Sphingolipids: Regulators of azole drug resistance and fungal pathogenicity

In recent years, the role of sphingolipids in pathogenic fungi, in terms of pathogenicity and resistance to azole drugs, has been a rapidly growing field. This review describes evidence about the roles of sphingolipids in azole resistance and fungal virulence. Sphingolipids can serve as signaling molecules that contribute to azole resistance through modulation of the expression of drug efflux pumps. They also contribute to azole resistance by participating in various microbial pathways such as the unfolded protein response (UPR), pH-responsive Rim pathway, and pleiotropic drug resistance (PDR) pathway. In addition, sphingolipid signaling and eisosomes also coordinately regulate sphingolipid biosynthesis in response to azole-induced membrane stress. Sphingolipids are important for fungal virulence, playing roles during growth in hosts under stressful conditions, maintenance of cell wall integrity, biofilm formation, and production of various virulence factors. Finally, we discuss the possibility of exploiting fungal sphingolipids for the development of new therapeutic strategies to treat infections caused by pathogenic fungi.     

Hybrid histidine kinases in pathogenic fungi

Histidine kinases (HK) sense and transduce via phosphorylation events many intra‐ and extracellular signals in bacteria, archaea, slime moulds and plants. HK are also widespread in the fungal kingdom, but their precise roles in the regulation of physiological processes remain largely obscure. Expanding genomic resources have recently given the opportunity to identify uncharacterised HK family members in yeasts and moulds and now allow proposing a complex classification of Basidiomycota, Ascomycota and lower fungi HK. A growing number of genetic approaches have progressively provided new insight into the role of several groups of HK in prominent fungal pathogens. In particular, a series of studies have revealed that members of group III HK, which occur in the highest number of fungal species and contain a unique N‐terminus region consisting of multiple HAMP domain repeats, regulate morphogenesis and virulence in various human, plant and insect pathogenic fungi. This research field is further supported by recent shape‐function studies providing clear correlation between structural properties and signalling states in group III HK. Since HK are absent in mammals, these represent interesting fungal target for the discovery of new antifungal drugs.     

Deciphering cell wall integrity signalling in Aspergillus fumigatus: identification and functional characterization of cell wall stress sensors and relevant Rho GTPases

The fungal cell wall, a conserved and highly dynamic structure, is essential for virulence and viability of fungal pathogens and is an important antifungal drug target. The cell wall integrity (CWI) signalling pathway regulates shape and biosynthesis of the cell wall. In this work we identified, localized and functionally characterized four putative CWI stress sensors of Aspergillus fumigatus, an airborne opportunistic human pathogen and the cause of invasive aspergillosis. We show that Wsc1 is specifically required for resistance to echinocandin antifungals. MidA is specifically required for elevated temperature tolerance and resistance to the cell wall perturbing agents congo red and calcofluor white. Wsc1, Wsc3 and MidA additionally have overlapping functions and are redundantly required for radial growth and conidiation. We have also analysed the roles of three Rho GTPases that have been implicated in CWI signalling in other fungi. We show that Rho1 is essential and that conditional downregulation of rho1 or deletion of rho2 or rho4 results in severely impaired CWI. Our data indicate significant functional differences between the CWI stress sensors of yeasts and moulds.     

Sphingolipids and glycerophospholipids – The “ying and yang” of lipotoxicity in metabolic diseases

Sphingolipids in general and ceramides in particular, contribute to pathophysiological mechanisms by modifying signalling and metabolic pathways. Here, we present the available evidence for a bidirectional homeostatic crosstalk between sphingolipids and glycerophospholipids, whose dysregulation contributes to lipotoxicity induced metabolic stress. The initial evidence for this crosstalk originates from simulated models designed to investigate the biophysical properties of sphingolipids in plasma membrane representations. In this review, we reinterpret some of the original findings and conceptualise them as a sort of “ying/yang” interaction model of opposed/complementary forces, which is consistent with the current knowledge of lipid homeostasis and pathophysiology. We also propose that the dysregulation of the balance between sphingolipids and glycerophospholipids results in a lipotoxic insult relevant in the pathophysiology of common metabolic diseases, typically characterised by their increased ceramide/sphingosine pools.     

