Mammalian epithelial surfaces are remarkable for their ability to provide critical physiologic functions in the face of frequent microbial challenges. The fact that these mucosal surfaces remain infection-free in the normal host suggests that highly effective mechanisms of host defense have evolved to protect these environmentally exposed tissues. Throughout the animal and plant kingdoms, endogenous genetically encoded antimicrobial peptides have been shown to be key elements in the response to epithelial compromise and microbial invasion. In mammals, a variety of such peptides have been identified, including the well-characterized defensins and cathelicidins. A major source of these host defense molecules is circulating phagocytic leukocytes. However, more recently, it has been shown that resident epithelial cells of the skin and respiratory, alimentary, and genitourinary tracts also synthesize and release antimicrobial peptides. Both in vitro and in vivo data support the hypothesis that these molecules are important contributors to intrinsic mucosal immunity. Alterations in their level of expression or biologic activity can predispose the organism to microbial infection. The regulatory and developmental aspects of antimicrobial peptide synthesis are discussed from a perspective that emphasizes the possible relevance to pediatric medicine.
A striking feature of evolution in the animal kingdom is the development of highly specialized epithelial surfaces. These sites of host-environment interaction provide vital physiologic functions including gas exchange, nutrient absorption, water conservation, and reproduction. The low incidence of infectious and inflammatory complications at epithelial surfaces suggests that local host immunity includes highly effective, broad-spectrum, noninflammatory antimicrobial defenses.
As a general paradigm, antimicrobial defenses can be divided into two systems: clonal (acquired) immunity and innate (nonacquired) immunity(1). The clonal immune system uses B and T lymphocytes to mediate and amplify antigen-specific humoral and cellular responses. These responses require days to weeks for maximal activity, involve somatic gene rearrangement, and lead to immunologic memory. Although the acquired immune system represents the "crowning accomplishment of vertebrate immunity"(2) and has been the focus of significant clinically related research (10 Nobel Prizes awarded), evidence now suggests that the function of the acquired immune system is intimately tied to and complemented by the evolutionarily ancient but less highly glorified innate immune system(3,4).
Innate immunity encompasses a complex of first-line host-defense elements. This host-defense system can provide for (i) recognition of microbial organisms as foreign(5–10); (ii) incapacitation and elimination of pathogens(11); and (iii) adjuvant magnification of the acquired immune response when such a response is warranted(3,4,12–14). The elements of innate immunity do not function in isolation, but interact to ensure that the magnitude of the host response reflects the severity of the microbial threat. Components of innate immunity range in complexity from simple inorganic molecules such as nitric oxide to phagocytic and natural killer cells.
In contrast to the acquired immune system in which an effective response involves gene rearrangements and is developed over a period of days, the innate system remains ever-ready or immediately inducible. In the innate immune system, the ability to discriminate self from nonself is "hard-wired" in the genome, a result of selective pressures of evolution to optimize both recognition and effector molecules. The working hypotheses for much current immunologic research highlight the role of the innate immune system in host defense of epithelial surfaces in humans and other animals (Fig. 1).
Recent investigations have uncovered a large and remarkably varied collection of antimicrobial peptides (arbitrarily defined as <100 amino acids in size) that comprise a widespread effector arm of the innate immune system. Antimicrobial peptides have been identified in organisms as diverse as humans, frogs, insects, plants, and protozoa. Their cellular origin includes granulocytes, platelets, specialized epithelial glands, wet mucosal epithelia including intestinal Paneth cells, fetal membranes, leaves, flowers, and seeds. Chemically divergent in structure (Fig. 2), they are microbicidal at micromolar concentrations against a wide range of target organisms. In this review, we will focus on the properties of those antimicrobial peptides known to be present in tissues of mammalian species (Fig. 3), especially as they may relate to human innate immunity at wet mucosal surfaces. The interested reader is directed to recent reviews that emphasize other aspects of antimicrobial peptide structure and biology(15–21).
Mechanism of activity. Antimicrobial peptides generally are microbicidal rather than static agents, with the most detailed information on their mechanism of action derived from anti-bacterial studies [for reviews, see(15,16,22)]. Bacterial killing occurs in minutes and in most cases requires bacterial cell growth. Addition of purified cecropin, magainin, or defensin peptides to artificial membrane systems leads to pore formation and membrane depolarization, suggesting a possible mechanism of their microbicidal activity(23–30). Other peptides, e.g. some members of the cathelicidin family, seem to act via disruption of bacterial energy metabolism or biosynthetic pathways(31,32).
Gene encoding. Unlike most other antibiotics in nature that are products of multienzyme cascades, these molecules are products of prototypical genes(18) (Fig. 4). Families of anti-microbial peptide genes are generally tightly clustered and typically have two or more exons. Families of structurally similar antimicrobial peptides map to syntenic chromosomal segments in different mammalian species, consistent with their distant evolution from an ancestral gene(33–41). In some cases, the genes are adjacent to repetitive sequence elements that may provide a mechanism for gene family expansion and diversification through homologous but unequal cross-over during meiosis(42). There has been only an isolated report of alternative splicing as a mechanism of gene diversification(43).
The primary translational product is a prepropeptide with a putative N-terminal endoplasmic reticulum targeting (signal) sequence that has been conserved within specific gene families. Adjacent to this signal peptide is a pro segment, which is often anionic in charge and may be important for neutralization/processing/folding of the cationic C-terminal peptide(44–47). A proposed function for the propeptide segment is that of rendering the cationic peptide inactive through charge interaction as a means of protecting the host cell. Some propeptides (e.g. neutrophil defensins; see Major Classes of Mammalian Antimicrobial Peptides, β-Defensins) are processed by post-translational cleavage steps leading to intracellular peptide storage in lysosomal-type granules, whereas others (e.g. cathelicidins; see Major Classes of Mammalian Antimicrobial Peptides, Cathelicidins) are stored as propeptides with posttranslational cleavage occurring extracellularly. The posttranslational processing steps include proteolytic cleavage that liberates the mature peptide and, in some cases, formation of intramolecular disulfide bonding, C-terminal amidation, and/or N-terminal formation of pyroglutamate.
Sites of expression. (i) Granules of mammalian neutrophils contain high levels of cysteine-rich defensins (humans, rabbits, rats, guinea pigs, and cattle; see Major Classes of Mammalian Antimicrobial Peptides, Defensins) and/or cathelicidins (cattle, sheep, rabbits, and pigs; see Major Classes of Mammalian Antimicrobial Peptides, Cathelicidins). The mouse is a notable exception in which neutrophil granules contain no defensins and only low levels of cathelicidins(48–50). (ii) Antimicrobial peptides present at epithelial surfaces are derived either from in situ synthesis by resident cells or from mobilized storage depots present in circulating cells (granulocytes, monocytes, platelets)(50,51). Examples of in situ synthesis include constitutive expression of α-defensins (in Paneth cell granules of mouse, rat, and human small intestine)(52) and inducible expression of β-defensins (in bovine airway and human skin)(53,54). Examples of systemic delivery include neutrophil localization and release of intragranular peptides in pig skin (PR-39)(55,56) and human lung (α-defensins)(57).
MAJOR CLASSES OF MAMMALIAN ANTIMICROBIAL PEPTIDES
Defensins. Mammalian defensins are cationic antimicrobial peptides characterized by the presence of three intramolecular disulfide bonds [for more detailed reviews, see(17,19,22)]. Defensins interact with and disrupt microbial membranes, resulting in cell death(23). In standardized assays conducted in the presence of low salt concentrations, defensins are microbicidal at 10-100 µg/mL (3-30 µM)(22). However, their activity is inhibited at higher salt concentrations (≥ ∼ 75 mM NaCl).
Mammalian defensins can be subdivided into two general classes, the α-defensins(17,22) and the β-defensins(21), based on (i) alternative spacing of their six cysteine residues; (ii) differences in the alignment of the disulfide bridges(58,59); and (iii) variation in the length of the pro segment. Both classes have been described in humans and rodents, whereas only α-defensins have been reported in rabbits and guinea pigs and only β-defensins have been described in cattle, sheep, and pigs. In humans and mice, genes encoding α- and β-defensin peptides map to the same chromosomal segment(38,41,60), consistent with their derivation from a common ancestral sequence. Despite their differences in sequence and disulfide bond pattern, the α- and β-defensins share a similar three-dimensional structure in solution(61).
