Transcriptomics technologies are the techniques used to study an organism’s transcriptome, the sum of all of its RNA transcripts. The information content of an organism is recorded in the DNA of its genome and expressed through transcription. Here, mRNA serves as a transient intermediary molecule in the information network, whilst noncoding RNAs perform additional diverse functions. A transcriptome captures a snapshot in time of the total transcripts present in a cell.The first attempts to study the whole transcriptome began in the early 1990s, and technological advances since the late 1990s have made transcriptomics a widespread discipline. Transcriptomics has been defined by repeated technological innovations that transform the field. There are two key contemporary techniques in the field: microarrays, which quantify a set of predetermined sequences, and RNA sequencing (RNA-Seq), which uses high-throughput sequencing to capture all sequences.Measuring the expression of an organism’s genes in different tissues, conditions, or time points gives information on how genes are regulated and reveals details of an organism’s biology. It can also help to infer the functions of previously unannotated genes. Transcriptomic analysis has enabled the study of how gene expression changes in different organisms and has been instrumental in the understanding of human disease. An analysis of gene expression in its entirety allows detection of broad coordinated trends which cannot be discerned by more targeted assays.
Transition metal ions are essential nutrients to all forms of life. Iron, copper, zinc, manganese, cobalt and nickel all have unique chemical and physical properties that make them attractive molecules for use in biological systems. Many of these same properties that allow these metals to provide essential biochemical activities and structural motifs to a multitude of proteins including enzymes and other cellular constituents also lead to a potential for cytotoxicity. Organisms have been required to evolve a number of systems for the efficient uptake, intracellular transport, protein loading and storage of metal ions to ensure that the needs of the cells can be met while minimizing the associated toxic effects. Disruptions in the cellular systems for handling transition metals are observed as a number of diseases ranging from hemochromatosis and anemias to neurodegenerative disorders including Alzheimer's and Parkinson's disease. The yeast Saccharomyces cerevisiae has proved useful as a model organism for the investigation of these processes and many of the genes and biological systems that function in yeast metal homeostasis are conserved throughout eukaryotes to humans. This review focuses on the biological roles of iron, copper, zinc, manganese, nickel and cobalt, the homeostatic mechanisms that function in S. cerevisiae and the human diseases in which these metals have been implicated.
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.
bIn recent decades, pathogenic fungi have become a serious threat to human health, leading to major efforts aimed at characterizing new agents for improved treatments. Promising in this context are antimicrobial peptides produced by animals and plants as part of innate immune systems. Here, we describe an antifungal defensin, NaD1, with activity against the major human pathogen Candida albicans, characterize the mechanism of killing, and identify protection strategies used by the fungus to survive defensin treatment. The mechanism involves interaction between NaD1 and the fungal cell surface followed by membrane permeabilization, entry into the cytoplasm, hyperproduction of reactive oxygen species, and killing induced by oxidative damage. By screening C. albicans mutant libraries, we identified that the high-osmolarity glycerol (HOG) pathway has a unique role in protection against NaD1, while several other stress-responsive pathways are dispensable. The involvement of the HOG pathway is consistent with induction of oxidative stress by NaD1. The HOG pathway has been reported to have a major role in protection of fungi against osmotic stress, but our data indicate that osmotic stress does not contribute significantly to the adverse effects of NaD1 on C. albicans. Our data, together with previous studies with human beta-defensins and salivary histatin 5, indicate that inhibition of the HOG pathway holds promise as a broad strategy for increasing the activity of antimicrobial peptides against C. albicans.
Extracellular vesicles (EVs) are membranous vesicles that are released by cells. In this study, the role of the Endosomal Sorting Complex Required for Transport (ESCRT) machinery in the biogenesis of yeast EVs was examined. Knockout of components of the ESCRT machinery altered the morphology and size of EVs as well as decreased the abundance of EVs. In contrast, strains with deletions in cell wall biosynthesis genes, produced more EVs than wildtype. Proteomic analysis highlighted the depletion of ESCRT components and enrichment of cell wall remodelling enzymes, glucan synthase subunit Fks1 and chitin synthase Chs3, in yeast EVs. Interestingly, EVs containing Fks1 and Chs3 rescued the yeast cells from antifungal molecules. However, EVs from fks1 ∆ or chs3 ∆ or the vps23 ∆ chs3 ∆ double knockout strain were unable to rescue the yeast cells as compared to vps23 ∆ EVs. Overall, we have identified a potential role for yeast EVs in cell wall remodelling.