Lipid signalling in plant responses to abiotic stress

Lipids are one of the major components of biological membranes including the plasma membrane, which is the interface between the cell and the environment. It has become clear that membrane lipids also serve as substrates for the generation of numerous signalling lipids such as phosphatidic acid, phosphoinositides, sphingolipids, lysophospholipids, oxylipins, N‐acylethanolamines, free fatty acids and others. The enzymatic production and metabolism of these signalling molecules are tightly regulated and can rapidly be activated upon abiotic stress signals. Abiotic stress like water deficit and temperature stress triggers lipid‐dependent signalling cascades, which control the expression of gene clusters and activate plant adaptation processes. Signalling lipids are able to recruit protein targets transiently to the membrane and thus affect conformation and activity of intracellular proteins and metabolites. In plants, knowledge is still scarce of lipid signalling targets and their physiological consequences. This review focuses on the generation of signalling lipids and their involvement in response to abiotic stress. We describe lipid‐binding proteins in the context of changing environmental conditions and compare different approaches to determine lipid–protein interactions, crucial for deciphering the signalling cascades.     

Consequences of lipid raft association on G-protein-coupled receptor function

GPCRs (G-protein-coupled receptors) play key roles in many cellular processes, and malfunction may lead to a range of pathologies, including psychiatric and neurological disorders. It is therefore not surprising that this group of receptors supplies a majority of the targets for pharmaceutical drug development. Despite their importance, the mechanisms that regulate their function and signalling still remain only partially understood. Recently, it has become evident that a subset of GPCRs is not homogeneously distributed in the plasma membrane, but localizes instead to specific membrane microdomains known as lipid rafts. Lipid rafts are characterized by their enrichment in cholesterol and sphingolipids, and have been suggested to serve as platforms for a range of cellular signalling complexes. In the present review, we will be discussing the effects of the lipid raft environment on trafficking, signalling and internalization of raft-associated GPCRs.     

Lipid metabolism in cancer

Lipids form a diverse group of water-insoluble molecules that include triacylglycerides, phosphoglycerides, sterols and sphingolipids. They play several important roles at cellular and organismal levels. Fatty acids are the major building blocks for the synthesis of triacylglycerides, which are mainly used for energy storage. Phosphoglycerides, together with sterols and sphingolipids, represent the major structural components of biological membranes. Lipids can also have important roles in signalling, functioning as second messengers and as hormones. There is increasing evidence that cancer cells show specific alterations in different aspects of lipid metabolism. These alterations can affect the availability of structural lipids for the synthesis of membranes, the synthesis and degradation of lipids that contribute to energy homeostasis and the abundance of lipids with signalling functions. Changes in lipid metabolism can affect numerous cellular processes, including cell growth, proliferation, differentiation and motility. This review will examine some of the alterations in lipid metabolism that have been reported in cancer, at both cellular and organismal levels, and discuss how they contribute to different aspects of tumourigenesis.     

Monitoring Diel Variations of Physiological Status and Bacterial Diversity in an Estuarine Microbial Mat: An Integrated Biomarker Analysis

Microbial mats are highly productive microbial systems and a source of not-yet characterized microorganisms and metabolic strategies. In this article, we introduced a lipid biomarker/microbial isolation approach to detect short-term variations of microbial diversity, physiological and redox status, and also characterize lipid biomarkers from specific microbial groups that can be further monitored. Phospholipid fractions (PLFA) were examined for plasmalogens, indicative of certain anaerobes. The glycolipid fraction was processed for polyhydroxyalkanoates (PHA) and the neutral lipid fraction was used to evaluate respiratory quinone content. Data demonstrate an increase in the metabolic stress, unbalanced growth, proportion of anaerobic bacteria and respiratory rate after the maximal photosynthetic activity. Higher accumulation of polyhydroxyalkanoates at the same sampling point also suggested a situation of carbon storage by heterotrophs closely related to photosynthetic microorganisms. Besides, the characterization of lipid biomarkers (plasmalogens, sphingolipids) from specific microbial groups provided clues about the dynamics and diversity of less-characterized mat members. In this case, lipid analyses were complemented by the isolation and characterization of anaerobic spore formers and sulfate reducers to obtain insight into their affiliation and lipid composition. The results revealed that temporal shifts in lipid biomarkers are indicative of an intense change in the physiology, redox condition, and community composition along the diel cycle, and support the hypothesis that interactions between heterotrophs and primary producers play an important role in the carbon flow in microbial mats. Dedicated to the memory of David C. White.     