α-Defensins. The α-defensins are 29-35 amino acids in length containing three disulfide bridges in a 1-6, 2-4, 3-5 alignment (Fig. 5)(58). Structural studies of α-defensins show a triple-stranded β-sheet and a β-hairpin loop containing cationic charged residues(62,63). α-Defensins are found in great abundance in intracellular granules of circulating neutrophils (5-18% of total protein)(64–66) and in granule-containing Paneth cells of the small intestine(67,68). The human α-defensin family consists of four defensins isolated from neutrophils, HD-1-HD-4 [often referred to as human neutrophil peptide (HNP)-1-HNP-4](65,69,70), and two defensins expressed in Paneth cells of the small intestine, HD-5(68,71,72) and HD-6(73,74). Recently, it has been shown that HD-5 is expressed also at multiple sites within the female reproductive tract, placenta, and fetal membranes(75–77).
The human α-defensins are synthesized as 93-100 amino acid prepropeptides with a predicted 19-amino acid signal peptide and a 41-51-amino acid anionic pro segment(66,78). The pro segment has been shown to be necessary for accurate processing and transport of the defensin peptide(44–46), but its removal is thought to be necessary for expression of antimicrobial activity(44,47). Human neutrophil defensins associate as amphiphilic dimers(28,63,79) and, when incubated with model membranes, form voltage-dependent channels that are weakly anion-selective(24). The induction of Cl--specific channels has been shown also upon incubation of murine α-defensins with cultured T84 gastrointestinal epithelial cells(80).
Individual α-defensins have unique spectra of antibacterial activity against both Gram-positive and Gram-negative species including intracellular and extracellular organisms [for review, see(22)], antiviral activity with effects on propagation of enveloped virus including members of the Herpes family(81), antifungal activity targeting Candida albicans(82–84), and antiparasitic activity including anti-Giardia(85) and antitreponemal effects(86,87).
β-Defensins. The β-defensins are 36-42 amino acids in length with six cysteines in a spacing pattern and a disulfide alignment (1-5, 2-4, 3-6)(59), differing from that of the α-defensins (Fig. 5). A subset of ruminant β-defensins has been shown to contain an N-terminal pyroglutamate residue(88). In cattle neutrophils, there are at least 13 different β-defensin peptides(88), although none have been reported in sheep and human neutrophils. β-defensins are synthesized at epithelial surfaces including the upper respiratory tract(42,89–92), nasal mucosa(42), tongue(93,94), kidney(90,95,96), pancreas(90), colon(97,98), female reproductive tract(96), and conjunctiva(92).
The β-defensins are synthesized as 64-68 amino acid pre-propeptides with the 26-32 amino acids at the N-terminus comprising putative signal and propeptide segments. The intracellular processing, storage, and release pathways remain to be defined. Limited data are available on the spectrum of β-defensin antimicrobial activity, although several peptides of the family have been shown to be active against Gram-positive and Gram-negative bacteria as well as C. albicans and Aspergillus fumigatus(41,54,88,89,93,96,99–101). In most tissues, the constitutive level of β-defensin expression seems to be low. However, expression of at least four different β-defensins has been shown to be inducible: β-defensins TAP(53), enteric β-defensin(97), lingual antimicrobial peptide (LAP)(92,93,102), and HBD-2(54). Bovine tracheal epithelial cells exposed to lipopolysaccharide up-regulate transcription of both the TAP and lingual antimicrobial peptide genes in a dose- and time-dependent fashion(102). NF-κB recognition elements are present upstream of the TAP coding sequences that may mediate the induction(42,53).
Two HBD peptides have been identified to date, HBD-1(95,96) and HBD-2(54). The former was identified initially as a 36-amino acid peptide purified from blood filtrate(95). The gene encoding HBD-1 has been shown to be expressed in epithelial tissues including kidney, lung (both upper respiratory tract and parenchyma), pancreas, testis, gingival tissue, and vagina(90,91,95,96,103,104). Several isoforms of HBD-1 that represent N-terminal extensions of the 36-amino acid peptide have been identified in urine and vaginal mucosal secretions(96). Urine concentrations are increased in pregnancy and isoform differences exist between males and females. The various isoforms may represent alternative processing of the prepropeptide and suggest that proteolytic cleavage may be an important site of biologic regulation. In contrast with the bovine airway β-defensins, HBD-1 expression does not seem to be inducible(90,96).
HBD-2 is a 41-amino acid peptide purified from lesional psoriatic skin based on its binding to a whole Escherichia coli affinity column. In addition to skin, expression of HBD-2 was detected also in the tracheal mucosa(54,105–107). HBD-2 is bactericidal in vitro against both Pseudomonas aeruginosa and C. albicans but relatively ineffective against Gram-positive Staphylococcus aureus. All three organisms induce HBD-2 transcription in foreskin-derived keratinocytes, as does the cytokine TNF-α.
