Proline-rich antimicrobial peptides: converging to a non-lytic mechanism of action

Proline-rich antimicrobial peptides are a group of cationic host defense peptides of vertebrates and invertebrates characterized by a high content of proline residues, often associated with arginine residues in repeated motifs. Those isolated from some mammalian and insect species, although not evolutionarily related, use a similar mechanism to selectively kill Gram-negative bacteria, with a low toxicity to animals. Unlike other types of antimicrobial peptides, their mode of action does not involve the lysis of bacterial membranes but entails penetration into susceptible cells, where they then act intracellularly. Some aspects of the transport system and cytoplasmic targets have been elucidated. These features make them attractive both as anti-infective lead compounds and as a new class of potential cell-penetrating peptides capable of internalising membrane-impermeant drugs into both bacterial and eukaryotic cells     

Lysozyme activates Enterococcus faecium to induce necrotic cell death in macrophages

Enterococci are commensal organisms in the alimentary tract. However, they can cause a variety of life-threatening infections, especially in nosocomial settings. We hypothesized that induction of cell death might enable these facultative pathogenic bacteria to evade the innate immune response and to cause infections of their host. We demonstrate that E. faecium when exposed to lysozyme induces cell death in macrophages in vitro and in vivo. Flow cytometric analyses of J774A.1 macrophages infected with E. faecium revealed loss of cell membrane integrity indicated by uptake of propidium iodide and decrease of the inner mitochondrial transmembrane potential ΔΨ_m. Inhibition of caspases, treatment of macrophages with cytochalasin D, or rifampicin did not prevent cells from dying, suggesting cell death mechanisms that are independent of caspase activation, bacterial uptake, and intracellular bacterial replication. Characteristics of necrotic cell death were demonstrated by both lack of procaspase 3 activation and cell shrinkage, electron microscopy, and release of lactate dehydrogenase. Pretreatment of E. faecium with lysozyme and subsequently with broad spectrum protease considerably reduced cell death, suggesting that a bacterial surface protein is causative for cell death induction. Moreover, in a mouse peritonitis model we demonstrated that E. faecium induces cell death of peritoneal macrophages in vivo. Altogether, our results show that enterococci, under specific conditions such as exposure to lysozyme, induce necrotic cell death in macrophages, which might contribute to disseminated infections by these facultative pathogenic bacteria.     

Properties and mechanisms of action of naturally occurring antifungal peptides

Antimicrobial peptides are a vital component of the innate immune system of all eukaryotic organisms and many of these peptides have potent antifungal activity. They have potential application in the control of fungal pathogens that are a serious threat to both human health and food security. Development of antifungal peptides as therapeutics requires an understanding of their mechanism of action on fungal cells. To date, most research on antimicrobial peptides has focused on their activity against bacteria. Several antimicrobial peptides specifically target fungal cells and are not active against bacteria. Others with broader specificity often have different mechanisms of action against bacteria and fungi. This review focuses on the mechanism of action of naturally occurring antifungal peptides from a diverse range of sources including plants, mammals, amphibians, insects, crabs, spiders, and fungi. While antimicrobial peptides were originally proposed to act via membrane permeabilization, the mechanism of antifungal activity for these peptides is generally more complex and often involves entry of the peptide into the cell.     

Bacterial resistance mechanisms against host defense peptides

Host defense peptides and proteins are important components of the innate host defense against pathogenic microorganisms. They target negatively charged bacterial surfaces and disrupt microbial cytoplasmic membranes, which ultimately leads to bacterial destruction. Throughout evolution, pathogens devised several mechanisms to protect themselves from deleterious damage of host defense peptides. These strategies include (a) inactivation and cleavage of host defense peptides by production of host defense binding proteins and proteases, (b) repulsion of the peptides by alteration of pathogen’s surface charge employing modifications by amino acids or amino sugars of anionic molecules (e.g., teichoic acids, lipid A and phospholipids), (c) alteration of bacterial membrane fluidity, and (d) expulsion of the peptides using multi drug pumps. Together with bacterial regulatory network(s) that regulate expression and activity of these mechanisms, they represent attractive targets for development of novel antibacterials.     

