 2023, sources of structurally novel antimicrobial compounds are critically needed. Many genes expressed in leech salivary cells encode previously unknown salivary components (Babenko et al. 2020).

The study does not address hirudotherapy directly: the hirunipins were predicted from a Hirudo nipponia transcriptome and tested as synthetic peptides in vitro, and it was not shown that they are secreted at the leech bite site.