Fungal pathogenicity

Successful penetration of living plant tissue by fungal pathogens is preceded by an exchange of signals between both organisms. Recent mutational approaches revealed the importance of cAMP-dependent signalling pathways for fungal development and virulence on their hosts.     

Biochemistry of insect epicuticle degradation by entomopathogenic fungi

The biochemical interaction between fungal pathogens and their insect host epicuticle was studied by examining fungal hydrocarbon degrading ability. As a contact insecticide, entomopathogenic fungi invade their host through the cuticle, covered by an outermost lipid layer mainly composed of highly stable, very long chain structures. Strains of Beauveria bassiana and Metarhizium anisopliae (Deuteromycotina: Hyphomycetes), pathogenic both to the blood-sucking bug Triatoma infestans (Hemiptera: Reduviidae) and the bean-weevil Acanthoscelides obtectus (Coleoptera, Bruchidae), were grown on different carbon sources. Alkane-grown cells showed a lipid pattern different from that of glucose-grown cells, evidenced by a major switch in the triacylglycerol and sterol components. Radiolabelled hydrocarbons were used to investigate the catabolic pathway and the by-product incorporation into fungal cellular components. The first oxidation round is presumably carried out by a cytochrome P450 enzyme system, the metabolites will traverse the peroxisomal membrane, and after successive transformations will eventually provide the appropriate fatty acyl CoA for complete degradation in the peroxisomes, the site of beta-oxidation in fungi. In this review, we will show the relationship between fungal ability to catabolize very long chain hydrocarbons and virulence parameters.     

The modulation of gap-junctional intercellular communication by lipid rafts

Lipid rafts are specific microdomains of plasma membrane which are enriched in cholesterol and sphingolipids. These domains seem to favour the interactions of particular proteins and the regulation of signalling pathways in the cells. Recent data have shown that among the proteins, which are preferentially localized in lipid rafts, are connexins that are the structural proteins of gap junctions. Since gap junctional intercellular communication is involved in various cellular processes and pathologies such as cancer, we were interested to review the various observations concerning this specific localization of connexins in lipid rafts and its consequences on gap junctional intercellular communication capacity. In particular, we will focus our discussion on the role of the lipid raft-connexin connection in cancer progression. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.     

Sphingolipid signalling domains floating on rafts or buried in caves?

Ceramide is a novel lipid mediator involved in regulating cell growth, cell differentiation and cell death. Many studies have focused on characterizing the stimulus-induced production of ceramide and identifying putative downstream molecular targets. However, little remains known about the localization of the regulated production of ceramide through sphingomyelin metabolism in the plasma membrane. Additionally, it is unclear whether a localized increase in ceramide concentration is necessary to facilitate downstream signalling events initiated by this lipid. Recent studies have suggested that detergent-insoluble plasma membrane domains may be highly localized sites for initiating signal transduction cascades by both tyrosine kinase and sphingolipid signalling pathways. These domains are typically enriched in both sphingolipids and cholesterol and have been proposed to form highly ordered lipid rafts floating in a sea of glycerophospholipids. Alternatively, upon integration of the cholesterol binding protein caveolin, these domains may also form small cave-like structures called caveolae. Emerging evidence suggests that the enhanced sphingomyelin content of these lipid domains make them potential substrate pools for sphingomyelinases to produce a high local concentration of ceramide. The subsequent formation of ceramide microdomains in the plasma membrane may be a critical factor in regulating downstream signalling through this lipid messenger.     

Sphingolipids as modulators of membrane proteins

The diversity of the transmembranome of higher eukaryotes is matched by an enormous diversity of sphingolipid classes and molecular species. The intrinsic properties of sphingolipids are not only suited for orchestrating lateral architectures of biological membranes, but their molecular distinctions also allow for the evolution of protein motifs specifically recognising and interacting with individual lipids. Although various reports suggest a role of sphingolipids in membrane protein function, only a few cases have determined the specificity of these interactions. In this review we discuss examples of specific protein–sphingolipid interactions for which a modulator-like dependency on sphingolipids was assigned to specific proteins. These novel functions of sphingolipids in specific protein–lipid assemblies contribute to the complexity of the sphingolipid classes and other molecular species observed in animal cells. This article is part of a Special Issue entitled New Frontiers in Sphingolipid Biology.     

