What Are Three Antimicrobial Proteins

Production of antimicrobial peptides and proteins is an important means of host defense in eukaryotes. The larger antimicrobial proteins, containing more than 100 amino acids, are often lytic enzymes, nutrient-binding proteins or contain sites that target specific microbial macromolecules. The smaller antimicrobial peptides act largely by disrupting the structure or function of microbial cell membranes. Hundreds of antimicrobial peptides have been found in the epithelial layers, phagocytic cells and body fluids of multicellular animals, from mollusks to humans. Some antimicrobial peptides are produced constitutively, others are induced in response to infection or inflammation. Studies of the regulation of antimicrobial peptide synthesis in Drosophila have been particularly fruitful, and have provided a new paradigm for the analysis of mammalian host defense responses. It now appears that the general patterns of antimicrobial responses of invertebrates have been preserved in vertebrates (“innate immunity”) where they contribute to host defense both independently and in complex interplay with adaptive immunity.

Multicellular organisms continually defend themselves against parasitization by potentially harmful microbes. In the absence of penetrating injury, the most common sites of initial encounter with microbes are the epithelial surfaces (skin, the moist surfaces of the eyes, nose, airways and the lungs, mouth and the digestive tract, and the urinary and reproductive systems). Because mechanisms requiring specific antigen recognition depend on clonal proliferation of immunocytes, and therefore take days to weeks to develop fully, the initial host resistance mechanisms must recognize or target microbe-specific class characteristics and employ mechanisms that are either constitutive or rapidly inducible. Some unique microbial molecular features are recognized by complementary receptors that trigger localized effector mechanisms (“pattern recognition”) while other structural or metabolic characteristics make the microbes selectively susceptible to the action of injurious antimicrobial substances including chemically highly reactive molecules, lytic enzymes, pore-forming molecules, or substances that sequester essential nutrients. Certain antimicrobial substances may be present constitutively; the local synthesis or release of others is provoked by invading microbes; and yet other antimicrobial substances can be brought into the area of invasion by mobile cells. Unlike innate immunity, adaptive immunity (antibodies and antigen-recognizing cytotoxic lymphocytes) is a late evolutionary development developed fully only in higher vertebrates. Specific antigen recognition by lymphocytes probably plays a limited role during the initial encounter but it is especially effective against persistent microbes or against microbes previously encountered by the host.

The innate antimicrobial properties of epithelial surfaces were noted a century ago by Metchnikoff, who emphasized the cleansing role of mechanical factors such as the continuous movement of the tear film across the frontal surface of the eye. Metchnikoff also observed that microbes that breached the epithelial surfaces were met by mobile cells (phagocytes) that ingested and killed the invaders. Having described the phagocytic killing of microbes, Metchnikoff surmised that microbicidal substances must be present in phagocytes and thought that these were “ferments” (enzymes). In the 1920s, Fleming discovered that the fluid coating the epithelia contained an antimicrobial enzyme which he named lysozyme, and showed that the same substance was also found in abundance in phagocytes. Later studies identified the main target of lysozyme as a sugar linkage in the peptidoglycan cell wall of bacteria.

Over the past 40 years, a number of additional antimicrobial substances produced by epithelia and phagocytes have been characterized, ranging in size from small inorganic molecules such as hydrogen peroxide to large protein complexes such as those generated by the activation of the complement cascade. Antimicrobial peptides are conventionally defined as polypeptide antimicrobial substances, encoded by genes and synthesized by ribosomes, with fewer than 100 amino acid residues. This definition distinguishes them from most (but not all) peptide antibiotics of bacteria and fungi, which are synthesized by specialized metabolic pathways and often incorporate exotic amino acids.

In invertebrates, the fluid portion of blood (hemolymph) as well as the granules of phagocytic cells (hemocytes) contain antimicrobial peptides (Boman et al., 1991; Iwanaga et al., 1994). Secretion of antimicrobial peptides from the fat body (equivalent to the liver in vertebrates) into hemolymph appears to be the dominant mechanism in injured or infected insects, while hemocytes may be the more important source in horseshoe crabs. Like in vertebrates, insect epithelia, most prominently the gut, secrete tissue-specific antimicrobial peptides (Richman and Kafatos, 1996; Richman et al., 1997), a response which is likely to be important in insect resistance to intestinal parasites.

