Carbon Chapter 100 LL-37 Carbon

History of AMPs

AMPs are evolutionarily conserved molecules found in organisms ranging from prokaryotes to humans. Lysozyme, the first reported human antimicrobial protein, was identified in 1922 from nasal mucus by Alexander Fleming. This observation was overshadowed when in 1928 Fleming discovered penicillin, and in the 1940s he, along with others, brought the therapeutic use of penicillin to fruition, for which he was awarded a share of the 1945 Nobel Prize for Medicine. Thus, the beginning of the 'Golden Age of antibiotics' in the 1940s led to a loss of interest in the therapeutic potential of natural AMPs, such as lysozyme. In the 1960s, the rise of multidrug-resistant microbial pathogens awakened the interest in AMPs as host defense molecules.

AMPs have been identified from all species of life that have been studied for this activity. AMP discovery likely started in plants, followed by the description in the 1960s of brombinin in frogs and lactoferrin from milk. In 1981, a landmark study by Hans Boman reported how injection of bacteria into the Cecropia silk moth induced a potent AMP. This discovery of 'cecropins' was the first major report of α-helical AMPs. Another important advance occurred when Zasloff and colleagues in 1987 isolated and characterized cationic AMPs, named 'magainins' from the African clawed frog Xenopus laevis. In the mid-1990s, Hoffmann's group showed that genetic ablation of AMP synthesis rendered the fruit fly susceptible to a massive fungal infection, providing the first evidence of the critical role of AMPs in insect host defense. Accelerated clinical and scientific interest proceeded with the discovery in our laboratory of AMPs in mammalian skin in 1994 and our subsequent generation of mice deficient in one of these cathelicidins that clearly demonstrated the relevance of AMPs to mammalian host defense. AMPs have since been extensively characterized and discovered in virtually all multicellular organisms that have been studied for this activity. Presently, more than 2,500 AMPs have been deposited in the Antimicrobial Peptide Database (http://aps.unmc.edu/AP/main.php). It is likely that this list represents only a small fraction of gene-encoded antibiotic proteins produced in nature.

Properties and diversity of AMPs

Cationic AMPs typically consist of between 10 and around 50 amino acid residues with an overall positive charge. These peptides frequently contain a distribution of basic amino acids and hydrophobic residues that align in three dimensions on opposing faces, therefore forming unique structures that are water soluble, positively charged and hydrophobic. Folded AMPs can be classified into groups based on their secondary structure: α-helical; β-sheet; and extended AMPs. Amphipathic α-helical AMPs include the frog magainin, and the extensively studied human cathelicidin peptide LL37. These peptides exhibit little secondary structure in aqueous solution but adopt the amphipathic α-helical architecture when they enter a non-polar environment, such as the bacterial membrane. Other AMPs, such as bactenecins and defensins, are characterized by two or more β-sheets that are stabilized by disulfide bonds. Lastly, the extended AMPs are peptides that do not possess a specific structural motif but rather are defined by a high content of specific residues, such as histidine, arginine, glycine or tryptophan. For example, histatins from humans are rich in histidine residues, and indolicidin from bovine leukocytes has multiple tryptophan and arginine residues.

Microbes also produce a variety of AMPs to limit the growth of other microorganisms and should be considered another normal source of AMPs. These peptides from microbes are quite distinct from the vertebrate AMPs because they can be synthesized from nonribosomal peptide synthase. Nonribosomal peptides often have cyclic or branched structures, can contain non-proteinogenic amino acids including D-amino acids, carry modifications like N-methyl and N-formyl groups, and can be glycosylated, acylated, halogenated, or hydroxylated. Some prominent examples of microbial AMPs include the cationic peptides polymyxin B (produced by Bacillus polymyxa) and the noncationic glycopeptide vancomycin (produced by Amycolatopsis orientalis), both of which are FDA-approved antibiotics. Polymyxin B is clinically effective against major multidrug-resistant Gram-negative bacteria such as Pseudomonas aeruginosa, Acinetobacter baumannii and Klebsiella pneumoniae. Vancomycin is an important first-line antibiotic for treatment of Gram-positive infections, including serious methicillin-resistant Staphylococcus aureus infections. Other notable AMPs from bacteria include toxins like cholera and diphtheria toxins, and bacteriolytic enzymes like lysostaphin and hemolysins, and bacteriocins and bacteriocin-like peptides. So far, over 250 bacteriocins have been characterized from bacteria, and some of them show a relatively broad spectrum of inhibition.

