Studies on the {\it in vivo} function of a phosphatidylinositol transfer protein from {\it Saccharomyces cerevisiae}

Phosphatidylinositol transfer proteins (PI-TP's) catalyze the transfer of phosphatidylinositol and phosphatidylcholine between membranes in vitro. However the in vivo function of these proteins is unknown. In this thesis we have used a combined biochemical and genetic approach to determine the importance of PI-TP in vivo. An oligonucleotide based on the amino terminal sequence of the PI-TP from Saccharomyces cerevisiae, was used to screen a yeast genomic library for the gene encoding PI-TP (PIT1 gene). Yeast strains transformed with the positive clones showed overproduction of transfer activities and transfer protein in the 100,000 x g supernatants. The 5$\sp\prime$ terminus of the PIT1 gene correlates with the predicted codons for residues 3-30 of the determined protein sequence. Tetrad analysis of a heterozygous diploid (PIT1/pit1::LEU2) revealed that the PIT1 gene is essential for cell growth. Non-viable spores could be rescued by transformation of the above diploid prior to sporulation, with a plasmid borne copy of the wild type gene. Sequencing of the entire PIT1 gene has revealed that the PIT1 gene is identical to the SEC14 gene. The sec14 ts mutant which exhibits conditional defects at the Golgi stage of protein secretion, is also temperature sensitive for PI-TP activity in vitro. These findings represent the first instance in which a physiological function has been assigned to any phospholipid transfer protein. ^

Phagocytic uptake of Encephalitozoon cuniculi by nonprofessional phagocytes

Encephalitozoon cuniculi is an obligate intracellular, spore-forming parasite belonging to the microsporidia that can cause disseminated infection in immunocompromised persons. E. cuniculi spores infect host cells by germination, i.e., by explosively everting the polar filament, through which the spore contents (sporoplasms) are subsequently injected into the cytoplasm. In addition, we observed intracellular, nongerminated spores in various nonprofessional phagocytes. In MRC5 cells, the number of internalized spores was approximately 10-fold higher than the number of injected sporoplasms. Compared to the rate of uptake by human monocyte-derived macrophages, internalization rates by A549 cells, MRC5 cells, and 293 cells were 0.6, 4.4, and 22.2%, respectively. The mechanism of uptake was studied in MRC5 cells. Killed spores were internalized at the same rate as live spores, indicating that nongerminated parasites do not actively participate in cell entry. Cytochalasin D inhibited uptake of spores by 95%, demonstrating an actin-dependent process. By electron and epifluorescence microscopy, intracellular spores were found in a tightly fitting membrane-bound compartment. The vacuole containing the spores was positive for the lysosomal membrane protein LAMP-1 and colocalized with the late endosomal-lysosomal content marker rhodamine dextran. Our results show that, in addition to the unique way in which microsporidia infect cells, E. cuniculi spores enter nonprofessional phagocytes by phagocytosis and traffic into a late endosomal-lysosomal compartment.

Sex-Specific Differences in Pathogen Susceptibility in Honey Bees (Apis mellifera)

Sex-related differences in susceptibility to pathogens are a common phenomenon in animals. In the eusocial Hymenoptera the two female castes, workers and queens, are diploid and males are haploid. The haploid susceptibility hypothesis predicts that haploid males are more susceptible to pathogen infections compared to females. Here we test this hypothesis using adult male (drone) and female (worker) honey bees (Apis mellifera), inoculated with the gut endoparasite Nosema ceranae and/or black queen cell virus (BQCV). These pathogens were chosen due to previously reported synergistic interactions between Nosema apis and BQCV. Our data do not support synergistic interactions between N. ceranae and BQCV and also suggest that BQCV has limited effect on both drone and worker health, regardless of the infection level. However, the data clearly show that, despite lower levels of N. ceranae spores in drones than in workers, Nosema-infected drones had both a higher mortality and a lower body mass than non-infected drones, across all treatment groups, while the mortality and body mass of worker bees were largely unaffected by N. ceranae infection, suggesting that drones are more susceptible to this pathogen than workers. In conclusion, the data reveal considerable sex-specific differences in pathogen susceptibility in honey bees and highlight the importance of ultimate measures for determining susceptibility, such as mortality and body quality, rather than mere infection levels

Endozoochory by slugs can increase bryophyte establishment and species richness

Herbivory can affect plant community composition and diversity by removing biomass and reducing light competition. Herbivory may particularly benefit low growing species such as bryophytes, which are frequently limited by light competition. Gastropods are important herbivores of seed plants and cryptogams, furthermore, they can disperse propagules such as seeds and spores via endozoochory. However, whether gastropod herbivory can reduce the dominance of vascular plants and thereby promote the germination and establishment of endozoochorously dispersed bryophyte spores has never been tested experimentally. Moreover, it is unclear whether these possible interacting effects can influence bryophyte species richness. Here, we tested for endozoochorous spore dispersal by slugs, in combination with sowing of vascular plants, in a fully factorial common garden experiment. Enclosures contained either slugs previously fed with bryophyte sporophytes, control slugs, or no slugs. After 21 days the bryophyte cover was on average 2.8 times higher (3.9 versus 1.4) and after eight months the bryophyte species richness 2.6 times higher (5.8 versus 2.2) in enclosures containing slugs previously fed with bryophyte sporophytes than in the other treatments. Furthermore, after eight months high vascular plant cover reduced bryophyte diversity. On average enclosures without seed sowing harboured 1.6 times more bryophyte species than the ones with seed sowing (4.2 versus 2.6), indicating competitive effects of vascular plants on bryophytes. Our findings suggest that slugs are important dispersal vectors for bryophytes and that they can increase bryophyte populations and maintain bryophyte diversity by reducing the dominance of vascular plants.

Interactions of climate and land use documented in the varved sediments of Seebergsee in the Swiss Alps

This paper presents a multiproxy high-resolution study of the past 2600 years for Seebergsee, a small Swiss lake with varved sediments at the present tree-line ecotone. The laminae were identified as varves by a numerical analysis of diatom counts in the thin-sections. The hypothesis of two diatom blooms per year was corroborated by the 210Pb and 137Cs chronology. A period of intensive pasturing during the ‘Little Ice Age’ between ad 1346 and ad 1595 is suggested by coprophilous fungal spores, as well as by pollen indicators of grazing, by the diatom-inferred total phosphorus, by geochemistry and by documentary data. The subsequent re-oligotrophication of the lake took about 88 years, as determined by the timelag between the decline of coprophile fungal spores and the restoration of pre-eutrophic nutrient conditions. According to previous studies of latewood densities from the same region, cold summers around ad 1600 limited the pasturing at this altitude. This demonstrated the socio-economic impact of a single climatic event. However, the variance partitioning between the effects of land use and climate, which was applied for the whole core, revealed that climate independent of land use and time explained only 1.32% of the diatom data, while land use independent of climate and time explained 15.7%. Clearly land use in‘ uenced the lake, but land use was not always driven by climate. Other factors beside climate, such as politics or the introduction of fertilizers in the seventeenth and eighteenth centuries also in‘ uenced the development of Alpine pasturing.

A 22,000 14C year BP sediment and pollen record of climate change from Laguna Miscanti (23°S), northern Chile

Lake sediments and pollen, spores and algae from the high-elevation endorheic Laguna Miscanti (22°45′S, 67°45′W, 4140 m a.s.l., 13.5 km2 water surface, 10 m deep) in the Atacama Desert of northern Chile provide information about abrupt and high amplitude changes in effective moisture. Although the lack of terrestrial organic macrofossils and the presence of a significant 14C reservoir effect make radiocarbon dating of lake sediments very difficult, we propose the following palaeoenvironmental history. An initial shallow freshwater lake (ca. 22,000 14C years BP) disappeared during the extremely dry conditions of the Last Glacial Maximum (LGM; 18,000 14C years BP). That section is devoid of pollen. The late-glacial lake transgression started around 12,000 14C years BP, peaked in two phases between ca. 11,000 and <9000 14C years BP, and terminated around 8000 14C years BP. Effective moisture increased more than three times compared to modern conditions (∼200 mm precipitation), and a relatively dense terrestrial vegetation was established. Very shallow hypersaline lacustrine conditions prevailed during the mid-Holocene until ca. 3600 14C years BP. However, numerous drying and wetting cycles suggest frequent changes in moisture, maybe even individual storms during the mid-Holocene. After several humid spells, modern conditions were reached at ca. 3000 14C years BP. Comparison between limnogeological data and pollen of terrestrial plants suggest century-scale response lags. Relatively constant concentrations of long-distance transported pollen from lowlands east of the Andes suggest similar atmospheric circulation patterns (mainly tropical summer rainfall) throughout the entire period of time. These findings compare favorably with other regional paleoenvironmental data.

