Oral Presentation The 3rd Prato Conference on Pore Forming Proteins 2015

Dual roles for a novel pore-forming peptide toxin in the human fungal pathogen Candida albicans (#33)

Jonathan Richardson 1 , David L Moyes 1 , Duncan Wilson 2 , Shirley Tang 1 , Sarah Höfs 2 , Mariana Blagojevic 1 , Nessim Kichik 1 , Simona Iancu 1 , Thomas Gutsmann 3 , Oliver Bader 4 , Ernesto Cota 5 , Bernhard Hube 2 , Julian R Naglik 1
  1. Department of Oral Immunology, King’s College London Dental Institute, King’s College London, London
  2. Department of Microbial Pathogenicity Mechanisms, Hans Knoell Institute (HKI), Beutenbergstrasse
  3. Division of Biophysics, Research Center Borstel, Parkallee
  4. Institute for Medical Microbiology and German National Reference Center for Systemic Mycose, Germany
  5. Division of Molecular Sciences, Imperial College London, London

Introduction: The existence of pore-forming peptides or proteins in human pathogenic fungi is unknown. The fungus Candida albicans is a commensal in the human population but can cause serious mucosal and life-threatening systemic infections in immunocompromised hosts. During disease, the fungus grows as invasive hyphae that damages mucosal tissue. Recently, we discovered a MAPK-based epithelial signalling mechanism that discriminates between the commensal (yeast) and pathogenic (hyphal) form of C.albicans. Here, we identify a novel pore-forming peptide in C.albicans, which activates this MAPK response. This 32 amino acid peptide becomes active after being generated from its parent protein, Ece1p, and is exclusively produced by the hyphal form.
Methods: Lactate dehydrogenase release was used as a reporter for cellular damage. Epithelial stimulation was assessed by quantification of secreted cytokines using microbead assays. Activation of MAPK signalling was demonstrated by Western blotting. Membrane intercalation and pore formation was determined by FRET, fluorescent chip technologies, patch clamping and calcium influx assays.
Results: This fungal peptide has dual function, activating epithelial immunity via MAPK signalling at low doses and inducing epithelial damage via pore formation at high doses. A C.albicans strain deficient in the peptide fragment is unable to activate epithelial immunity or cellular damage in vitro and is avirulent in a murine model of oral candidiasis. The peptide is amphipathic, has a predicted α-helical structure, and can damage different human epithelial cells. Membrane intercalation and pore formation is phosphatidylserine-dependent and requires an intact positively-charged C-terminal KR motif in the peptide. Pore formation is rapid and results in an inward current due to calcium influx.
Conclusion: This work identifies the first putative pore-forming peptide toxin in a human pathogenic fungus and appears to be targeted by the host to enable discrimination between the commensal and pathogenic state of this medically important pathogen.