AMPs vary substantially even among mammalian species. For example, while α-defensins represent the most prevalent host defense peptide in human neutrophils, these AMPs are completely absent in neutrophils from mice and cattle. Indeed, defensins are considered to be amongst the most rapidly evolving group of mammalian proteins; hundreds have been identified, and substantial variation of defensins has been found even among primate species. In contrast, much less variation is seen in the cathelicidin family. Only a few cathelicidin AMP members have been identified in most species. Among the first mammalian cathelicidins described were the bactenecins (Bac5 and 7), which were isolated from bovine neutrophils, and rabbit CAP18 from granulocytes. Although multiple cathelicidins are present in cattle, buffalo, horse, pig, chicken and fish, only a single gene for cathelicidin has been found in humans, most primates, mice and rats. It has been speculated that the gene duplication seen in livestock, such as pigs and cows, may be the result of selective pressures for disease resistance that came from domestication. Overall, however, it is important to recognize that the general term AMP has been applied to the products of a highly diverse set of genes that are grouped under this term only because of observations that the protein they encode can kill or inhibit growth of a microbe. Indeed, as many of these observations of function have only been made in highly artificial in vitro systems, it remains debatable that all molecules called AMPs are really antimicrobial.

Mechanisms of action of AMPs

The ability of AMPs to kill bacteria usually depends upon their ability to interact with bacterial membranes or cell walls. Generally, AMPs exhibit a net positive charge and a high ratio of hydrophobic amino acids, allowing them to selectively bind to negatively charged bacterial membranes. Binding of AMPs to the bacterial membrane leads to non-enzymatic disruption. Selectivity for specific species is due to differences in the membrane composition of different microbes and cell types.

Defensins and cathelicidins comprise the major families of membrane-disrupting peptides in vertebrates. The electrostatic interaction between defensin's cationic residue clusters and negatively charged phospholipid groups can form pores in the bacterial membrane that destroy membrane integrity, promoting lysis of the targeted microbes (Figure 1). Like defensins, most cathelicidins also kill bacteria by membrane disruption. The human cathelicidin peptide LL37 is cationic, α-helical and binds to membranes through electrostatic interactions, followed by peptide insertion and membrane disruption. Some AMPs also disrupt bacterial membranes through enzymatic digestion. For example, lysozyme hydrolyses the beta-glycosidic linkage between N-acetylmuramic acid and N-acetyl glucosamine in the peptidoglycan of bacterial cell walls, and phospholipase A2 (PLA2) secreted from human platelets kills bacteria by hydrolyzing bacterial membrane phospholipids. Nonetheless, some peptides have been described as being able to cross the lipid bilayer without causing any damage, but kill bacteria by inhibiting intracellular functions, such as blocking enzyme activity or inhibiting protein and nucleic acid synthesis. In addition to direct antimicrobial activities, some AMPs are also able to inhibit biofilm formation and disrupt existing biofilms.

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Figure 1. Biological function of antimicrobial peptides.

AMPs bind to bacterial membranes through electrostatic interactions either to disrupt the membrane or to enter the bacterium to inhibit intracellular function. Some AMPs also modulate host immunity by recruiting/activating immunocytes or by influencing Toll-like receptor (TLR) recognition of microbial products and nucleic acids released upon tissue damage. DC, dendritic cell; LPS, lipopolysaccharide; LTA, lipoteichoic acid; MAVS, mitochondrial antiviral signaling protein.

Epithelial surfaces are colonized by many commensal, often beneficial, microorganisms that are critical for maintaining homeostasis. It has been a puzzle that adaptive immune responses appear to target all bacteria, yet healthy individuals maintain and permit survival of bacteria, even when exposed to a host of environmental disturbances. It has been suggested that bacteria reduce their susceptibility to AMPs by hindering peptide attachment to the outer membrane. A recent report by Cullen et al. in Science has reported that microbiome stability may be maintained by a single gene that mediates dephosphorylation of LPS, thereby reducing net negative surface charge. This allows bacteria to resist AMPs by decreasing their interactions with the bacterial cell surface. Although probably not the whole story for all commensal microbes at all epithelia, this illustrates one potential system to explain the survival of microbes without virulence.

