Activation of stress signalling pathways enhances tolerance of fungi to chemical fungicides and antifungal proteins

Fungal disease is an increasing problem in both agriculture and human health. Treatment of human fungal disease involves the use of chemical fungicides, which generally target the integrity of the fungal plasma membrane or cell wall. Chemical fungicides used for the treatment of plant disease, have more diverse mechanisms of action including inhibition of sterol biosynthesis, microtubule assembly and the mitochondrial respiratory chain. However, these treatments have limitations, including toxicity and the emergence of resistance. This has led to increased interest in the use of antimicrobial peptides for the treatment of fungal disease in both plants and humans. Antimicrobial peptides are a diverse group of molecules with differing mechanisms of action, many of which remain poorly understood. Furthermore, it is becoming increasingly apparent that stress response pathways are involved in the tolerance of fungi to both chemical fungicides and antimicrobial peptides. These signalling pathways such as the cell wall integrity and high-osmolarity glycerol pathway are triggered by stimuli, such as cell wall instability, changes in osmolarity and production of reactive oxygen species. Here we review stress signalling induced by treatment of fungi with chemical fungicides and antifungal peptides. Study of these pathways gives insight into how these molecules exert their antifungal effect and also into the mechanisms used by fungi to tolerate sub-lethal treatment by these molecules. Inactivation of stress response pathways represents a potential method of increasing the efficacy of antifungal molecules.     

Antifungal proteins: targets,mechanisms and prospective applications

All organisms have evolved several defence systems in order to protect themselves against bacteria, fungi and viruses. Higher organisms have developed a complex network of humoral and cellular responses, called adaptive immunity. A second defence system, innate immunity, was discovered in the early 1980s, consisting of small cationic peptides with a broad antimicrobial spectrum. These proteins act immediately at sites of infection or inflammation. The production of proteins with antimicrobial activity was not limited to higher organisms but was also found in insects, plants and microorganisms. During the last 2decades a broad range of proteins with very different structural features have been isolated and characterised from differing organisms ranging from bacteria to human beings. Over 500cationic membrane-acting proteins with antimicrobial and antifungal activities have been identified to date. Apart from these proteins, a very large number of antifungal proteins active on the fungal cell wall, on enzymes of the cell wall synthesis machinery, the plasma membrane and on intracellular targets have been characterised.Received 17 June 2003; received after revision 4 August 2003; accepted 18 August 2003     

Structural diversity and species distribution of host-defense peptides in frog skin secretions

Cationic peptides that adopt an amphipathic α-helical conformation in a membrane-mimetic environment are synthesized in the skins of many frog species. These peptides often display cytolytic activities against bacteria and fungi consistent with the idea that they play a role in the host’s system of defense against pathogenic microorganisms, but their importance in the survival strategy of the animal is not clearly understood. Despite the common misconception that antimicrobial peptides are synthesized in the skins of all anurans, the species distribution is sporadic, suggesting that their production may confer some evolutionary advantage to the organism but is not necessary for survival. 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.     

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     

Multifunctional cationic host defence peptides and their clinical applications

With the rapid rise in the emergence of bacterial strains resistant to multiple classes of antimicrobial agents, there is an urgent need to develop novel antimicrobial therapies to combat these pathogens. Cationic host defence peptides (HDPs) and synthetic derivatives termed innate defence regulators (IDRs) represent a promising alternative approach in the treatment of microbial-related diseases. Cationic HDPs (also termed antimicrobial peptides) have emerged from their origins as nature’s antibiotics and are widely distributed in organisms from insects to plants to mammals and non-mammalian vertebrates. Although their original and primary function was proposed to be direct antimicrobial activity against bacteria, fungi, parasites and/or viruses, cationic HDPs are becoming increasingly recognized as multifunctional mediators, with both antimicrobial activity and diverse immunomodulatory properties. Here we provide an overview of the antimicrobial and immunomodulatory activities of cationic HDPs, and discuss their potential application as beneficial therapeutics in overcoming infectious diseases.     

Host defense peptides as new weapons in cancer treatment

In the last decade intensive research has been conducted to determine the role of innate immunity host defense peptides (also termed antimicrobial peptides) in the killing of prokaryotic and eukaryotic cells. Many antimicrobial peptides damage the cellular membrane as part of their killing mechanism. However, it is not clear what makes cancer cells more susceptible to some of these peptides, and what the molecular mechanisms underlying these activities are. Two general mechanisms were suggested: (i) plasma membrane disruption via micellization or pore formation, and (ii) induction of apoptosis via mitochondrial membrane disruption. To be clinically used, these peptides need to combine high and specific anticancer activity with stability in serum. Although so far very limited, new studies have paved the way for promising anticancer host defense peptides with a new mode of action and with a broad spectrum of anticancer activity.     

The Penicillium chrysogenum antifungal protein PAF, a promising tool for the development of new antifungal therapies and fungal cell biology studies

In recent years the interest in antimicrobial proteins and peptides and their mode of action has been rapidly increasing due to their potential to prevent and combat microbial infections in all areas of life. A detailed knowledge about the function of such proteins is the most important requirement to consider them for future application. Our research in recent years has been focused on the low molecular weight, cysteine-rich and cationic antifungal protein PAF from Penicillium chrysogenum, which inhibits the growth of opportunistic zoo-pathogens including Aspergillus fumigatus, numerous plant-pathogenic fungi and the model organism Aspergillus nidulans. So far, the experimental results indicate that PAF elicits hyperpolarization of the plasma membrane and the activation of ion channels, followed by an increase in reactive oxygen species in the cell and the induction of an apoptosis-like phenotype. Detailed knowledge about the molecular mechanism of action of antifungal proteins such as PAF contributes to the development of new antimicrobial strategies that are urgently needed. Received 09 August 2007; received after revision 17 September 2007; accepted 19 September 2007     

Plant thionins – the structural perspective

Thionins belong to a rapidly growing family of biologically active peptides in the plant kingdom. Thionins are small (∼5 kDA), cysteine-rich peptides with toxic and antimicrobial properties. They show a broad cellular toxicity against wide range of organisms and eukaryotic cell lines; while possessing some selectivity. Thionins are believed to be involved in protection against plant pathogens, including bacteria and fungi, by working directly at the membrane. The direct mechanism of action is still surrounded by controversy. Here the results of structural studies are reviewed and confronted with recent results of biophysical studies aimed at defining the function of thionins. The proposed toxicity mechanisms are reviewed and the attempt to reconcile competing hypotheses with a wealth of structural and functional studies is made. Received 3 December 2005; received after revision 6 February 2006; accepted 18 March 2006     

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.     

Vernix caseosa as a multi-component defence system based on polypeptides, lipids and their interactions

Vernix caseosa is a white cream-like substance that covers the skin of the foetus and the newborn baby. Recently, we discovered antimicrobial peptides/proteins such as LL-37 in vernix, suggesting host defence functions of vernix. In a proteomic approach, we have continued to characterize proteins in vernix and have identified 20 proteins, plus additional variant forms. The novel proteins identified, considered to be involved in host defence, are cystatin A, UGRP-1, and calgranulin A, B and C. These proteins add protective functions to vernix such as antifungal activity, opsonizing capacity, protease inhibition and parasite inactivation. The composition of the lipids in vernix has also been characterized and among these compounds the free fatty acids were found to exhibit antimicrobial activity. Interestingly, the vernix lipids enhance the antimicrobial activity of LL-37 in vitro, indicating interactions between lipids and antimicrobial peptides in vernix. In conclusion, vernix is a balanced cream of compounds involved in host defence, protecting the foetus and newborn against infection.     

Mononuclear phagocyte-mediated antifungal immunity: the role of chemotactic receptors and ligands

Over the past two decades, fungal infections have emerged as significant causes of morbidity and mortality in patients with hematological malignancies, hematopoietic stem cell or solid organ transplantation and acquired immunodeficiency syndrome. Besides neutrophils and CD4⁺ T lymphocytes, which have long been known to play an indispensable role in promoting protective antifungal immunity, mononuclear phagocytes are now being increasingly recognized as critical mediators of host defense against fungi. Thus, a recent surge of research studies has focused on understanding the mechanisms by which resident and recruited monocytes, macrophages and dendritic cells accumulate and become activated at the sites of fungal infection. Herein, we critically review how a variety of G-protein coupled chemoattractant receptors and their ligands mediate mononuclear phagocyte recruitment and effector function during infection by the most common human fungal pathogens.     

New antimicrobial activity for the catecholamine release-inhibitory peptide from chromogranin A

Catestatin (bCGA_344–364), an endogenous peptide of bovine chromogranin A, was initially characterized for its effect on the inhibition of catecholamine release from chromaffin cells. Catestatin and its active domain (bCGA_344–358) were identified in chromaffin cells and in secretion medium. The present study identified a potent antimicrobial activity of bCGA_344–358 in the lowmicromolar range against bacteria, fungi and yeasts, without showing any haemolytic activity. Confocal laser microscopy demonstrated penetration of the rhodaminated peptide into the cell membranes of fungi and yeasts and its intracellular accumulation. Time-lapse videomicroscopy showed arrest of fungal growth upon penetration of the labelled peptide into a fungal filament. We identified several catestatin-containing fragments in the stimulated secretion medium of human polymorphonuclear neutrophils, suggesting the N-terminal sequence of catestatin (bCGA_344–358) (named cateslytin) as a novel component of innate immunity.Received 21 October 2004; received after revision 6 December 2004; accepted 7 December 2004     

Snake venoms: attractive antimicrobial proteinaceous compounds for therapeutic purposes

Gram-positive and -negative bacteria are dangerous pathogens that may cause human infection diseases, especially due to the increasingly high prevalence of antibiotic resistance, which is becoming one of the most alarming clinical problems. In the search for novel antimicrobial compounds, snake venoms represent a rich source for such compounds, which are produced by specialized glands in the snake’s jawbone. Several venom compounds have been used for antimicrobial effects. Among them are phospholipases A₂, which hydrolyze phospholipids and could act on bacterial cell surfaces. Moreover, metalloproteinases and l-amino acid oxidases, which represent important enzyme classes with antimicrobial properties, are investigated in this study. Finally, antimicrobial peptides from multiple classes are also found in snake venoms and will be mentioned. All these molecules have demonstrated an interesting alternative for controlling microorganisms that are resistant to conventional antibiotics, contributing in medicine due to their differential mechanisms of action and versatility. In this review, snake venom antimicrobial compounds will be focused on, including their enormous biotechnological applications for drug development.     

Fungitoxic properties of Rosa chinensis Jacq

Summary During a systematic survey of higher plants for their fungitoxicity, the flowers of Rosa chinensis Jacq. were found to exhibit strong antifungal properties. On chemical investigation the antifungal principle was isolated as a shining, needle-shaped crystalline substance. It was identified as gallic acid. It exhibited fungistatic action against as many as 17 fungi at 3% concentration.Authors are thankful to Prof. K. S. Bhargava for laboratory facilities.     

The mode of antifungal action of plant, insect and human defensins

Defensins are small (~5 kDa), basic, cysteine-rich antimicrobial peptides that fulfill an important role in the innate immunity of their host by combating pathogenic invading micro-organisms. Defensins can inhibit the growth or virulence of microorganisms directly or can do so indirectly by enhancing the host's immune system. Because of their wide distribution in nature, defensins are believed to be ancient molecules with a common ancestor that arose more than a billion years ago. This review summarizes current knowledge concerning the mode of antifungal action of plant, insect and human defensins.     

