Resistance to the Plant Defensin NaD1 Features Modifications to the Cell Wall and Osmo-Regulation Pathways of Yeast

Over the last few decades, the emergence of resistance to commonly used antifungal molecules has become a major barrier to effective treatment of recurrent life-threatening fungal diseases. Resistance combined with the increased incidence of fungal diseases has created the need for new antifungals, such as the plant defensin NaD1, with different mechanisms of action to broaden treatment options. Antimicrobial peptides produced in plants and animals are promising new molecules in the arsenal of antifungal agents because they have different mechanisms of action to current antifungals and are often targeted specifically to fungal pathogens (van der Weerden et al., 2013). A key step in the development of novel antifungals is an understanding of the potential for the fungus to develop resistance. Here, we have used the prototypic plant defensin NaD1 in serial passages with the model fungus Saccharomyces cerevisiae to examine the evolution of resistance to plant antifungal peptides. The yeast strains did develop tolerance to NaD1, but it occurred more slowly than to the clinically used antifungal caspofungin. Sequencing the genomes of the strains with increased tolerance failed to identify any ‘hotspot’ mutations associated with increased tolerance to NaD1 and led to the identification of 12 genes that are involved in resistance. Characterization of the strains with increased tolerance to NaD1 also revealed changes in tolerance to abiotic stressors. Resistance developed slowly via an accumulation of single nucleotide mutations and had a fitness penalty associated with it. One of the genes identified FPS1, revealed that there is a common mechanism of resistance to NaD1 that involves the osmotic stress response pathway. These data indicate that it is more difficult to generate resistance to antimicrobial peptides such as NaD1 compared to small molecule antifungals

Nicotiana alata defensin chimeras reveal differences in the mechanism of fungal and tumor cell killing and an enhanced antifungal variant

The plant defensin NaD1 is a potent antifungal molecule that also targets tumor cells with a high efficiency. We examined the features of NaD1 that contribute to these two activities by producing a series of chimeras with NaD2, a defensin that has relatively poor activity against fungi and no activity against tumor cells. All plant defensins have a common tertiary structure known as a cysteine-stabilized alpha-beta motif which consists of an alpha helix and a triple-stranded beta-sheet stabilized by four disulfide bonds. The chimeras were produced by replacing loops 1 to 7, the sequences between each of the conserved cysteine residues on NaD1, with the corresponding loops from NaD2. The loop 5 swap replaced the sequence motif (SKILRR) that mediates tight binding with phosphatidylinositol 4,5-bisphosphate [PI(4,5)P-2] and is essential for the potent cytotoxic effect of NaD1 on tumor cells. Consistent with previous reports, there was a strong correlation between PI(4,5)P-2 binding and the tumor cell killing activity of all of the chimeras. However, this correlation did not extend to antifungal activity. Some of the loop swap chimeras were efficient antifungal molecules, even though they bound poorly to PI(4,5)P-2, suggesting that additional mechanisms operate against fungal cells. Unexpectedly, the loop 1B swap chimera was 10 times more active than NaD1 against filamentous fungi. This led to the conclusion that defensin loops have evolved as modular components that combine to make antifungal molecules with variable mechanisms of action and that artificial combinations of loops can increase antifungal activity compared to that of the natural variants

Extracellular Vesicles from Fusarium graminearum Contain Protein Effectors Expressed during Infection of Corn.

Fusarium graminearum (Fgr) is a devastating filamentous fungal pathogen that causes diseases in cereals, while producing mycotoxins that are toxic for humans and animals, and render grains unusable. Low efficiency in managing Fgr poses a constant need for identifying novel control mechanisms. Evidence that fungal extracellular vesicles (EVs) from pathogenic yeast have a role in human disease led us to question whether this is also true for fungal plant pathogens. We separated EVs from Fgr and performed a proteomic analysis to determine if EVs carry proteins with potential roles in pathogenesis. We revealed that protein effectors, which are crucial for fungal virulence, were detected in EV preparations and some of them did not contain predicted secretion signals. Furthermore, a transcriptomic analysis of corn (Zea mays) plants infected by Fgr revealed that the genes of some of the effectors were highly expressed in vivo, suggesting that the Fgr EVs are a mechanism for the unconventional secretion of effectors and virulence factors. Our results expand the knowledge on fungal EVs in plant pathogenesis and cross-kingdom communication, and may contribute to the discovery of new antifungals