Defensins are a class of ubiquitously expressed cationic antimicrobial peptides (CAPs) that play an important role in innate defense. Plant defensins are active against a broad range of microbial pathogens and act via multiple mechanisms, including cell membrane permeabilization. The cytolytic activity of defensins has been proposed to involve interaction with specific lipid components in the target cell wall or membrane and defensin oligomerization. Indeed, the defensin Nicotiana alata defensin 1 (NaD1) binds to a broad range of membrane phosphatidylinositol phosphates and forms an oligomeric complex with phosphatidylinositol (4,5)-bisphosphate (PIP2) that facilitates membrane lysis of both mammalian tumor and fungal cells. Here, we report that the tomato defensin TPP3 has a unique lipid binding profile that is specific for PIP2 with which it forms an oligomeric complex that is critical for cytolytic activity. Structural characterization of TPP3 by X-ray crystallography and site-directed mutagenesis demonstrated that it forms a dimer in a "cationic grip" conformation that specifically accommodates the head group of PIP2 to mediate cooperative higher-order oligomerization and subsequent membrane permeabilization. These findings suggest that certain plant defensins are innate immune receptors for phospholipids and adopt conserved dimeric configurations to mediate PIP2 binding and membrane permeabilization. This mechanism of innate defense may be conserved across defensins from different species. P lant defensins are small (ϳ5 kDa), cysteine-rich, cationic peptides that belong to the broad class of innate defense molecules known as cationic antimicrobial peptides (CAPs). Plant defensins play a major role in plant innate immunity and have been identified in all analyzed plant species to date, as either constitutively expressed or induced defense molecules that are produced in response to pathogenic attack or environmental stress (1). The tertiary structure of all plant defensins is highly conserved, comprising a triple-stranded antiparallel ␤-sheet and a single ␣-helix stabilized by four disulfide bridges, known as the "cysteine-stabilized alpha-beta" or "CS␣␤" motif (2). Despite this conserved three-dimensional structure, there is a high degree of variation in the primary sequence of plant defensins, particularly at intervening loop regions, which are typically important for activity (3).Many plant defensins have antifungal activity, but other functions have been reported, including antibacterial activity, ion channel blocking, protein synthesis inhibition, and trypsin and ␣-amylase inhibition as well as roles in heavy metal tolerance, plant development, and pollen tube guidance (3-5). They can be divided into two classes based on whether or not a C-terminal propeptide (CTPP) (of ϳ33 amino acids) is present (2, 6). This domain is involved in vacuolar targeting and protects the plant cells from phytotoxicity during transit through the secretory pathway (7). Defensins expressed with the additional CTPP domain are kn...
Extracellular vesicles (EVs) represent a system for the coordinated secretion of a variety of molecular cargo including proteins, lipids, nucleic acids, and metabolites. They have an essential role in intercellular communication in multicellular organisms and have more recently been implicated in host-pathogen interactions. Study of the role for EVs in fungal biology has focused on pathogenic yeasts that are major pathogens in humans. In this study we have expanded the investigation of fungal EVs to plant pathogens, specifically the major cotton pathogen Fusarium oxysporum f. sp. vasinfectum. EVs isolated from F. oxysporum f. sp. vasinfectum culture medium have a morphology and size distribution similar to EVs from yeasts such as Candida albicans and Cryptococcus neoformans. A unique feature of the EVs from F. oxysporum f. sp. vasinfectum is their purple color, which is predicted to arise from a napthoquinone pigment being packaged into the EVs. Proteomic analysis of F. oxysporum f. sp. vasinfectum EVs revealed that they are enriched in proteins that function in synthesis of polyketides as well as proteases and proteins that function in basic cellular processes. Infiltration of F. oxysporum f. sp. vasinfectum EVs into the leaves of cotton or N. benthamiana plants led to a phytotoxic response. These observations lead to the hypothesis that F. oxysporum f. sp. vasinfectum EVs are likely to play a crucial role in the infection process.
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