title: Studies on Antifungal Potential, Primary Characterization and Mode of Action of a De Novo Cytoplasmic Protein (EAF) from Human Commensal Escherichia coli Against Aspergillus spp.
abstract: A de novo protein named as EAF (Escherichia antifungal protein) from the cytoplasmic pool of an Escherichia coli strain (MTCC 1652), has been purified to homogeneity using anion exchange (Q-XL Sepharose) and cation exchange (SP-Sepharose) chromatography. The MIC (minimum inhibitory concentration) values of purified protein against A. fumigatus (the major pathogenic species) were found to be comparable with standard drugs i.e. 3.90 µg/ml, 3.90 µg/ml and 1.25 µg/disc via microbroth dilution assay (MDA), percentage spore germination inhibition (PSGI) and disc diffusion assay (DDA) respectively. Toxicity results confirmed that it causes no haemolysis against human RBCs upto a concentration of 1000.0 µg/ml as compared to Amphotericin B (conventional antifungal drug) that causes hundred percent haemolysis at a concentration of 37.50 µg/ml only.The purified protein demonstrated a molecular mass of 28 kDa on SDS-PAGE which was further authenticated by MALDI-TOF. Proteomic and bioinformatics studies deciphered its significant homology (72 %) with chain A-D-ribose binding protein (cluster 2 sugar binding periplasmic proteins; sequence homologues of transcription regulatory proteins) from E. coli. Single dimensional page analysis of A. fumigatusproteins with due effect of EAF (at MIC50) revealed the inhibition of two major proteins; a heat shock protein 70-Hsp70 (68 kDa); having role in protein folding and functioning andphenylanalyl-t RNA synthetase PodG subunit protein (74 kDa); involved in growth polarity in fungi. Scanning electron microscopic studies depicted homologous results. We suggest that EAF most likely belongs to a new group of proteins with potent antifungal characteristics, negligible toxicity and targeting vital proteins of fungal metabolism.
authors.forename: Meenakshi
authors.initials: M
authors.lastname: Balhara
authors.forename: Sonam
authors.initials: S
authors.lastname: Ruhil
authors.forename: Sandeep
authors.initials: S
authors.lastname: Dhankhar
authors.affiliation: Centre for Biotechnology, M.D. University, Rohtak, India. anil.chhillar@gmail.com.
authors.forename: Anil K
authors.initials: AK
authors.lastname: Chhillar
mesh_terms: D000595:Amino Acid Sequence; D000935:Antifungal Agents / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D001230:Aspergillus / Q000187:drug effects*; D004912:Erythrocytes; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D006801:Humans; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology
chemical_list: D000935:Antifungal Agents; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins