Metka Lenassi

HIV Nef is secreted in exosomes and triggers apoptosis in bystander CD4+ T cells.

The HIV accessory protein negative factor (Nef) is one of the earliest and most abundantly expressed viral proteins. It is also found in the serum of infected individuals (Caby MP, Lankar D, Vincendeau‐Scherrer C, Raposo G, Bonnerot C. Exosomal‐like vesicles are present in human blood plasma. Int Immunol 2005;17:879–887). Extracellular Nef protein has deleterious effects on CD4+ T cells (James CO, Huang MB, Khan M, Garcia‐Barrio M, Powell MD, Bond VC. Extracellular Nef protein targets CD4+ T cells for apoptosis by interacting with CXCR4 surface receptors. J Virol 2004;78:3099–3109), the primary targets of HIV, and can suppress immunoglobulin class switching in bystander B cells (Qiao X, He B, Chiu A, Knowles DM, Chadburn A, Cerutti A. Human immunodeficiency virus 1 Nef suppresses CD40‐dependent immunoglobulin class switching in bystander B cells. Nat Immunol 2006;7:302–310). Nevertheless, the mode of exit of Nef from infected cells remains a conundrum. We found that Nef stimulates its own export via the release of exosomes from all cells examined. Depending on its intracellular location, these Nef exosomes form at the plasma membrane, late endosomes or both compartments in Jurkat, SupT1 and primary T cells, respectively. Nef release through exosomes is conserved also during HIV‐1 infection of peripheral blood lymphocytes (PBLs). Released Nef exosomes cause activation‐induced cell death of resting PBLs in vitro. Thus, HIV‐infected cells export Nef in bioactive vesicles, which facilitate the depletion of CD4+ T cells that is a hallmark of acquired immunodeficiency syndrome (AIDS).

Adaptation of extremely halotolerant black yeast Hortaea werneckii to increased osmolarity: a molecular perspective at a glance.

Halophilic adaptations have been studied almost exclusively on prokaryotic microorganisms. Discovery of the black yeast Hortaea werneckii as the dominant fungal species in hypersaline waters enabled the introduction of a new model organism to study the mechanisms of salt tolerance in eukaryotes. Its strategies of cellular osmotic adaptations on the physiological and molecular level revealed novel, intricate mechanisms to combat fluctuating salinity. H. werneckii is an extremely halotolerant eukaryotic microorganism and thus a promising source of transgenes for osmotolerance improvement of industrially important yeasts, as well as in crops.

Whole Genome Duplication and Enrichment of Metal Cation Transporters Revealed by De Novo Genome Sequencing of Extremely Halotolerant Black Yeast Hortaea werneckii

Hortaea werneckii, ascomycetous yeast from the order Capnodiales, shows an exceptional adaptability to osmotically stressful conditions. To investigate this unusual phenotype we obtained a draft genomic sequence of a H. werneckii strain isolated from hypersaline water of solar saltern. Two of its most striking characteristics that may be associated with a halotolerant lifestyle are the large genetic redundancy and the expansion of genes encoding metal cation transporters. Although no sexual state of H. werneckii has yet been described, a mating locus with characteristics of heterothallic fungi was found. The total assembly size of the genome is 51.6 Mb, larger than most phylogenetically related fungi, coding for almost twice the usual number of predicted genes (23333). The genome appears to have experienced a relatively recent whole genome duplication, and contains two highly identical gene copies of almost every protein. This is consistent with some previous studies that reported increases in genomic DNA content triggered by exposure to salt stress. In hypersaline conditions transmembrane ion transport is of utmost importance. The analysis of predicted metal cation transporters showed that most types of transporters experienced several gene duplications at various points during their evolution. Consequently they are present in much higher numbers than expected. The resulting diversity of transporters presents interesting biotechnological opportunities for improvement of halotolerance of salt-sensitive species. The involvement of plasma P-type H+ ATPases in adaptation to different concentrations of salt was indicated by their salt dependent transcription. This was not the case with vacuolar H+ ATPases, which were transcribed constitutively. The availability of this genomic sequence is expected to promote the research of H. werneckii. Studying its extreme halotolerance will not only contribute to our understanding of life in hypersaline environments, but should also identify targets for improving the salt- and osmotolerance of economically important plants and microorganisms.