The Sushi peptides: structural characterization and mode of action against Gram-negative bacteria

The compositional difference in microbial and human cell membranes allows antimicrobial peptides to preferentially bind microbes. Peptides which specifically target lipopolysaccharide (LPS) and palmitoyl-oleoyl-phosphatidylglycerol (POPG) are efficient antibiotics. From the core LPS-binding region of Factor C, two 34-mer Sushi peptides, S1 and S3, were derived. S1 functions as a monomer, while S3 is active as a dimer. Both S1 and S3 display detergent-like properties in disrupting LPS aggregates, with specificity for POPG resulting from electrostatic and hydrophobic forces between the peptides and the bacterial lipids. During interaction with POPG, the S1 transitioned from a random coil to an α-helix, while S3 resumed a mixture of α-helix and β-sheet structures. The unsaturated nature of POPG confers fluidity and enhances insertion of the peptides into the lipid bilayer, causing maximal disruption of the bacterial membrane. These parameters should be considered in designing and developing new generations of peptide antibiotics with LPS-neutralizing capability. Received 2 October 2007; received after revision 2 November 2007; accepted 4 December 2007 J. L. Ding, B. Ho: Co-senior authors.     

Peptide aptamers: exchange of the thioredoxin-A scaffold by alternative platform proteins and its influence on target protein binding

Peptide aptamers have emerged as powerful new tools for molecular medicine. They can specifically bind to and functionally inactivate a given target molecule under intracellular conditions. Typically, peptide aptamers are generated by screening a randomized peptide expression library, displayed from the Escherichia coli thioredoxin A (TrxA) protein. Here, we transferred peptide moieties from defined TrxA-based peptide aptamers to alternative scaffold proteins, such as the green fluorescent protein and staphylococcal nuclease. Yeast and mammalian two-hybrid assays as well as in vitro binding analyses show that the TrxA scaffold can be a major determinant for the binding of peptide aptamers. In addition, we demonstrate that TrxA can correctly display peptide sequences that correspond to the binding domains of natural interaction partners. Therefore, sequence analyses of TrxA-based peptide aptamers, isolated by two-hybrid screening from randomized expression libraries, should also be useful to find cellular binding partners for a given target protein, by homology. Received 1 August 2002; received after revision 17 September 2002; accepted 19 September 2002 RID="*" ID="*"Corresponding author.     

Phagocytes,phagocytose et défense de l'organisme contre les infections

Summary After the evocation of classic notions about morphology and physiology of free and fixed phagocytes, and their role in the defense of organism against infections, writers give new observations on two principal points.1. They show that polysaccharids, to which the grampositive bacteria owe their specificity, are the most important agents responsable of the migration of polymorphonuclear leucocytes in inflamed connective tissue.2. They show that bacterial endotoxins, nature of which is glucido-lipido-polypeptidic, and which are also antigens O complets of gram-negative micro-organisms, appear able to stop the coming of polymorphonuclears in inflamed connective tissues. The endotoxins kill not leukocytes; they are not leucocidins, but they prevent diapedesis through a mechanism still obscure. For this reason, they favour the development of infections.Ultimately, writers tell how the two great theories of immunity, cellular theory ofMetchnikoff and humoral theory of german authors, can mary together: in vivo, phagocytes have the first and also the last word to kill bacteria, but their work is very much easier after opsonization with antibodies.     

Gramicidin S and polymyxins: the revival of cationic cyclic peptide antibiotics

Gramicidin S and polymyxins are small cationic cyclic peptides and act as potent antibiotics against Gram-negative and Gram-positive bacteria by perturbing integrity of the bacterial membranes. Screening of a natural antibiotics library with bacterial membrane vesicles identified gramicidin S as an inhibitor of cytochrome bd quinol oxidase and an alternative NADH dehydrogenase (NDH-2) and polymyxin B as an inhibitor of NDH-2 and malate: quinone oxidoreductase. Our studies showed that cationic cyclic peptide antibiotics have novel molecular targets in the membrane and interfere ligand binding on the hydrophobic surface of enzymes. Improvement of the toxicity and optimization of the structures and clinical uses are urgently needed for their effective application in combating drug-resistant bacteria.     

