Quorum sensing in \u3ci\u3eCandida albicans\u3c/i\u3e: farnesol versus farnesoic acid

Candida albicans is a clinically important dimorphic fungus that exhibits either a budding yeast or a mycelial-hyphal or pseudohyphal growth, depending on environmental conditions. The yeast-to-mycelia morphologic transition, which is generally regarded as an important virulence determinant, depends on the inoculum size of liquid cultures. The yeast form is favored when cultures are inoculated at \u3e 106 cells∙mL–1, whereas the mycelial form is favored at inoculum densities \u3c 106∙mL–1. Farnesoic acid (FA) and farnesol (FOH) are two related sesquiterpene quorum- sensing molecules that, upon accumulation, prevent the yeast-to-mycelial conversion. Oh et al. showed that C. albicans strain ATCC 10231 excretes FA, while Hornby et al. showed that C. albicans A72 and SC5314 excrete FOH. Subsequent work indicated that 10231 was the only isolate of C. albicans that fails to produce detectable FOH. Moreover, when tested on C. albicans A72, FA had only 3.2% of the hyphal-inhibitory activity relative to FOH. These observations raised two questions: 1., Do FOH and FA block mycelial development via the same molecular mechanism; and 2., What is the biochemical or physiologic difference in strain 10231 that underlies excretion of FA and not FOH? Do farnesol and farnesoic acid share a common mechanism of action? An apparent paradox in the regulation of hyphal morphogenesis by CaPho81p Why does C. albicans strain 10231 secrete farnesoic acid, while other C. albicans strains secrete farnesol

Mitochondria as a Potential Antifungal Target for Isocyanide Compounds

The discovery of antibiotics and antifungals greatly impacted medicine and human health, allowing the effective treatment of infections that were previously deadly. However, due to routine and sometimes excessive usage of these compounds, the development of antimicrobial resistance has created a need for new antibiotic and antifungal compounds. Isocyanide compounds have been shown to have antibacterial, antifungal, and anti-cancer properties, but very little is known about their biochemical effects. Our research aims to understand the mechanism of action of isocyanide compounds. We have conducted a genetic screen of a Saccharomyces gene-deletion (“knockout”) collection on media containing an easily synthesized model isocyanide compound, para-nitrophenyl isocyanide (p-NPIC). This allowed us to identify genes which, when deleted, render the mutant strains resistant or hypersensitive to the compound. Based on our genetic screen for hypersensitive mutants, we hypothesize that the isocyanides impact mitochondrial function, specifically altering the function of the Cu++-containing respiratory complex, Cytochrome C Oxidase (Complex IV). Our findings provide new information on the mechanism(s) of action of this class of antimicrobials and will help guide the development of new molecules based on lead-compounds such as p-NPIC

Molecular machinery of auxin synthesis,secretion, and perception in the unicellular chlorophyte alga Chlorella sorokiniana UTEX 1230

Indole-3-acetic acid is a ubiquitous small molecule found in all domains of life. It is the predominant and most active auxin in seed plants, where it coordinates a variety of complex growth and development processes. The potential origin of auxin signaling in algae remains a matter of some controversy. In order to clarify the evolutionary context of algal auxin signaling, we undertook a genomic survey to assess whether auxin acts as a signaling molecule in the emerging model chlorophyte Chlorella sorokiniana UTEX 1230. C. sorokiniana produces the auxin indole-3-acetic acid (IAA), which was present in both the cell pellet and in the supernatant at a concentration of ~ 1 nM, and its genome encodes orthologs of genes related to auxin synthesis, transport, and signaling in higher plants. Candidate orthologs for the canonical AUX/IAA signaling pathway were not found; however, auxin-binding protein 1 (ABP1), an alternate auxin receptor, is present and highly conserved at essential auxin binding and zinc coordinating residues. Additionally, candidate orthologs for PIN proteins, responsible for intercellular, vectorial auxin transport in higher plants, were not found, but PILs (PIN-Like) proteins, a recently discovered family that mediates intracellular auxin transport, were identified. The distribution of auxin related gene in this unicellular chlorophyte demonstrates that a core suite of auxin signaling components was present early in the evolution of plants. Understanding the simplified auxin signaling pathways in chlorophytes will aid in understanding phytohormone signaling and crosstalk in seed plants, and in understanding the diversification and integration of developmental signals during the evolution of multicellular plants

A functional genomic screen in Saccharomyces cerevisiae reveals divergent mechanisms of resistance to different alkylphosphocholine chemotherapeutic agents

