Biochemical characterization of an anti-Candida factor produced by Enterococcus faecalis

Abstract

Background: Because Candida albicans is resistant to several antifungal antibiotics, there is a need to identify other less toxic natural products, particularly antimicrobial proteins, peptides or bacteriocin like inhibitory substances. An attempt has been made to purify and characterise an anti-Candida compound produced by Enterococcus faecalis.

Results: An anti-Candida protein (ACP) produced by E. faecalis active against 8 C. albicans strains was characterised and partially purified. The ACP showed a broad-spectrum activity against multidrug resistant C. albicans MTCC 183, MTCC 7315, MTCC 3958, NCIM 3557, NCIM 3471 and DI. It was completely inactivated by treatment with proteinase K and partially by pronase E.The ACP retained biological stability after heat-treatment at 90°C for 20 min, maintained activity over a pH range 6-10, and remained active after treatment with α-amylase, lipase, organic solvents, and detergents. The antimicrobial activity of the E. faecalis strain was found exclusively in the extracellular filtrate produced in the late logarithmic growth phase. The highest activity (1600 AU mL-1) against C. albicans MTCC 183 was recorded at 48 h of incubation, and activity decreased thereafter. The peptide showed very low haemagglutination and haemolytic activities against human red blood cells. The antimicrobial substance was purified by salt-fractionation and chromatography.Partially purified ACP had a molecular weight of approximately 43 KDa in Tricine-PAGE analysis. The 12 amino acid N terminal sequence was obtained by Edman degradation. The peptide was de novo sequenced by ESI-MS, and the deduced combined sequence when compared to other bacteriocins and antimicrobial peptide had no significant sequence similarity.

Conclusions: The inhibitory activity of the test strain is due to the synthesis of an antimicrobial protein. To our knowledge, this is the first report on the isolation of a promising non-haemolytic anti-Candida protein from E. faecalis that might be used to treat candidiasis especially in immunocompromised patients.

Figure 1

a. Biological activity of ACP against C. albicans (MTCC 7315). b. Biological activity of ACP against C. albicans (MTCC 183) after 85% ammonium sulfate fractionation, The zone of inhibition was detected in 85% palette dissolved in 20 mmol sodium phosphate buffer pH 8.0, but activity was not detected in supernatant. c. Mild biological activity of ACP against wild type C. albicans (DI) isolated from a diabetic patient in BITS Goa. d and e. Different concentration of dialyzed concentrate of ACP showing zone of inhibition against a lawn of C. albicans MTCC 183.

Figure 2

Kinetics of anti-mycotic protein and biomass production of E. faecalis.

Figure 3

Chromatogram of antimycotic protein ACP produced by E. faecalis on DEAE Sepharose, absorbance of fractions taken at 280 nm. Fractions (31–35) showing biological activity.

Figure 4

Tricine-PAGE of ACP purification fractions and gel overlay with C. albicans (MTCC 183). Lane 1, molecular weight marker. Lane 2, dialyzed concentrate after 85% ammonium sulfate fractionation. Lane 3, pooled active fractions collected through DEAE Sepharose matrix. Lane 4, silver stained fractions after gel filtration using Sephadex-G 75. Lane 5, Inhibition zone by antimycotic protein (ACP) on the overlay gel.

Figure 5

a. De novo spectra for peptide 718.29 m/z, WLPPAGLLGRCGR. b. De novo spectra for peptide 1,039.72 m/z, WFRPWLLWLQSGAQYK. c. De novo spectra for peptide 601.24 m/z, WLGNLFGLPGK. d. Combined de novo sequence of ACP having 3 peptide residues of m/z ratio 718, 1039 and 601.

Figure 6

Antimycotic effect of ACP on the growth of C. albicans (MTCC 183, 3958, 7315, and DI), analyzed by a microbroth dilution assay. Well (a) medium only, well (b) ACP in the medium only, well (c) Grown C. albicans in the medium. Rows A–D, normal growth of Candida albicans, wells treated with different concentrations of ACP.

Figure 7

Haemolytic activity of the dialyzed concentrate containing ACP against human erythrocyte cells.

Figure 8

Haemagglutination activity of ACP with different concentration.