Induction of a Cryphonectria parasitica cellobiohydrolase I gene is suppressed by hypovirus infection and regulated by a GTP-binding-protein-linked signaling pathway involved in fungal pathogenesis.

Extracellular cellulase activity is readily induced when the chestnut blight fungus Cryphonectria parasitica is grown on cellulose substrate as the sole carbon source. However, an isogenic C. parasitica strain rendered hypovirulent due to hypovirus infection failed to secrete detectable cellulase activity when grown under parallel conditions. Efforts to identify C. parasitica cellulase-encoding genes resulted in the cloning of a cellobiohydrolase (exoglucanase, EC 3.2.1.91) gene designated chb-1. Northern blot analysis revealed an increase in cbh-1 transcript accumulation in a virus-free virulent C. parasitica strain concomitant with the induction of extracellular cellulase activity. In contrast, induction of cbh-1 transcript accumulation was suppressed in an isogenic hypovirus-infected strain. Significantly, virus-free C. parasitica strains rendered hypovirulent by transgenic cosuppression of a GTP-binding protein alpha subunit were also found to be deficient in the induction of cbh-1 transcript accumulation.

Fungal metabolic model for human type I hereditary tyrosinaemia.

Type I hereditary tyrosinaemia (HT1) is a severe human inborn disease resulting from loss of fumaryl-acetoacetate hydrolase (Fah). Homozygous disruption of the gene encoding Fah in mice causes neonatal lethality, seriously limiting use of this animal as a model. We report here that fahA, the gene encoding Fah in the fungus Aspergillus nidulans, encodes a polypeptide showing 47.1% identity to its human homologue, fahA disruption results in secretion of succinylacetone (a diagnostic compound for human type I tyrosinaemia) and phenylalanine toxicity. We have isolated spontaneous suppressor mutations preventing this toxicity, presumably representing loss-of-function mutations in genes acting upstream of fahA in the phenylalanine catabolic pathway. Analysis of a class of these mutations demonstrates that loss of homogentisate dioxygenase (leading to alkaptonuria in humans) prevents the effects of a Fah deficiency. Our results strongly suggest human homogentisate dioxygenase as a target for HT1 therapy and illustrate the usefulness of this fungus as an alternative to animal models for certain aspects of human metabolic diseases.

NDR1, a locus of Arabidopsis thaliana that is required for disease resistance to both a bacterial and a fungal pathogen.

We have employed Arabidopsis thaliana as a model host plant to genetically dissect the molecular pathways leading to disease resistance. A. thaliana accession Col-0 is susceptible to the bacterial pathogen Pseudomonas syringae pv. tomato strain DC3000 but resistant in a race-specific manner to DC3000 carrying any one of the cloned avirulence genes avrB, avrRpm1, avrRpt2, and avrPph3. Fast-neutron-mutagenized Col-0 M2 seed was screened to identify mutants susceptible to DC3000(avrB). Disease assays and analysis of in planta bacterial growth identified one mutant, ndr1-1 (nonrace-specific disease resistance), that was susceptible to DC3000 expressing any one of the four avirulence genes tested. Interestingly, a hypersensitive-like response was still induced by several of the strains. The ndr1-1 mutation also rendered the plant susceptible to several avirulent isolates of the fungal pathogen Peronospora parasitica. Genetic analysis of ndr1-1 demonstrated that the mutation segregated as a single recessive locus, located on chromosome III. Characterization of the ndr1-1 mutation suggests that a common step exists in pathways of resistance to two unrelated pathogens.

A cytoplasmically transmissible hypovirulence phenotype associated with mitochondrial DNA mutations in the chestnut blight fungus Cryphonectria parasitica.

Mutations causing mitochondrial defects were induced in a virulent strain of the chestnut blight fungus Cryphonectria parasitica (Murr.) Barr. Virulence on apples and chestnut trees was reduced in four of six extensively characterized mutants. Relative to the virulent progenitor, the attenuated mutants had reduced growth rates, abnormal colony morphologies, and few asexual spores, and they resembled virus-infected strains. The respiratory defects and attenuated virulence phenotypes (hypovirulence) were transmitted from two mutants to a virulent strain by hyphal contact. The infectious transmission of hypovirulence occurred independently of the transfer of nuclei, did not involve a virus, and dynamically reflects fungal diseases caused by mitochondrial mutations. In these mutants, mitochondrial mutations are further implicated in generation of the attenuated state by (i) uniparental (maternal) inheritance of the trait, (ii) presence of high levels of cyanide-insensitive mitochondrial alternative oxidase activity, (iii) cytochrome deficiencies, and (iv) structural abnormalities in the mtDNA. Hence, cytoplasmically transmissible hypovirulence phenotypes found in virus-free strains of C. parasitica from recovering trees may be caused by mutant forms of mtDNA.