Almost all antimicrobial peptides are cationic and amphipathic. The simplest antimicrobial peptide structures whose mechanism of action has been investigated are either α-helices or β-hairpins. Both types of peptides can form transmembrane channels. The length of a simple α-helix is approximately 1.5 Å per amino acid residue whereas that of a β-hairpin is roughly 3.5 Å per two residues. Since the hydrocarbon core of the phospholipid membrane is roughly 30 Å across it takes about twenty amino acids to span the membrane by either an α-helical or β-hairpin peptide. Indeed, the simplest antimicrobial peptides of these two classes are the frog skin peptide magainin (23 amino acids) (Bechinger et al., 1993; Ludtke et al., 1996) and the pig leukocyte peptide protegrin (16–18 amino acids) (Aumelas et al., 1996; Fahrner et al., 1996). Smaller natural antimicrobial peptides exist (e.g., the 12 amino acid cyclic dodecapeptide (Romeo et al., 1988)) but their structure in membranes and their mechanism of action have not been extensively investigated. More recently, it has been demonstrated that even smaller artificial peptides (6 or 8 amino acids) can generate pores in membranes by assembling into nanotubes (Fernandez-Lopez et al., 2001). It is possible that naturally occurring small peptides that employ similar mechanisms will be discovered.

There are three major hypotheses about how the disruption of membrane integrity kills the target microbes. The loss of microbial viability may be due to the cumulative effects of energy drain due to the equilibration of intracellular and extracellular ion concentrations through the disrupted membrane. Alternatively, antimicrobial peptides may enter the target cell through the disrupted membrane, bind to as yet unknown intracellular molecules and interfere with their metabolic function. Finally, some peptides may generate pores that admit water but do not allow osmotically active substances to pass. The entry of water generates osmotic pressure that eventually stretches and breaks the microbial membrane (Lehrer et al., unpublished). Either way, repair processes may limit or reverse these lesions when peptide concentrations are low or limited in time. Prolonged exposure to higher concentrations of antimicrobial peptides overwhelms the repair capacity of the microbe and the damage becomes irreversible.

The assembly of membrane pores by magainins (Ludtke et al., 1996; Matsuzaki, 1998; Shai, 1999) and tachyplesins (β-hairpin peptides from horseshoe crab hemocytes) (Matsuzaki et al., 1991) is favored by membranes that are rich in anionic phospholipids, a characteristic property of bacterial membranes. Conversely, the cell membranes of animals are rich in neutral phospholipids and cholesterol, substances that inhibit the incorporation of these peptides into membranes and the formation of pores. This mechanism explains why the concentrations necessary to kill eukaryotic cells are much higher than those required for killing most bacteria. Current evidence favors similar mechanisms of action for other peptides commonly found in the animal and plant kingdoms (Lohner et al., 1997).

Defensins (Ganz and Lehrer, 1995) are particularly abundant and widely distributed antimicrobial peptides characterized by a cationic β-sheet rich amphipathic structure stabilized by a conserved three-disulfide motif. They range in size from 29 to 47 amino acids, and are abundant in many vertebrate granulocytes, Paneth cells (specialized granule-rich intestinal host defense cells), and on epithelial surfaces. Like the simpler magainins and protegrins, defensins also form pores in target membranes. There is evidence that the permeabilization of target cells is nonlethal unless followed by defensin entry into the cell and additional intracellular damage (Lichtenstein, 1991).