Expression, regulation and processing of AMPs

AMPs have been identified at most sites of the human body that are normally exposed to microbes, such as the skin, intestinal mucosa, oral mucosa, lung, eye and reproductive tract. It became clear that, while some AMPs are constitutively expressed, the majority of these peptides are induced during infection or by inflammation or injury. The specific sites of expression and the strict regulation of AMP expression are key to understanding how they work. This may explain why AMPs, as evolutionarily ancient gene products, remain as effective antibiotics, while pharmaceutically derived antibiotics can rapidly become useless due to the development of bacterial resistance.

Keratinocytes, the predominant cell type in the epidermis, are continuously exposed to microorganisms from the external environment, therefore operating as a front line of defense against the invasion of pathogenic microbes. A variety of AMPs are secreted from keratinocytes upon stimulation. One family of AMPs produced by these cells are β-defensins. While human β-defensin 1 (hBD1) expression is primarily constitutive, hBD2 and hBD3 are inducible during wounding or infection. Another potent keratinocyte-derived AMP family is the cathelicidins. This family was named based on the conserved amino-terminal protein domain which was called 'cathelin' because it was first purified for its capacity to inhibit cathepsin L. Cathelicidins correspond to the first AMP observed in mammalian skin, but it is now clear that several cell types produce cathelicidin, including resident skin cells such as keratinocytes, eccrine sweat glands and sebocytes, and bone-marrow-derived cells found within the skin, including neutrophils, mast cells and dendritic cells. We have also recently found that differentiating adipocytes are an abundant source of cathelicidin during S. aureus infections, suggesting that fat cells play a critical role in host defense and are the deepest layer within the skin that has barrier function. An overview of AMPs from the skin is shown in Figure 2.

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Figure 2. The strategy of 'layered' AMPs used by human skin.

A representative but incomplete list of AMPs expressed by human skin. Epidermal keratinocytes constitutively express human β-defensin 1 (hBD1) and RNase7, whereas other AMPs are either downregulated in atopic dermatitis (AD) or upregulated during infection and inflammation or in psoriasis. Commensal bacteria, such as S. epidermidis, produce a number of AMPs, such as phenol-soluble modulins (PSMs). In the dermis, sebaceous glands and eccrine sweat glands also secrete AMPs. Immunocytes, including neutrophils, dendritic cells (DC), T cells, mast cells and monocytes, are recruited to skin upon infection or inflammation, and AMPs produced by those cells are listed. In the dermal adipose layer, cathelicidin is produced during the differentiation of adipocytes (Ad) from preadipocytes (pAd).

The human intestine also interacts with a large burden of commensal as well as potentially pathogenic microorganisms. Intestinal AMPs can fend off ingested pathogens, and they also play a crucial role in maintaining a healthy balanced relationship between the host and the extreme number of commensal inhabitants. Paneth cells are the major source of AMPs in the small intestine, producing α-defensins HD-5 and HD-6, lysozyme C, secretory phospholipase 2 (sPLA2), angiogenin 4 (ANG4) and lectins. HD-5 and HD-6 are the most abundant AMPs in Paneth cells and are constitutively expressed at high levels in these cells. In contrast to α-defensins, the expression of β-defensins is more variable throughout the gastrointestinal tract. Six human β-defensins and five mouse β-defensins (mBDs) have been identified and characterized. While hBD1 and its ortholog mBD1 are constitutively expressed in enterocytes, hBD2, hBD3 and hBD4 are induced during infection or inflammation. Cathelicidins constitute another major class of AMPs expressed by the intestinal epithelium at the upper crypts of the colon. Antimicrobial activity has also been observed in a number of epithelium-derived chemokines, including CCL20/MIP3α and an additional 17 chemokines. Other peptide molecules, including elafin (a peptidase inhibitor), secretory leukocyte protease inhibitor (SLPI), bactericidal/permeability-increasing protein (BPI) and REG3a are all anti-infective molecules present in the intestinal mucosa.

AMPs produced by the commensal microorganisms resident on epithelial surfaces of vertebrates also contribute to the antimicrobial shield of these structures. Staphylococcus epidermidis, the most abundant skin commensal bacterium, produces several AMPs, including epidermin (Staphylococcin 1580), Pep5 and epilancin K7, all of which belong to a unique group of AMPs known as lantibiotics because these peptides contain the thioether amino acids lanthionine and/or methyllanthionine. Our group has shown that S. epidermidis also produces the phenol-soluble modulins PSMγ and PSMδ, which are AMPs that selectively exhibit bactericidal activity against skin pathogens, such as S. aureus, group A Streptococcus and Escherichia coli, but are not active against S. epidermidis, their host organism. Emerging data have also shown that many other potent and selective AMPs are secreted on the epidermal surface by the commensal bacteria on the skin and may be both diagnostically and therapeutically useful.

