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Table 1. Some representative mammalian peptides

Peptide

Class

Amino acid sequence*

HNP-1 (α-defensin)

β-sheet

AC1YC2RIPAC3lAGERRYGTC3lYQGRLWAFC2C1

HBD-2 (β-defensin)

β-sheet

MRVLYLLFSFLFIFLMPLPGVFGGIGDPVTC1LKSGAIC2HPVFC3PRRYKQIGTC2GLPGTKC1C3KKP

Protegrin

β-sheet

RGGRLC1YC2RRRFC1VC2VGR

Indolicidin

Extended

ILPWKWPWWPWRR-NH2

Bac5

Extended

RFRPPIRRPPIRPPFYPPFRPPIRPPIFPPIRPPFRPPLGPFP

Bactenicin

Loop

RLC1RIVVIRVC1R

LL-37

α-helical

LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES

Cecropin P1

α-helical

SWLSKTAKKLENSAKKRISEGIAIAIQGGPR

* Single-letter amino acid code.

Subscript numbers represent amino acids that are joined by cysteine disulfides.

kidney, salivary glands, small intestine, and liver. This is in contrast to HBD-1, which is constitutively expressed mainly in the urogenital tract and kidney. Several other inducible β-defensins are produced in the epithelia, including tracheal antimicrobial pep tide (TAP), isolated from bovine respiratory mucosa, lingual antimicrobial peptide, and the enteric β-defensin (5). TAP is expressed in columnar epithelial cells of the conducting airways, and the mRNA of this β-defensin peptide is upregulated in response to LPS. The production of HBD-2 by human epithelium resembles the ancient defense mechanisms of plants and insects (2), because there is evidence in these situations for the involvement of the transcription factor NFκB and Toll receptors. Such a mechanism elicits an immediate antimicrobial response to the same microorganisms that have had contact with the epithelial cells, and these responses are related to, but completely independent of, the leukocyte-dependent immune defense mechanisms. Other obvious differences between the cationic antimicrobial peptide response and the immune response in animals are the highly specific nature of immune responses, the relative slowness of immune responses because of the requirement for clonal cell expansion, and the self vs. nonself discrimination built into the immune response. In contrast, inducible peptide responses result in an ability to act against a wide spectrum of pathogens, occur within minutes rather than days, and lack real-self vs. nonself discrimination (although the peptides appear to have relatively low activity against host cells).

Evidence for Their Role in Host Defenses

The evidence for a role of cationic antimicrobial peptides in innate host defenses has become quite convincing. It includes data demonstrating that some peptides are inducible by bacterial products and convincing animal model and transgenic animal experiments indicating that the peptides protect against infection in experimental animals. The inducibility of peptides has been summarized above. The kinetics of induction are highly suggestive of a role in early defenses against infection.

The animal model data demonstrating protection by a variety of different peptides applied both topically and systemically were summarized recently (6). Such studies have confirmed the antibacterial (vs. both Gram-negative and Gram-positive bacteria), antifungal, synergistic and antiendotoxic (see below) nature of antimicrobial peptides. These results have been confirmed by studies showing systemic protection by nisin against Streptococcus pneumoniae infections of mice (7), local protection by the protegrin-related peptide IB367 against polymicrobic oral mucosaitis in hamsters (8), protection against lethal Pseudomonas aeruginosa infections of burn-wound sites in mice by peptide D4B (9), and protection by LL-37 (CAP 18)-derived peptides against lethal endotoxemia and P. aeruginosa infection in mice (10). Our own recent studies have demonstrated that peptides (including the fish peptide pleurocidin) protect Coho salmon against lethal vibriosis (Aeromonas salmonicida infection), when administered continuously at low levels by using a device called an osmotic pump.

An alternative method of doing these types of studies involves a “gene-therapy” treatment of mice with an adenovirus vector containing the DNA for the human peptide LL-37 (11). Such mice showed a dramatic increase in serum and lung LL-37 and demonstrated significantly fewer bacteria and a lower inflammatory response after sublethal challenge and a dramatic increase in resistance to endotoxin and Escherichia coli challenges.

Although the role of such peptides in defense against infections has been emphasized, many other intriguing properties have been ascribed to selected cationic peptides, including induction of the wound-repair proteoglycans termed Syndecans (12), stimulation of nonopsonic phagocytosis (13), chemoattraction of IL-8-stimulated neutrophils (14), and penetration of the blood–brain barrier (15).

It is well known (1) that cationic antimicrobial peptides are major components of certain phagocytic cells, especially neutrophils and alveolar macrophages. They appear to be involved in nonoxidative killing by such cells (16). Although oxidative killing of bacteria by phagocytes is often emphasized, nonoxidative killing can be very effective, because neutrophils from chronic granulomatis disease patients, which lack an oxidative response, are still able to kill most bacteria (16). Indeed, such patients are only substantially more susceptible to infections by Burkholderia cepacia, one of the bacteria that are naturally resistant to cationic antimicrobial peptide action (17).

Mechanism of Action

Cationic antimicrobial peptides have been described as membrane-active agents (1). This is certainly true for many peptides, but we and others have recently described data that indicate that the membrane is not necessarily the target for many, or perhaps even most, cationic peptides (3, 18, 19 and 20). To summarize the proposed mechanism of action for Gram-negative bacteria, the peptides interact with and cross both cell envelope membranes and then kill cells by a multihit mechanism that involves action on more than one anionic target. The initial uptake across the outer membrane is via self-promoted uptake (3, 13) proposed by us 20 years ago to explain uptake of polycationic antibiotics like aminoglycosides and polymyxins across the outer membrane. In this mechanism, the peptides initially interact with the polyanionic surface LPS and competitively displace the divalent cations that bridge and partly neutralize the LPS. This causes disruption of the outer membrane (visualized as surface blebbing), and it is through these disrupted outer membranes that peptide molecules are proposed to pass (i.e., the peptides self promote their own uptake). Next, the peptides associate with the negatively charged phospholipid membrane and insert into the membrane so they are oriented parallel to the membrane. It is then proposed that the peptide molecules, when they reach a certain critical concentration, form informal transmembrane channels that we have termed aggregate channels but that others ( 21) call



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