Vegetation and fire history of coastal north-eastern Sardinia (Italy) under changing Holocene climates and land use

Little is known about the vegetation and fire history of Sardinia, and especially the long-term history of the thermo-Mediterranean belt that encompasses its entire coastal lowlands. A new sedimentary record from a coastal lake based on pollen, spores, macrofossils and microscopic charcoal analysis is used to reconstruct the vegetation and fire history in north-eastern Sardinia. During the mid-Holocene (c. 8,100–5,300 cal bp), the vegetation around Stagno di Sa Curcurica was characterised by dense Erica scoparia and E. arborea stands, which were favoured by high fire activity. Fire incidence declined and evergreen broadleaved forests of Quercus ilex expanded at the beginning of the late Holocene. We relate the observed vegetation and fire dynamics to climatic change, specifically moister and cooler summers and drier and milder winters after 5,300 cal bp. Agricultural activities occurred since the Neolithic and intensified after c. 7,000 cal bp. Around 2,750 cal bp, a further decline of fire incidence and Erica communities occurred, while Quercus ilex expanded and open-land communities became more abundant. This vegetation shift coincided with the historically documented beginning of Phoenician period, which was followed by Punic and Roman civilizations in Sardinia. The vegetational change at around 2,750 cal bp was possibly advantaged by a further shift to moister and cooler summers and drier and milder winters. Triggers for climate changes at 5,300 and 2,750 cal bp may have been gradual, orbitally-induced changes in summer and winter insolation, as well as centennial-scale atmospheric reorganizations. Open evergreen broadleaved forests persisted until the twentieth century, when they were partly substituted by widespread artificial pine plantations. Our results imply that highly flammable Erica vegetation, as reconstructed for the mid-Holocene, could re-emerge as a dominant vegetation type due to increasing drought and fire, as anticipated under global change conditions.

Is fern endozoochory widespread among fern-eating herbivores?

Endozoochory is an important dispersal mechanism for seed plants and has recently been demonstrated to occur also in spore plants, such as ferns, which are commonly consumed by herbivores. However, it is not known whether fern species from particular habitats are differentially preferred by herbivores and whether their spores differ in their ability to survive the gut passage of herbivores. Such differences would suggest adaptation to endozoochorous dispersal, as it is known for seed plants. Moreover, it is unclear whether herbivore species differ in their efficiency to disperse fern spores. In a factorial experiment, we fed fertile leaflets of 13 fern species from different forest and open habitats to three polyphagous herbivore species and recorded the germination of spores from feces after 46 and 81 days. Fern spores germinated in 66 % of all samples after 46 days. At this stage, germination success differed among fern and herbivore species, but was independent of the ferns’ habitat. Interestingly, after 81 days fern spores germinated in 85 % of all samples and earlier significant differences in germination success among fern and herbivore species were not sustained. The overall high germination success and the absence of differences among fern species from different habitats together with the consistency across three tested herbivores strongly imply endozoochorous dispersal to be a taxonomically widespread phenomenon among fern-eating herbivores, which all might act as potential dispersal vectors. © 2015, Springer Science+Business Media Dordrecht.