In contrast to vertebrates, insects lack an adaptive immune system, but have a robust innate immune system that effectively kills invading microbes, mainly by humoral responses. This involves the rapid synthesis and release of different types of AMPs. Secretion of AMPs from the fat body (the insect equivalent of the vertebrate liver) into hemolymph (the fluid portion of insect blood) is the dominant mechanism for host defense in infected insects. Furthermore, as in vertebrates, insect gut epithelia also secretes tissue-specific AMPs, a response that is important in insect resistance to intestinal parasites (Figure 3). Insect defensins are structurally characterized by two or three β-sheets stabilized by disulfide bonds formed between cysteine residues. Three-dimensional structural analyses have revealed that there is a closer relationship between invertebrate defensins and vertebrate β-defensins than between vertebrate α- and β-defensins, suggesting that defensins are ancient molecules that are conserved across the eukaryotic kingdom. Cecropins are cysteine-free cationic peptides that adopt an α-helical structure in a hydrophobic environment. Cecropin was originally found in the pupae of the Hyalophora cecropia moth and the first identified genes encoding Drosophila AMPs were the cecropin genes. To date, eight distinct classes of AMPs have been identified in Drosophila melanogaster, and these can be further classified into three groups based on their main biological targets: Drosophila defensins are active against Gram-positive bacteria; cecropins, attacins, diptericins, and drosocin are active against Gram-negative bacteria; and drosomycin and metchnikowin have activity against filamentous fungi. Andropin, the only Drosophila AMP that is not induced during infection, is expressed specifically in male flies during mating to protect the reproductive tract. Extensive genetic analysis in Drosophila has shown that production of AMPs is controlled by the Toll and the Imd signaling pathways. The Toll pathway is activated primarily by Gram-positive bacteria or fungi, whereas the Imd pathway responds to Gram-negative bacterial infection. The common downstream feature of both pathways is activation of the fly homologues of the NF-κB signaling pathway, involving degradation of the IκB homolog Cactus followed by nuclear translocation of the NF-κB homologs Dorsal/Dif, leading to induction of the effector AMP genes.

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Figure 3. Local and systemic AMP response in Drosophila.

Drosophila produces AMPs in response to microbial infection, either locally by epithelial cells or systemically by the fat body which secretes AMPs into hemolymph. Activation of the Toll pathway by Gram-positive bacteria or fungi, or the Imd pathway by Gram-negative bacteria, triggers NFκB activation followed by induction of AMPs, the effector molecules that mediate microbial lysis. The table on the right lists the main AMPs and their antimicrobial targets in Drosophila.

Many AMPs are derived from larger precursor proteins with a signal sequence at the amino terminus and the antimicrobial peptide domain at the carboxyl terminus. The active peptide is generated by post-translational modification through proteolytic cleavage. The precursor protein holds the carboxy-terminal peptide in an inactive form before it is further processed and prepared for release when needed. Because of the potential detrimental effect of mature AMPs on mammalian cell membranes, the processing of many AMPs is also regulated by storage in secretory granules. α-defensins are stored in the secretory granule of the intestinal Paneth cell as inactive pro-peptides (∼10 kDa), which are cleaved by trypsin in humans or by matrix metalloproteinase 7 (MMP7) in mouse to generate the mature α-defensin peptides (∼4.5 kDa) with antimicrobial activity. The human cathelicidin hCAP18 precursor protein is cleaved by serine proteinases to its active 37 amino acid peptide (LL37). These proteases appear to be specific to the cell type expressing the AMP because proteinase 3 and elastase are active in the granules of neutrophils whereas kallikrein-related peptidase is active in the lamellar bodies of epidermal keratinocytes.

