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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

RNase 7 is secreted in vivo on the body surface.Standardized areas of various body locations on healthy volunteers (n = 10) were rinsed with 10 mM sodium phosphate buffer containing 150 mM NaCl, pH 7.4 to determine the concentration of RNase 7 at the skin surface by ELISA. Data represent the amount of soluble RNase7/cm2. Each symbol represents data from a single volunteer.
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pone-0006424-g003: RNase 7 is secreted in vivo on the body surface.Standardized areas of various body locations on healthy volunteers (n = 10) were rinsed with 10 mM sodium phosphate buffer containing 150 mM NaCl, pH 7.4 to determine the concentration of RNase 7 at the skin surface by ELISA. Data represent the amount of soluble RNase7/cm2. Each symbol represents data from a single volunteer.

Mentions: To study whether RNase 7 is secreted by keratinocytes, we analyzed keratinocyte culture supernatants as well as cell extracts by ELISA. High amounts (3–18 ng/1.5×105 cells) of RNase 7 were detected in the supernatants of primary keratinocytes cultured for 16 h indicating that RNase 7 is efficiently secreted by the keratinocytes (Fig. 2B). A comparison of RNase 7 amounts present in the culture supernatants and cell extracts derived from primary keratinocytes revealed that the main portion of RNase 7 is released (Fig. 2C). To determine whether RNase 7 is secreted in vivo, we investigated the presence of RNase 7 at various skin surface sites. We rinsed standardized 0.5-cm2 skin areas of healthy human donors with 500 µl 10 mM sodium phosphate buffer containing 150 mM NaCl, pH 7.4 and analyzed the washing fluids for their content of RNase 7 by ELISA. RNase 7 amounts at skin surfaces depended on the donor (n = 10), the skin area, and previous washings for body care. RNase 7 amounts were mainly in the range between 0.3 and 3 ng⋅cm−2 (Fig. 3). In addition, we investigated the concentration of RNase 7 in perianal swabs derived from six individuals. In all six samples RNase 7 was detectable (range of 3.9–12.2 ng⋅ml−1; 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)

RNase 7 is secreted in vivo on the body surface.Standardized areas of various body locations on healthy volunteers (n = 10) were rinsed with 10 mM sodium phosphate buffer containing 150 mM NaCl, pH 7.4 to determine the concentration of RNase 7 at the skin surface by ELISA. Data represent the amount of soluble RNase7/cm2. Each symbol represents data from a single volunteer.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0006424-g003: RNase 7 is secreted in vivo on the body surface.Standardized areas of various body locations on healthy volunteers (n = 10) were rinsed with 10 mM sodium phosphate buffer containing 150 mM NaCl, pH 7.4 to determine the concentration of RNase 7 at the skin surface by ELISA. Data represent the amount of soluble RNase7/cm2. Each symbol represents data from a single volunteer.
Mentions: To study whether RNase 7 is secreted by keratinocytes, we analyzed keratinocyte culture supernatants as well as cell extracts by ELISA. High amounts (3–18 ng/1.5×105 cells) of RNase 7 were detected in the supernatants of primary keratinocytes cultured for 16 h indicating that RNase 7 is efficiently secreted by the keratinocytes (Fig. 2B). A comparison of RNase 7 amounts present in the culture supernatants and cell extracts derived from primary keratinocytes revealed that the main portion of RNase 7 is released (Fig. 2C). To determine whether RNase 7 is secreted in vivo, we investigated the presence of RNase 7 at various skin surface sites. We rinsed standardized 0.5-cm2 skin areas of healthy human donors with 500 µl 10 mM sodium phosphate buffer containing 150 mM NaCl, pH 7.4 and analyzed the washing fluids for their content of RNase 7 by ELISA. RNase 7 amounts at skin surfaces depended on the donor (n = 10), the skin area, and previous washings for body care. RNase 7 amounts were mainly in the range between 0.3 and 3 ng⋅cm−2 (Fig. 3). In addition, we investigated the concentration of RNase 7 in perianal swabs derived from six individuals. In all six samples RNase 7 was detectable (range of 3.9–12.2 ng⋅ml−1; 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