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A21

ANTIMICROBIAL PEPTIDES AND THE MICROBIOME

Michael Zasloff94

Antimicrobial peptides are widely distributed throughout nature, present in all animals and plants. In general they are short (less than 50 amino acids), cationic, and amphipathic. Most can target a broad range of microbes, including bacteria, fungi, viruses, and protozoa. Because they generally act by disturbing membrane permeability, most are microbicidal and kill the target within seconds to minutes (Zasloff, 2002b).

Antimicrobial peptides have traditionally been considered components of the innate immune system that protect the “milieu interieur” from microbial invasion. Indeed, there is a large body of data that unequivocally supports this role for antimicrobial peptides. In humans, for example, areas of the body that we regard as normally “sterile,” such as the urinary tract and the distal divisions of the airway, are kept free of microbes, in states of health, by the orchestrated elaboration of suites of antimicrobial peptides and proteins (Zasloff, 2002b, 2007). In the setting of wounds, we still believe that effective repair and healing requires the elimination of microbes within the wound environment, and antimicrobial peptides and proteins again play a role here (Lai and Gallo, 2009; Sorensen et al., 2003).

In the context of this meeting, the question arises as to whether antimicrobial peptides and proteins influence the commensal microbiome of humans, or function solely to prevent microbial invasion.

Lessons from a Frog

The African clawed frog, Xenopus laevis, is an aquatic creature. Its world as such is somewhat indeterminate with respect to the micro-organisms it can encounter. The skin of this amphibian, like that of other frogs, is invested with granular glands, neuroendocrine structures that synthesize at least a dozen antimicrobial peptides, along with proteins that create a hydrophobic gel on the skin surface when the gland discharges its contents. The wound is subsequently covered with a hydrophobic salve containing a cocktail of antimicrobial peptides at a concentration about 50–100 fold greater than required to kill all micro-organisms that might interfere with healing (Zasloff, 1987).

Yet, the skin of a healthy Xenopus laevis, like other amphibians, is populated by microbes, including organisms that cause lethal systemic infections in this animal (Culp, 2007). Aeromonas hydrophila, for instance, causes “red-leg,” a devastating hemorrhagic septic infection. Surprisingly, A. hydrophila is relatively

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APPENDIX A 489 A21 Antimicrobial Peptides and the Microbiome Michael Zasloff 94 Antimicrobial peptides are widely distributed throughout nature, present in all animals and plants. In general they are short (less than 50 amino acids), cationic, and amphipathic. Most can target a broad range of microbes, including bacteria, fungi, viruses, and protozoa. Because they generally act by disturbing membrane permeability, most are microbicidal and kill the target within seconds to minutes (Zasloff, 2002b). Antimicrobial peptides have traditionally been considered components of the innate immune system that protect the “milieu interieur” from microbial invasion. Indeed, there is a large body of data that unequivocally supports this role for anti­ microbial peptides. In humans, for example, areas of the body that we regard as normally “sterile,” such as the urinary tract and the distal divisions of the airway, are kept free of microbes, in states of health, by the orchestrated elaboration of suites of antimicrobial peptides and proteins (Zasloff, 2002b, 2007). In the setting of wounds, we still believe that effective repair and healing requires the elimina- tion of microbes within the wound environment, and antimicrobial peptides and proteins again play a role here (Lai and Gallo, 2009; Sorensen et al., 2003). In the context of this meeting, the question arises as to whether antimicrobial peptides and proteins influence the commensal microbiome of humans, or func- tion solely to prevent microbial invasion. Lessons from a Frog The African clawed frog, Xenopus laevis, is an aquatic creature. Its world as such is somewhat indeterminate with respect to the micro-organisms it can encounter. The skin of this amphibian, like that of other frogs, is invested with granular glands, neuroendocrine structures that synthesize at least a dozen anti- microbial peptides, along with proteins that create a hydrophobic gel on the skin surface when the gland discharges its contents. The wound is subsequently cov- ered with a hydrophobic salve containing a cocktail of antimicrobial peptides at a concentration about 50–100 fold greater than required to kill all micro-organisms that might interfere with healing (Zasloff, 1987). Yet, the skin of a healthy Xenopus laevis, like other amphibians, is populated by microbes, including organisms that cause lethal systemic infections in this animal (Culp, 2007). Aeromonas hydrophila, for instance, causes “red-leg,” a devastating hemorrhagic septic infection. Surprisingly, A. hydrophila is relatively 94   MedStar Georgetown Transplant Institute, Georgetown University, Washington, DC.

