Enzymatic Mechanisms Involved in Evasion of Fungi to the Oxidative Stress: Focus on <Emphasis Type="Italic">Scedosporium apiospermum</Emphasis>

The airways of patients with cystic fibrosis (CF) are frequently colonized by various filamentous fungi, mainly Aspergillus fumigatus and Scedosporium species. To establish within the respiratory tract and cause an infection, these opportunistic fungi express pathogenic factors allowing adherence to the host tissues, uptake of extracellular iron, or evasion to the host immune response. During the colonization process, inhaled conidia and the subsequent hyphae are exposed to reactive oxygen species (ROS) and reactive nitrogen species (RNS) released by phagocytic cells, which cause in the fungal cells an oxidative stress and a nitrosative stress, respectively. To cope with these constraints, fungal pathogens have developed various mechanisms that protect the fungus against ROS and RNS, including enzymatic antioxidant systems. In this review, we summarize the different works performed on ROS- and RNS-detoxifying enzymes in fungi commonly encountered in the airways of CF patients and highlight their role in pathogenesis of the airway colonization or respiratory infections. The potential of these enzymes as serodiagnostic tools is also emphasized. In addition, taking advantage of the recent availability of the whole genome sequence of S. apiospermum, we identified the various genes encoding ROS- and RNS-detoxifying enzymes, which pave the way for future investigations on the role of these enzymes in pathogenesis of these emerging species since they may constitute new therapeutics targets.     

Type I signal peptidase: an overview

The signal hypothesis suggests that proteins contain information within their amino acid sequences for protein targeting to the membrane. These distinct targeting sequences are cleaved by specific enzymes known as signal peptidases. There are various type of signal peptidases known such as type I, type II, and type IV. Type I signal peptidases are indispensable enzymes, which catalyze the cleavage of the amino-terminal signal-peptide sequences from preproteins, which are translocated across biological membranes. These enzymes belong to a novel group of serine proteases, which generally utilize a Ser-Lys or Ser-His catalytic dyad instead of the prototypical Ser-His-Asp triad. Despite having no distinct consensus sequence other than a commonly found 'Ala-X-Ala' motif preceding the cleavage site, signal sequences are recognized by type I signal peptidase with high fidelity. Type I signal peptidases have been found in bacteria, archaea, fungi, plants, and animals. In this review, I present an overview of bacterial type I signal peptidases and describe some of their properties in detail.     

Soluble neutral metallopeptidases: physiological regulators of peptide action.

Classically, the pre- and post-secretory processing of peptide signals appears to be mediated primarily by subtilisin-like peptidases in secretory vesicles and/or membrane-associated neutral endopeptidases in the extracellular environment. This article presents both biochemical and physiological evidence to support a role for soluble neutral metallopeptidases in the mediation of cell-to-cell communication by the selective generation and termination of peptide signals. These soluble peptidases have been implicated in the normal and disease-state processing of peptides involved in neurological, endocrine and cardiovascular functions. In this context, specific inhibitors of these enzymes could selectively modulate peptide levels and thus have considerable therapeutic potential. The aim of this review is to discuss the design and development of specific inhibitors of soluble neutral metallopeptidases that have been instrumental in identifying the roles of these enzymes. It will also review the evidence and present a case for the involvement of soluble neutral metallopeptidases in the regulation of peptide signalling in both central nervous system (CNS) and peripheral tissues.     

Extracellular hydrolytic enzymes produced by entomopathogenic fungi--role in an infection process

Entomopathogenic fungi have a great potential in biological control of insect pest population. Fungal pathogens are promising source of insecticides and notable alterative to chemical pesticides. These fungi possess a unique mechanism of insects paralysis. As natural enemies of insects they attack direct host cuticle via a combination of mechanical pressure and cuticle-degrading enzymes. Entomopathogenic fungi produce several proteo-, chitino- and lipolytic enzymes, which are accepted as key factors in insect mycosis. The role of extracellular enzymes in pathogenesis is still not well understood. Profound understanding the mechanisms of insect paralysis by entomopathogenic fungi will help in the production of safer for environment and more efficiency mycoinsecticides.     