Temporins, anti-infective peptides with expanding properties

Antimicrobial peptides are effector molecules of the innate immune response of all pluricellular organisms, providing them with first-line defence against pathogens. Amphibian skin secretions represent one of the richest natural sources for such peptide antibiotics, and temporins, a large family of antimicrobial peptides from frog skin, are among the smallest ones found in nature to date. Their functional role and modes of action have been described, along with their interesting and unique properties. These properties make temporins good molecules for an in-depth understanding of host defence peptides in general. Furthermore, they are attractive templates for the future design of new therapeutics against infectious diseases with new modes of action, urgently needed due to the increasing resistance of microorganisms to the available drugs. Received 8 November 2005; received after revision 19 December 2005; accepted 18 January 2006     

Lipid complexes with cationic peptides and OAKs; their role in antimicrobial action and in the delivery of antimicrobial agents

Antimicrobial agents are toxic to bacteria by a variety of mechanisms. One mechanism that is very dependent on the lipid composition of the bacterial membrane is the clustering of anionic lipid by cationic antimicrobial agents. Certain species of oligo-acyl-lysine (OAK) antimicrobial agents are particularly effective in clustering anionic lipids in mixtures mimicking the composition of bacterial membranes. The clustering of anionic lipids by certain cationic antimicrobial agents contributes to the anti-bacterial action of these agents. Bacterial membrane lipids are a determining factor, resulting in some species of bacteria being more susceptible than others. In addition, lipids can be used to increase the effectiveness of antimicrobial agents when administered in vivo. Therefore, we review some of the structures in which lipid mixtures can assemble, to more effectively be utilized as antimicrobial delivery systems. We describe in more detail the complexes formed between mixtures of lipids mimicking bacterial membranes and an OAK and their usefulness in synergizing with antibiotics to overcome bacterial multidrug resistance.     

Antimicrobial peptides: natural templates for synthetic membrane-active compounds

The innate immunity of multicellular organisms relies in large part on the action of antimicrobial peptides (AMPs) to resist microbial invasion. Crafted by evolution into an extremely diversified array of sequences and folds, AMPs do share a common amphiphilic 3-D arrangement. This feature is directly linked with a common mechanism of action that predominantly (although not exclusively) develops upon interaction of peptides with cell membranes of target cells. This minireview reports on current understanding of the modes of interaction of AMPs with biological and model membranes, especially focusing on recent insights into the folding and oligomerization requirements of peptides to bind and insert into lipid membranes and exert their antibiotic effects. Given the potential of AMPs to be developed into a new class of anti-infective agents, emphasis is placed on how the information on peptide-membrane interactions could direct the design and selection of improved biomimetic synthetic peptides with antibiotic properties.     

Tumor cell membrane-targeting cationic antimicrobial peptides: novel insights into mechanisms of action and therapeutic prospects

There is an ongoing need for effective and targeted cancer treatments that can overcome the detrimental side effects presented by current treatment options. One class of novel anticancer molecules with therapeutic potential currently under investigation are cationic antimicrobial peptides (CAPs). CAPs are small innate immunity peptides found ubiquitously throughout nature that are typically membrane-active against a wide range of pathogenic microbes. A number of CAPs can also target mammalian cells and often display selective activity towards tumor cells, making them attractive candidates as novel anticancer agents warranting further investigation. This current and comprehensive review describes key examples of naturally occurring membrane-targeting CAPs and their modified derivatives that have demonstrated anticancer activity, across multiple species of origin and structural subfamilies. In addition, we address recent advances made in the field and the ongoing challenges faced in translating experimental findings into clinically relevant treatments.     

Cathelicidins - a family of multifunctional antimicrobial peptides

One component of host defence at mucosal surfaces are epithelial-derived antimicrobial peptides. Cathelicidins are one family of antimicrobial peptides characterized by conserved pro-peptide sequences that have been identified in several mammalian species. LL-37/hCAP-18 is the only cathelicidin found in humans and is expressed in inflammatory and epithelial cells. Besides their direct antimicrobial function, cathelicidins have multiple roles as mediators of inflammation influencing diverse processes such as cell proliferation and migration, immune modulation, wound healing, angiogenesis and the release of cytokines and histamine. Finally, cathelicidin antimicrobial peptides qualify as prototypes of innovative drugs that may be used to treat infection and/or modulate the immune response. This review provides an overview of antimicrobial peptides of the cathelicidin family, the structures of their genes and peptides and their biological functions.