Bacterial phospholipases C.

A variety of pathogenic bacteria produce phospholipases C, and since the discovery in 1944 that a bacterial toxin (Clostridium perfringens alpha-toxin) possessed an enzymatic activity, there has been considerable interest in this class of proteins. Initial speculation that all phospholipases C would have lethal properties has not been substantiated. Most of the characterized enzymes fall into one of four groups of structurally related proteins: the zinc-metallophospholipases C, the sphingomyelinases, the phosphatidylinositol-hydrolyzing enzymes, and the pseudomonad phospholipases C. The zinc-metallophospholipases C have been most intensively studied, and lethal toxins within this group possess an additional domain. The toxic phospholipases C can interact with eukaryotic cell membranes and hydrolyze phosphatidylcholine and sphingomyelin, leading to cell lysis. However, measurement of the cytolytic potential or lethality of phospholipases C may not accurately indicate their roles in the pathogenesis of disease. Subcytolytic concentrations of phospholipase C can perturb host cells by activating the arachidonic acid cascade or protein kinase C. Nonlethal phospholipases C, such as the Listeria monocytogenes PLC-A, appear to enhance the release of the organism from the host cell phagosome. Since some phospholipases C play important roles in the pathogenesis of disease, they could form components of vaccines. A greater understanding of the modes of action and structure-function relationships of phospholipases C will facilitate the interpretation of studies in which these enzymes are used as membrane probes and will enhance the use of these proteins as models for eukaryotic phospholipases C.     

Characterisation of Legionella pneumophila phospholipases and their impact on host cells

Phospholipases are a diverse class of enzymes produced both by eukaryotic hosts and their pathogens. Major insights into action pathways of bacterial phospholipases have been provided during the last years. On the one hand bacterial phospholipases act as potent membrane destructors and on the other hand they manipulate and initiate host signalling paths, such as chemokine expression or the inflammatory cascade. Reaction products of bacterial phospholipases may potentially influence many more host cell processes, such as cell respreading, lamellopodia formation, cell migration and membrane traffic. Phospholipases play a dominant role in the biology of the lung pathogen Legionella pneumophila. So far, 15 different phospholipase A-encoding genes have been identified in the L. pneumophila genome. These phospholipases can be divided into three major groups, the GDSL, the patatin-like and the PlaB-like enzymes. The first two lipase families are also found in higher plants (such as flowering plants) and the second family shows similarities to eukaryotic cytosolic phospholipases A. Therefore, when those enzymes are injected or secreted by the bacterium into the host cell they may mimic eukaryotic phospholipases. The current knowledge on L. pneumophila phospholipases is summarised here with emphasis on their activity, mode of secretion, localisation, expression and importance for host cell infections.     

Candida albicans adhesins: Biochemical aspects and virulence

The recognition of host cells by the pathogenic yeast, Candida albicans, is probably an essential step in the pathogenesis of disease development. The interaction of yeast and hyphal mannoproteins with host cell receptors has been studied by a number of laboratories. C. albicans recognizes a variety of host cells as well as host cell extracellular matrix proteins. This observation is not unexpected given the number of sites within and on the body which can be colonized and infected by the organism. Indeed, it would appear that C. albicans has evolved a number of ways in which it recognizes the host. This statement is made with the qualification that the organism uses other processes to infect, such as morphogenesis, phenotypic switching and the production of invasive enzymes, including secreted aspartyl proteases and phosholipases. Recognition of epithelial cells is accomplished through cell surface mannoproteins (adhesins) which bind to carbohydrate-containing receptors. The number of such mannoproteins is not known; pro adhesins exist. The organism also binds to keratinocytes, endothelial cells and matrix proteins, such as fibronectin, laminin, collagen and entactin, and, as such, appears to have a integrin-like cell surface adhesin. In most cases, the adhesin for each of these host proteins is a mannoprotein. The biochemistry of the candidal adhesins has been extensively studied. However, molecular analyses of the encoding genes is only now being studied. Thus, until clean, genetic analyses are complete and strains lacking an adhesin function are constructed, a direct role for the adhesins in pathogenesis can only be inferred. At present, spontaneous, non-adhering strains of the organism have been described which are avirulent in animal models of candidiasis. However, these data only suggest a role for adherence; future studies should be directed towards resolving questions about the role of these proteins in pathogenesis.     