Peptidase activity and toxicity of strains of Pseudomonas aeruginosa

Pseudomonas aeruginosa is a microorganism of the terrestrial and aquatic environment which may cause pulmonary, urinary and corneal infections. The pathogenesis of Pseudomonas-induced diseases seems to be linked to the production of extracellular substances such as exotoxins, hemolysins, leukocidins and hydrolytic enzymes, including various peptidases. Anyway there is not a clear view on the role other proteases have in the mechanism of pathogenesis, and even if their activity may always be associated to the toxicity of the microorganism. We have therefore determined the activity of a number of eso- and endopeptidases in 17 strains of P. aeruginosa isolated from patients with urinary pathologies. Four of these peptidase activities, namely elastase, neutral protease, aminopeptidase I and leucine aminopeptidase show a positive correlation with three parameters selected as indices of toxicity, i.e. the mucoid appearance, the gentamicin resistance and the adhesivity of colonies.     

The secretome of Pleurotus sapidus

Due to their unique capability to attack lignified biopolymers, extracellular enzymes of white-rot fungi enjoy an increasing interest in various fields of white biotechnology. The edible fungus Pleurotus sapidus was selected as a model organism for the analysis of the secretome by means of 2-DE. For enzyme production, the fungus was grown in submerged cultures either on peanut shells or on glass wool as a carrier material. Identification of the secreted enzymes was performed by tryptic digestion, ESI-MS/MS ab initio sequencing, and homology searches against public databases. The spectrum of secreted enzymes comprised various types of hydrolases and lignolytic enzymes of the manganese peroxidase/versatile peroxidase family. While peptidases were secreted mainly by the cultures grown on peanut shells, versatile peroxidase type enzymes dominated in the cultures grown on glass wool.     

White-rot fungi and their enzymes for the treatment of industrial dye effluents

White-rot fungi produce various isoforms of extracellular oxidases including laccase, Mn peroxidase and lignin peroxidase (LiP), which are involved in the degradation of lignin in their natural lignocellulosic substrates. This ligninolytic system of white-rot fungi (WRF) is directly involved in the degradation of various xenobiotic compounds and dyes. This review summarizes the state of the art in the research and prospective use of WRF and their enzymes (lignin-modifying enzymes, LME) for the treatment of industrial effluents, particularly dye containing effluents. The textile industry, by far the most avid user of synthetic dyes, is in need of ecoefficient solutions for its colored effluents. The decolorization and detoxification potential of WRF can be harnessed thanks to emerging knowledge of the physiology of these organisms as well as of the biocatalysis and stability characteristics of their enzymes. This knowledge will need to be transformed into reliable and robust waste treatment processes.     

The peptidases from fungi and viruses

Fungi and viruses encode a variety of peptidases having a plethora of functions. Many fungal peptidases are extracellular and are likely used to degrade proteins in their environment. Viral peptidases are processing enzymes, intimately involved in the virus infectious cycle. The viral RNA genome is translated by the host-cell machinery into a large polyprotein that is cleaved by the viral peptidases into mature capsid proteins, non-structural proteins and enzymes. I review the structure and catalytic mechanism of scytalidoglutamic peptidase isolated from the wood-destroying fungus Scytalidium lignicolum. This enzyme has a unique beta-sandwich fold and a novel catalytic mechanism based on a glutamate, a glutamine and a nucleophilic water molecule. Hepatitis A virus (HAV) 3C peptidase was the first structure identified for a viral 3C enzyme that exhibited the three-dimensional fold of the chymotrypsin family of serine peptidases but had a cysteine sulfur atom instead of the serine oxygen as the nucleophile. The structure of HAV 3C was unusual in that the Asp residue expected as the third member of the catalytic triad did not interact with the general base His. The present structure is of a beta-lactone-inhibited version of HAV 3C that has a restored catalytic triad.     