Size Characterization and Quantification of Exosomes by Asymmetrical-Flow Field-Flow Fractionation

In the past few years extracellular vesicles called exosomes have gained huge interest of scientific community since they show a great potential for human diagnostic and therapeutic applications. However, an ongoing challenge is accurate size characterization and quantification of exosomes because of the lack of reliable characterization techniques. In this work, the emphasis was focused on a method development to size-separate, characterize, and quantify small amounts of exosomes by asymmetrical-flow field-flow fractionation (AF4) technique coupled to a multidetection system (UV and MALS). Batch DLS (dynamic light-scattering) and NTA (nanoparticle tracking analysis) analyses of unfractionated exosomes were also conducted to evaluate their shape and internal structure, as well as their number density. The results show significant influence of cross-flow conditions and channel thickness on fractionation quality of exosomes, whereas the focusing time has less impact. The AF4/UV-MALS and DLS results display the presence of two particles subpopulations, that is, the larger exosomes and the smaller vesicle-like particles, which coeluted in AF4 together with impurities in early eluting peak. Compared to DLS and AF4-MALS results, NTA somewhat overestimates the size and the number density for larger exosome population, but it discriminates the smaller particle population.

Fungal adaptation to extremely high salt concentrations.

Hypersaline environments support substantial microbial communities of selected halotolerant and halophilic organisms, including fungi from various orders. In hypersaline water of solar salterns, the black yeast Hortaea werneckii is by far the most successful fungal representative. It has an outstanding ability to overcome the turgor loss and sodium toxicity that are typical for hypersaline environments, which facilitates its growth even in solutions that are almost saturated with NaCl. We propose a model of cellular responses to high salt concentrations that integrates the current knowledge of H. werneckii adaptations. The negative impact of a hyperosmolar environment is counteracted by an increase in the energy supply that is needed to drive the energy-demanding export of ions and synthesis of compatible solutes. Changes in membrane lipid composition and cell-wall structure maintain the integrity and functioning of the stressed cells. Understanding the salt responses of H. werneckii and other fungi (e.g., the halophilic Wallemia ichthyophaga) will extend our knowledge of fungal stress tolerance and promote the use of the currently unexploited biotechnological potential of fungi that live in hypersaline environments.

Adaptation to high salt concentrations in halotolerant/halophilic fungi: a molecular perspective

Molecular studies of salt tolerance of eukaryotic microorganisms have until recently been limited to the bakers yeast Saccharomyces cerevisiae and a few other moderately halotolerant yeast. Discovery of the extremely halotolerant and adaptable fungus Hortaea werneckii and the obligate halophile Wallemia ichthyophaga introduced two new model organisms into studies on the mechanisms of salt tolerance in eukaryotes. H. werneckii is unique in its adaptability to fluctuations in salt concentrations, as it can grow without NaCl as well as in the presence of up to 5 M NaCl. On the other hand, W. ichthyophaga requires at least 1.5 M NaCl for growth, but also grows in up to 5 M NaCl. Our studies have revealed the novel and intricate molecular mechanisms used by these fungi to combat high salt concentrations, which differ in many aspects between the extremely halotolerant H. werneckii and the halophilic W. ichthyophaga. Specifically, the high osmolarity glycerol signaling pathway that is important for sensing and responding to increased salt concentrations is here compared between H. werneckii and W. ichthyophaga. In both of these fungi, the key signaling components are conserved, but there are structural and regulation differences between these pathways in H. werneckii and W. ichthyophaga. We also address differences that have been revealed from analysis of their newly sequenced genomes. The most striking characteristics associated with H. werneckii are the large genetic redundancy, the expansion of genes encoding metal cation transporters, and a relatively recent whole genome duplication. In contrast, the genome of W. ichthyophaga is very compact, as only 4884 protein-coding genes are predicted, which cover almost three quarters of the sequence. Importantly, there has been a significant increase in their hydrophobins, cell-wall proteins that have multiple cellular functions.

Novel group VII histidine kinase HwHhk7B from the halophilic fungi Hortaea werneckii has a putative role in osmosensing

Histidine kinases (HKs) are abundant among prokaryotes and have been characterized in fungi and plants, although not yet in animals. These enzymes regulate diverse processes, including adaptation to osmotic stress and virulence of plant and animal pathogens. Here, we report the cloning, characterization and phylogenetic analysis of HwHHK7A and HwHHK7B, HK genes from the fungi Hortaea werneckii, a proposed model system for studying salt tolerance in eukaryotes. The two HwHhk7 isoforms are 96.7% identical in amino-acid sequence and have a typical eukaryotic hybrid HK domain composition. On the bases of the conserved sequence of the H box, they are classified into the group VII ascomycete HKs. For the HwHhk7B protein, the autokinase activity was demonstrated in vitro. The salt-responsive expression of the HwHHK7 genes and the increased osmotolerance of a wild-type Saccharomyces cerevisiae strain expressing the HwHHK7B gene lead us to speculate that these newly identified HKs have roles in osmosensing.