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

Transition metal tolerance and the Saccharomyces cerevisiae genome

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 leads 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. The yeast Saccharomyces cerevisiae has been used as model organism for the investigation of these systems and a majority of the genes and biological systems that function in yeast metal homeostasis are conserved throughout eukaryotes to humans. Traditionally, genomic studies in metal homeostasis focus on the response to one, or in some cases two, metals. Here, I have used high density yeast arrays of a S. cerevisiae deletion collection to study the genes required for tolerance to six transition metals in parallel and I have used this data to examine the role of genes not only in the homeostasis of individual metals but to also gain insight into cellular transition metal homeostasis as a whole. Genes and pathways with novel function in the homeostasis of a particular metal have been identified along with the systems that function with broad spectrum metal specificity. Data generated in this screen has also be combined with previously published data sets that examine different aspects of yeast biology in an attempt to delve deeper in to the cellular machinery that allows yeast, and potentially the cells of other organisms, to maintain the balance between metal ions as essential nutrients as opposed to toxic moieties. Metallochaperones represent a relatively recent emerging class of proteins that play a central role in maintaining this balance. As part of the analysis of the high density array screens, putative chaperones have been identified. Additionally a yeast 2 hybrid screen using a cytoplasmic domain of the S. cerevisiae high affinity iron transporter Ftr1p has been performed to with the goal of discovering candidate iron chaperones. As a whole, the research discussed in this thesis has shed light on a number of new features of the homeostatic mechanisms that function in S. cerevisiae and will provide the basis for further investigation into the interactions between cells and metal ions eventually leading to implications in human health and disease.Medicine, Faculty ofMedical Genetics, Department ofGraduat

The nature and extent of corporate restructuring within Europe's single market: Cutting through the hype

Although we have long crossed the '1992' political threshold it is the economic forces that will determine the true extent of European integration. This article reports on the results of a survey on corporate restructuring activity, undertaken by some of Europe's leading companies, major agents for this integration process. This article looks at different structural and organisational dimensions of restructuring across a range of value added activities, and finds that in all cases a significant amount of restructuring has taken place. Whilst there has been a major push to organise activities on a pan-European basis, the change is by no means universal with some activities, such as procurement developing along more global lines whilst the sales and service activity has become more localised. Successful European restructuring is much more complex than simple choices about the degree of rationalisation or centralisation. In designing the right portfolio of restructuring activities management should give critical attention to twc factors: the dynamics of competition in the specific industries in which a company operates and its stage of organisational development in Europe.

Identification and mechanism of action of the plant defensin NaD1 as a new member of the antifungal drug arsenal against Candida albicans

In 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

SUR7 deletion in Candida albicans impacts extracellular vesicle features and delivery of virulence factors

Abstract Extracellular vesicles (EVs) from human fungal pathogens have been implicated in fungal virulence, yet little is known about their role in the host‐pathogen interaction. Progress has been hampered by the lack of a specific marker for fungal EVs that can be used to monitor EV isolation and tracking in biological systems. Here we report the effect of a SUR7 gene knockout on the production, properties, and role of EVs in the virulence of Candida albicans. Sur7 is a component of the membrane compartment of Can1 (MCC) complex and is enriched in the EVs from C. albicans and other fungal species. MCC is a plasma membrane complex which together with the eisosome, a cytoplasmic protein complex, is a key regulator in plasma membrane organisation and plasma membrane associated processes. The SUR7 knockout strain produces smaller EVs than the wild‐type (WT) with different protein and carbohydrate cargos. Furthermore, proteins with known roles in Candida pathogenesis were present in WT EVs and absent or diminished in the sur7Δ EVs. We demonstrate that the reduced virulence of the sur7Δ cells can be partially restored with EVs from a WT strain. These findings demonstrate the importance of Sur7‐like proteins in the biogenesis of EVs in fungi and enhance our understanding of the role of fungal EVs in human pathogenesis

The Plant Defensin NaD1 Enters the Cytoplasm of Candida albicans via Endocytosis

Antimicrobial peptides are widespread in nature and are produced by many organisms as a first line of defence against pathogens. These peptides have a broad range of biological activities, such as antibacterial or antifungal activities and act with varied mechanisms of action. A large number of the peptides are amphipathic α-helices which act by disrupting plasma membranes and allowing leakage of intracellular contents. However, some peptides have more complex mechanisms of action that require internalisation into the target organisms’ cytoplasm. The method by which these peptides enter the cytoplasm varies, with some requiring the energy dependent processes of endocytosis or polyamine transport and others entering via passive transport. Here we describe the mechanism that the antimicrobial peptide, the plant defensin NaD1, uses to transverse the fungal membrane and gain access to the fungal cytoplasm. By inhibiting ATP synthesis and using an inhibitor of actin polymerisation, we show that NaD1 is internalised into C. albicans yeast cells by the energy-dependent process of endocytosis

Transcriptomic databases.

<p>Transcriptomic databases.</p