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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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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.
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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.

Mentions: Based on the de novo sequence the combined peptides having 40 amino acid residues were assembled. Individual peptides having m/z 718, 1039 and 601 were found. The combined peptide did not contain any charged acidic residues (Asp, Glu). Hydrophobic amino acids constituted (42.5%, excluding Gly). The peptides did not significantly match any known proteins present in the MASCOT and BLASTp databases. The amino acid sequence of ACP (40 residues) obtained from peptide fragments after digestion of the antimycotic protein with trypsin was analyzed by MS/MS spectra using PEAKS Studio Version 4.5 SP2 [Bioinformatics Solutions] with subsequent de-novo sequencing. The peaks obtained are indicated in the sequence below, and overlapping residues are shown in bold. The de novo spectra for peptides are given in Figure 5a, b, and c.


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

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

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

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

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.
Mentions: Based on the de novo sequence the combined peptides having 40 amino acid residues were assembled. Individual peptides having m/z 718, 1039 and 601 were found. The combined peptide did not contain any charged acidic residues (Asp, Glu). Hydrophobic amino acids constituted (42.5%, excluding Gly). The peptides did not significantly match any known proteins present in the MASCOT and BLASTp databases. The amino acid sequence of ACP (40 residues) obtained from peptide fragments after digestion of the antimycotic protein with trypsin was analyzed by MS/MS spectra using PEAKS Studio Version 4.5 SP2 [Bioinformatics Solutions] with subsequent de-novo sequencing. The peaks obtained are indicated in the sequence below, and overlapping residues are shown in bold. The de novo spectra for peptides are given in Figure 5a, b, and c.

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