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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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RNase 7 contributes to the killing activity of human stratum corneum extracts against E. faecium.(A) RNase 7 (12.5 µg⋅ml−1) was tested in a microdilution assay against E. faecium (ATCC 6057) alone (R7) or in the presence of 10 mg⋅ml−1 RNase 7 antibodies (R7+R7-Ab). Application of the RNase 7 antibodies completely blocked the E. faecium-killing activity of RNase 7. As a control, RNase 7 was incubated with irrelevant goat antibodies (R7+irr.Ab). Both, RNase 7 antibodies (R7-Ab) alone as well as irrelevant antibodies (irr. Ab) alone did not influence the growth of E. faecium. (B) The killing activity of skin-extracts derived from stratum corneum against E. faecium was tested. 2 h incubation of E. faecium with stratum corneum extract revealed high killing activity of the extract against E. faecium (s.c.). In contrast, the application of RNase 7-blocking antibodies to the stratum corneum extract significantly reduced the killing activity of the extract (s.c.+ R7-specific Ab; p<0.01, Student's t-test). Incubation of the stratum corneum extract with the irrelevant antibodies did not affect the killing activity of the extracts (s.c.+ irrelevant Ab). Data show means±S.D. of triplicate samples. A representative result of three independent experiments is shown.
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pone-0006424-g007: RNase 7 contributes to the killing activity of human stratum corneum extracts against E. faecium.(A) RNase 7 (12.5 µg⋅ml−1) was tested in a microdilution assay against E. faecium (ATCC 6057) alone (R7) or in the presence of 10 mg⋅ml−1 RNase 7 antibodies (R7+R7-Ab). Application of the RNase 7 antibodies completely blocked the E. faecium-killing activity of RNase 7. As a control, RNase 7 was incubated with irrelevant goat antibodies (R7+irr.Ab). Both, RNase 7 antibodies (R7-Ab) alone as well as irrelevant antibodies (irr. Ab) alone did not influence the growth of E. faecium. (B) The killing activity of skin-extracts derived from stratum corneum against E. faecium was tested. 2 h incubation of E. faecium with stratum corneum extract revealed high killing activity of the extract against E. faecium (s.c.). In contrast, the application of RNase 7-blocking antibodies to the stratum corneum extract significantly reduced the killing activity of the extract (s.c.+ R7-specific Ab; p<0.01, Student's t-test). Incubation of the stratum corneum extract with the irrelevant antibodies did not affect the killing activity of the extracts (s.c.+ irrelevant Ab). Data show means±S.D. of triplicate samples. A representative result of three independent experiments is shown.

Mentions: To analyze whether RNase 7 contributes to the Enterococcus–killing activity of healthy skin, we determined whether antibodies to RNase 7 affected the killing of E. faecium in skin extracts derived from stratum corneum. First, we analyzed whether RNase 7 antibodies neutralized the antibacterial activity of RNase 7 against E. faecium. For this purpose we tested the activity of RNase 7 against E. faecium in an antibacterial microdilution assay in the presence of RNase 7 antibodies. Fig. 7A shows that application of the RNase 7 antibodies completely blocked the E. faecium-killing activity of RNase 7. Antimicrobial activity was not inhibited when equivalent concentrations of irrelevant antibodies (antibodies derived from goat preimmune serum) were used. Having established that the RNase 7-specific antibodies neutralized the antimicrobial effect of RNase 7, we used this approach to investigate the role of RNase 7 for the killing activity of human skin extracts. The application of the RNase 7-blocking antibodies to skin extracts derived from stratum corneum before inoculation with E. faecium resulted in a substantial increase in E. faecium growth (Fig. 7B).


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 contributes to the killing activity of human stratum corneum extracts against E. faecium.(A) RNase 7 (12.5 µg⋅ml−1) was tested in a microdilution assay against E. faecium (ATCC 6057) alone (R7) or in the presence of 10 mg⋅ml−1 RNase 7 antibodies (R7+R7-Ab). Application of the RNase 7 antibodies completely blocked the E. faecium-killing activity of RNase 7. As a control, RNase 7 was incubated with irrelevant goat antibodies (R7+irr.Ab). Both, RNase 7 antibodies (R7-Ab) alone as well as irrelevant antibodies (irr. Ab) alone did not influence the growth of E. faecium. (B) The killing activity of skin-extracts derived from stratum corneum against E. faecium was tested. 2 h incubation of E. faecium with stratum corneum extract revealed high killing activity of the extract against E. faecium (s.c.). In contrast, the application of RNase 7-blocking antibodies to the stratum corneum extract significantly reduced the killing activity of the extract (s.c.+ R7-specific Ab; p<0.01, Student's t-test). Incubation of the stratum corneum extract with the irrelevant antibodies did not affect the killing activity of the extracts (s.c.+ irrelevant Ab). Data show means±S.D. of triplicate samples. A representative result of three independent experiments is shown.
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Related In: Results  -  Collection

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pone-0006424-g007: RNase 7 contributes to the killing activity of human stratum corneum extracts against E. faecium.(A) RNase 7 (12.5 µg⋅ml−1) was tested in a microdilution assay against E. faecium (ATCC 6057) alone (R7) or in the presence of 10 mg⋅ml−1 RNase 7 antibodies (R7+R7-Ab). Application of the RNase 7 antibodies completely blocked the E. faecium-killing activity of RNase 7. As a control, RNase 7 was incubated with irrelevant goat antibodies (R7+irr.Ab). Both, RNase 7 antibodies (R7-Ab) alone as well as irrelevant antibodies (irr. Ab) alone did not influence the growth of E. faecium. (B) The killing activity of skin-extracts derived from stratum corneum against E. faecium was tested. 2 h incubation of E. faecium with stratum corneum extract revealed high killing activity of the extract against E. faecium (s.c.). In contrast, the application of RNase 7-blocking antibodies to the stratum corneum extract significantly reduced the killing activity of the extract (s.c.+ R7-specific Ab; p<0.01, Student's t-test). Incubation of the stratum corneum extract with the irrelevant antibodies did not affect the killing activity of the extracts (s.c.+ irrelevant Ab). Data show means±S.D. of triplicate samples. A representative result of three independent experiments is shown.
Mentions: To analyze whether RNase 7 contributes to the Enterococcus–killing activity of healthy skin, we determined whether antibodies to RNase 7 affected the killing of E. faecium in skin extracts derived from stratum corneum. First, we analyzed whether RNase 7 antibodies neutralized the antibacterial activity of RNase 7 against E. faecium. For this purpose we tested the activity of RNase 7 against E. faecium in an antibacterial microdilution assay in the presence of RNase 7 antibodies. Fig. 7A shows that application of the RNase 7 antibodies completely blocked the E. faecium-killing activity of RNase 7. Antimicrobial activity was not inhibited when equivalent concentrations of irrelevant antibodies (antibodies derived from goat preimmune serum) were used. Having established that the RNase 7-specific antibodies neutralized the antimicrobial effect of RNase 7, we used this approach to investigate the role of RNase 7 for the killing activity of human skin extracts. The application of the RNase 7-blocking antibodies to skin extracts derived from stratum corneum before inoculation with E. faecium resulted in a substantial increase in E. faecium growth (Fig. 7B).

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