The Clostridium perfringens alpha-toxin

The gene encoding the alpha-(cpa) is present in all strains of Clostridium perfringens, and the purified alpha-toxin has been shown to be a zinc-containing phospholipase C enzyme, which is preferentially active towards phosphatidylcholine and sphingomyelin. The alpha-toxin is haemolytic as a result if its ability to hydrolyse cell membrane phospholipids and this activity distinguishes it from many other related zinc-metallophospholipases C. Recent studies have shown that the alpha-toxin is the major virulence determinant in cases of gas gangrene, and the toxin might play a role in several other diseases of animals and man as diverse as necrotic enteritis in chickens and Crohn's disease in man. In gas gangrene the toxin appears to have three major roles in the pathogenesis of disease. First, it is able to cause mistrafficking of neutrophils, such that they do not enter infected tissues. Second, the toxin is able to cause vasoconstriction and platelet aggregation which might reduce the blood supply to infected tissues. Finally, the toxin is able to detrimentally modulate host cell metabolism by activating the arachidonic acid cascade and protein kinase C. The molecular structure of the alpha-toxin reveals a two domain protein. The amino-terminal domain contains the phospholipase C active site which contains zinc ions. The carboxyterminal domain is a paralogue of lipid binding domains found in eukaryotes and appears to bind phospholipids in a calcium-dependent manner. Immunisation with the non-toxic carboxyterminal domain induces protection against the alpha-toxin and gas gangrene and this polypeptide might be exploited as a vaccine. Other workers have exploited the entire toxin as the basis of an anti-tumour system.     

Biochemical Diversity in the Trypanosoma congolense Trans-sialidase Family

Trans-sialidases are key enzymes in the life cycle of African trypanosomes in both, mammalian host and insect vector and have been associated with the disease trypanosomiasis, namely sleeping sickness and nagana. Besides the previously reported TconTS1, we have identified three additional active trans-sialidases, TconTS2, TconTS3 and TconTS4, and three trans-sialidase like genes in Trypanosoma congolense. At least TconTS1, TconTS2 and TconTS4 are found in the bloodstream of infected animals. We have characterised the enzymatic properties of recombinant proteins expressed in eukaryotic fibroblasts using fetuin as model blood glycoprotein donor substrate. One of the recombinant trans-sialidases, TconTS2, had the highest specific activity reported thus far with very low sialidase activity. The active trans-sialidases share all the amino acids critical for the catalytic reaction with few variations in the predicted binding site for the leaving or acceptor glycan. However, these differences cannot explain the orders of magnitudes between their transfer activities, which must be due to other unidentified structural features of the proteins or substrates selectivity. Interestingly, the phylogenetic relationships between the lectin domains correlate with their specific trans-sialylation activities. This raises the question whether and how the lectin domains regulate the trans-sialidase reaction. The identification and enzymatic characterisation of the trans-sialidase family in T. congolense will contribute significantly towards the understanding of the roles of these enzymes in the pathogenesis of Animal African Trypanosomiasis.     

Surface-associated proteins of Staphylococcus aureus: Their possible roles in virulence

Abstract A class of proteins that are associated with the cell surface of Gram-positive bacteria has been recognised. Common structural features which are implicated in the proper secretion and attachment of these proteins to the cell surface occur in the C-termini. N-terminal domains interact with the host by binding to soluble host proteins, to matrix proteins or to host cells. They probably have important roles in pathogenicity by allowing bacteria to avoid host defences and by acting as adhesins. Four such proteins of Staphylococcus aureus have been characterised: protein A (immunoglobulin binding protein), fibronectin binding proteins, collagen binding protein and the fibrinogen binding protein (clumping factor). Site-specific mutants are being used to define their roles in pathogenesis in in vitro and in vivo models of adherence and infection.     