In invertebrates and plants, organisms that lack adaptive immunity, antimicrobial peptides constitute a major component of host defense (Fritig et al., 1998; Meister et al., 1997). Many of the plant and invertebrate peptides (e.g., insect defensins and plant defensins) structurally and functionally resemble their vertebrate counterparts but a comprehensive evolutionary lineage has not yet been established. Both plants and invertebrates induce the synthesis of antimicrobial peptides in response to infection. The signaling pathways that mediate this response are similar to the acute phase response in animals and employ similar transcriptional regulators, most prominently the rel/NF-κB family. In vertebrates, antimicrobial peptide synthesis is either constitutive or inducible by microbial macromolecules and/or cytokines. The epithelial β-defensin of the bovine trachea, the tracheal antimicrobial peptide (TAP), is synthesized in the airway epithelia when these are exposed to inhaled bacteria or lipopolysaccharide (Diamond et al., 1996). This response is initiated by lipopolysaccharide receptors that ultimately signal to transcriptional regulators including the NF-κB complex, acting on NF-κB binding motifs in the promoter of the TAP gene. In addition to transcriptional regulation of synthesis, stimulus-dependent degranulation provides an additional level of responsiveness and specificity. Thus the granulocytes of many vertebrates contain antimicrobial defensin peptides in their phagocytic granules and another class of antimicrobial peptides, cathelicidins, in granules destined for extracellular secretion (Rice et al., 1987; Sorensen et al., 1997). Intestinal Paneth cells, positioned at the bottom of narrow crypts in the small intestine, release their defensin-rich granules (Ouellette and Selsted, 1996) upon stimulation by cholinergic or bacterial stimuli, both of which are associated with food ingestion (Qu et al., 1996).

All known antimicrobial peptides are synthesized as larger precursors, containing one or multiple copies of the active peptide segment which are released by proteolytic processing. In the simplest cases the cotranslational removal of an N-terminal signal peptide frees the active moiety but more commonly one or more anionic propieces are also removed during processing (Valore and Ganz, 1992; Terry et al., 1988; Zasloff, 1987). Perhaps the most intriguing and as yet unexplained processing pattern is seen with cathelicidins, a group of peptides with a conserved 100 amino acid domain that is frequently proteolytically cleaved from the highly variable C-terminal antimicrobial domain (Zanetti et al., 1995). In phagocytes, the cathelicidins are commonly stored as inactive precursors in secretory granules. In many cases, the processing enzyme is neutrophil elastase contained in a separate set of storage granules. During phagocytosis, this binary system combines to generate active antimicrobial peptides. The function of the highly conserved cathelin domain is not yet known.

Many antimicrobial peptides display activity against gram-positive and gram-negative bacteria, yeasts and fungi, and even certain enveloped viruses and protozoa. Other peptides are more restricted in their spectrum. Even minor variations in peptide structure can influence activity, and a systematic understanding of the relationship between peptide structure and activity is an important area for future investigations. Evidence is accumulating that many peptides act synergistically with larger polypeptides whose antimicrobial activity is enzymatic (e.g., lysozyme) or is dependent on specific recognition of bacterial macromolecules (e.g., the bactericidal permeability-inducing protein, BPI) (Levy et al., 1994). Synergistic interactions between two antimicrobial peptides in the frog skin, magainin 2 and PGLa, have also been reported (Westerhoff et al., 1995). In addition to their action on microbes, some antimicrobial peptides can function as regulatory molecules in the host. For example, in vitro studies suggest that defensins can attract phagocytes and lymphocytes to sites of infection, inhibit the release of cortisol from adrenal cells, induce the proliferation of fibroblasts and modify ionic fluxes in epithelial cells (Ganz and Lehrer, 1995).

• 1) Human α-defensin (Human Neutrophil Peptide, HNP) …
• 2) Human α-defensin 5 (HD-5) and Human α-defensin 6 (HD-6) …
• 3) Mouse α-defensin (Cryptdin) …
• 4) Human β-defensin 1 (hBD-1) …
• 5) Human β-defensin (hBD-2, -3, -4) …
• 6) θ-defensin.