The alkylphosphocholine (APC) class of antineoplastic and antiprotozoal drugs, such as edelfosine and miltefosine, are structural mimics of lyso-phosphatidylcholine (lyso-PC), and are inhibitory to the yeast Saccharomyces cerevisiae at low micromolar concentrations. Cytotoxic effects related to inhibition of phospholipid synthesis, induction of an unfolded protein response, inhibition of oxidative phosphorylation, and disruption of lipid rafts have been attributed to members of this drug class, however, the molecular mechanisms of action of these drugs remain incompletely understood. Cytostatic and cytotoxic effects of the APCs exhibit variability with regard to chemical structure, leading to differences in effectiveness against different organisms or cell types. We now report the comprehensive identification of S. cerevisiae titratableessential gene and haploid nonessential gene deletion mutants that are resistant to the APC drug miltefosine (hexadecyl-O-phosphocholine). Fifty-eight strains out of~5600 tested displayed robust and reproducible resistance to miltefosine. This gene set was heavily enriched in functions associated with vesicular transport steps, especially those involving endocytosis and retrograde transport of endosome derived vesicles to the Golgi or vacuole, suggesting a role for these trafficking pathways in transport of miltefosine to potential sites of action in the endoplasmic reticulum and mitochondrion. In addition, we identified mutants with defects in phosphatidylinositol-4-phosphate synthesis (TetO::STT4) and hydrolysis (sac1Δ), an oxysterol binding protein homolog (osh2Δ), a number of ER-resident proteins, and multiple components of the eisosome. These findings suggest that ER-plasma membrane contact sites and retrograde vesicle transport are involved in the interorganelle transport of lyso-PtdCho and related lyso-phospholipid-like analogs to their intracellular sites of cytotoxic activity

Integration of biology, ecology and engineering for sustainable algal‑based biofuel and bioproduct biorefinery

Despite years of concerted research efforts, an industrial-scale technology has yet to emerge for production and conversion of algal biomass into biofuels and bioproducts. The objective of this review is to explore the ways of possible integration of biology, ecology and engineering for sustainable large algal cultivation and biofuel production systems. Beside the costs of nutrients, such as nitrogen and phosphorous, and fresh water, upstream technologies which are not ready for commercialization both impede economic feasibility and conflict with the ecological benefits in the sector. Focusing mainly on the engineering side of chemical conversion of algae to biodiesel has also become obstacle. However, to reduce the costs, one potential strategy has been progressing steadily to synergistically link algal aquaculture to the governmentally mandated reduction of nitrogen and phosphorous concentrations in municipal wastewater. Recent research also supports the suppositions of scalability and cost reduction. Noticeably, less is known of the economic impact of conversion of the whole algae-based biorefinery sector with additional biochemical and thermochemical processes and integration with ecological constraints. This review finds that a biorefinery approach with integrated biology, ecology, and engineering could lead to a feasible algal-based technology for variety of biofuels and bioproducts

Synthesis, secretion, and perception of abscisic acid regulates stress responses in \u3ci\u3eChlorella sorokiniana\u3c/i\u3e

Abscisic acid (ABA) is a phytohormone that has been extensively characterized in higher plants for its roles in seed and bud dormancy, leaf abscission, and stress responses. Genomic studies have identified orthologs for ABA-related genes throughout the Viridiplantae, including in unicellular algae; however, the role of ABA in algal physiology has not been characterized, and the existence of such a role has been a matter of dispute. In this study, we demonstrate that ABA is involved in regulating algal stress responses. Chlorella sorokiniana strain UTEX 1230 contains genes orthologous to those of higher plants which are essential for ABA biosynthesis, sensing, and degradation. RNAseq-based transcriptomic studies reveal that treatment with ABA induces dramatic changes in gene expression profiles, including the induction of a subset of genes involved in DNA replication and repair, a phenomenon which has been demonstrated in higher plants. Pretreatment of C. sorokiniana cultures with ABA exerts a protective effect on cell viability in response to ultraviolet radiation. Additionally, C. sorokiniana produces and secretes biologically relevant amounts of both ABA and the oxylipin 12-oxo-phytodienoic acid (OPDA) into the growth medium in response to abiotic stressors. Taken together, these phenomena suggest that ABA signaling evolved as an intercellular stress response signaling molecule in eukaryotic microalgae prior to the evolution of multicellularity and colonization of land

Mitochondria as a Potential Antifungal Target for Isocyanide Compounds

The discovery of antibiotics and antifungals greatly impacted medicine and human health, allowing the effective treatment of infections that were previously deadly. However, due to routine and sometimes excessive usage of these compounds, the development of antimicrobial resistance has created a need for new antibiotic and antifungal compounds. Isocyanide compounds have been shown to have antibacterial, antifungal, and anti-cancer properties, but very little is known about their biochemical effects. Our research aims to understand the mechanism of action of isocyanide compounds. We have conducted a genetic screen of a Saccharomyces gene-deletion (“knockout”) collection on media containing an easily synthesized model isocyanide compound, para-nitrophenyl isocyanide (p-NPIC). This allowed us to identify genes which, when deleted, render the mutant strains resistant or hypersensitive to the compound. Based on our genetic screen for hypersensitive mutants, we hypothesize that the isocyanides impact mitochondrial function, specifically altering the function of the Cu++-containing respiratory complex, Cytochrome C Oxidase (Complex IV). Our findings provide new information on the mechanism(s) of action of this class of antimicrobials and will help guide the development of new molecules based on lead-compounds such as p-NPIC

Comparative genomics, transcriptomics, and physiology distinguish symbiotic from free-living \u3ci\u3eChlorella\u3c/i\u3e strains