The peptidoglycan recognition proteins LCa and LCx

Infection of bacteria triggers innate immune defense reactions in Drosophila. So far, the only bacterial component known to be recognized by the insect innate immune system is peptidoglycan, one of the most abundant constituents of the bacterial cell wall. Insects use peptidoglycan recognition proteins to detect peptidoglycan and to activate innate immune responses. Such specialized peptidoglycan receptors appear to have evolved from phage enzymes that hydrolyze bacterial cell walls. They are able to bind specific peptidoglycan molecules with distinct chemical moieties and activate innate immune pathways by interacting with other signaling proteins. Recent X-ray crystallographic studies of the peptidoglycan recognition proteins LCa, and LCx bound to peptidoglycan have provided structural insights into recognition of peptidoglycan and activation of innate immunity in insects. Received 28 December 2006; received after revision 2 February 2007; accepted 21 February 2007     

Aminoglycoside antibiotics: old drugs and new therapeutic approaches

Aminoglycoside antibiotics kill bacteria by binding to the ribosomal decoding site and reducing fidelity of protein synthesis. Since the discovery of these natural products over 50 years ago, aminoglycosides have provided a mainstay of antibacterial therapy of serious Gram-negative infections. In recent years, aminoglycosides have become important tools to study molecular recognition of ribonucleic acid (RNA). In an ingenious exploitation of the aminoglycosides’ mechanism of action, it has been speculated that drug-induced readthrough of premature stop codons in mutated messenger RNAs might be used to treat patients suffering from certain heritable genetic disorders. Received 23 January 2007; received after revision 25 February 2007; accepted 29 March 2007     

Isolation of peptides blocking the function of anti-apoptotic Livin protein

Livin (ML-IAP) is a cancer-associated member of the inhibitor of apoptosis protein (IAP) family. By yeast two-hybrid screening of a randomized peptide expression library, we isolated short linear peptides that specifically bind to Livin, but not to other IAPs. Intracellular expression of the peptides sensitized livin-expressing cancer cells toward different pro-apoptotic stimuli. The bioactive peptides neither showed sequence homologies to Smac-derived IAP inhibitors, nor did they interfere with the binding of Livin to Smac. Intracellular expression of the peptides did not affect the levels or the subcellular distribution of Livin. Growth of livin-expressing tumor cells was inhibited in colony formation assays by the Livin-targeting peptides. These findings provide evidence that the targeted inhibition of Livin by peptides represents a viable approach for the apoptotic sensitization and growth inhibition of tumor cells. The inhibitory peptides isolated here could form a novel basis for the development of therapeutically useful Livin inhibitors.     

Autonomous translocation and intracellular trafficking of the cell-penetrating and immune-suppressive effector protein YopM

Extracellular Gram-negative pathogenic bacteria target essential cytoplasmic processes of eukaryotic cells by using effector protein delivery systems such as the type III secretion system (T3SS). These secretion systems directly inject effector proteins into the host cell cytoplasm. Among the T3SS-dependent Yop proteins of pathogenic Yersinia, the function of the effector protein YopM remains enigmatic. In a recent study, we demonstrated that recombinant YopM from Yersinia enterocolitica enters host cells autonomously without the presence of bacteria and thus identified YopM as a novel bacterial cell-penetrating protein. Following entry YopM down-regulates expression of pro-inflammatory cytokines such as tumor necrosis factor α. These properties earmark YopM for further development as a novel anti-inflammatory therapeutic. To elucidate the uptake and intracellular targeting mechanisms of this bacterial cell-penetrating protein, we analyzed possible routes of internalization employing ultra-cryo electron microscopy. Our results reveal that under physiological conditions, YopM enters cells predominantly by exploiting endocytic pathways. Interestingly, YopM was detected free in the cytosol and inside the nucleus. We could not observe any colocalization of YopM with secretory membranes, which excludes retrograde transport as the mechanism for cytosolic release. However, our findings indicate that direct membrane penetration and/or an endosomal escape of YopM contribute to the cytosolic and nuclear localization of the protein. Surprisingly, even when endocytosis is blocked, YopM was found to be associated with endosomes. This suggests an intracellular endosome-associated transport of YopM.     

