RNase 7 Contributes to the Cutaneous Defense against Enterococcus faecium

Abstract

BackgroundHuman 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 FindingsAbsolute 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/SignificanceOur 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.

Two Human Host Defense Ribonucleases against Mycobacteria, the Eosinophil Cationic Protein (RNase 3) and RNase 7

“…The bactericidal activity of RNase 3 and RNase 7 has been extensively documented against a wide range of Gram-negative and Grampositive bacteria (30,33,38,60). Here, both the eosinophil-secreted RNase 3 and the skin-derived RNase 7 were envisaged as good candidates to contribute to the host defense against mycobacterial infections.…”

“…RNase 3 and RNase 7 ( Fig. 1) are two representative members of the vertebrate-secreted RNase superfamily with a well-characterized cytotoxic action against a variety of pathogens (29)(30)(31)(32)(33). RNase 3, also called the eosinophil cationic protein (ECP), is a small highly cationic protein secreted by eosinophil secondary granules with potent antibacterial and antiparasitic activities (34,35).…”

“…Complementarily, we have analyzed RNase 7 as an antimicrobial protein secreted by a variety of epithelial tissues (32,33,(46)(47)(48)(49). In particular, RNase 7 is abundantly secreted by keratinocytes and mainly contributes to the skin barrier protection (49,50).…”

Contribution of Structural Domains to the Activity of Ribonuclease 7 against Uropathogenic Bacteria

“…Recombinant peptides were expressed and purified as described previously (13). Briefly, each construct was transformed into E. coli BL21 AI (Invitrogen) to allow for L-arabinose-inducible expression.…”

“…The supernatant was applied to a Ni 2ϩ charged Hi-Trap Chelating HP column (General Electric, Piscataway, NJ). After being washed with wash buffer (20 mM NaPO 4 [pH 7.4], 500 mM NaCl) containing 20 mM imidazole and then 50 mM imidazole, the recombinant peptide was eluted with 500 mM imidazole (13). Purified peptides were dialyzed against DN(0) buffer (25 mM Tris [pH 7.0], 0.1 mM EDTA, 10% glycerol), and the peptide concentrations were determined with a Bradford protein assay (Bio-Rad, Hercules, CA) and confirmed with a bicinchoninic acid assay (Pierce, Rockford, IL).…”