Science Bite - 3 min Oral Presentation (Students and ECRs only) Lorne Infection and Immunity 2020

Combating pathogenic infections and cancer by using defensins to target phosphoinositides (#65)

Guneet Bindra 1 , Cassandra Humble 1 , Scott Williams 1 , Fung Lay 1 , Kha Phan 1 , Mark Hulett 1
  1. Department of Biochemistry and Genetics, La Trobe Institute for Moelcular Science, Melbourne, VIC, Australia

Defensins are small cysteine-rich innate immune peptides with antimicrobial and anticancer activity. Plant defensins such as NaD1 [1] and TPP3 [2], as well as human β-defensins HBD2 [3] and HBD3 [4], have been shown to exert their activity by interacting with specific anionic membrane phospholipids, known as phosphoinositides. Phosphoinositides are minor membrane lipids that are crucial for growth, proliferation and survival of the cell and are often dysregulated in microbes and tumour cells [5, 6], and may be targeted for therapeutic application. In this study, human β-defensins HBD9 and HBD14 have been characterised by determining their phosphoinositide-binding specificity and cytotoxic activity against fungal and tumour cells.


HBD9 and HBD14 were successfully expressed and purified using pHUE vector in BL21 CodonPlus (RIL) cells. The quality, purity and integrity of the defensins were confirmed by SDS-PAGE, immunoblot and mass spectrometry analyses. Protein-lipid overlay assay revealed that HBD9 binds specifically to phosphatidylinositol-4-phosphate (PI4P), whereas HBD14 bound to a broad range of different phospholipids, including phosphatidic acid (PA) and phosphatidylinositol-4,5-bisphoaphate (PI(4,5)P₂). HBD9 was shown to be active against human tumour cell lines (PC3, HeLa, MDA-MB-231 and MCF-7) and the human fungal pathogen Candida albicans strains (ATCC10231 and ATCC90028) at low micromolar concentrations (0.3-1.3 μM). HBD14 was also active against the tumour cell lines and C. albicans but at higher micromolar concentrations (15-72 μM). Membrane permeabilisation assay showed uptake of the membrane impermeable dye, propidium iodide (PI) in MDA-MB-231 and HeLa cells treated with HBD9 and HBD14, demonstrating that these defensins act by destabilising membranes.


The data presented in this study provides proof of concept that defensins have a conserved mode of action, where they exert their activity by binding to membrane phospholipids, causing membrane disruption and cell death.

  1. [1] Poon IKH, Baxter AA, Lay FT, Mills GD, Adda CG, Payne JA, Phan TK, Ryan GF, White JA, Veneer PK, van der Weerden NL, Anderson MA, Kvansakul M and Hulett MD (2014) “Phosphoinositide-mediated oligomerisation of a defensin induces cell lysis”, eLife, 3:e01808
  2. [2] Baxter AA, Richter V, Lay FT, Poon IK, Adda CG, Veneer PK, Phan TK, Bleackley MR, Anderson MA, Kvansakul M and Hulett MD (2015) “ The Tomato Defensin TPP3 Binds Phosphatidylinositol (4,5)-Bisphosphate via a Conserved Dimeric Cationic Grip Conformation To Mediate Cell Lysis”, Mol Cell Biol, 35(11):1964-78.
  3. [3] Jarva M, Phan TK, Lay FT, Caria S, Kvansakul M and Hulett MD (2018) “Human β-defensin 2 kills Candida albicans through phosphatidylinositol 4,5-bisphosphate–mediated membrane permeabilisation”, Science Advances, 4(7).
  4. [4] Phan TK, Lay FT, Poon IK, Hind MG, Kvansakul M and Hulett MD (2018) “Human β-defensin 3 contains an oncolytic motif that binds PI(4,5)P2 to mediate tumour cell permeabilisation”, Oncotarget, 7(2):2054-69.
  5. [5] Bunney TD and Katan M (2010) “Phosphoinositide signalling in cancer: beyond PI3K and PTEN”, Nat Rev Cancer, 10: 342-52.
  6. [6] Phan TK, Williams SA, Bindra GK, Lay FT, Poon IKH, Hulett MD (2019) Phosphoinositides: multipurpose cellular lipids with emerging roles in cell death. Cell death and differentiation 26: 781-793