Oxylipins and oxylipin synthesis pathways in fungi

Oxylipins are a family of oxygenated fatty acids that are very diverse with regard to origin, structure, and functions. These compounds are found in almost all living beings and serve both as autoregulators of the development of organisms and as communication molecules. The autoregulatory role of oxylipins in fungi is to control the development, reproduction, synthesis of secondary metabolites (including mycotoxins), and adaptive responses. The role of oxylipins in the regulation of pathogenesis accounts for an important aspect of research on the biological activity of these compounds. The synthetic pathways and functions of oxylipins of fungi, the differences between fungal oxylipins and oxylipins from bacteria, higher plants, and mammals, and the role of oxylipins in the interaction of fungi with other organisms are considered in the present review.     

Bacterial tyrosine-kinases: Structure–function analysis and therapeutic potential

Since the characterization of genes encoding Ser/Thr-kinases and Tyr-kinases in bacteria, in 1991 and 1997, respectively, a growing body of evidence has been reported showing the important role of these enzymes in the regulation of bacterial physiology. While most Ser/Thr-kinases share structural similarity with their eukaryotic counterparts, it seems that bacteria have developed their own Tyr-kinases to catalyze protein phosphorylation on tyrosine. Different types of Tyr-kinases have been identified in bacteria and a large number of them are similar to ATP-binding proteins with Walker motifs. These enzymes have been grouped in the same family (BY-kinases) and the crystal structures of two of them have been recently characterized. Phosphoproteome analysis suggest that BY-kinases are involved in several cellular processes and to date, the best-characterized role of BY-kinases concerns the control of extracellular polysaccharide synthesis. Knowing the role of these compounds in the virulence of bacterial pathogens, BY-kinases can be considered as promising targets to combat some diseases. Here, we review the current knowledge on BY-kinases and discuss their potential for the development of new antibiotics.     

Thermophilic fungi: their physiology and enzymes.

Thermophilic fungi are a small assemblage in mycota that have a minimum temperature of growth at or above 20 degrees C and a maximum temperature of growth extending up to 60 to 62 degrees C. As the only representatives of eukaryotic organisms that can grow at temperatures above 45 degrees C, the thermophilic fungi are valuable experimental systems for investigations of mechanisms that allow growth at moderately high temperature yet limit their growth beyond 60 to 62 degrees C. Although widespread in terrestrial habitats, they have remained underexplored compared to thermophilic species of eubacteria and archaea. However, thermophilic fungi are potential sources of enzymes with scientific and commercial interests. This review, for the first time, compiles information on the physiology and enzymes of thermophilic fungi. Thermophilic fungi can be grown in minimal media with metabolic rates and growth yields comparable to those of mesophilic fungi. Studies of their growth kinetics, respiration, mixed-substrate utilization, nutrient uptake, and protein breakdown rate have provided some basic information not only on thermophilic fungi but also on filamentous fungi in general. Some species have the ability to grow at ambient temperatures if cultures are initiated with germinated spores or mycelial inoculum or if a nutritionally rich medium is used. Thermophilic fungi have a powerful ability to degrade polysaccharide constituents of biomass. The properties of their enzymes show differences not only among species but also among strains of the same species. Their extracellular enzymes display temperature optima for activity that are close to or above the optimum temperature for the growth of organism and, in general, are more heat stable than those of the mesophilic fungi. Some extracellular enzymes from thermophilic fungi are being produced commercially, and a few others have commercial prospects. Genes of thermophilic fungi encoding lipase, protease, xylanase, and cellulase have been cloned and overexpressed in heterologous fungi, and pure crystalline proteins have been obtained for elucidation of the mechanisms of their intrinsic thermostability and catalysis. By contrast, the thermal stability of the few intracellular enzymes that have been purified is comparable to or, in some cases, lower than that of enzymes from the mesophilic fungi. Although rigorous data are lacking, it appears that eukaryotic thermophily involves several mechanisms of stabilization of enzymes or optimization of their activity, with different mechanisms operating for different enzymes.     