Bacterial signal peptide recognizes HeLa cell mitochondrial import receptors and functions as a mitochondrial leader sequence

Phage display was used to identify new components of the mammalian mitochondrial receptor complex using Tom20 as a binding partner. Two peptides were identified. One had partial identity (SMLTVMA) with a bacterial signal peptide from Toho-1, a periplasmic protein. The other had partial identity with a mitochondrial inner membrane glutamate carrier. The bacterial signal peptide could carry a protein into mitochondria both in vivo and in vitro. The first six residues of the sequence, SMLTVM, were necessary for import but the two adjacent arginine residues in the 30-amino-acid leader were not critical for import. The signal peptides of Escherichia coli β-lactamase and Bacillsus subtilis lipase could not carry proteins into mitochondria. Presumably, the Toho-1 leader can adopt a structure compatible for recognition by the import apparatus.Received 29 April 2005; received after revision 8 June 2005; accepted 17 June 2005     

Anti herpes simplex virus activity of lactoferrin/lactoferricin – an example of antiviral activity of antimicrobial protein/peptide

One of the most common viral infections in humans is caused by the herpes simplex virus (HSV). It was first effectively treated in the 1970s with the introduction of acyclovir, which is still the most commonly used treatment. Naturally occurring antimicrobial proteins and peptides have also been shown to possess antiviral activity against HSV. This review will focus on the anti-HSV activity of one such protein, lactoferrin, and a small peptide fragment from its N-terminal domain, lactoferricin. Both components have been shown to effectively block entry of HSV into the host cell. In addition to blocking HSV entry, the peptides appear to have immune stimulatory activity, although this is still somewhat controversial. Mode of action studies and knowledge about the anti-HSV activity of lactoferricin have also been successfully employed in the design of new, more specific HSV blockers. Received 25 May 2005; received after revision 24 August 2005; accepted 6 September 2005     

Fluorescence spectroscopy and molecular dynamics simulations in studies on the mechanism of membrane destabilization by antimicrobial peptides

Since their initial discovery, 30 years ago, antimicrobial peptides (AMPs) have been intensely investigated as a possible solution to the increasing problem of drug-resistant bacteria. The interaction of antimicrobial peptides with the cellular membrane of bacteria is the key step of their mechanism of action. Fluorescence spectroscopy can provide several structural details on peptide–membrane systems, such as partition free energy, aggregation state, peptide position and orientation in the bilayer, and the effects of the peptides on the membrane order. However, these “low-resolution” structural data are hardly sufficient to define the structural requirements for the pore formation process. Molecular dynamics simulations, on the other hand, provide atomic-level information on the structure and dynamics of the peptide–membrane system, but they need to be validated experimentally. In this review we summarize the information that can be obtained by both approaches, highlighting their versatility and complementarity, suggesting that their synergistic application could lead to a new level of insight into the mechanism of membrane destabilization by AMPs.     

Perforin and its role in T lymphocyte-mediated cytolysis

The killing mediated by cytotoxic T lymphocytes (CTL) represents an important mechanism in the immune defence against tumors and virus infections. The lytic mechanism has been proposed to consist of a polarized secretion of granule-stored molecules, occurring on effector-target cell contact. By electron microscopy, membrane deposited, pore-like lesions are detected on the target cell membrane during cytolysis by CTL. These structures resembled strikingly pores formed during complement attack.Granules of CTL isolated by nitrogen cavitation and Percoll gradient centrifugation were shown to retain cytotoxic activity. Further purification of proteins stored in these granules led to the discovery of a membranolytic protein named perforin which was capable of polymerizing into pore-like structures. In addition to this cytolytic protein, a set of serine esterases was found as well as lysosomal enzymes and proteoglycans, whose function are not yet clearly defined. The role of perforin in the cytotoxic process is currently being explored by ablating the active gene in mice.     