Surface signaling in pathogenesis.

Surface signaling plays a major role in fungal infection. Topographical features of the plant surface and chemicals on the surface can trigger germination of fungal spores and differentiation of the germ tubes into appressoria. Ethylene, the fruit-ripening hormone, triggers germination of conidia, branching of hyphae, and multiple appressoria formation in Colletotrichum, thus allowing fungi to time their infection to coincide with ripening of the host. Genes uniquely expressed during appressoria formation induced by topography and surface chemicals have been isolated. Disruption of some of them has been shown to decrease virulence on the hosts. Penetration of the cuticle by the fungus is assisted by fungal cutinase secreted at the penetration structure of the fungus. Disruption of cutinase gene in Fusarium solani pisi drastically decreased its virulence. Small amounts of cutinase carried by spores of virulent pathogens, upon contact with plant surface, release small amounts of cutin monomers that trigger cutinase gene expression. The promoter elements involved in this process in F. solani pisi were identified, and transcription factors that bind these elements were cloned. One of them, cutinase transcription factor 1, expressed in Escherichia coli, is phosphorylated. Several protein kinases from F. solani pisi were cloned. The kinase involved in phosphorylation of specific transcription factors and the precise role of phosphorylation in regulating cutinase gene transcription remain to be elucidated.

Retinal microglia are activated by systemic fungal infection

Purpose: To determine whether systemic fungal infection could cause activation of retinal microglia and therefore could be potentially harmful for patients with retinal degenerative diseases. Methods: Activation of retinal microglia was measured in a model of sublethal invasive candidiasis in C57BL/6J mice by (i) confocal immunofluorescence and (ii) flow cytometry analysis, using anti-CD11b, anti-Iba1, anti-MHCII and anti-CD45 antibodies. Results: Systemic fungal infection causes activation of retinal microglia, with phenotypic changes in morphology, surface markers expression, and microglial re-location in retinal layers. Conclusions: As an excessive or prolonged microglial activation may lead to chronic inflammation with severe pathological side effects, causing or worsening the course of retinal dystrophies, a systemic infection may represent a risk factor to be considered in patients with ocular neurodegenerative diseases, such as diabetic retinopathy, glaucoma, age-related macular degeneration or retinitis pigmentosa.

Carbon and nitrogen limitation increase chitosan antifungal activity in Neurospora crassa and fungal human pathogens

Chitosan permeabilizes plasma membrane and kills sensitive filamentous fungi and yeast. Membrane fluidity and cell energy determine chitosan sensitivity in fungi. A five-fold reduction of both glucose (main carbon (C) source) and nitrogen (N) increased 2-fold Neurospora crassa sensitivity to chitosan. We linked this increase with production of intracellular reactive oxygen species (ROS) and plasma membrane permeabilization. Releasing N. crassa from nutrient limitation reduced chitosan antifungal activity in spite of high ROS intracellular levels. With lactate instead of glucose, C and N limitation increased N. crassa sensitivity to chitosan further (4-fold) than what glucose did. Nutrient limitation also increased sensitivity of filamentous fungi and yeast human pathogens to chitosan. For Fusarium proliferatum, lowering 100-fold C and N content in the growth medium, increased 16-fold chitosan sensitivity. Similar results were found for Candida spp. (including fluconazole resistant strains) and Cryptococcus spp. Severe C and N limitation increased chitosan antifungal activity for all pathogens tested. Chitosan at 100 μg ml-1 was lethal for most fungal human pathogens tested but non-toxic to HEK293 and COS7 mammalian cell lines. Besides, chitosan increased 90% survival of Galleria mellonella larvae infected with C. albicans. These results are of paramount for developing chitosan as antifungal.

Chitosan enhances parasitism of Meloidogyne javanica eggs by the nematophagous fungus Pochonia chlamydosporia

Pochonia chlamydosporia (Pc), a nematophagous fungus and root endophyte, uses appressoria and extracellular enzymes, principally proteases, to infect the eggs of plant parasitic nematodes (PPN). Unlike other fungi, Pc is resistant to chitosan, a deacetylated form of chitin, used in agriculture as a biopesticide to control plant pathogens. In the present work, we show that chitosan increases Meloidogyne javanica egg parasitism by P. chlamydosporia. Using antibodies specific to the Pc enzymes VCP1 (a subtilisin), and SCP1 (a serine carboxypeptidase), we demonstrate chitosan elicitation of the fungal proteases during the parasitic process. Chitosan increases VCP1 immuno-labelling in the cell wall of Pc conidia, hyphal tips of germinating spores, and in appressoria on infected M. javanica eggs. These results support the role of proteases in egg parasitism by the fungus and their activation by chitosan. Phylogenetic analysis of the Pc genome reveals a large diversity of subtilisins (S8) and serine carboxypeptidases (S10). The VCP1 group in the S8 tree shows evidence of gene duplication indicating recent adaptations to nutrient sources. Our results demonstrate that chitosan enhances Pc infectivity of nematode eggs through increased proteolytic activities and appressoria formation and might be used to improve the efficacy of M. javanica biocontrol.

