RNase 7 Contributes to the Cutaneous Defense against Enterococcus faecium

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 an

Generation of RNase 7 specific antibodies and ELISA.

<p>(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.</p

Abundant expression and secretion of RNase 7 in primary keratinocytes.

<p>(A) Transcript levels quantified by real-time PCR of various antimicrobial proteins expressed in primary keratinocytes. Shown is the range of data obtained in three independent experiments measured in triplicate. (B) Secretion of antimicrobial proteins from keratinocytes cultured for 16 h. Supernatants of primary keratinocytes were analyzed for RNase 7, psoriasin and hBD-2 by ELISA. Shown is the range of data obtained by three independent experiments measured in triplicate. (C) Comparison of RNase 7 protein present in cell extracts with supernatants from primary keratinocytes cultured for 16 h. Shown are the results of three independent cell culture samples (columns represent means±S.D.).</p

RNase 7 is secreted <i>in vivo</i> on the body surface.

<p>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.</p

Antimicrobial activity of RNase 7 against <i>E. faecium</i> at various pH and high salt conditions.

<p>The antimicrobial activity of RNase 7 (1.6 µg⋅ml⁻¹) was tested in a microdilution assay against <i>E. faecium</i> (ATCC 6057) at various pH conditions or in the presence of 150 mM NaCl. Results are from triplicate determinations and presented as the mean±S.D.</p

Bactericidal activity of RNase 7 requires no enzymatic activity.

<p>(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 <i>E. faecium</i> (ATCC 6057). Data are means±S.D.</p

Epidermal keratinocytes express RNase 7 <i>in vivo</i>.

<p>(A & B) Immunostaining of RNase 7 expression in human normal skin using affinity-purified RNase 7 antibodies. Strong RNase 7 immunoreactivity was detected in the epidermis with highest activity in the uppermost epidermal layers. Sebaecous glands and hair follicles also stained positively. (A) 10×magnification, (B) 20 X magnification of the indicated area of panel A. (C) Negative control using preimmune serum. (E: Epidermis, SC: Stratum Corneum, HF: Hair Follicle, I: Infundibulum, SG: Sebaceous Gland, DP: Dermal Papilla, ORS: Outer Root Sheath, IRS: Inner Root Sheath)</p

RNase 7 contributes to the killing activity of human stratum corneum extracts against <i>E. faecium</i>.

<p>(A) RNase 7 (12.5 µg⋅ml⁻¹) was tested in a microdilution assay against <i>E. faecium</i> (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 <i>E. faecium</i>-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 <i>E. faecium</i>. (B) The killing activity of skin-extracts derived from stratum corneum against <i>E. faecium</i> was tested. 2 h incubation of <i>E. faecium</i> with stratum corneum extract revealed high killing activity of the extract against <i>E. faecium</i> (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 <i>t</i>-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.</p