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490 MICROBIAL ECOLOGY IN STATES OF HEALTH AND DISEASE resistant to the action of the skin’s antimicrobial peptides (Rollins-Smith et al., 2002), as are several of the other gram-negative bacteria present on the skin that are also associated with red leg, including Morganella and Serratia spe- cies (­ asloff, 1987). The population of its skin by organisms that are relatively Z resistant to the action of the skin peptides would seem to be a straightforward consequence of the selective pressure imposed by the antimicrobial arsenal. It is likely that continuous low level discharge of the widely distributed granular glands maintains a relatively restricted diversity of resistant bacteria on its skin. In addition, several of the amphibian skin commensals are known to themselves secrete antimicrobial agents that must act to further reduce species diversity (Harris, 2006). Invasion by these organisms resulting in disease occurs with low probability, generally believed to occur when homeostasis is disturbed (“stress”), possibly resulting in a breakdown of the full defensive capacity of the skin. Thus a bal- ance of sort has evolved in the frog between itself and its selected commensals: organisms that comprise the skin microbiome can survive low-level assault by the antimicrobial peptides released onto the surface, but which, should these defenses fail, can nonetheless cause disease. That these granular glands are positioned to deal with commensal microbes can be inferred from a fascinating simple experiment. If the granular glands of a frog are fully discharged by an appropriate dose of noradrenaline (or an electrical discharge), and the animal is placed into a tank of sterile water containing broad- spectrum antibiotics, antimicrobial peptides will not reappear within the granular glands. In contrast, if the animals are returned to a tank containing its normal com- mensal microbes the glands fully regenerate within days (Mangoni et al., 2001). Human Epidermis and Its Microbial Inhabitants The micro-organisms that populate human skin, with whom we have co- evolved, also exhibit evidence of the pressure exerted on their survival by anti- microbial peptides. The best example is seen in the case Staphylococcus aureus. Human epidermis is invested with an array of cationic antimicrobial peptides and proteins, most of which are transcribed initially by the more basal keratinocyte layers (Gallo et al., 2011; Schroder and Harder, 2006). Some are secreted consti- tutively, like HBD1, while others are expressed after injury or infection, such as LL-37, human defensins 2 and 3. These peptides, like most cationic anti­ icrobial m peptides, target the cytoplasmic membrane of microbes through electrostatic at- traction, since the bacteria display negatively charged phospholipids on the outer leaflet of their cytoplasmic membrane (Zasloff, 2002b). Once bound to the mem- brane, antimicrobial peptides cause damage (by a variety of mechanism) and g ­ enerally kill the microbe. In response, the modern strains S. aureus appear to have developed a means of enzymatically reducing the net negative surface charge of its cytoplasmic membrane by coupling phosphatidyl glycerol with lysine (Andra

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APPENDIX A 491 et al., 2011). Presumably the existing strains of S. aureus have developed a degree of resistance to cationic antimicrobial peptides that permits them to survive on the skin, but not sufficient to normally resist the action of defensins that are present within the neutrophils and other phagocytic cells, or induced in the skin in high concentration on injury. Thus a détente of sorts exists between host and microbe. Organisms within our microbiome that exist in proximity to sites of secretion of antimicrobial proteins and peptides have evolved mechanisms of relative resis- tance to their action. Human Diseases Where Failed Antimicrobial Defenses Lead to an Altered Microbiome Atopic Dermatitis: A Failure to Contain the Growth of S. aureus Several of these antimicrobial proteins keep certain organisms at a very low relative abundance on the skin surface. Psoriasin and RNAse 7 are two very abun- dant proteins that are constitutively secreted onto the skin (Glaser et al., 2011; Koten et al., 2009). Both of these antimicrobial agents were first discovered in a survey of antimicrobial substances present in the isolated skin scales from indi- viduals suffering from psoriasis. Christophers, an astute clinician, had noted that although psoriatic lesions are inflamed and physically defective “barriers,” they rarely suffer bacterial infection (Glaser et al., 2005). Schroder and colleagues, following up on this clinical clue, surmised that the skin of the psoriatic might compensate for the barrier defect by over expressing antimicrobial peptides and proteins (Glaser et al., 2005; Harder and Schroder, 2005). Indeed, this is the case, now realized to be a consequence of the intense expression of IL-17 by lympho- cytes within the dermis (Martin et al., 2013). It was from psoriatic scales that several human antimicrobial peptides were first purified and identified (Harder and Schroder, 2005). Psoriasin is active against E. coli, while RNase 7 is most active against E. faecalis. If either organism is applied to the surface of unwashed human adult skin, within several minutes these bacteria die. If an antibody that inactivates either of the antimicrobial proteins is applied to the skin prior to application of bacteria, the corresponding microbes remain viable on the skin (Glaser et al., 2005; Koten et al., 2009). These studies teach us that the microbiome on the skin surface is constrained on the undamaged skin of a “healthy” human by the pres- ence of certain antimicrobial agents. In addition, we should appreciate that ex- cessive washing of the skin with strong detergents will remove the anti­ icrobial m shield and possibly permit the establishment of an alternate microbiome. Following injury the orchestration of batteries of antimicrobial peptides is initiated, releasing molecules not normally seen on healthy skin. It is not surpris- ing that the skin microbiome of the injury site changes in the setting of acute injury (Zeeuwen et al., 2012).

