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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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Generation of RNase 7 specific antibodies and ELISA.(A) A goat was immunized with a combination of natural and recombinant RNase 7, and serum was purified with an RNase 7 affinity column. The specificity of the RNase 7 affinity-purified polyclonal antibodies was verified by Western-Blot analysis. 100 µg of stratum corneum extract and different amounts of natural skin-derived RNase 7 and recombinant RNase 7 were subjected to Western-Blot analysis using the affinity-purified polyclonal antibodies raised against RNase 7. (B) The RNase 7 antibodies were used to establish an ELISA as described in the experimental procedures. A representative standard curve of the RNase 7 ELISA is shown. The detection limit of the ELISA was a concentration of 0.3 ng⋅ml−1 RNase 7.
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pone-0006424-g001: Generation of RNase 7 specific antibodies and ELISA.(A) A goat was immunized with a combination of natural and recombinant RNase 7, and serum was purified with an RNase 7 affinity column. The specificity of the RNase 7 affinity-purified polyclonal antibodies was verified by Western-Blot analysis. 100 µg of stratum corneum extract and different amounts of natural skin-derived RNase 7 and recombinant RNase 7 were subjected to Western-Blot analysis using the affinity-purified polyclonal antibodies raised against RNase 7. (B) The RNase 7 antibodies were used to establish an ELISA as described in the experimental procedures. A representative standard curve of the RNase 7 ELISA is shown. The detection limit of the ELISA was a concentration of 0.3 ng⋅ml−1 RNase 7.

Mentions: First, we successfully expressed RNase 7 in recombinant form in E. coli. The serum of a goat immunized by a mixture of natural and recombinant RNase 7 showed high RNase 7 immunoreactivity. Purification of RNase 7 antibodies from the serum using an RNase 7 affinity column led to the isolation of RNase 7 specific antibodies. These antibodies specifically detected RNase 7 in stratum corneum extracts (Fig. 1A). For quantitative analyses and to determine how RNase 7 is secreted in vivo at different skin sites, we developed an RNase 7-specific enzyme-linked immunosorbent assay (ELISA) using the RNase 7-specific polyclonal antibodies. Fig. 1B shows a representative standard curve using different concentrations of RNase 7. The detection limit of the ELISA was at a concentration of 0.3 ng⋅ml−1. The specificity of the RNase 7 antibodies was further verified by testing other cationic antimicrobial proteins such as lysozyme, hBD-2, hBD-3 and the closely related (78% identity) RNase 8 [29]. All these proteins were not detected by the RNase 7 ELISA (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)

Generation of RNase 7 specific antibodies and ELISA.(A) A goat was immunized with a combination of natural and recombinant RNase 7, and serum was purified with an RNase 7 affinity column. The specificity of the RNase 7 affinity-purified polyclonal antibodies was verified by Western-Blot analysis. 100 µg of stratum corneum extract and different amounts of natural skin-derived RNase 7 and recombinant RNase 7 were subjected to Western-Blot analysis using the affinity-purified polyclonal antibodies raised against RNase 7. (B) The RNase 7 antibodies were used to establish an ELISA as described in the experimental procedures. A representative standard curve of the RNase 7 ELISA is shown. The detection limit of the ELISA was a concentration of 0.3 ng⋅ml−1 RNase 7.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0006424-g001: Generation of RNase 7 specific antibodies and ELISA.(A) A goat was immunized with a combination of natural and recombinant RNase 7, and serum was purified with an RNase 7 affinity column. The specificity of the RNase 7 affinity-purified polyclonal antibodies was verified by Western-Blot analysis. 100 µg of stratum corneum extract and different amounts of natural skin-derived RNase 7 and recombinant RNase 7 were subjected to Western-Blot analysis using the affinity-purified polyclonal antibodies raised against RNase 7. (B) The RNase 7 antibodies were used to establish an ELISA as described in the experimental procedures. A representative standard curve of the RNase 7 ELISA is shown. The detection limit of the ELISA was a concentration of 0.3 ng⋅ml−1 RNase 7.
Mentions: First, we successfully expressed RNase 7 in recombinant form in E. coli. The serum of a goat immunized by a mixture of natural and recombinant RNase 7 showed high RNase 7 immunoreactivity. Purification of RNase 7 antibodies from the serum using an RNase 7 affinity column led to the isolation of RNase 7 specific antibodies. These antibodies specifically detected RNase 7 in stratum corneum extracts (Fig. 1A). For quantitative analyses and to determine how RNase 7 is secreted in vivo at different skin sites, we developed an RNase 7-specific enzyme-linked immunosorbent assay (ELISA) using the RNase 7-specific polyclonal antibodies. Fig. 1B shows a representative standard curve using different concentrations of RNase 7. The detection limit of the ELISA was at a concentration of 0.3 ng⋅ml−1. The specificity of the RNase 7 antibodies was further verified by testing other cationic antimicrobial proteins such as lysozyme, hBD-2, hBD-3 and the closely related (78% identity) RNase 8 [29]. All these proteins were not detected by the RNase 7 ELISA (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