Physiological relevance of the endogenous mono(ADP-ribosyl)ation of cellular proteins

The mono(ADP-ribosyl)ation reaction is a post-translational modification that is catalysed by both bacterial toxins and eukaryotic enzymes, and that results in the transfer of ADP-ribose from betaNAD+ to various acceptor proteins. In mammals, both intracellular and extracellular reactions have been described; the latter are due to glycosylphosphatidylinositol-anchored or secreted enzymes that are able to modify their targets, which include the purinergic receptor P2X7, the defensins and the integrins. Intracellular mono(ADP-ribosyl)ation modifies proteins that have roles in cell signalling and metabolism, such as the chaperone GRP78/BiP, the beta-subunit of heterotrimeric G-proteins and glutamate dehydrogenase. The molecular identification of the intracellular enzymes, however, is still missing. A better molecular understanding of this reaction will help in the full definition of its role in cell physiology and pathology.     

Avirulence proteins from haustoria-forming pathogens

A major insight that has emerged in the study of haustoria-forming plant pathogens over the last few years is that these eukaryotic biotrophs deliver suites of secreted proteins into host cells during infection. This insight has largely derived from successful efforts to identify avirulence (Avr) genes and their products from these pathogens. These Avr genes, identified from a rust and a powdery mildew fungus and three oomycete species, encode small proteins that are recognized by resistance proteins in the host plant cytoplasm, suggesting that they are transported inside plant cells during infection. These Avr proteins probably represent examples of fungal and oomycete effector proteins with important roles in subverting host cell biology during infection. In this respect, they represent a new opportunity to understand the basis of disease caused by these biotrophic pathogens. Elucidating how these pathogen proteins gain entry into plant cells and their biological function will be key questions for future research.     

What a Difference a Dalton Makes: Bacterial Virulence Factors Modulate Eukaryotic Host Cell Signaling Systems via Deamidation

SUMMARY

Pathogenic bacteria commonly deploy enzymes to promote virulence. These enzymes can modulate the functions of host cell targets. While the actions of some enzymes can be very obvious (e.g., digesting plant cell walls), others have more subtle activities. Depending on the lifestyle of the bacteria, these subtle modifications can be crucially important for pathogenesis. In particular, if bacteria rely on a living host, subtle mechanisms to alter host cellular function are likely to dominate. Several bacterial virulence factors have evolved to use enzymatic deamidation as a subtle posttranslational mechanism to modify the functions of host protein targets. Deamidation is the irreversible conversion of the amino acids glutamine and asparagine to glutamic acid and aspartic acid, respectively. Interestingly, all currently characterized bacterial deamidases affect the function of the target protein by modifying a single glutamine residue in the sequence. Deamidation of target host proteins can disrupt host signaling and downstream processes by either activating or inactivating the target. Despite the subtlety of this modification, it has been shown to cause dramatic, context-dependent effects on host cells. Several crystal structures of bacterial deamidases have been solved. All are members of the papain-like superfamily and display a cysteine-based catalytic triad. However, these proteins form distinct structural subfamilies and feature combinations of modular domains of various functions. Based on the diverse pathogens that use deamidation as a mechanism to promote virulence and the recent identification of multiple deamidases, it is clear that this enzymatic activity is emerging as an important and widespread feature in bacterial pathogenesis.     