4. The β Family: α-Defensins

Alpha-defensins. Structurally, α-defensins consist of three β-strands that form a β-sheet. In the crystal, a dimeric structure is found for human HNP-1, where two copies of the molecule pack together. HNP-2 and HNP-3 have a similar structure. Thus, it is primarily due to the single amino acid difference in these defensins ( ) that influences peptide activity. With a more hydrophobic sequence, HNP-4 is more potent against E. coli and C. albicans than other human α-defensins [23]. Using a kinetic 96-well turbidimetric procedure, the relative potencies of six human α-defensins were compared. In the case of Gram-positive S. aureus, the activity is in the following order: HNP-2 > HNP-1 > HNP-3 > HNP-4. In contrast, their relative potencies against Gram-negative E. coli is HNP-4 > HNP-2 > HNP-1 = HNP-3 [224]. Thus, the antibacterial activities of these defensins are also bacteria dependent. This likely reflects the distinct differences in membranes of these organisms. The poor antibacterial activity of HNP-3 is not surprising considering the presence of an acidic aspartate at the N-terminus of the peptide, making it unfavorable to target the negatively charged surface of bacteria. HD-5 displayed a rather potent activity, which is comparable to HNP-2 against S. aureus and HNP-4 against E. coli. The higher activities of HNP-4 and HD-5 against E. coli are correlated with their higher net charge of +4 ( ). HD-6 has a poor antibacterial activity. In the crystal, it forms a tetrameic structure ( B) [123].

4. The αβ Family: β-Defensins, Antimicrobial Chemokines, RNases, and RegIIIα

Beta-defensins. Human β-defensins comprise both α and β structures in the same 3D fold. C shows the NMR structure of human β-defensin 3 (hBD-3), which starts with a helical structure followed by three beta strands [225]. NMR translational diffusion studies revealed a dimer for hBD-3, but a monomer for both hBD-1 and hBD-2 in solution. The stronger antibacterial activity of hBD-3 than either hBD-1 or hBD-2 was attributed to the dimeric structure as well as higher charge density on the protein surface [225]. Interestingly, the disulfide-linked form of hBD-1 is poorly active and became highly potent against bacteria and fungus C. albicans under reduced conditions where the disulfide-linked structure was disrupted [226]. It seems that the folded hBD-1 is the stored form, which can be transformed into an active form when needed.

Antimicrobial chemokines. Chemokines interact with receptors to realize chemotactic functions. They share a similar fold consisting of a three-stranded sheet followed by one α-helix at the C-terminus [227,228,229]. The N-terminal region is frequently disordered ( A–C).

This can be best seen using a superimposed structural ensemble for CCL20 or CCL27 [230,231] determined by NMR ( D,E). The β-sheet appears to separate the N-terminal domain that interacts with cell receptors and the C-terminal domain that contains the antimicrobial helix for targeting bacterial membranes ( F) [232]. Some chemokines can also form oligomers. The dimeric forms of CCL20, CCL13, CXCL1, and CXCL10 [233,234,235,236] are shown in panels G to J of . The dimer interface is normally composed of the C-terminal helix and strand 3. In the case of CCL13, however, it is the N-terminal region that occupies the interface ( H). Yung et al. found a direct binding of antimicrobial chemokines CXCL9 (net charge of +20) and CXCL10 (net charge +11) to the cell wall of S. aureus likely via the positively charged patches on these protein surfaces [237].

Under certain situations, the antimicrobial peptide is generated by further processing of a precursor protein. For example, antimicrobial thrombocidin-1 (TC-1) is produced by truncating two residues from the C-terminus of the parent protein NAP-2 (i.e., neutrophil-activating peptide-2), which is poorly active. NMR analysis revealed that the C-terminus of TC-1 is mobile. In contrast, the C-terminus of NAP-2 is less mobile. It was proposed that the additional two residues locked the C-terminus via electrostatic interactions [238]. The additional Asp residue could have masked the positively-charged surface of the C-terminal helix that targets bacterial membranes. Likewise, insertion of an acidic Glu to the N-terminal region of GF-17, a peptide corresponding to the major antimicrobial region of human cathelicidin LL-37 [179], substantially reduced the peptide activity [239].