Perforin and its role in T lymphocyte-mediated cytolysis.

The killing mediated by cytotoxic T lymphocytes (CTL) represents an important mechanism in the immune defence against tumors and virus infections. The lytic mechanism has been proposed to consist of a polarized secretion of granule-stored molecules, occurring on effector-target cell contact. By electron microscopy, membrane deposited, pore-like lesions are detected on the target cell membrane during cytolysis by CTL. These structures resembled strikingly pores formed during complement attack. Granules of CTL isolated by nitrogen cavitation and Percoll gradient centrifugation were shown to retain cytotoxic activity. Further purification of proteins stored in these granules led to the discovery of a membranolytic protein named perforin which was capable of polymerizing into pore-like structures. In addition to this cytolytic protein, a set of serine esterases was found as well as lysosomal enzymes and proteoglycans, whose function are not yet clearly defined. The role of perforin in the cytotoxic process is currently being explored by ablating the active gene in mice.     

Timing is everything: the regulation of type III secretion

Type Three Secretion Systems (T3SSs) are essential virulence determinants of many Gram-negative bacteria. The T3SS is an injection device that can transfer bacterial virulence proteins directly into host cells. The apparatus is made up of a basal body that spans both bacterial membranes and an extracellular needle that possesses a channel that is thought to act as a conduit for protein secretion. Contact with a host-cell membrane triggers the insertion of a pore into the target membrane, and effectors are translocated through this pore into the host cell. To assemble a functional T3SS, specific substrates must be targeted to the apparatus in the correct order. Recently, there have been many developments in our structural and functional understanding of the proteins involved in the regulation of secretion. Here we review the current understanding of protein components of the system thought to be involved in switching between different stages of secretion.     

Identification of the bioactive peptide PEC-60 in brain

PEC-60 is a 60-residue peptide originally isolated from pig intestine. It inhibits glucose-induced insulin secretion from perfused pancreas in a hormonal manner and also has biological activity in the immune system. PEC-60-like immunoreactive material has been reported in catecholamine neurons of the central and peripheral nervous systems, but the peptide has not been identified from that material. We have now isolated PEC-60 from pig and rat brains with a method that combines column purification procedures with the specificity of a radioimmunoassay and the sensitivity of mass spectrometry to directly identify the peptide. The results show that PEC-60, like many other peptides, is expressed in the gastrointestinal tract and the central nervous system. The specific regional brain distribution and interaction with classical neurotransmitters raise the possibility that PEC-60may play a role in the central nervous system disorders involving dopamine dysregulation. Received 6 December 2002; received after revision 10 December 2002; accepted 11 December 2002 RID="*" ID="*"Corresponding author.     

Epithelial antimicrobial peptides: innate local host response elements

Multicellular organisms have to survive in an environment laden with numerous microorganisms, which represent a potential hazard to life. Different strategies have been developed to ward off infections by preventing microorganisms from entering surfaces and by preventing the attack of microorganisms that have already entered the epithelia. Therefore, it is not surprising that epithelia are equipped with various antimicrobial substances that act rapidly to kill a broad range of microorganisms. This review summarizes our present knowledge about epithelial peptide antibiotics produced in plants, invertebrates, and vertebrates including humans. There is now strong evidence that in addition to constitutively secreted peptide antibiotics, others are induced upon contact with microorganisms or by proinflammatory cytokines. beta-Defensins represent one family of vertebrate antimicrobial peptides, members of which are inducible and have recently been identified in humans. The defensin-characteristic local expression pattern may indicate that specialized surfaces express a characteristic surface antimicrobial peptide pattern that might define the characteristic microflora as well as the density of microorganisms present on the surface.