Genes involved in protein glycosylation determine the activity and cell internalization of the antifungal peptide PAF26 in Saccharomyces cerevisiae.
We have previously characterized the synthetic hexapeptide PAF26 as a cell-penetrating and non-lytic antifungal peptide that is active against Saccharomyces cerevisiae and filamentous fungi. Numerous cell wall (CW) proteins are glycosylated in fungi and many of these play important roles in fungal pathogenesis. In this study, we screened a collection of S. cerevisiae deletion mutants for protein glycosylation genes whose deletion altered the sensitivity to PAF26. Increased tolerance to PAF26 was observed in mutants with the following disrupted genes: PMT1-6, EOS1, ALG5, MNN1, MNN4 and MNN5. Significantly, genes coding for protein O-mannosyltransferase 2 (Pmt2p), which is responsible for the addition of the first mannosyl residue of O-linked carbohydrates, and for Eos1p, an enzyme involved in N-linked glycosylation of proteins, showed resistance to PAF26 and defects in CW integrity. Microscopic studies on the S. cerevisiae Δeos1 deletion mutant demonstrated a blockage of peptide internalization by cells. Protoplasts lacking CWs interacted with the peptide, but were more resistant to peptide killing than cells possessing CWs due to a blockage in PAF26 internalization. Interestingly, protoplasts obtained from Δeos1 behaved similarly to those of the parental strain. Collectively, these observations demonstrate that the CW is a positive factor that determines the internalization of the PAF26, and that Eos1p exerts its activity through the glycosylation of specific protein(s) involved in peptide internalization.
Food Science Department, Instituto de Agroquímica y Tecnología de Alimentos (IATA), Consejo Superior de Investigaciones Científicas (CSIC), Avda Agustín Escardino 7, 46980 Paterna, Valencia, Spain; Fungal Cell Biology Group, Institute of Cell Biology, University of Edinburgh, Rutherford Building, Edinburgh EH9 3JH, UK.
Eleonora
E
Harries
Lourdes
L
Carmona
Alberto
A
Muñoz
José I
JI
Ibeas
Nick D
ND
Read
Mónica
M
Gandía
Jose F
JF
Marcos
D000935:Antifungal Agents / Q000378:metabolism / Q000494:pharmacology*; D002473:Cell Wall / Q000187:drug effects / Q000378:metabolism; D006031:Glycosylation / Q000187:drug effects; D009842:Oligopeptides / Q000378:metabolism / Q000494:pharmacology*; D012441:Saccharomyces cerevisiae / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
D000935:Antifungal Agents; D009842:Oligopeptides; C459185:PAF26 peptide; D029701:Saccharomyces cerevisiae Proteins
Antimicrobial peptide; Cell wall; Cell-penetrating peptide; Histatins; Melittin; Protein glycosylation