Long-distance spore dispersal in wood-inhabiting Basidiomycetes

Eight species of wood-inhabiting basidiomycetes (Laurilia sulcata, Peniophora aurantiaca, Resinicium bicolor, Scytinostroma galactinum, Terana caerulea, Trichaptum abietinum, T. biforme and T. fuscoviolaceum) were used in a spore-trapping test to evaluate their individual ability for long-distance spore dispersal. Petri dishes with single spore mycelia were used as baits. In the experiment, carried out at the Botanical Institute in Göteborg, spores from the air were regularly captured. Surprisingly, spores were captured from species whose nearest known natural occurrence was located quite far from Göteborg. The closest population of Peniophora aurantiaca is about 1000 km south of Göteborg. The results from this experiment support the hypothesis that fungal spores are widely and efficiently dispersed. Such a broad and extensive dispersal ability is of vital importance, especially for wood-inhabiting species which are highly dependent on a substrate which is onlv temporarily available.

The palaeolimnology of Soppensee (Central Switzerlabnd), as evidenced by diatom, pollen, and fossil-pigment analyses

The development of Soppensee (Central Switzerland, 596 m a.s.l.) has been reconstructed using algal remains such as diatoms, chlorophytes and fossil pigments, as well as the pollen and spores of macrophytes. Sediment accumulation in Soppensee began at the end of the last glacial period, approximately 15,000 yrs ago. During the Oldest Dryas biozone (> 12,700 radiocarbon yrs B.P.) the lake had low primary productivity. After reforestation with birch and later pine, around 12,700 B.P., phases of summer anoxia occurred in the lake. These anoxic conditions were most probably caused by additional carbon input from the catchment, as well as longer phases of stratification due to reduced wind exposure caused by the sheltering effect of increased tree cover. From the Younger Dryas biozone (10,800 to 10,000 radiocarbon yrs B.P.) onwards, Soppensee became meromictic for several millennia.

Comprehensive environmental study in high risk areas for cancer patients comparing air and water Aspergillus species samples

Background. Nosocomial invasive aspergillosis (a highly fatal disease) is an increasing problem for immunocompromised patients. Aspergillus spp. can be transmitted via air (most commonly) and by water. ^ The hypothesis for this prospective study was that there is an association between patient occupancy, housekeeping practices, patients, visitors, and Aspergillus spp. loading. Rooms were sampled as not terminally cleaned (dirty) and terminally cleaned (clean). The secondary hypothesis was that Aspergillus spp. positive samples collected from more than one sampling location within the same patient room represent the same isolate. ^ Methods. Between April and October 2004, 2873 environmental samples (713 air, 607 water, 1256 surface and 297 spore traps) were collected in and around 209 “clean” and “dirty” patient rooms in a large cancer center hospital. Water sources included aerosolized water from patient room showerheads, sinks, drains, and toilets. Bioaerosol samples were from the patient room and from the running shower, flushing toilet, and outside the building. The surface samples included sink and shower drains, showerheads, and air grills. Aspergillus spp. positive samples were also sent for PCR, molecular typing (n = 89). ^ Results. All water samples were negative for Aspergillus spp. There were a total of 130 positive culturable samples (5.1%). The predominant species found was Aspergillus niger. Of the positive culturable samples, 106 (14.9%) were air and 24 (3.8%) were surface. There were 147 spore trap samples, and 49.5% were positive for Aspergillus/Penicillum spp. Of the culturable positive samples sent for PCR, 16 were indistinguishable matches. There was no significant relationship between air and water samples and positive samples from the same room. ^ Conclusion. Primarily patients, visitors and staff bring the Aspergillus spp. into the hospital. The high number of A. niger samples suggests the spores are entering the hospital from outdoors. Eliminating the materials brought to the patient floors from the outside, requiring employees, staff, and visitors to wear cover up over their street clothes, and improved cleaning procedures could further reduce positive samples. Mold strains change frequently; it is probably more significant to understand pathogenicity of viable spores than to commit resources on molecular strain testing on environmental samples alone. ^

Ligand-stimulated internalization of a Dictyostelium discoideum G protein-coupled cAMP receptor

The social amoeba, Dictyostelium discoideum, undergoes a remarkable starvation-induced program of development that transforms a population of unicellular amoebae into a fruiting body composed of resistant spores suspended on a stalk. During this development, secreted cAMP drives chemotaxis of the amoebae, leading to their aggregation, and subsequent differentiation and morphogenesis. Four sequentially expressed G protein-coupled receptors (GPCRs) for cAMP play critical roles in this process. The first of these, cAR1, is essential for aggregation as it mediates chemotaxis as well as the propagation of secreted cAMP waves throughout aggregating populations. Ligand-induced internalization has been shown to regulate a variety of GPCRs. However, little was known at the outset of this study about the role of internalization in the regulation of cAR1 function or, for that matter, in developmental systems in general. For this study, cAMP-induced cAR1 internalization was assessed by measuring (1) the reduction of cell surface binding sites for [ 3H]cAMP and (2) the redistribution of YFP-tagged receptors to the cell's interior, cAMP was found to induce little or no loss of ligand binding (LLB) in vegetative cells. However, the ability to induce LLB increased progressively over the initial 6 hrs of development, reaching ∼70% in cells undergoing aggregation. Despite these reductions in surface binding, detectable cAR1-YFP redistribution could be induced by cAMP only after the cells reached the mound stage (10 hrs) and was found to occur naturally by the ensuing slug stage (18 hrs). Site-directed substitution of a cluster of 5 serines in the receptor's cytoplasmic tail that was previously shown to be the principal site of cAMP-induced cAR1 phosphorylation impaired both LLB and receptor redistribution and furthermore resulted in mound-stage developmental arrest, suggesting that phosphorylation of cAR1 is a prerequisite for its internalization and that cAR1 internalization is required for post-aggregative development. To assess the involvement of clathrin mediated endocytosis, Dictyostelium cells lacking the clathrin light chain gene (clc-) or either of two dynamin genes were examined and found to be defective in LLB and, in the case of clc- cells, also cAR1 redistribution and turnover. Furthermore, cAR1 overexpression in clc- cells (like the serine mutant in wild-type cells) promoted developmental arrest in mounds. The mound-arrest phenotype was also recapitulated in a wild-type background by the specific expression of cAR1 in prestalk cells (but not prespore cells), suggesting that development depends critically on internalization and clearance of cAR1 from these cells. Persistent cAR1 expression following aggregation was found to be associated with aberrant expression of prestalk and prespore genes, which may adversely affect development in the prestalk cell lineage. The PI3 kinase-TORC2 signal transduction pathway, known to be important for Dictyostelium chemotaxis and internalization of yeast pheromone receptors, was examined using chemical inhibitors and null cells and found to be necessary for cAR1 internalization. In conclusion, cAR1 was shown to be similar to other GPCRs in that its internalization depends on phosphorylation of cytoplasmic domain serines, utilizes clathrin and dynamin, and involves the TORC2 complex. In addition, the findings presented here that cAR1 internalization is both developmentally regulated and required for normal development represent a novel regulatory paradigm that might pertain to other GPCRs known to play important roles in the development of humans and other metazoans. ^