RNases. The structures of RNase 3 ( , panels A and B) and RNase 5 (panels C and D) were solved by both X-ray diffraction and multi-dimensional NMR spectroscopy. Although RNase 3 is dimeric in the crystal, the protein fold determined by the two techniques is similar. In addition, NMR studies revealed two conformations for His114 of RNase 5 in solution [240]. The structures of RNase 2 and RNase 7 are given in (panels E and F). Unlike chemokines discussed above, the antimicrobial region has been mapped to the N-terminus of RNase 7 [130,241]. In particular, a cluster of lysines were identified as key elements for antibacterial activity (bold in ). It is proposed recently that the N-terminal antimicrobial function is conserved in the ribonuclease family [242]. One may wonder why a protein is created for bacterial defense if only part of the chain is required to kill pathogens. One possibility is the stability gain as part of the protein. Another possibility is that a folded protein structure allows for the incorporation of a variety of active sites on the protein surface. In certain cases, such functional sites may be overlapping [243]. In the case of RNase 7, the adjacent active site and antimicrobial residues allows us to propose a yet-to-be-proved “peel-and-kill” model. In other words, binding to bacteria by the cationic amino acids is followed by digestion of pathogenic nucleic acids. The multiple active sites also enable functional regulation. For example, an endogenous molecule can bind to RNase 7 and regulates its antimicrobial activity [244]. This could be one of the unique features of antimicrobial proteins distinct from small antimicrobial peptides.

Antimicrobial lectin RegIIIα. RegIIIα (or HIP/PAP) is a C-type lectin that binds peptidoglycan carbohydrates of Gram-positive bacterial cell walls. The structural basis of this binding has been elucidated ( D) [245]. Different from other C-type calcium-dependent lectins, the binding of RegIIIα to peptidoglycans is calcium independent (i.e., lacking calcium-binding motif). The binding, however, requires the “EPN” motif and depends on sugar chain length. However, it seems that this peptidoglycan binding serves as an early recognition step for the peptide action as it can create a pore on bacterial membranes. The structure of the oligomeric form of the protein has recently been determined by combining X-ray structure and electron microscopy data, providing insight into the lethal step of bacterial killing by this intestine lectin [246].

APPs in the Blood and Bloodstream Infection

APPs consistently circulate in the bloodstream, they are transported freely within the plasma, and provide an ongoing low-level non-specific immune defense against potential invasive pathogens. Cellular expression and secretion of some APPs, including defensins (73), LL-37 (74), and BPI can be mediated by TLRs (6). In infants with BSI with bacterial etiology, plasma BPI concentrations are higher than those in healthy infants, which indicates that BPI transcription and/or cellular secretion is upregulated during infection (41, 75). Additionally, healthy uninfected neonates born to mothers who have suffered from an amniotic infection demonstrate higher levels of LF, BPI, HNP-1, HNP-2, and HNP-3 in the cord plasma (25). Maternal plasma LL-37 levels appear to be the most important predictor of infant plasma LL-37 levels, and although the source of these APPs in cord blood is not known, it is possible that these higher levels may not be a reflection of the functional status of the infant’s own immune system, but an example of maternally derived transplacentally transferred immune protection (76).

However, generally, intracellular levels of APPs are lower in neonates than in later life: LL-37 and BPI levels are reduced in neonatal whole blood and neutrophils when compared with adults (17, 31, 32, 77), and BPI deficiency of neutrophils in neonates is associated with reduced bacterial-killing capacity (41). It is yet to be established whether an infant’s intrinsic intracellular or plasma levels of APPs influence an individual’s risk of developing a BSI, or indeed the clinical outcome following BSI. Measuring serum, plasma or even resting-state intracellular levels of APPs have obvious limitations in understanding the importance of differences between neonates and adults. Indeed, more relevant perhaps is identification of functional impairments of the innate immune response in neonates, such as defective NET formation resulting in impaired bacterial killing in vitro (78). Further characterization of the functional capacities of peripheral blood neutrophils in term and preterm infants will undoubtedly yield insights into understanding neonatal BSI and developing strategies for its prevention and cure.

FAQ

What are the 3 antimicrobial proteins?

What are the antimicrobial proteins?

• Peptide.
• Lysozyme.
• Defensin.
• Lantibiotics.
• Antimicrobial Peptides.
• Neutrophil.
• Enzymes.
• Proteins.

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