The role of cationic antimicrobial peptides in innate host defences

Cationic antimicrobial peptides are found in all living species. A single animal can contain >24 different antimicrobial peptides, which fall into four structural classes. These peptides are produced in large quantities at sites of infection and/or inflammation and can have broad-spectrum antibacterial, antifungal, antiviral, antiprotozoan and antisepsis properties. In addition, they interact directly with host cells to modulate the inflammatory process and innate defences.     

Synthetic oligoureas of metaphenylenediamine mimic host defence peptides in their antimicrobial behaviour

Oligomeric ureas of m-phenylenediamine target anionic DMPG (dimyristoylphosphatidylglycerol) and possess promise as antimicrobial agents. Their similar size, shape and hydrophobicity to helical antimicrobial peptides (AMPs) may be important for activity to exist and the ability of these compounds to insert into a well ordered lipid environment.     

The role of Paneth cells and their antimicrobial peptides in innate host defense

The intestinal epithelium is the largest surface area that is exposed to various pathogens in the environment, however, in contrast to the colon the number of bacteria that colonize the small intestine is extremely low. Paneth cells, one of four major epithelial cell lineages in the small intestine, reside at the base of the crypts and have apically oriented secretory granules. These granules contain high levels of antimicrobial peptides that belong to the alpha-defensin family. Paneth cells secrete these microbicidal granules that contain alpha-defensins when exposed ex vivo to bacteria or their antigens, and recent evidence reveals that antimicrobial peptides, particularly alpha-defensins, that are present in Paneth cells contribute to intestinal innate host defense.     

The dermaseptin superfamily: A gene-based combinatorial library of antimicrobial peptides

Skin secretions of hylid frogs show amazing levels of interspecific and intraspecific diversity and are comprised of a cocktail of genetically-related, but markedly diverse antimicrobial peptides that are grouped into a superfamily, termed the dermaseptins, comprising several families: dermaseptins (sensu stricto), phylloseptins, plasticins, dermatoxins, phylloxins, hyposins, caerins, and aureins. Dermaseptin gene superfamily evolution is characterized by repeated gene duplications and focal hypermutations of the mature peptide coding sequence, followed by positive (diversifying) selection. We review here molecular mechanisms leading to these vast combinatorial peptide libraries, and structural and functional properties of antimicrobial peptides of the dermaseptin and plasticin families, as well as those of dermaseptin S9, an amyloidogenic peptide with antimicrobial and chemoattractant activities.     

A family of helminth molecules that modulate innate cell responses via molecular mimicry of host antimicrobial peptides

Over the last decade a significant number of studies have highlighted the central role of host antimicrobial (or defence) peptides in modulating the response of innate immune cells to pathogen-associated ligands. In humans, the most widely studied antimicrobial peptide is LL-37, a 37-residue peptide containing an amphipathic helix that is released via proteolytic cleavage of the precursor protein CAP18. Owing to its ability to protect against lethal endotoxaemia and clinically-relevant bacterial infections, LL-37 and its derivatives are seen as attractive candidates for anti-sepsis therapies. We have identified a novel family of molecules secreted by parasitic helminths (helminth defence molecules; HDMs) that exhibit similar biochemical and functional characteristics to human defence peptides, particularly CAP18. The HDM secreted by Fasciola hepatica (FhHDM-1) adopts a predominantly α-helical structure in solution. Processing of FhHDM-1 by F. hepatica cathepsin L1 releases a 34-residue C-terminal fragment containing a conserved amphipathic helix. This is analogous to the proteolytic processing of CAP18 to release LL-37, which modulates innate cell activation by classical toll-like receptor (TLR) ligands such as lipopolysaccharide (LPS). We show that full-length recombinant FhHDM-1 and a peptide analogue of the amphipathic C-terminus bind directly to LPS in a concentration-dependent manner, reducing its interaction with both LPS-binding protein (LBP) and the surface of macrophages. Furthermore, FhHDM-1 and the amphipathic C-terminal peptide protect mice against LPS-induced inflammation by significantly reducing the release of inflammatory mediators from macrophages. We propose that HDMs, by mimicking the function of host defence peptides, represent a novel family of innate cell modulators with therapeutic potential in anti-sepsis treatments and prevention of inflammation.     