Cysteine cathepsins in human cancer

Proteases play causal roles in the malignant progression of human tumors. This review centers on the roles in this process of cysteine cathepsins, i.e., peptidases belonging to the papain family (C1) of the CA clan of cysteine proteases. Cysteine cathepsins, most likely along with matrix metalloproteases (MMPs) and serine proteases, degrade the extracellular matrix, thereby facilitating growth and invasion into surrounding tissue and vasculature. Studies on tumor tissues and cell lines have shown changes in expression, activity and distribution of cysteine cathepsins in numerous human cancers. Molecular, immunologic and pharmacological strategies to modulate expression and activity of cysteine cathepsins have provided evidence for a causal role for these enzymes in tumor progression and invasion. Clinically, the levels, activities and localization of cysteine cathepsins and their endogenous inhibitors have been shown to be of diagnostic and prognostic value. Understanding the roles that cysteine proteases play in cancer could lead to the development of more efficacious therapies.     

Solid-state and membrane-surface liquid cultures of micromycetes: Specific features of their development and enzyme production (a Review)

Specific features in the development of micromycetes, typical mechanisms of their enzyme production, and conditions providing for an increase in enzyme secretion by the microscopic fungi in solid-state (on natural substrates and inert carriers) and membrane-surface liquid cultures are considered. The prospects and advantages of these fermentation methods for the production of extracellular enzymes are discussed and compared with submerged cultures.     

Type I signal peptidases of Gram-positive bacteria

Proteins that are exported from the cytoplasm to the periplasm and outer membrane of Gram-negative bacteria, or the cell wall and growth medium of Gram-positive bacteria, are generally synthesized as precursors with a cleavable signal peptide. During or shortly after pre-protein translocation across the cytoplasmic membrane, the signal peptide is removed by signal peptidases. Importantly, pre-protein processing by signal peptidases is essential for bacterial growth and viability. This review is focused on the signal peptidases of Gram-positive bacteria, Bacillus and Streptomyces species in particular. Evolutionary concepts, current knowledge of the catalytic mechanism, substrate specificity requirements and structural aspects are addressed. As major insights in signal peptidase function and structure have been obtained from studies on the signal peptidase LepB of Escherichia coli, similarities and differences between this enzyme and known Gram-positive signal peptidases are highlighted. Notably, while the incentive for previous research on Gram-positive signal peptidases was largely based on their role in the biotechnologically important process of protein secretion, present-day interest in these essential enzymes is primarily derived from the idea that they may serve as targets for novel anti-microbials.     

Thermophilic Fungi: Their Physiology and Enzymes

Thermophilic fungi are a small assemblage in mycota that have a minimum temperature of growth at or above 20°C and a maximum temperature of growth extending up to 60 to 62°C. As the only representatives of eukaryotic organisms that can grow at temperatures above 45°C, the thermophilic fungi are valuable experimental systems for investigations of mechanisms that allow growth at moderately high temperature yet limit their growth beyond 60 to 62°C. Although widespread in terrestrial habitats, they have remained underexplored compared to thermophilic species of eubacteria and archaea. However, thermophilic fungi are potential sources of enzymes with scientific and commercial interests. This review, for the first time, compiles information on the physiology and enzymes of thermophilic fungi. Thermophilic fungi can be grown in minimal media with metabolic rates and growth yields comparable to those of mesophilic fungi. Studies of their growth kinetics, respiration, mixed-substrate utilization, nutrient uptake, and protein breakdown rate have provided some basic information not only on thermophilic fungi but also on filamentous fungi in general. Some species have the ability to grow at ambient temperatures if cultures are initiated with germinated spores or mycelial inoculum or if a nutritionally rich medium is used. Thermophilic fungi have a powerful ability to degrade polysaccharide constituents of biomass. The properties of their enzymes show differences not only among species but also among strains of the same species. Their extracellular enzymes display temperature optima for activity that are close to or above the optimum temperature for the growth of organism and, in general, are more heat stable than those of the mesophilic fungi. Some extracellular enzymes from thermophilic fungi are being produced commercially, and a few others have commercial prospects. Genes of thermophilic fungi encoding lipase, protease, xylanase, and cellulase have been cloned and overexpressed in heterologous fungi, and pure crystalline proteins have been obtained for elucidation of the mechanisms of their intrinsic thermostability and catalysis. By contrast, the thermal stability of the few intracellular enzymes that have been purified is comparable to or, in some cases, lower than that of enzymes from the mesophilic fungi. Although rigorous data are lacking, it appears that eukaryotic thermophily involves several mechanisms of stabilization of enzymes or optimization of their activity, with different mechanisms operating for different enzymes.     