Immunoregulatory and non-antimicrobial functions of AMPs

One of the key advantages of AMPs over pharmaceutical antibiotics is the ability of some of these peptides to also modulate immune responses. For example, in addition to their direct antimicrobial activity, AMPs can protect the host by a range of mechanisms: chemotactic activity, attracting leukocytes; modulation of host-cell responsiveness to TLR ligands; stimulation of angiogenesis; enhancement of leukocyte/monocyte activation and differentiation; and modulation of the expression of proinflammatory cytokines/chemokines (Figure 1). For example, LL37 has been shown to attract neutrophils, monocytes, mast cells and T cells. Different groups of AMPs appear to have distinct chemotactic activities from each other. Human α-defensins selectively chemoattract CD4+ CD45RA+ naïve T cells but not CD4+ CD45RO+ memory T cells, whereas β-defensins are chemotactic for both CD4+ CD45RO+ resting memory T cells and immature dendritic cells. LL37 can suppress TLR signaling and bacterial-induced inflammation by binding to bacterial factors including lipoteichoic acid or lipopolysaccharide. On the other hand, LL37 can also promote inflammation by binding to nucleic acids to facilitate recognition of self-DNA by TLR9, self-RNA by TLR7 and TLR8, or double-stranded RNA by TLR3. A recent study from our lab has also found that LL37 enables dsRNA recognition by the MAVS/RIG-I pattern recognition signaling pathways in keratinocytes, thus further amplifiying the response to self-RNA, a known 'danger' signal. These interactions indicate that, during infection, AMPs can kill pathogens, initiate immunocyte recruitment/activation, and at the same time neutralize an excessive inflammatory response at the site of infection. However, sustained expression of AMPs could increase the host's responsiveness to self-nucleic acids, leading to the development of autoimmune disorders, such as psoriasis and rosacea.

AMPs and human disease

AMPs are pivotal for host defense and have several relevant associations with human disease. One indirect effect of the AMPs is their role in shaping the composition of the commensal microbiota and thereby contributing to the maintenance of skin and intestinal homeostasis. Disruption of the stability of the microbiome can lead to severe health consequences, including obesity, recurrent infections, and diseases such as inflammatory bowel syndrome and atopic dermatitis.

Numerous clinical studies have linked chronic intestinal inflammation to defective antibacterial mechanisms, possibly due to altered expression and secretion of AMPs. For example, Crohn's disease of the ileum has been associated with a deficiency in Paneth cell α-defensins. Crohn's disease of the colon is associated with a low-gene copy number polymorphism of the human β-defensin locus, resulting in a reduced epithelial induction of β-defensin AMPs in the colonic mucosa. Defensin deficiency weakens the antimicrobial defense properties of the intestinal mucosa, leading to alterations in the composition of intestinal flora that ultimately may promote bacterial invasion of the mucosa and predispose to the chronic inflammation of Crohn's disease. Similarly, abnormal expression of AMPs has also been linked to the development of atopic dermatitis, a common and chronic relapsing inflammatory skin disease. Atopic dermatitis patients usually bear abnormally high burdens of S. aureus on their skin, and this correlates with reduced expression of AMPs, such as LL37 and β-defensins (hBD2 and hBD3).

In contrast, overproduction of AMPs can directly initiate inflammatory diseases. For example, patients with psoriasis, a chronic auto-inflammatory skin disease characterized by high levels of cytokines produced by Th1 and Th17 T helper cells, express excessive amounts of AMPs, such as cathelicidin, hBD2, hBD3 and S100A8/9 in their skin. In rosacea, another auto-inflammatory skin disease mainly affecting the central portions of the face, proteolytic activation of cathelicidin protein is enhanced due to elevated expression of cutaneous proteinases such as KLK5 and KLK7. These observations implicating AMPs in skin disease pathogenesis have provided new opportunities for disease therapy.

Conclusions

All living organisms are constantly threatened by large numbers of microorganisms seeking to exploit the same environmental space. To cope with this substantial microbial threat, most cells produce natural antibiotic-like molecules that directly kill or inhibit the growth of foreign microorganisms. Since the discovery of lysozyme more than 90 years ago, more than 2,500 AMPs have been identified in single-celled organisms, plants, insects and animals. These molecules have selective antimicrobial activity against a wide range of organisms, largely due to their relatively strong electronic interaction with negatively charged membranes. In vertebrate species, some AMPs also promote host defense by modulating host cellular immunity. Furthermore, in mammals it has recently become clear that AMPs not only fend off pathogens but also shape the composition of the microbiome, and this is important for the maintenance of many aspects of health. The growing problem of resistance to and overuse of conventional antibiotics has stimulated interest in the development of AMPs as the next generation anti-infectives and as methods to more selectively combat pathogens. Although several limitations remain, control of endogenous AMP production may represent the next major paradigm for treatment of a broad range of human and animal diseases.

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