The RNase H-like superfamily: new members,comparative structural analysis and evolutionary classification

Ribonuclease H-like (RNHL) superfamily, also called the retroviral integrase superfamily, groups together numerous enzymes involved in nucleic acid metabolism and implicated in many biological processes, including replication, homologous recombination, DNA repair, transposition and RNA interference. The RNHL superfamily proteins show extensive divergence of sequences and structures. We conducted database searches to identify members of the RNHL superfamily (including those previously unknown), yielding >60 000 unique domain sequences. Our analysis led to the identification of new RNHL superfamily members, such as RRXRR (PF14239), DUF460 (PF04312, COG2433), DUF3010 (PF11215), DUF429 (PF04250 and COG2410, COG4328, COG4923), DUF1092 (PF06485), COG5558, OrfB_IS605 (PF01385, COG0675) and Peptidase_A17 (PF05380). Based on the clustering analysis we grouped all identified RNHL domain sequences into 152 families. Phylogenetic studies revealed relationships between these families, and suggested a possible history of the evolution of RNHL fold and its active site. Our results revealed clear division of the RNHL superfamily into exonucleases and endonucleases. Structural analyses of features characteristic for particular groups revealed a correlation between the orientation of the C-terminal helix with the exonuclease/endonuclease function and the architecture of the active site. Our analysis provides a comprehensive picture of sequence-structure-function relationships in the RNHL superfamily that may guide functional studies of the previously uncharacterized protein families.     

Human RNase 7: a new cationic ribonuclease of the RNase A superfamily

Here we report on the expression and function of RNase 7, one of the final RNase A superfamily ribonucleases identified in the human genome sequence. The human RNase 7 gene is expressed in various somatic tissues including the liver, kidney, skeletal muscle and heart. Recombinant RNase 7 is ribonucleolytically active against yeast tRNA, as expected from the presence of eight conserved cysteines and the catalytic histidine–lysine– histidine triad which are signature motifs of this superfamily. The protein is atypically cationic with an isoelectric point (pI) of 10.5. Expression of recombinant RNase 7 in Escherichia coli completely inhibits the growth of the host bacteria, similar to what has been observed for the cationic RNase, eosinophil cationic protein (ECP/RNase 3, pI 11.4). An in vitro assay demonstrates dose-dependent cytotoxicity of RNase 7 against bacteria E.coli, Pseudomonas aeruginosa and Staphylococcus aureus. While RNase 7 and ECP/RNase 3 are both cationic and share this particular aspect of functional similarity, their protein sequence identity is only 40%. Of particular interest, ECP/RNase 3’s cationicity is based on an (over)abundance of arginine residues, whereas RNase 7 includes an excess of lysine. This difference, in conjunction with the independent origins and different expression patterns, suggests that RNase 7 and ECP/RNase 3 may have been recruited to target different pathogens in vivo, if their physiological functions are indeed host defenses.     

NODs in defence: from vulnerable antimicrobial peptides to chronic inflammation

Defensins and cathelicidins are prevalent and essential gastrointestinal cationic antimicrobial peptides (CAPs). However, these defensive peptides are not infallible because certain enteropathogens can overcome their protective function. Furthermore, impaired defensin synthesis has been linked to the occurrence of Crohn's disease (CD), a chronic inflammatory bowel disease. Recently, defective bacterial sensing through NOD1 and NOD2 has been related to reduced defensin production, CD predisposition and susceptibility to enteric infection. Hence, we propose that microbial sensors at the gut interface monitor the levels of these effector peptides, which might function as "danger" signals to confer tolerance and alert immunocytes. Further work is required to clarify how gastrointestinal CAPs are regulated and to assess their role in maintaining epithelial homeostasis and triggering adaptive immunity.     

Parameters involved in antimicrobial and endotoxin detoxification activities of antimicrobial peptides

Endotoxin [lipopolysaccharide (LPS)] covers more than 90% of the outer monolayer of the outer membrane of Gram-negative bacteria, and it plays a dual role in its pathogenesis: as a protective barrier against antibiotics and as an effector molecule, which is recognized by and activates the innate immune system. The ability of host-defense antimicrobial peptides to bind LPS on intact bacteria and in suspension has been implicated in their antimicrobial and LPS detoxification activities. However, the mechanisms involved and the properties of the peptides that enable them to traverse the LPS barrier or to neutralize LPS endotoxic activity are not yet fully understood. Here we investigated a series of antimicrobial peptides and their analogues with drastically altered sequences and structures, all of which share the same amino acid composition (K 6L 9). The list includes both all- l-amino acid peptides and their diastereomers (composed of both l- and d-amino acids). The peptides were investigated functionally for their antibacterial activity and their ability to block LPS-dependent TNF-alpha secretion by macrophages. Fluorescence spectroscopy and transmission electron microscopy were used to detect their ability to bind LPS and to affect its oligomeric state. Their secondary structure was characterized in solution, in LPS suspension, and in LPS multibilayers by using CD and FTIR spectroscopy. Our data reveal specific biophysical properties of the peptides that are required to kill bacteria and/or to detoxify LPS. Besides shedding light on the mechanisms of these two important functions, the information gathered should assist in the development of AMPs with potent antimicrobial and LPS detoxification activities.     