The proteotytic systems of lactic acid bacteria

Proteolysis in dairy lactic acid bacteria has been studied in great detail by genetic, biochemical and ultrastructural methods. From these studies the picture emerges that the proteolytic systems of lactococci and lactobacilli are remarkably similar in their components and mode of action. The proteolytic system consists of an extracellularly located serine-proteinase, transport systems specific for di-tripeptides and oligopeptides (> 3 residues), and a multitude of intracellular peptidases. This review describes the properties and regulation of individual components as well as studies that have led to identification of their cellular localization. Targeted mutational techniques developed in recent years have made it possible to investigate the role of individual and combinations of enzymes in vivo. Based on these results as well as in vitro studies of the enzymes and transporters, a model for the proteolytic pathway is proposed. The main features are: (i) proteinases have a broad specificity and are capable of releasing a large number of different oligopeptides, of which a large fraction falls in the range of 4 to 8 amino acid residues; (ii) oligopeptide transport is the main route for nitrogen entry into the cell; (iii) all peptidases are located intracellularly and concerted action of peptidases is required for complete degradation of accumulated peptides.     

Bodo sp., a Free-Living Flagellate, Expresses Divergent Proteolytic Activities from the Closely Related Parasitic Trypanosomatids

ABSTRACT. We report the characterization of cell-associated and extracellular peptidases of Bodo sp., a free-living flagellate of the Bodonidae family, order Kinetoplastida, which is considered ancestral to the trypanosomatids. This bodonid isolate is phylogenetically related to Bodo caudatus and Bodo curvifilus . The proteolytic activity profiles of Bodo sp. were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis containing co-polymerized gelatin, casein, hemoglobin, or bovine serum albumin as substrates. The enzymatic complex degraded gelatin better in acidic pH, and under these conditions four proteolytic bands (120, 100, 90, and 75 kDa) were detected in the cellular or extracellular extracts. Two peptidases (250 and 200 kDa) were exclusively detected with the substrate casein. All these enzymes belong to the serine peptidase class, based on inhibition by aprotinin and phenylmethylsulfonyl fluoride. This is the first biochemical characterization of peptidases in a free-living Bodo sp., potentially providing insight into the physiology of these protozoa and the evolutionary importance of peptidases to the order Kinetoplastida as some of these enzymes are important virulence factors in pathogenic trypanosomatids.     

LOVe across kingdoms: Blue light perception vital for growth and development in plant–fungal interactions

Blue light plays an important role in the growth and development of fungi. Environmental cues enable plant pathogenic fungi to synchronise essential metabolic pathways to that of their hosts to gain a competitive advantage. Phylogenetic analysis of the LOV domain present in blue light receptors across all three kingdoms suggests that these receptors in fungal lineages have undergone convergent evolution to use the same domain for control and regulation of similar cellular and metabolic processes. In this review, the genetic basis of blue light photoperception in fungi, and the functions it regulates, will be discussed. Furthermore, the evolution of the light sensing domain and its role in pathogenesis is hypothesised concluding with how knowledge of conserved LOV domains may be exploited for fungal disease control in crop plants.