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RNase 7 contributes to the cutaneous defense against Enterococcus faecium.

Köten B, Simanski M, Gläser R, Podschun R, Schröder JM, Harder J - PLoS ONE (2009)

Bottom Line: RNase 7 was still active against E. faecium at low pH (5.5) or high NaCl (150 mM) concentration and the bactericidal activity of RNase 7 against E. faecium required no ribonuclease activity as shown by recombinant RNase 7 lacking enzymatic activity.Treatment of the skin extract with an RNase 7 specific antibody, which neutralizes the antimicrobial activity of RNase 7, diminished its E. faecium killing activity.Our data indicate that RNase 7 contributes to the E. faecium-killing activity of skin extracts and suggest an important role for RNase 7 in the protection of human skin against E. faecium colonization.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany.

ABSTRACT

Background: Human skin is able to mount a fast response against invading microorganisms by the release of antimicrobial proteins such as the ribonuclease RNase 7. Because RNase 7 exhibits high activity against Enterococcus faecium the aim of this study was to further explore the role of RNase 7 in the cutaneous innate defense system against E. faecium.

Methodology/principal findings: Absolute quantification using real-time PCR and ELISA revealed that primary keratinocytes expressed high levels of RNase 7. Immunohistochemistry showed RNase 7 expression in all epidermal layers of the skin with an intensification in the upper more differentiated layers. Furthermore, RNase 7 was secreted by keratinocytes in vitro and in vivo in a site-dependent way. RNase 7 was still active against E. faecium at low pH (5.5) or high NaCl (150 mM) concentration and the bactericidal activity of RNase 7 against E. faecium required no ribonuclease activity as shown by recombinant RNase 7 lacking enzymatic activity. To further explore the role of RNase 7 in cutaneous defense against E. faecium, we investigated whether RNase 7 contributes to the E. faecium killing activity of skin extracts derived from stratum corneum. Treatment of the skin extract with an RNase 7 specific antibody, which neutralizes the antimicrobial activity of RNase 7, diminished its E. faecium killing activity.

Conclusions/significance: Our data indicate that RNase 7 contributes to the E. faecium-killing activity of skin extracts and suggest an important role for RNase 7 in the protection of human skin against E. faecium colonization.

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Related in: MedlinePlus

Bactericidal activity of RNase 7 requires no enzymatic activity.(A) Shown are the ribonuclease activities of wildtype non-mutated recombinant RNase 7 (wt R7) and mutated recombinant RNase 7 containing the double mutation, histidin-123 to aspartate and lysine-38 to arginine (mut R7). n.d. = not detectable. (B) The antimicrobial activity of wildtype recombinant RNase 7 (wt R7) and ribonuclease-deficient RNase 7 (mut R7) were tested at concentrations of 20 and 1.5 µg⋅ml−1 against E. faecium (ATCC 6057). Data are means±S.D.
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pone-0006424-g006: Bactericidal activity of RNase 7 requires no enzymatic activity.(A) Shown are the ribonuclease activities of wildtype non-mutated recombinant RNase 7 (wt R7) and mutated recombinant RNase 7 containing the double mutation, histidin-123 to aspartate and lysine-38 to arginine (mut R7). n.d. = not detectable. (B) The antimicrobial activity of wildtype recombinant RNase 7 (wt R7) and ribonuclease-deficient RNase 7 (mut R7) were tested at concentrations of 20 and 1.5 µg⋅ml−1 against E. faecium (ATCC 6057). Data are means±S.D.

Mentions: To assess whether the ribonuclease activity of RNase 7 might be responsible for its antibacterial activity, we expressed recombinant RNase 7 containing two mutated amino acids in its active site required for enzymatic activity. As shown in Fig. 6A, mutated RNase 7 exhibited no ribonuclease activity whereas the recombinant wildtype RNase 7 showed high ribonuclease activity. However, when tested against E. faecium, no differences in the killing activity of wildtype and mutated RNase 7 were observed, suggesting that the ribonuclease activity of RNase 7 is not necessary for its antibacterial activity (Fig. 6B). Similar results were obtained with E. coli (not shown).


