Small proline-rich proteins 1 and 2 function as antimicrobial proteins in the skin

Human skin prevents the entry of foreign pathogens while also accommodating a myriad of commensal microorganisms. A key contributor to skin immunity is the sebaceous gland, yet our understanding of how sebocytes function in host defense is incomplete. Here we show that sebaceous glands respond to bacterial stimuli by generating small proline-rich proteins, SPRR1B and SPRR2A, which display bactericidal functions in skin. Further, LPS injected into mouse skin triggers the expression of the mouse SPRR orthologous genes, Sprr1a and Sprr2a. Both mouse and human SPRR proteins display potent bactericidal activity against methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa. Thus, Sprr1a;Sprr2a mice are more susceptible to skin infection with these bacteria. Lastly, our findings demonstrate that SPRR proteins exert their bactericidal activity through binding and disruption of the bacterial membrane. These findings provide insight into the regulation of SPRR proteins in skin and how the sebaceous gland contributes to host defense. Introduction The skin is the human body’s largest organ, with direct contact with the external environment (Belkaid and Segre, 2014). As a result, the skin surface continuously encounters a diverse microbial community including bacteria, fungi, viruses and parasites (Duerkop and Hooper, 2013; Findley et al., 2013; Oh et al., 2016). When host defense is impaired, skin infection results. Thus, skin and soft tissue infections pose a considerable public health threat (Dryden, 2009). The majority of infections of the skin are caused by Staphylococcus aureus (S. aureus) (Stulberg et al., 2002). Additionally, Pseudomonas aeruginosa (P. aeruginosa) infections in burn patients are one of the most common causes of mortality (Huebinger et al., 2016). Adding to the challenges of treating infections posed by these pathogens has been the development of antibiotic resistant strains of bacteria such as methicillin resistant Staphylococcus aureus (MRSA) (Klevens et al., 2007). Skin antimicrobial proteins (AMPs) play an essential role in defending the host from the invasion of pathogens (Nakatsuji and Gallo, 2012; Zhang and Gallo, 2016). Mammalian antimicrobial proteins are evolutionarily ancient immune effectors that rapidly kill bacteria by targeting bacterial cell wall or cell membrane structures (Mishra et al., 2018; Mukherjee et al., 2014). Several distinct antimicrobial protein families, such as β-defensins, cathelicidins, resistin and S100 proteins, have been identified and characterized in skin (Gallo and Hooper, 2012; Harris et al., 2019). However, we still have a limited understanding of the arsenal of antimicrobial proteins expressed by the skin, the regulatory networks that control the expression of AMPs, and how AMPs function to protect mammalian skin surfaces. Even less is known about the contribution that skin appendages make to host defense. Sebaceous glands (SGs) are specialized epithelial cells that cover the entire skin surface except the palms and soles. SGs excrete a lipid-rich and waxy substance called sebum to the skin surface (Fischer et al., 2017; Zouboulis et al., 2016, 2020). SGs are believed to contribute to the antimicrobial functions of the skin (Gallo and Hooper, 2012), yet few current studies have examined the role of SGs in skin host defense. Here we show the impact of the bacterial cell wall component lipopolysaccharide (LPS) on human sebocytes and demonstrate that Gram-negative bacteria stimulate the expression of members of the small proline-rich protein (SPRR) family. SPRR proteins were originally identified as markers of terminal differentiation and function as substrates of transglutaminase in the crosslinked cornified envelope present at the skin surface (Cabral et al., 2001; Candi et al., 2005). In this study, we demonstrate an additional function of SPRR proteins, as antimicrobial proteins. Sprr1a;Sprr2a mice are more susceptible to topical MRSA and P. aeruginosa skin infection, revealing that SPRR proteins protects against pathogenic bacterial infections of the skin. We also show mechanistically that the bactericidal activity of SPRR proteins is mediated through the binding and disruption of bacterial membranes. Taken together our findings support a novel antimicrobial function of SPRR proteins in skin and provide insight into the role of sebaceous glands in host defense.