INTERACTION OF BACILLUS ANTHRACIS EXOSPORIUM PROTEIN BCLA WITH COMPLEMENT FACTOR H AND SPORE PERSISTENCE IN THE LUNG

Anthrax outbreaks in the United States and Europe and its potential use as a bioweapon have made Bacillus anthracis an interest of study. Anthrax infections are caused by the entry of B. anthracis spores into the host via the respiratory system, the gastrointestinal tract, cuts or wounds in the skin, and injection. Among these four forms, inhalational anthrax has the highest lethality rate and persistence of spores in the lungs of animals following pulmonary exposure has been noted for decades. However, details or mechanisms of spore persistence were not known. In this study, we investigated spore persistence in a mouse model. The results suggest that B. anthracis spores have special properties that promote persistence in the lung, and that there may be multiple mechanisms contributing to spore persistence. Moreover, recent discoveries from our laboratory suggest that spores evolved a sophisticated mechanism to interact with the host complement system. The complement system is a crucial part of the host defense mechanism against foreign microorganisms. Knowledge of the specific interactions that occur between the complement system and B. anthracis was limited. Studies performed in our laboratory have suggested that spores of B. anthracis can target specific proteins, such as Factor H (fH) of the complement system. Spores of B. anthracis are enclosed by an exosporium, which consists of a basal layer surrounded by a nap of hair-like filaments. The major structural component of the filaments is called Bacillus collagen-like protein of anthracis (BclA), which comprises a central collagen-like region and a globular C-terminal domain. BclA is the first point of contact with the innate system of an infected host. In this study, we investigated the molecular details of BclA-fH interaction with respect to the specific binding mechanism and the functional significance of this interaction in a murine model of anthrax infection. We hypothesized that the recruitment of fH to the spore surface by BclA limits the extent of complement activation and promotes pathogen survival and persistence in the infected host. Findings from this study are significant to understanding how to treat post-exposure prophylaxis and improve our knowledge of spores with the host immune system.

Functional analysis of GNA -1, a Galphai superfamily member found in the filamentous fungus Neurospora crassa

Heterotrimeric GTP-binding proteins, G proteins, are integral components of eukaryotic signaling systems linking extracellular signals to intracellular responses. Through coupling to seven-transmembrane helix receptors, G proteins convey primary signaling events into multi-leveled cascades of intracellular activity by regulating downstream enzymes, collectively called effectors. The effector enzymes regulated by G proteins include adenylyl cyclase, cAMP phosphodiesterase, phospolipase C-β, mitogen-activated protein kinases, and ion channels. ^ Neurospora crassa is a multicellular, filamentous fungus that is capable of both asexual and sexual reproduction by elaboration of specialized, developmentally controlled structures that give rise to either asexual or sexual spores, respectively. N. crassa possesses at least three heterotrimeric Gα proteins (GNA-1–3) and one Gβ subunit (GNB-1). GNA-1 was the first microbial protein that could be classified in the Gαi superfamily based on its amino acid identity and demonstration that it is a substrate for ADP-ribosylation by pertussis toxin. ^ Experiments were designed to identify the signal transduction pathways and the effector enzymes regulated by GNA-1. Targeted gene-replacement of gna-1 revealed that GNA-1 controls multiple developmental pathways including both asexual and sexual reproduction, maintenance of growth, and resistance to osmotic stress. The Gαi and Gαz members of the Gαi superfamily negatively regulate adenylyl cyclase activity in mammalian cells; therefore, adenylyl cyclase and cAMP levels were measured in Δgna-1 strains and also in strains that were deleted for both gna-1 and gna-2, a second Gα in N. crassa shown to have overlapping functions with GNA-1. Direct measurements of adenylyl cyclase activity revealed that GNA-1, but not GNA-2, was responsible for GTP-stimulated adenylyl cyclase activity in N. crassa. Furthermore, anti-GNA-1 IgG could specifically inhibit GTP-stimulated adenylyl cyclase activity in wild-type strain extracts. These studies also provided evidence that N. crassa possesses feedback mechanisms that control steady-state cAMP levels through indirect regulation of cAMP-phosphodiesterase activity; mutations in gna-1 and gna-2 were additive in their effect on lowering cAMP-phosphodiesterase activity under growth conditions where steady-state cAMP levels were normal but GTP-stimulated adenylyl cyclase activity was reduced 90% in comparison to control strains. ^ Genetic and biochemical epistasis experiments utilizing a Δ gna-1 cr-1 mutant suggest that GNA-1 is essential for female fertility in a cAMP-independent pathway. Furthermore, deletion of gna-1 in a cr-1 background exacerbated many of the defects already observed in the cr-1 strain including more severe growth restriction and developmental defects. However, deletion of gna-1 had no effect on the increased thermotolerance of cr-1, which has been attributed to loss of cAMP. cr-1 possesses GNA-1 protein, and crude membrane fractions from this strain reconstituted GTP-stimulated adenylyl cyclase activity in Δgna-1 membrane fractions. These studies provide direct evidence for the involvement of Gα proteins in the regulation of adenylyl cyclase activity in eukaryotic microbes. ^