Abundance of RNase4 and RNase5 mRNA and protein in host defence related tissues and secretions in cattle

Members of the RNaseA family are present in various tissues and secretions but their function is not well understood. Some of the RNases are proposed to participate in host defence. RNase4 and RNase5 are present in cows' milk and have antimicrobial activity. However, their presence in many tissues and secretions has not been characterised. We hypothesised that these two RNases are present in a range of tissues and secretions where they could contribute to host defence. We therefore, determined the relative abundance of RNase4 and RNase5 mRNA as well as protein levels in a range of host defence related and other tissues as well as a range of secretions in cattle, using real time PCR and western blotting. The two RNases were found to be expressed in liver, lung, pancreas, mammary gland, placenta, endometrium, small intestine, seminal vesicle, salivary gland, kidney, spleen, lymph node, skin as well as testes. Corresponding proteins were also detected in many of the above tissues, as well as in seminal fluid, mammary secretions and saliva. This study provides evidence for the presence of RNase4 and RNase5 in a range of tissues and secretions, as well as some major organs in cattle. The data are consistent with the idea that these proteins could contribute to host defence in these locations. This work contributes to growing body of data suggesting that these proteins contribute to the physiology of the organism in a more complex way than acting merely as digestive enzymes.     

An alloy of zinc and innate immunity: Galvanising host defence against infection

Innate immune cells such as macrophages and neutrophils initiate protective inflammatory responses and engage antimicrobial responses to provide frontline defence against invading pathogens. These cells can both restrict the availability of certain transition metals that are essential for microbial growth and direct toxic concentrations of metals towards pathogens as antimicrobial responses. Zinc is important for the structure and function of many proteins, however excess zinc can be cytotoxic. In recent years, several studies have revealed that innate immune cells can deliver toxic concentrations of zinc to intracellular pathogens. In this review, we discuss the importance of zinc status during infectious disease and the evidence for zinc intoxication as an innate immune antimicrobial response. Evidence for pathogen subversion of this response is also examined. The likely mechanisms, including the involvement of specific zinc transporters that facilitate delivery of zinc by innate immune cells for metal ion poisoning of pathogens are also considered. Precise mechanisms by which excess levels of zinc can be toxic to microorganisms are then discussed, particularly in the context of synergy with other antimicrobial responses. Finally, we highlight key unanswered questions in this emerging field, which may offer new opportunities for exploiting innate immune responses for anti‐infective development.     

Lactoferrin and host defence: an overview of its immuno-modulating and anti-inflammatory properties

Lactoferrin is a member of the transferrin family of iron-binding glycoproteins that is abundantly expressed and secreted from glandular epithelial cells. In secretions, such as milk and fluids of the intestinal tract, lactoferrin is an important component of the first line of host defence. During the inflammatory process, lactoferrin, a prominent component of the secondary granules of neutrophils (PMNs), is released in infected tissues and in blood and then it is rapidly cleared by the liver. In addition to the antimicrobial properties of lactoferrin, a set of studies has focused on its ability to modulate the inflammatory process and the overall immune response. Though many in vitro and in vivo studies report clear regulation of the immune response and protective effect against infection and septic shock by lactoferrin, elucidation of all the cellular and molecular mechanisms of action is far from being achieved. At the cellular level, lactoferrin modulates the migration, maturation and function of immune cells. At the molecular level and in addition to iron binding, interactions of lactoferrin with a plethora of compounds, either soluble or membrane molecules, account for its modulatory properties. This paper reviews our current understanding of the cellular and molecular mechanisms that explain the regulatory properties of lactoferrin in host defence.     

The role of the interleukin-1/Toll-like receptor superfamily in inflammation and host defence

The IL-1 receptor/Toll-like receptor superfamily comprises a diverse family of cell surface receptors defined by a characteristic conserved sequence in their cytosolic regions, termed the Toll/IL-1 receptor domain, which function in inflammation and host defence against microbial pathogens. Members include receptors for the proinflammatory cytokines IL-1 and IL-18 and Toll-like receptors 2 and 4, which are involved in host responses to Gram-positive and Gram-negative bacteria, respectively. Signalling pathways activated by these receptors are conserved and the superfamily represents a pan-genomic system involved in the host response to infection and injury.     

PEGylation enhances the antibacterial and therapeutic potential of amphibian host defence peptides

