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Lls (Steppan et al., 2001; Pine et al., 2018), but is undescribed in skin epithelium. Immunofluorescence analysis of mouse skin revealed that RELM was expressed by keratinocytes and sebocytes inside the epidermis (Figure 1D, Figure S2C). Although the mouse genome encodes 4 RELM family members, the human genome encodes only two RELM proteins: Resistin-like molecule (RELM), that is expressed in the intestine (Rajala et al., 2003), and Resistin (RETN), that is expressed in keratinocytes and sebaceous glands with the skin (Harrison et al., 2007). Immunofluorescence and fluorescence in situ hybridization (FISH) evaluation confirmed that, like mouse RELM (mRELM), human RETN (hRETN) is expressed by epidermal keratinocytes (Figure 1E,1F, S2C). The place of RELM expression in monocytes, adipocytes, keratinocytes and sebaceous glands is shared with other cutaneous antimicrobial peptides which include cathelicidin (Braff et al., 2005; Chronnell et al., 2001; Zhang et al., 2015; Zhang and Gallo, 2016) (Figure 1F), suggesting that mRELM and hRETN may well function in antimicrobial defense with the skin. RELM kills bacteria by disrupting their membranes We next tested the ability of mRELM and hRETN to kill bacteria. We produced recombinant mRELM and hRETN in Escherichia coli and purified folded, monomeric protein (Figure S3A). We added the purified proteins to a panel of commensal and pathogenic bacteria that included each Gram-positive and Gram-negative species (Fig. 2A,B). Each mRELM and hRETN caused a dose-dependent reduction in the viability of strains from the Gram-positive species Streptococcus pyogenes (Figure 2A) along with the Gramnegative species Pseudomonas aeruginosa (99 decline in viability soon after a 2 hour exposure to 2.five M of either protein) (Figure 2B). The viability from the intestinal Gram-negative bacterial species Citrobacter rodentium and Escherichia coli K12 also declined, but a lot much less markedly (Figure 2B). Propionibacterium acnes, a Gram-positive commensal species linked to acne in human skin (Holland et al., 1998; Beylot et al., 2014; Coughlin et al., 2017), was highly susceptible to hRETN but significantly less so to mRELM (Figure 2B). Thus, mRELM and hRETN are bactericidal for Gram-positive and Gram-negative bacteria at lowCell Host Microbe. Author manuscript; available in PMC 2020 June 12.Harris et al.Pagemicromolar concentrations, related to other skin antimicrobial proteins (Figure S3B) (Ganz and Lehrer, 1995; Durr et al., 2006). Interestingly, while a strain on the Gram-positive skin commensal Staphylococcus epidermidis was susceptible to mRELM and hRETN, a strain with the pathogen Staphylococcus aureus was resistant (Figure 2B). This Caspase 7 Inhibitor custom synthesis recommended that S. aureus may well have distinct attributes that defend it from mRELM-and hRETN-mediated killing. A one of a kind feature of S. aureus is its ability to make staphyloxanthin (STX), a yellow pigment that intercalates into the S. aureus membrane and protects it from attack by pore-forming antimicrobial proteins (Mishra et al., 2011; Liu et al., 2005). Certainly, S. aureus mutants lacking STX (DCRTM) had been much more susceptible to killing by mRELM and hRETN (Figure 2C), indicating that STX is expected for S. aureus resistance to RELM bactericidal activity. The requirement of STX for S. aureus resistance to mRELM recommended that mRELM bactericidal CaMK II Activator supplier activity may involve membrane permeabilization. This notion was also supported by our prior getting that mRELM killed Gram-negative bacteria by forming pores that permeabilized bacterial.