RNase 7 contributes to the cutaneous defense against Enterococcus faecium.

Köten B, Simanski M, Gläser R, Podschun R, Schröder JM, Harder J - PLoS ONE (2009)

Bactericidal activity of RNase 7 requires no enzymatic activity.(A) Shown are the ribonuclease activities of wildtype non-mutated recombinant RNase 7 (wt R7) and mutated recombinant RNase 7 containing the double mutation, histidin-123 to aspartate and lysine-38 to arginine (mut R7). n.d. = not detectable. (B) The antimicrobial activity of wildtype recombinant RNase 7 (wt R7) and ribonuclease-deficient RNase 7 (mut R7) were tested at concentrations of 20 and 1.5 µg⋅ml−1 against E. faecium (ATCC 6057). Data are means±S.D.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2712763&req=5

pone-0006424-g006: Bactericidal activity of RNase 7 requires no enzymatic activity.(A) Shown are the ribonuclease activities of wildtype non-mutated recombinant RNase 7 (wt R7) and mutated recombinant RNase 7 containing the double mutation, histidin-123 to aspartate and lysine-38 to arginine (mut R7). n.d. = not detectable. (B) The antimicrobial activity of wildtype recombinant RNase 7 (wt R7) and ribonuclease-deficient RNase 7 (mut R7) were tested at concentrations of 20 and 1.5 µg⋅ml−1 against E. faecium (ATCC 6057). Data are means±S.D.
Mentions: To assess whether the ribonuclease activity of RNase 7 might be responsible for its antibacterial activity, we expressed recombinant RNase 7 containing two mutated amino acids in its active site required for enzymatic activity. As shown in Fig. 6A, mutated RNase 7 exhibited no ribonuclease activity whereas the recombinant wildtype RNase 7 showed high ribonuclease activity. However, when tested against E. faecium, no differences in the killing activity of wildtype and mutated RNase 7 were observed, suggesting that the ribonuclease activity of RNase 7 is not necessary for its antibacterial activity (Fig. 6B). Similar results were obtained with E. coli (not shown).

Bottom Line: RNase 7 was still active against E. faecium at low pH (5.5) or high NaCl (150 mM) concentration and the bactericidal activity of RNase 7 against E. faecium required no ribonuclease activity as shown by recombinant RNase 7 lacking enzymatic activity.Treatment of the skin extract with an RNase 7 specific antibody, which neutralizes the antimicrobial activity of RNase 7, diminished its E. faecium killing activity.Our data indicate that RNase 7 contributes to the E. faecium-killing activity of skin extracts and suggest an important role for RNase 7 in the protection of human skin against E. faecium colonization.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany.

ABSTRACT

Background: Human skin is able to mount a fast response against invading microorganisms by the release of antimicrobial proteins such as the ribonuclease RNase 7. Because RNase 7 exhibits high activity against Enterococcus faecium the aim of this study was to further explore the role of RNase 7 in the cutaneous innate defense system against E. faecium.

Methodology/principal findings: Absolute quantification using real-time PCR and ELISA revealed that primary keratinocytes expressed high levels of RNase 7. Immunohistochemistry showed RNase 7 expression in all epidermal layers of the skin with an intensification in the upper more differentiated layers. Furthermore, RNase 7 was secreted by keratinocytes in vitro and in vivo in a site-dependent way. RNase 7 was still active against E. faecium at low pH (5.5) or high NaCl (150 mM) concentration and the bactericidal activity of RNase 7 against E. faecium required no ribonuclease activity as shown by recombinant RNase 7 lacking enzymatic activity. To further explore the role of RNase 7 in cutaneous defense against E. faecium, we investigated whether RNase 7 contributes to the E. faecium killing activity of skin extracts derived from stratum corneum. Treatment of the skin extract with an RNase 7 specific antibody, which neutralizes the antimicrobial activity of RNase 7, diminished its E. faecium killing activity.

Conclusions/significance: Our data indicate that RNase 7 contributes to the E. faecium-killing activity of skin extracts and suggest an important role for RNase 7 in the protection of human skin against E. faecium colonization.

Show MeSH
Related in: MedlinePlus