CONCISE COMMUNICATION DOI 10.1111/j.1365-2133.2007.07886.x DNase1L2 suppresses biofilm formation by Pseudomonas aeruginosa and Staphylococcus aureus L. Eckhart, H. Fischer, K.B. Barken,* T. Tolker-Nielsen* and E. Tschachler Department of Dermatology, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna, Austria *Centre for BioScience Technology, BioCentrum-DTU, Technical University of Denmark, 2800 Lyngby, Denmark Correspondence Erwin Tschachler. E-mail: erwin.tschachler@meduniwien.ac.at Accepted for publication 26 December 2006 Key words antimicrobial defence, biofilm, deoxyribonuclease, Pseudomonas aeruginosa, Staphylococcus aureus, stratum corneum Conflicts of interest None declared. Summary Background The formation of biofilms, which is an important step in bacterial colon- ization, can be inhibited by deoxyribonuclease (DNase)-mediated breakdown of extracellular DNA. We have recently demonstrated that epidermal keratinocytes strongly express DNase1-like 2 (DNase1L2) in a differentiation-associated manner. Objectives To determine whether enzymatically active DNase1L2 is present in human stratum corneum and whether it is able to suppress bacterial biofilm formation. Methods DNase1L2 was extracted from normal human stratum corneum, immuno- captured and incubated with plasmid DNA. DNA hydrolysis was monitored by gel electrophoresis and ethidium bromide staining. The effect of DNase1L2 on biofilm formation was assayed by cultivation of Pseudomonas aeruginosa and Staphylo- coccus aureus in the presence or absence of purified recombinant DNase1L2 in microtitre plates and subsequent quantification of biofilm-forming bacteria by crystal violet staining. Results DNase1L2 was found to be present in an enzymatically active form in the stratum corneum of human skin. In an in vitro assay, purified recombinant DNase1L2 efficiently suppressed the formation of biofilms by P. aeruginosa and S. aureus. Conclusions Our data suggest that DNase1L2 is a novel component of the innate antimicrobial defence of the epidermis. The outermost layer of human skin, i.e. the stratum corneum, consists of cornified cell envelopes devoid of organelles. 1 These corneocytes are formed by terminal differentiation of epidermal keratinocytes and, together with extracellular lipids, establish the barrier of the body to water loss, chemical and mechanical insults, and microbial infection. 2 The proper bar- rier function of the stratum corneum is highly dependent on the expression and activity of a wide variety of specialized structural proteins, lipids and enzymes as well as elements of the innate immune defence such as antimicrobial peptides. 2,3 Both the living layers of the epidermis and the stratum cor- neum contain deoxyribonucleases (DNases). 4 Recently, we have identified DNase1-like 2 (DNase1L2) as the first DNase that is specifically expressed during terminal differentiation of keratinocytes. 5 Furthermore, we demonstrated that DNase1L2 is essential for the breakdown of nuclear DNA during corneo- cyte formation in skin equivalent models. 5 The fate and poten- tial activity of DNase1L2 in the outermost layers of the epidermis, however, remained elusive. Two DNases have been shown to have antibiofilm activity at least in vitro. Biofilms are matrix-enclosed bacterial popula- tions adherent to each other and/or to surfaces, and the bio- film mode of growth plays an important role in bacterial pathogenicity. 6 Bovine DNase1 was shown to suppress Pseudo- monas aeruginosa, Streptococcus intermedius and S. mutans biofilm for- mation, 7,8 and the streptococcal DNase streptodornase, a component of a therapeutic agent for suppurative tissue, was shown to suppress P. aeruginosa biofilm formation. 9 The antibio- film activity of these DNases involves degradation of extracel- lular DNA, which is an essential component of biofilms of the aforementioned species. 8,10,11 As DNase1L2 is expressed at a body site exposed to biofilm-forming bacteria such as staphy- lococci, 12,13 we reasoned that it may interfere with the forma- tion of such biofilms. To test this hypothesis, we investigated if DNase1L2 is pre- sent in its catalytically active form in the superficial layers of human skin and if it can suppress bacterial biofilm formation. Materials and methods Deoxyribonucleases Recombinant human DNase1L2 was produced in the methylo- tropic yeast Pichia pastoris and purified from the culture Ó 2007 The Authors 1342 Journal Compilation Ó 2007 British Association of Dermatologists • British Journal of Dermatology 2007 156, pp1342–1345