Fungal Contamination of Sandpits from Recreational Parks and Schools: a Potential Risk for Human Health

Sandpits used by children are frequently visited by wild life which constitutes a source of fungal pathogens and allergenic fungi. This study aimed to take an unannounced snapshot of the urban levels of fungal contaminants in sands, using for this purpose two public recreational parks, three elementary schools and two kindergartens. All samples were from Lisbon and neighboring municipalities and were tested for fungi of clinical interest. Potentially pathogenic fungi were isolated from all samples besides one. Fusarium dimerum (32.4%) was found to be the dominant species in one park and Chrysonilia spp. in the other (46.6%). Fourteen different species and genera were detected and no dermatophytes were found. Of a total of 14 species and genera, the fungi most isolated from the samples of the elementary schools were Penicillium spp. (74%), Cladophialophora spp. (38%) and Cladosporium spp. (90%). Five dominant species and genera were isolated from the kindergartens. Penicillium spp. was the only genus isolated in one, though with remarkably high counts (32500 colony forming units per gram). In the other kindergarten Penicillium spp. were also the most abundant species, occupying 69% of all the fungi found. All of the samples exceeded the Maximum Recommended Value (MRV) for beach sand defined by Brandão et al. 2011, which are currently the only quantitative guidelines available for the same matrix. The fungi found confirm the potential risk of exposure of children to keratinophilic fungi and demonstrates that regular cleaning or replacing of sand needs to be implemented in order to minimize contamination.

Oral fungal microflora associated with implant-supported VS. mucous-supported dentures

Poster presented at the 1st International Congress of CiiEM - From Basic Sciences to Clinical Research, 27-28 November 2015, Egas Moniz, Caparica, Portugal.

Age, pollen and spores of ODP Site 175-1082

The present-day condition of bipolar glaciation characterized by rapid and large climate fluctuations began at the end of the Pliocene with the intensification of the Northern Hemisphere continental glaciations. The global cooling steps of the late Pliocene have been documented in numerous studies of Ocean Drilling Program (ODP) sites from the Northern Hemisphere. However, the interactions between oceans and between land and ocean during these cooling steps are poorly known. In particular, data from the Southern Hemisphere are lacking. Therefore I investigated the pollen of ODP Site 1082 in the southeast Atlantic Ocean in order to obtain a high-resolution record of vegetation change in Namibia between 3.4 and 1.8 Ma. Four phases of vegetation development are inferred that are connected to global climate change. (1) Before 3 Ma, extensive, rather open grass-rich savannahs with mopane trees existed in Namibia, but the extension of desert and semidesert vegetation was still restricted. (2) Increase of winter rainfall dependent Renosterveld-like vegetation occurred between 3.1 and 2.2 Ma connected to strong advection of polar waters along the Namibian coast and a northward shift of the Polar Front Zone in the Southern Ocean. (3) Climatically induced fluctuations became stronger between 2.7 and 2.2 Ma and semiarid areas extended during glacial periods probably as the result of an increased pole-equator thermal gradient and consequently globally enhanced atmospheric circulation. (4) Aridification and climatic variability further increased after 2.2 Ma, when the Polar Front Zone migrated southward and the influence of Atlantic moisture brought by the westerlies to southern Africa declined. It is concluded that the positions of the frontal systems in the Southern Ocean which determine the locations of the high-pressure cells over the South Atlantic and the southern Indian Ocean have a strong influence on the climate of southern Africa in contrast to the climate of northwest and central Africa, which is dominated by the Saharan low-pressure cell.

Pollen and spores in bottom sediments of the Mediterranean Sea

The paper presents results of palynological analysis of deposits from core VITYAZ4779 (length 6.5 m) that was collected from depth 3090 m in the eastern Mediterranean Sea. These palynological data reveal four distinct strata of sediments, each of which was accumulated under different physiographic conditions.