Patatin‐like phospholipases in microbial infections with emerging roles in fatty acid metabolism and immune regulation by Apicomplexa

Emerging lipidomic technologies have enabled researchers to dissect the complex roles of phospholipases in lipid metabolism, cellular signaling and immune regulation. Host phospholipase products are involved in stimulating and resolving the inflammatory response to pathogens. While many pathogen‐derived phospholipases also manipulate the immune response, they have recently been shown to be involved in lipid remodeling and scavenging during replication. Animal and plant hosts as well as many pathogens contain a family of patatin‐like phospholipases, which have been shown to have phospholipase A₂ activity. Proteins containing patatin‐like phospholipase domains have been identified in protozoan parasites within the Apicomplexa phylum. These parasites are the causative agents of some of the most widespread human diseases. Malaria, caused by Plasmodium spp., kills nearly half a million people worldwide each year. Toxoplasma and Cryptosporidium infect millions of people each year with lethal consequences in immunocompromised populations. Parasite‐derived patatin‐like phospholipases are likely effective drug targets and progress in the tools available to the Apicomplexan field will allow for a closer look at the interplay of lipid metabolism and immune regulation during host infection.     

Structure and function of clostridial phospholipases C

A range of clostridial species produce phospholipases C. The zinc metallo phospholipases C have related sequences but different properties. All of these enzymes may be arranged, like alpha-toxin as two-domain proteins. Differences in enzymatic, haemolytic and toxic properties may be explained by differences in amino acids at key positions.     

Deubiquitinating enzymes--the importance of driving in reverse along the ubiquitin-proteasome pathway

Ubiquitination of proteins is now recognized to target proteins for degradation by the proteasome and for internalization into the lysosomal system, as well as to modify functions of some target proteins. Although much progress has been made in characterizing enzymes that link ubiquitin to proteins, our understanding of deubiquitinating enzymes is less developed. These enzymes are involved in processing the products of ubiquitin genes which all encode fusion proteins, in negatively regulating the functions of ubiquitination (editing), in regenerating free ubiquitin after proteins have been targeted to the proteasome or lysosome (recycling) and in salvaging ubiquitin from possible adducts formed with small molecule nucleophiles in the cell. A large number of genes encode deubiquitinating enzymes suggesting that many have highly specific and regulated functions. Indeed, recent findings provide strong support for the concept that ubiquitination is regulated by both specific pathways of ubiquitination and deubiquitination. Interestingly, many of these enzymes are localized to subcellular structures or to molecular complexes. These localizations play important roles in determining specificity of function and can have major influences on their catalytic activities. Future studies, particularly aimed at characterizing the interacting partners and potential substrates in these complexes as well as at determining the effects of loss of function of specific deubiquitinating enzymes will rapidly advance our understanding of the important roles of these enzymes as biological regulators.     

GPI-anchored proteins: now you see 'em, now you don't.

Many cell surface proteins are attached to membranes via covalent glycosylphosphatidylinositol (GPI) anchors that are posttranslationally linked to the carboxy-terminus of the protein. Removal of the GPI lipid moieties by enzymes such as GPI-specific phospholipases or by chemical treatments generates a soluble form of the protein that no longer associates with lipid bilayers. We have found that the removal of lipid moieties from the anchor can also have a second, unexpected effect on the antigenicity of a variety of GPI-anchored surface molecules, suggesting that they undergo major conformational changes. Several antibodies raised against GPI-anchored proteins from protozoa and mammalian cells were no longer capable of binding the corresponding antigens once the lipid moieties had been removed. Conversely, antibodies raised against soluble (delipidated) forms reacted poorly with intact GPI-anchored proteins, but showed enhanced binding after treatment with phospholipases. In the light of these findings, we have reevaluated a number of publications on GPI-anchored proteins. Many of the results are best explained by lipid-dependent changes in antigenicity, indicating this might be a widespread phenomenon. Since many pathogen surface proteins are GPI-anchored, researchers should be aware that the presence or absence of the GPI lipid moieties may have a major impact on the host immune response to infection or vaccination.     

