Lls (Steppan et al., 2001; Pine et al., 2018), but is undescribed in skin epithelium. Immunofluorescence evaluation of mouse skin revealed that RELM was expressed by keratinocytes and sebocytes within the epidermis (Figure 1D, Figure S2C). Even though the mouse genome encodes four RELM members of the family, the human genome encodes only two RELM proteins: Resistin-like molecule (RELM), which is expressed in the intestine (Rajala et al., 2003), and Resistin (RETN), which is expressed in keratinocytes and sebaceous glands in the skin (Harrison et al., 2007). Immunofluorescence and fluorescence in situ hybridization (FISH) analysis confirmed that, like mouse RELM (mRELM), human RETN (hRETN) is expressed by epidermal keratinocytes (Figure 1E,1F, S2C). The place of RELM expression in mAChR3 Antagonist drug monocytes, adipocytes, keratinocytes and sebaceous glands is shared with other cutaneous antimicrobial peptides like 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 on the skin. RELM kills bacteria by disrupting their membranes We subsequent tested the capacity 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 integrated both Caspase 6 Inhibitor supplier Gram-positive and Gram-negative species (Fig. 2A,B). Each mRELM and hRETN triggered a dose-dependent reduction in the viability of strains of 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 of your intestinal Gram-negative bacterial species Citrobacter rodentium and Escherichia coli K12 also declined, but substantially 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 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; accessible in PMC 2020 June 12.Harris et al.Pagemicromolar concentrations, similar to other skin antimicrobial proteins (Figure S3B) (Ganz and Lehrer, 1995; Durr et al., 2006). Interestingly, while a strain from the Gram-positive skin commensal Staphylococcus epidermidis was susceptible to mRELM and hRETN, a strain on the pathogen Staphylococcus aureus was resistant (Figure 2B). This suggested that S. aureus might have certain attributes that shield it from mRELM-and hRETN-mediated killing. A distinctive feature of S. aureus is its capability to produce staphyloxanthin (STX), a yellow pigment that intercalates in to 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 more susceptible to killing by mRELM and hRETN (Figure 2C), indicating that STX is necessary for S. aureus resistance to RELM bactericidal activity. The requirement of STX for S. aureus resistance to mRELM suggested that mRELM bactericidal activity may involve membrane permeabilization. This concept was also supported by our prior obtaining that mRELM killed Gram-negative bacteria by forming pores that permeabilized bacterial.