Melanin is crucial for growth of the black yeast Hortaea werneckii in its natural hypersaline environment.

Melanin has an important role in the ability of fungi to survive extreme conditions, like the high NaCl concentrations that are typical of hypersaline environments. The black fungus Hortaea werneckii that has been isolated from such environments has 1,8-dihydroxynaphthalene-melanin incorporated into the cell wall, which minimises the loss of glycerol at low NaCl concentrations. To further explore the role of melanin in the extremely halotolerant character of H. werneckii, we studied the effects of several melanin biosynthesis inhibitors on its growth, pigmentation and cell morphology. The most potent inhibitors were a 2,3-dihydrobenzofuran derivative and tricyclazole, which restricted the growth of H. werneckii on high-salinity media, as shown by growth curves and plate-drop assays. These inhibitors promoted release of the pigments from the H. werneckii cell surface and changed the medium colour. Inhibitor-treated H. werneckii cells exposed to high salinity showed both decreased and increased cell lengths. We speculate that this absence of melanin perturbs the integrity of the cell wall in H. werneckii, which affects its cell division and exposes it to the harmful effects of high NaCl concentrations. Surprisingly, melanin had no effect on H. werneckii survival under H₂O₂ oxidative stress.

Identification and characterization of putative osmosensors, HwSho1A and HwSho1B, from the extremely halotolerant black yeast Hortaea werneckii.

In Saccharomyces cerevisiae, the Sho1 protein is one of two potential osmosensors that can activate the kinase cascade of the HOG pathway in response to increased extracellular osmolarity. Two novel SHO1-like genes, HwSHO1A and HwSHO1B, have been cloned from the saltern-inhabiting, extremely halotolerant black yeast Hortaea werneckii. The HwSho1 protein isoforms are 93.8% identical in their amino-acid sequences, and have a conserved SH3 domain. When the HwSHO1 genes were transferred into S. cerevisae cells lacking the SHO1 gene, both of the HwSho1 isoforms fully complemented the function of the native S. cerevisiae Sho1 protein. Through microscopic and biochemical validation, we demonstrate that in S. cerevisiae, both of the HwSho1 proteins have characteristic subcellular localizations similar to the S. cerevisiae Sho1 protein, and they can both activate the HOG pathway under conditions of osmotic stress. To a lower extent, crosstalk to the mating pathway expressing HwSho1 proteins is conserved in the PBS2 deleted S. cerevisiae strain. These data show that the HwSho1 proteins from H. werneckii are true functional homologs of the Sho1 protein of S. cerevisiae.

Insertion of a specific fungal 3'-phosphoadenosine-5'-phosphatase motif into a plant homologue improves halotolerance and drought tolerance of plants.

Soil salinity and drought are among the most serious agricultural and environmental problems of today. Therefore, investigations of plant resistance to abiotic stress have received a lot of attention in recent years. In this study, we identified the complete coding sequence of a 3′-phosphoadenosine-5′-phosphatase protein, ApHal2, from the halotolerant yeast Aureobasidium pullulans. Expression of the ApHAL2 gene in a Saccharomyces cerevisiae hal2 mutant complemented the mutant auxotrophy for methionine, and rescued the growth of the hal2 mutant in media with high NaCl concentrations. A 21-amino-acids-long region of the ApHal2 enzyme was inserted into the Arabidopsis thaliana homologue of Hal2, the SAL1 phosphatase. The inserted sequence included the META motif, which has previously been implicated in increased sodium tolerance of the Hal2 homologue from a related fungal species. Transgenic Arabidopsis plants overexpressing this modified SAL1 (mSAL1) showed improved halotolerance and drought tolerance. In a medium with an elevated salt concentration, mSAL1-expressing plants were twice as likely to have roots in a higher length category in comparison with the wild-type Arabidopsis and with plants overexpressing the native SAL1, and had 5% to 10% larger leaf surface area under moderate and severe salt stress, respectively. Similarly, after moderate drought exposure, the mSAL1-expressing plants showed 14% increased dry weight after revitalisation, with no increase in dry weight of the wild-type plants. With severe drought, plants overexpressing native SAL1 had the worst rehydration success, consistent with the recently proposed role of SAL1 in severe drought. This was not observed for plants expressing mSAL1. Therefore, the presence of this fungal META motif sequence is beneficial under conditions of increased salinity and moderate drought, and shows no drawbacks for plant survival under severe drought. This demonstrates that adaptations of extremotolerant fungi should be considered as a valuable resource for improving stress-tolerance in plant breeding in the future.