Multiple phosphoinositide-specific phospholipases C in oat roots: characterization and partial purification

Phosphoinositide-specific phospholipases C are critical enzymes in the transduction of hormonal and environmental signals into animal cells. Several different isozymes of phospholipase C are known in mammalian systems which differ in their expression and regulation. Elucidation of the regulation of these phospholipases C has greatly advanced our understanding of the control of mammalian cell growth and development. Plant cells, too, contain phospholipases C specific for phosphoinositides, and there is evidence that they may be involved in plant cell responses to environmental stimuli. This paper reports that there are at least four variants of phosphoinositide-specific phospholipase C in the roots of oat seedlings, two cytosolic and two plasma membrane-associated. The two cytosolic and two plasma membrane variants can be separated on the basis of their affinity for binding to heparin. Both the cytosolic and the plasma membrane heparin-binding forms have apparent molecular weights of about 50–70 kDa by size exclusion chromatography. The two heparin-binding forms have been partially purified. The partially purified enzymes are activated by micromolar calcium and are specific for phosphorylated phosphoinositides; in their substrate specificities they resemble mammalian phospholipases C ɛ.     

New insights into the functions of anthrax toxin

Anthrax is the disease caused by the Gram-positive bacterium Bacillus anthracis. Two toxins secreted by B. anthracis - lethal toxin (LT) and oedema toxin (OT) - contribute significantly to virulence. Although these toxins have been studied for half a century, recent evidence indicates that LT and OT have several roles during infection not previously ascribed to them. Research on toxin-induced effects other than cytolysis of target cells has revealed that LT and OT influence cell types previously thought to be insensitive to toxin. Multiple host factors that confer sensitivity to anthrax toxin have been identified recently, and evidence indicates that the toxins probably contribute to colonisation and invasion of the host. Additionally, the toxins are now known to cause a wide spectrum of tissue and organ pathophysiologies associated with anthrax. Taken together, these new findings indicate that anthrax-toxin-associated pathogenesis is much more complex than has been traditionally recognised.     

Phospholipase D in cell signalling and its relationship to phospholipase C

Phospholipases C and D are phosphodiesterases which act on phospholipid head groups. Although the presence of these enzymes in living organisms has long been known, it is only recently that their role in cell signal transduction has been appreciated. The new developments on phospholipases D (PLD) are especially noteworthy, since these enzymes catalyze a novel pathway for second messenger generation. In a variety of mammalian cell systems, several biological or chemical agents have recently been shown to stimulate PLD activity. Depending on the system, activation of PLD has been suggested to be either dependent on, or independent of, Ca2+ and protein kinase C. PLD primarily hydrolyses phosphatidylcholine (PC) but phosphatidylinositol and phosphatidylethanolamine have also been reported as substrates. Different forms of endogenous PLD may also exist in cells. Exogenous addition of PLD causes alterations in cellular functions. In many instances, Ca2+ mobilizing agonists may stimulate both PLC and PLD pathways. Interestingly, several metabolites of these two enzymes are second messengers and are common to both pathways (e.g. phosphatidic acid, diglyceride). This has raised the issue of the interrelationship between these pathways. The regulation of either PLC or PLD by cellular components, e.g. guanine nucleotide binding proteins or protein kinases, is under intense investigation. These recent advances are providing novel information on the significance of phospholipase C and D mediated phospholipid turnover in cellular signalling. This review highlights some of these new discoveries and emerging issues, as well as challenges for future research on phospholipases.     

Genome-wide analysis of eukaryotic twin CX9C proteins

Twin CX(9)C proteins are eukaryotic proteins that derive their name from their characteristic motif, consisting of two pairs of cysteines that form two disulfide bonds stabilizing a coiled coil-helix-coiled coil-helix (CHCH) fold. The best characterized of these proteins are Cox17, a copper chaperone acting in cytochrome c oxidase biogenesis, and Mia40, the central component of a system for protein import into the mitochondrial inter-membrane space (IMS). However, the range of possible functions for these proteins is unclear. Here, we performed a systematic search of twin CX(9)C proteins in eukaryotic organisms, and classified them into groups of putative homologues, by combining bioinformatics methods with literature analysis. Our results suggest that the functions of most twin CX(9)C proteins vary around the common theme of playing a scaffolding role, which can tie their observed roles in mitochondrial structure and function. This study will enhance the present annotation of eukaryotic proteomes, and will provide a rational basis for future experimental work aimed at a deeper understanding of this remarkable class of proteins.