Cathelicidins. The cathelicidins are a remarkably diverse collection of molecules that derive from prepropeptides sharing a highly conserved N-terminal propeptide segment (Fig. 6)(108). The conserved propeptide segment of approximately 100 amino acids shares sequence similarity with the porcine protein cathelin, a putative cysteine protease inhibitor, and hence the family name(109–111). Cathelicidins are stored in neutrophil granules as propeptides (nonantimicrobial)(112,113), with neutrophil activation leading to elastase-mediated endoproteolytic cleavage and generation of the C-terminal antimicrobial peptide(114,115).
The cathelicidin gene families of pigs, cattle, and sheep are large and diverse(109,111,116–129), whereas those of humans and mice are limited to one or two genes(130,131). The human cathelicidin, referred to alternatively as FALL-39/hCAP18/LL-37/CAMP, in its mature form is a 37-amino acid amphiphilic α-helical peptide(132). Expression of LL-37 has been detected not only in neutrophils(132) but also in testes(130) and respiratory epithelia(133) and in keratinocytes at sites of inflammation(134).
Others. Antimicrobial peptides have been shown in platelets(135), salivary secretions(136), amniotic fluid(137), and lung fluid(138,139). As circulating blood elements, platelets would be an ideal cell type for the delivery of antimicrobial agents to sites of vascular compromise accompanying epithelial injury. Preliminary characterization of the amniotic-fluid and lung-fluid activity showed the presence of a unique, Zn-dependent antimicrobial anionic peptide(138,139).
ONTOGENY OF ANTIMICROBIAL PEPTIDE EXPRESSION
Antimicrobial peptide gene expression is developmentally regulated and occurs in tissue patterns that are species-specific. As examples, human α-defensins are expressed in neutrophils and Paneth cells but not in macrophages, whereas rabbit α-defensins are found in both neutrophils and macrophages and mouse α-defensins are present in Paneth cells and testes. Further, macrophage expression of rabbit α-defensins is absent in neonates but present in adults and is detectable in lung-derived but not in peritoneal-derived cells(140).
Paneth cell α-defensins. Human α-defensins HD-5 and HD-6 are expressed in the developing fetus as early as 13.5-wk gestation (detected by RT-PCR), with HD-5 present in both small intestine and colon and HD-6 present only in small intestine(74). By 17-wk gestation, PCR-based detection of both isoforms is limited to the small intestine, and at 24 wk, enteric defensin expression has reached levels detectable by Northern blot hybridization. At this point in gestation (late second trimester), both the number of Paneth cells and the level of enteric α-defensin transcription are significantly lower than in the adult. In contrast, mouse α-defensins of the small intestine (cryptdins) are expressed at low levels before birth, with a rapid increase during the weaning period(141–143). Interestingly, transgenic mice carrying the HD-5 gene express the peptide in Paneth cells in the same developmental sequence as the endogenous mouse genes (Salzman N, Bevins CL, Huttner KM 1996 Abstract 132/E-53. American Society of Microbiology, New Orleans, LA).
β-Defensins. HBD-1 is developmentally regulated in lung parenchyma, with detection by RNase-protection assay in postnatal and adult samples but not in 15- and 22-wk gestation fetal tissue(91). The cattle β-defensin TAP is developmentally regulated antenatally as well, as assayed by Northern blot hybridization on airway RNA samples from 4-mo gestation, 6-mo gestation, and adult animals(42). In sheep, β-defensin expression was detected by nested RT-PCR at d 115 of gestation (145-150 d term) in trachea, tongue, stomach, and uterus, and by Northern analysis at d 127 in ileum and colon(98). Using primer pairs specific for the two sheep β-defensin isoforms, we found that tissues express both SBD-1 and SBD-2 at 115 and 127 d of gestation (Fig. 7). In contrast, SBD-1 is the predominant or exclusive β-defensin in all adult tissues assayed with the exception of SBD-2 in the ileum (based on sequencing of multiple cDNA species).
Cathelicidins. Developmental regulation of the human cathelicidin LL-37 remains to be investigated. However, expression of the mouse cathelicidin CRAMP was detected by Northern blot hybridization both in adult bone marrow and in whole embryo extracts as early as embryonic d 12(131).