Pollen analysis of surface sediments from the Gulf of Guinea and offshore NW-Africa

The areas of marine pollen deposition are related to the pollen source areas by aeolian and fluvial transport regimes, whereas wind transport is much more important than river transport. Pollen distribution patterns of Pinus, Artemisia, Chenopodiaceae-Amaranthaceae, and Asteraceae Tubuliflorae trace atmospheric transport by the northeast trades. Pollen transport by the African Easterly Jet is reflected in the pollen distribution patterns of Chenopodiaceae-Amaranthaceae, Asteraceae Tubuliflorae, and Mitracarpus. Grass pollen distribution registers the latitudinal extension of Sahel, savannas and dry open forests. Marine pollen distribution patterns of Combretaceae-Melastomataceae, Alchornea, and Elaeis reflect the extension of wooded grasslands and transitional forests. Pollen from the Guinean-Congolian/Zambezian forest and from the Sudanian/Guinean vegetation zones mark the northernmost extension of the tropical rain forest. Rhizophora pollen in marine sediments traces the distribution of mangrove swamps. Only near the continent, pollen of Rhizophora, Mitracarpus, Chenopodiaceae-Amaranthaceae, and pollen from the Sudanian and Guinean vegetation zones are transported by the Upwelling Under Current and the Equatorial Under Current, where those currents act as bottom currents. The distribution of pollen in marine sediments, reflecting the position of major climatic zones (desert, dry tropics, humid tropics), can be used in tracing climatic changes in the past.

Changes in the vegetation and trade winds in equatorial Northwest Africa 140,000-70,000 yr B.P., as deduced from two marine pollen records

The deep-sea cores M 16415-2 and M 16416-2 at about 9°N off Sierra Leone were analysed palynologically for the time interval 140,000-70,000 yr B.P. Results were presented in absolute (pollen concentration and pollen influx) and relative diagrams (pollen percentage). In a previous study it was evidenced that in northwest Africa pollen is mainly transported to the Atlantic by wind, so that the efficiency of aeolian pollen transport (pollen flux) could be used to evaluate changes in the intensity of the northeast trade winds. The glacial episodes (represented by the oxygen isotope stages 6 and 4) are characterized by strong northeast trade winds, whereas the last interglacial (stage 5) is characterized by weak trade winds. The pollen influx diagram shows that the intensity of the trade winds increased slightly during the relatively cool intervals of stage 5 (viz. 5.4 and 5.2). Tropical forest had maximally expanded around 124,000 yr B.P. (stage 5.5), around 98,000 yr B.P. (transition of stage 5.3 to 5.2), and around 70,000 yr B.P. (first part of stage 4): an increasing delay of the response of tropical forest to global intervals with maximum temperature is apparent during the last interglacial. As tropical forests need continuous humidity, the record of tropical forest monitors changes in climatic humidity south of the Sahara. During the last interglacial, the southern boundary of the Sahara shifted only little: expansions and contractions of the tropical forest area are correlated with contra-oscillations of the grass-dominated savanna zone. Great latitudinal shifts of the desert savanna boundary, on the contrary, occurred during the penultimate glacial interglacial transition (around 128,000 yr B.P.) to the north, and during the last interglacial-glacial transition (around 65,000 yr B.P.) to the south.

Marine fungi from the Northwest African upwelling areas and from the Atlantic off Portugal

Quantitative data on lower marine Phycomycetes (fungi) found in the upwelling waters off the West African coast during cruises No. 13 (1968), 19 (1970), 36 (1975) and 44 (1977) of R.V. "Meteor" are reported. The distribution of the total fungi numbers is presented and, as far as possible, the evaluation of the material up to species level is given. Several provisionally named forms and groups of morphologically related, undescribed fungi are included. A correlation between the number of fungi in sediments and the water depth and distance from the coast line is postulated. There are typical distributions of the lower marine fungi in water bodies and sediments. Different values within replicates of the stations in different years show that there is a sequence in development of fungal populations induced by changes in the water bodies. Surface water far from the coast has low numbers of fungi; numbers increase to a maximum nearer to the coast. In the vicinity of the coast the values decrease. The numbers of fungi in the deep sediments are low below 1,200 m. However, there are isolated areas of higher fungal activities, indicated by some deeper grab samples. During two cruises, the "overlying water" in the grab samples was investigated. It was evident that the numbers of fungi lost by stirring of the sediment when the grab was brought up to the surface were small, relatively and absolutely. The seamount "Josephine Bank" has been investigated for the first time with respect to lower marine fungi; the populations are low in the sediments, but one sample of the surface water had a higher number than the water in the surroundings. In some hydrographic series there was a peculiar depth distribution. An increase occurred at a depth greater than 1,000 m. The results are discussed and some correlations to the aging of the fungal populations in the water masses are constructed.