Lipid metabolism in Cryptococcus neoformans

In recent years, lipids have been shown to act as signalling molecules not only in mammalian cells but also in many other eukaryotes. Whereas in mammalian cells lipids regulate cellular functions that play crucial roles in the regulation of pathobiological processes, such as cancer, cardiovascular and neurodegenerative disorders, and inflammation, in the fungus Cryptococcus neoformans lipids play key roles in the regulation of pathogenic traits required for the development of cryptococcosis, an infectious disease particularly frequent in immunocompromised individuals. In this minireview we discuss recent advances in the understanding of lipid metabolism in this important human pathogen, highlighting the potential of fungal lipid enzymatic pathways as promising new drug targets.     

Signalling pathways in the pathogenesis of Cryptococcus

Efficient communication with the environment is critical for all living organisms. Fungi utilize complex signalling systems to sense their environments and control proliferation, development and in some cases virulence. Well-studied signalling pathways include the protein kinase A/cyclic AMP (cAMP), protein kinase C (PKC)/mitogen-activated protein kinase (MAPK), lipid signalling cascades, and the calcium–calcineurin signalling pathway. The human pathogenic basidiomycetous fungus Cryptococcus neoformans deploys sensitive signalling systems to survive in the human host, leading to life-threatening meningoencephalitis. Known virulence traits of this fungus, including the antioxidant melanin production, the antiphagocytic polysaccharide capsule and the ability to grow at 37°C, are orchestrated by complex signalling networks, whose understanding is crucial to better treat, diagnose and prevent cryptococcosis.     

Chemical synthesis of fluorescent glycero- and sphingolipids

Glycero- and sphingolipids have been shown to be building blocks of membranes and lipoproteins, metabolites and important intermediaries in the signalling cascades involved in stress responses, proliferation of cells and also apoptosis. Investigations into the exact functions of these lipids have found that they are fundamentally more important than previously thought and that they are intricately involved in the processes of many significant metabolic pathways and diseases. Investigation of these functions requires the detection of the lipids in their natural environment within membranes. To this end, fluorescent labelling has become one of the preferred means in which to study these essential components due to the relative ease of detection. This review will look at the novel compounds that have been synthesised recently through various methodologies including classical lipid synthesis as well as the innovative application of organometallic chemistry. This field has expanded with the advancements in fluorescence detection and these lipids are being used as specific probes for an extensive range of applications in order to ascertain the mechanisms and signalling capabilities of this very important class of biological compounds.     

Lipid signalling in disease

Signalling lipids such as eicosanoids, phosphoinositides, sphingolipids and fatty acids control important cellular processes, including cell proliferation, apoptosis, metabolism and migration. Extracellular signals from cytokines, growth factors and nutrients control the activity of a key set of lipid-modifying enzymes: phospholipases, prostaglandin synthase, 5-lipoxygenase, phosphoinositide 3-kinase, sphingosine kinase and sphingomyelinase. These enzymes and their downstream targets constitute a complex lipid signalling network with multiple nodes of interaction and cross-regulation. Imbalances in this network contribute to the pathogenesis of human disease. Although the function of a particular signalling lipid is traditionally studied in isolation, this review attempts a more integrated overview of the key role of these signalling lipids in inflammation, cancer and metabolic disease, and discusses emerging strategies for therapeutic intervention.     

Lipid rafts as platforms for sphingosine 1-phosphate metabolism and signalling

Spontaneous segregation of cholesterol and sphingolipids as a liquid-ordered phase leads to their clustering in selected membrane areas, the lipid rafts. These specialized membrane domains enriched in gangliosides, sphingomyelin, cholesterol and selected proteins involved in signal transduction, organize and determine the function of multiprotein complexes involved in several aspects of signal transduction, thus regulating cell homeostasis. Sphingosine 1-phosphate, an important biologically active mediator, is involved in several signal transduction processes regulating a plethora of cell functions and, not only several of its downstream effectors tend to localize in lipid rafts, some of the enzymes involved in its pathway, of receptors involved in its signalling and its transporters have been often found in these membrane microdomains. Considering this, in this review we address what is currently known regarding the relationship between sphingosine 1-phosphate metabolism and signalling and plasma membrane lipid rafts.