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

Shekh RM, Roy U - BMC Microbiol. (2012)

Bottom Line: 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 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Sciences, Birla Institute of Technology and Science BITS Pilani KK Birla Goa Campus, NH-17B, Goa, 403726, India.

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.

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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.
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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.

Mentions: The highest antifungal activity against different C. albicans strains was present mainly in the fraction precipitated with 85% ammonium sulfate (Figure 1b). Fractions precipitated with 30% and 50% ammonium sulfate exhibited weak inhibition. The supernatant obtained after 85% ammonium sulfate precipitation clearly did not exhibit any antifungal activity. The antifungal substance present in the 85% cut-off also inhibited germ tube formation in C albicans NCIM 3471 (data not shown). As is clear from Table 3, ammonium sulfate precipitation resulted in an approximate 2-fold increase in specific activity. After ion- exchange chromatography using DEAE Sepharose, the adjacent fractions 31–35 in the chromatogram, showed biological activity (Figure 3), and the specific activity increased 17-fold. After gel filtration, the recovery was approximately 22-fold. Based on the purification steps summarised in Table 3, it was concluded that the total active antimycotic protein recovered was 0.45% only.


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

Shekh RM, Roy U - BMC Microbiol. (2012)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3585888&req=5

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.
Mentions: The highest antifungal activity against different C. albicans strains was present mainly in the fraction precipitated with 85% ammonium sulfate (Figure 1b). Fractions precipitated with 30% and 50% ammonium sulfate exhibited weak inhibition. The supernatant obtained after 85% ammonium sulfate precipitation clearly did not exhibit any antifungal activity. The antifungal substance present in the 85% cut-off also inhibited germ tube formation in C albicans NCIM 3471 (data not shown). As is clear from Table 3, ammonium sulfate precipitation resulted in an approximate 2-fold increase in specific activity. After ion- exchange chromatography using DEAE Sepharose, the adjacent fractions 31–35 in the chromatogram, showed biological activity (Figure 3), and the specific activity increased 17-fold. After gel filtration, the recovery was approximately 22-fold. Based on the purification steps summarised in Table 3, it was concluded that the total active antimycotic protein recovered was 0.45% only.

Bottom Line: 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 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Sciences, Birla Institute of Technology and Science BITS Pilani KK Birla Goa Campus, NH-17B, Goa, 403726, India.

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.

Show MeSH
Related in: MedlinePlus