Aurein 2.1, aurein 2.6 and aurein 3.1 are amphibian host defence peptides that kill bacteria via the use of lytic amphiphilic α-helical structures. The C-terminal PEGylation of these peptides led to decreased antibacterial activity (Minimum Lethal Concentration (MLCs) ↓ circa one and a half to threefold), reduced levels of amphiphilic α-helical structure in solvents (α-helicity ↓ circa 15.0%) and lower surface activity (Δπ ↓ > 1.5 mN m^?1). This PEGylation of aureins also led to decreased levels of amphiphilic α-helical structure in the presence of anionic membranes and zwitterionic membranes (α-helicity↓ > 10.0%) as well as reduced levels of penetration (Δπ ↓ > 3.0 mN m^?1) and lysis (lysis ↓ > 10.0%) of these membranes. Based on these data, it was proposed that the antibacterial action of PEGylated aureins involved the adoption of α-helical structures that promote the lysis of bacterial membranes, but with lower efficacy than their native counterparts. However, PEGylation also reduced the haemolytic activity of native aureins to negligible levels (haemolysis ↓ from circa 10% to 3% or less) and improved their relative therapeutic indices (RTIs ↑ circa three to sixfold). Based on these data, it is proposed that PEGylated aureins possess the potential for therapeutic development; for example, to combat infections due to multi-drug resistant strains of S. aureus, designated as high priority by the World Health Organization.     

Macrophage inflammatory proteins 1 and 2: members of a novel superfamily of cytokines

A number of studies of inflammation and of cell growth and transformation have recently converged by defining two related families of cytokines. The first, represented by macrophage inflammatory protein 1, is composed of several gene products that have been identified in activated T cells, macrophages, and fibroblasts. The biological activities of this family are still being characterized but so far include effects on neutrophils, monocytes, and hematopoietic cells. The second, represented by macrophage inflammatory protein 2, includes platelet products such as platelet factor 4 and beta-thromboglobulin as well as several other recently described gene products that have effects on a number of cell types including neutrophils, fibroblasts, hematopoietic cells, and melanoma cells. The two families are structurally related and may have evolved from a common ancestral gene that duplicated and then diverged. Their differential control and expression in a wide variety of cell types suggests that they may have multiple functions in regulating inflammation and cell growth.     

Viral and host proteins involved in picornavirus life cycle

Picornaviruses cause several diseases, not only in humans but also in various animal hosts. For instance, human enteroviruses can cause hand-foot-and-mouth disease, herpangina, myocarditis, acute flaccid paralysis, acute hemorrhagic conjunctivitis, severe neurological complications, including brainstem encephalitis, meningitis and poliomyelitis, and even death. The interaction between the virus and the host is important for viral replication, virulence and pathogenicity. This article reviews studies of the functions of viral and host factors that are involved in the life cycle of picornavirus. The interactions of viral capsid proteins with host cell receptors is discussed first, and the mechanisms by which the viral and host cell factors are involved in viral replication, viral translation and the switch from translation to RNA replication are then addressed. Understanding how cellular proteins interact with viral RNA or viral proteins, as well as the roles of each in viral infection, will provide insights for the design of novel antiviral agents based on these interactions.     

Expression of antimicrobial peptides and proteins in etanercept-treated psoriasis patients

Recent papers highlight the role of dysregulated expression of antimicrobial peptides and proteins (AMPs) in the pathogenesis of psoriasis. Etanercept, a blocker of the pro-inflammatory cytokine tumour necrosis factor-α (TNF-α), is effective in the treatment of psoriasis. We aimed to evaluate the expression profiles of AMPs in psoriatic skin before and after a 6-week course of etanercept therapy. We included 12 psoriasis patients who underwent medium-dose etanercept treatment for 6weeks. At baseline and at the end of therapy immunohistochemistry from lesional skin was performed for psoriasin, LL-37, and human ?-defensin 2 (hBD-2). After 6-week treatment, the modified psoriasis area and severity index significantly decreased from 37.5±5.9 to 14±13.4. Lesional immunoreactivity scores of psoriasin, LL-37, and hBD-2 also significantly decreased after a 6-week course of etanercept. We have demonstrated that etanercept-induced improvement of psoriasic lesions is associated with a significant decline of AMP protein expression.     

The contribution of skin antimicrobial peptides to the system of innate immunity in anurans

Cationic peptides with the propensity to adopt an amphipathic ??-helical conformation in a membrane-mimetic environment are synthesized in the skins of many species of anurans (frogs and toads). These peptides frequently display cytolytic activities against a range of pathogenic bacteria and fungi consistent with the idea that they play a role in the host's system of innate immunity. However, the importance of the peptides in the survival strategy of the animal is not clearly understood. It is a common misconception that antimicrobial peptides are synthesized in the skins of all anurans. In fact, the species distribution is sporadic suggesting that their production may confer some evolutionary advantage to the organism but is not necessary for survival. Although growth inhibitory activity against the chytrid fungus Batrachochytrium dendrobatidis, responsible for anuran population declines worldwide, has been demonstrated in vitro, the ability of frog skin antimicrobial peptides to protect the animal in the wild appears to be limited and there is no clear correlation between their production by a species and its resistance to fatal chytridiomycosis. The low potency of many frog skin antimicrobial peptides is consistent with the hypothesis that cutaneous symbiotic bacteria may provide the major system of defense against pathogenic microorganisms in the environment with antimicrobial peptides assuming a supplementary role in some species.