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492 MICROBIAL ECOLOGY IN STATES OF HEALTH AND DISEASE One of the common complications of atopic dermatitis is infection of the skin lesions by S. aureus. The damaged epidermis is unable to restrain the inva- sion of this microbe due to a failure of expression of antimicrobial peptides and proteins such as LL-37 (Ong et al., 2002; Zasloff, 2002a). The precise mechanism responsible for the depressed expression of epidermal antimicrobial defenses is not entirely understood, although several of the cytokines expressed by lympho- cytes within the dermis appear to play a role (Ong et al., 2002). Cystic Fibrosis: A Failure to Prevent a Microbiome from Establishing Itself in the Airway There are areas of the body that are generally regarded as “sterile,” such as the bronchi and more distal branches of the airway. Few, if any microorganisms can normally be seen microscopically in fluids sampled from a healthy human airway. The epithelial lining of the normal human airway distal to the trachea is covered by a micron-thick fluid layer secreted by the underlying cells. The height of the fluid layer, its ionic composition, and pH are maintained by the action of epithelial ion and water channels. Into to this fluid layer the epithelial cells secrete antimicrobial proteins and peptides, such as lysozyme, lipocalin, lacto- ferrin, LL-37, and human beta defensins. The cocktail of antimicrobial peptides and proteins present within the airway surface fluid layer creates a protective barrier that has the capacity to rapidly kill most microbes that are inhaled. In this anatomical compartment of humans, antimicrobial proteins and peptides actively suppress the establishment of a microbiome. In cystic fibrosis, however, the airway becomes populated by a dense micro­ biome that chronically colonizes the bronchial tree during the life of the af- fected individual. Organisms such as Ps. aeruginosa, S. aureus, and Burkholderia ­cepacia can together reach densities of 1010 cells/gram of sputum despite chronic intensive antibiotic therapy. In CF the antimicrobial activity of the surface fluid layer is depressed (Goldman et al., 1997; Smith et al., 1996) and rather than restrict the growth of inhaled bacteria provides a growth medium. The inflamma- tion that occurs within the airway of the individual with CF can be explained as being a secondary response to the presence of bacteria in the airway. The influx of neutrophils that characterizes the inflammatory response represents an attempt by the immune system to defend the airway from the CF microbiome, a futile response that ultimately destroys the physical structure of the bronchi. In CF, a pathological microbiome establishes itself in the airway. The etiology of the chronic infections in CF has been elucidated through the study of a genetically engineered pig in which the porcine CFTR has been replaced with a common human CFTR mutation (Ostedgaard et al., 2011). These animals will develop pulmonary inflammation and bronchial infection within several months of life (Ostedgaard et al., 2011). Longitudinal study of these animals from birth reveals that they have a defect in the capacity of their airway

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APPENDIX A 493 surface fluid to kill bacteria. The cause of this defect appears to be a failure of the epithelium to maintain the normal pH of the airway fluid, permitting it to become excessively acidic (Pezzulo et al., 2012), an effect of a perturbation caused by the defective CFTR. By simply making a sample of airway fluid more alkaline, antimicrobial activity is restored. Antimicrobial peptides and proteins normally prevent a microbiome from establishing itself in the airway. Crohn’s Disease: Unable to Keep the Microbiome at a Distance The human gastrointestinal tract is home to a complex microbiome, which differs in density and diversity throughout the various regions. An area of great interest and considerable investigation are the mechanisms that exist that permit us to contain great numbers of microbes within an organ, such as the ileum or colon that is lined by a single celled layer, and yet normally appears relatively free of inflammation. Like the airway, the surface of the epithelium of the small and large intes- tine is covered by a thin layer of fluid, secreted from the underlying epithelial cells. This layer is itself covered by a layer of mucous, secreted by goblet cells. M ­ icrobes present in the lumen of the intestine, were they to attempt to invade the epithelial layer, would first come in contact with the mucous layer, and then as they penetrated deeper, would enter the fluid layer. As in the airway, antimicrobial agents are secreted into the fluid layer. Some of these, such as beta-defensins, are the products of the common enterocyte. In the small ­ntestine, specialized Paneth i cells that lie at the base of the crypts, secrete high concentrations of a cocktail of antimicrobial peptides and proteins that flood the overlying surface fluid layer. As a consequence of the mucous barrier, the bactericidal submucous fluid layer, and the rapid regeneration of the epithelial layer, bacteria generally cannot gain a foot- hold on the epithelial surface, nor invade the layer and enter the lamina propria. Although the GI tract harbors a complex microbiome, the organisms are normally kept at bay from the epithelium through the action of these defenses (Salzman et al., 2007). Thus in the human intestine, antimicrobial peptides and proteins create an antimicrobial shield that permits containment of the intestinal microbiome. In Crohn’s disease the normal barrier defenses of the small intestine fail. Commensal organisms are no longer spatially restricted, and can access the epithe- lium and the lamina propria of the intestine (Swidsinski et al., 2005). This occurs in part through a failure of the antimicrobial defenses of the Paneth cell. Normally abundant antimicrobial peptides, such as human defensin 5, are present in reduced amounts, resulting in the reduced antibacterial strength of the antimicrobial barrier (Wehkamp et al., 2005a). As in cystic fibrosis, the failure of antimicrobial defense of the barrier results in microbes gaining access to the epithelium, subsequent invasion, and secondary inflammation (Wehkamp et al., 2005b). The secretion of high local concentrations of antimicrobial peptides by the Paneth cells likely influences the diversity of bacteria that find themselves in