Most animal–microbe symbiotic interactions must be advantageous to the host and provide nutritional benefits to the endosymbiont. When the host provides nutrients, it can gain the capacity to control the interaction, promote self-growth, and increase its fitness. Chlorella-like green algae engage in symbiotic relationships with certain protozoans, a partnership that significantly impacts the physiology of both organisms. Consequently, it is often challenging to grow axenic Chlorella cultures after isolation from the host because they are nutrient fastidious and often susceptible to virus infection. We hypothesize that the establishment of a symbiotic relationship resulted in natural selection for nutritional and metabolic traits that differentiate symbiotic algae from their free-living counterparts. Here, we compare metabolic capabilities of 5 symbiotic and 4 free-living Chlorella strains by determining growth levels on combinations of nitrogen and carbon sources. Data analysis by hierarchical clustering revealed clear separation of the symbiotic and free-living Chlorella into two distinct clades. Symbiotic algae did not grow on nitrate but did grow on two symbiont-specific amino acids (Asn and Ser) on which the free-living strains did not grow. The use of these amino acids was exclusively affected by the presence/absence of Ca2+ in the medium, and the differences were magnified if galactose was provided rather than sucrose or glucose. In addition, Chlorella variabilis NC64A genomic and differential expression analysis confirmed the presence of abundant amino acid transporter protein motifs, some of which were expressed constitutively both axenically and within the host. Significantly, all 5 symbiotic strains exhibited similar physiological phenotypes even though they were isolated as symbionts from different host organisms. Such similarities indicate a parallel coevolution of shared metabolic pathways across multiple independent symbiotic events. Collectively, our results suggest that physiological changes drive the Chlorella symbiotic phenotype and contribute to their natural fitness. Includes Supplementary materials

Comparative genomics, transcriptomics, and physiology distinguish symbiotic from free-living \u3ci\u3eChlorella\u3c/i\u3e strains

Most animal–microbe symbiotic interactions must be advantageous to the host and provide nutritional benefits to the endosymbiont. When the host provides nutrients, it can gain the capacity to control the interaction, promote self-growth, and increase its fitness. Chlorella-like green algae engage in symbiotic relationships with certain protozoans, a partnership that significantly impacts the physiology of both organisms. Consequently, it is often challenging to grow axenic Chlorella cultures after isolation from the host because they are nutrient fastidious and often susceptible to virus infection. We hypothesize that the establishment of a symbiotic relationship resulted in natural selection for nutritional and metabolic traits that differentiate symbiotic algae from their free-living counterparts. Here, we compare metabolic capabilities of 5 symbiotic and 4 free-living Chlorella strains by determining growth levels on combinations of nitrogen and carbon sources. Data analysis by hierarchical clustering revealed clear separation of the symbiotic and free-living Chlorella into two distinct clades. Symbiotic algae did not grow on nitrate but did grow on two symbiont-specific amino acids (Asn and Ser) on which the free-living strains did not grow. The use of these amino acids was exclusively affected by the presence/absence of Ca2+ in the medium, and the differences were magnified if galactose was provided rather than sucrose or glucose. In addition, Chlorella variabilis NC64A genomic and differential expression analysis confirmed the presence of abundant amino acid transporter protein motifs, some of which were expressed constitutively both axenically and within the host. Significantly, all 5 symbiotic strains exhibited similar physiological phenotypes even though they were isolated as symbionts from different host organisms. Such similarities indicate a parallel coevolution of shared metabolic pathways across multiple independent symbiotic events. Collectively, our results suggest that physiological changes drive the Chlorella symbiotic phenotype and contribute to their natural fitness. Includes Supplementary materials

Characterization of a novel polyextremotolerant fungus, \u3ci\u3eExophiala viscosa\u3c/i\u3e, with insights into its melanin regulation and ecological niche

Black yeasts are polyextremotolerant fungi that contain high amounts of melanin in their cell wall and maintain a primar yeast form. These fungi grow in xeric, nutrient depletes environments which implies that they require highly flexible metabolisms and have been suggested to contain the ability to form lichen-like mutualisms with nearby algae and bacteria. However, the exact ecological niche and interactions between these fungi and their surrounding community are not well understood. We have isolated 2 novel black yeasts from the genus Exophiala that were recovered from dryland biological soil crusts. Despite notable differences in colony and cellular morphology, both fungi appear to be members of the same species, which has been named Exophiala viscosa (i.e. E. viscosa JF 03-3 Goopy and E. viscosa JF 03-4F Slimy). A combination of whole genome sequencing, phenotypic experiments, and melanin regulation experiments have been performed on these isolates to fully characterize these fungi and help decipher their fundamental niche within the biological soil crust consortium. Our results reveal that E. viscosa is capable of utilizing a wide variety of carbon and nitrogen sources potentially derived from symbiotic microbes, can withstand many forms of abiotic stresses, and excretes melanin which can potentially provide ultraviolet resistance to the biological soil crust community. Besides the identification of a novel species within the genus Exophiala, our study also provides new insight into the regulation of melanin production in polyextremotolerant fungi