STRATEGY OF ANTIMICROBIAL PEPTIDES AS AGENTS OF HOST DEFENSE
The ubiquity of antimicrobial peptide expression in every plant and animal species examined suggests that these molecules function as highly effective host-defense elements(15). Facing a resurgence of antibiotic-resistant organisms in the clinical arena, what might we learn from the strategies used by these peptides that are conserved through evolution?
1. Target microbial structures essential for survival and not readily altered by simple genetic changes. The majority of antimicrobial peptides work by selectively disrupting the membrane of target microorganisms through channel or pore formation(23–30). Attempts to select out resistant bacterial strains from sensitive ones have been unsuccessful, suggesting that the bacterial target represents a structure fundamental for survival.
2. Use a combination of agents to increase effectiveness. Within most epithelial tissues studied to date, one can show the presence of multiple peptides from a single family or peptides and proteins from distinct families with unrelated structures and overlapping spectra of antimicrobial activity. Specific examples of antibacterial synergy between differing antimicrobial peptides or between antimicrobial peptides and other host-defense proteins have been reported(83,144,145).
3. Develop multifunctional agents. In addition to their direct microbicidal activity, antimicrobial peptides are agents that can modulate inflammatory response, wound repair, cell division, and adaptive immune response. One intriguing example is the porcine cathelicidin PR-39 that can localize to the site of skin wounds, sterilize the break in epithelial integrity, and additionally stimulate the wound-repair process(55). Other examples include defensin-mediated inactivation of serine proteinase inhibitors(146), defensin and cathelicidin chemotactic activity(147–149), defensin stimulation of epithelial cell proliferation(150), defensin antagonism of ACTH-stimulated glucocorticoid release(151,152), defensin inhibition of fibrinolysis(153), and enhancement of binding plasminogen and lipoprotein (a) to endothelial cells(153,154).
A LOOK TO THE FUTURE
As pediatricians, what role will antimicrobial peptides play in the well-being of our patients? Considering the rapidity with which new antimicrobial peptides are being discovered and how little we yet know of their in vivo properties, it may be premature to overly speculate on details. However, with the current information, we predict the following:
Antimicrobial peptides will be found in nearly all human mucosal secretions, contributing to host defense in the respiratory, gastrointestinal, and genitourinary tracts as well as throughout the oropharynx and ocular surfaces.
Insufficient expression of antimicrobial peptides will predispose to disease. A deficiency in antimicrobial peptide levels (or activity) may contribute to patient subpopulations being at higher risk for neonatal sepsis, otitis media, conjunctivitis, dental caries, nasopharynx carriage of potential pathogens, urinary tract infections, etc. Antimicrobial peptide levels may be reduced in our youngest and least mature patients, the preterm infants, secondary to their developmental state. Alternatively, changes in the local environment in which these peptides should function may impair antimicrobial activity and compromise host defense, as proposed in CF airway surface fluid(166,167).
Common therapeutic interventions and pharmacologic agents may alter natural defenses involving antimicrobial peptides. As examples, in the intubated patient, air-borne pathogens can bypass the antimicrobial peptide-rich upper airway epithelium. In the gastrointestinal tract, surgical resection or bypass of the distal ileum may lead to altered lumenal bacteria from omission of Paneth cell α-defensins. Further, glucocorticoids, used frequently in clinical practice, block inducible expression of immune and inflammation-associated genes (via inhibition of NF-κB signaling(171)) and may result in reduced host immunity. In one model system, frogs exposed to systemic or topical glucocorticoids suffered a 93% reduction in antimicrobial peptide expression, resulting in at least a 10-fold increase in oral bacterial counts(172).
Antimicrobial peptides may earn a place in our armamentarium of topical or systemic agents used in treating infections. Clinical trials of a magainin derivative (Xenopus laevis antimicrobial peptide), a recombinant bactericidal permeability-increasing protein fragment (rabbit), and a protegrin (pig cathelicidin) are underway as novel therapeutic agents(173).
airway epithelial cells
- CAMP and CRAMP
cathelin-related antimicrobial peptide
cystic fibrosis transmembrane regulator
39-amino acid cathelicidin-associated peptide with amino terminal sequence: Phe-Ala-Leu-Leu
37-amino acid cathelin-associated peptide with amino terminal sequence: Leu-Leu
nuclear factor kappa B
39-amino acid cathelin-associated peptide with amino terminal sequence: pro-arg
reverse transcriptase polymerase chain reaction
tracheal antimicrobial peptide
About this article
Molecular Biology Reports (2013)