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494 MICROBIAL ECOLOGY IN STATES OF HEALTH AND DISEASE contact with the mucous layer, rather than the planktonic microbes that live within the intestinal lumen, where the concentrations of antimicrobial peptides would be too low to exert antimicrobial activity. In mice engineered to express human defensin 5 in the small intestine, the bacteria that populate the mucous layer of the small intestine differ from those seen in the wild-type animals, reflecting the selective pressure imposed by the human antimicrobial peptide (Salzman et al., 2003). In individuals with Crohn’s disease genetic polymorphisms that influence levels of Paneth cell antimicrobial peptide expression appear to be associated with differences in the commensal microbiome of the ileum (Zhang et al., 2012). It is likely that as in the skin, the antimicrobial agents secreted from the intestinal wall influence the diversity of the organisms that populate the immediate luminal surface and ultimately the inflammatory state of the intestine. The ability of humans to coexist with environmental microbes and to support a diverse microbiome is in part a consequence of the existence of antimicrobial peptides and proteins. The antimicrobial barrier is generally clinically “silent” in states of health, creating a chemical barrier without the need for a degree of inflammation that we recognize clinically by the classic signs of “redness, heat, and swelling.” At the same time, these substances exert selective pressure on the organisms that comprise our microbiome, influencing microbial ecology. Many questions regarding the antimicrobial barrier still remain poorly explored and likely would provide insights into a deeper understanding of our microbiome. For example: Does the antimicrobial barrier change with age, acquired illness, or nutritional status? Are there significant genetic differences in the strength of the barrier between individuals? What can we do to strengthen this barrier, and what practices should we avoid? Hopefully these and other questions will be answered in the future. References Andra, J., T. Goldmann, C. M. Ernst, A. Peschel, and T. Gutsmann. 2011. Multiple peptide re- sistance factor (MPRF)-mediated resistance of Staphylococcus aureus against antimicrobial peptides coincides with a modulated peptide interaction with artificial membranes comprising ­lysyl-phosphatidylglycerol. Journal of Biological Chemistry 286(21):18692-18700. Culp, C. E., Falkinham, J.O., Belden, L.K. 2007. Identification of thf natural bactfrial microflora on the skin of eastern newts, bullfrog tadpoles and redback salamanders. Herpetologia 63:66-71. Gallo, R. L., M. Kulesz-Martin, and J. R. Bickenbach. 2011. Montagna symposium 2010: Small ­ olecules: Skin as the first line of defense. Journal of Investigative Dermatology m 131(11):2166-2168. Glaser, R., J. Harder, H. Lange, J. Bartels, E. Christophers, and J. M. Schroder. 2005. Antimicrobial psoriasin (s100a7) protects human skin from Escherichia coli infection. Nature Immunology 6(1):57-64. Glaser, R., B. Koten, M. Wittersheim, and J. Harder. 2011. Psoriasin: Key molecule of the cutaneous barrier? Journal der Deutschen Dermatologischen Gesellschaft 9(11):897-902. Goldman, M. J., G. M. Anderson, E. D. Stolzenberg, U. P. Kari, M. Zasloff, and J. M. Wilson. 1997. Human beta-defensin-1 is a salt-sensitive antibiotic in lung that is inactivated in cystic fibrosis. Cell 88(4):553-560.

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