Investigation of the role of Lip5 - a member of the secreted lipase gene family - in the virulence of Candida albicans

Candida albicans (Ca) is the most common species isolated from invasive candidiasis. It has been shown that secreted lipases of Ca contribute to the virulence of the fungus during infection. In this study, we generated heterozygous and homozygous lipase 5 (LIP5) mutants in Ca by the caSAT1-flipper technique. Notably, the Southern-hybridization results indicated a yet unknown allelic heterozygosity in LIP5 in our laboratory strain. Quantitative reverse transcription-PCR experiments demonstrated the absence of LIP5 expression in the homozygous knockout mutants. However, the knock out mutants showed no alteration in the rate of fungal growth, cell and colony morphology under temperature, pH or osmotic stress in comparison to wild type cells. In vitro infection studies using the J774.2 murine macrophage-like cell line revealed no alteration in the virulence of mutant cells. Thus, we conclude that the deletion of LIP5, somewhat surprisingly, has no effect on the physiology or virulence of Ca in our experimental setting

Antivirális és rákellenes hatóanyagok sejtbejutásáért felelős humán hCNT1 nukleozid transzporter szubsztrátkötő centrumának meghatározása új kísérleti rendszer kifejlesztésével = Determination of substrate binding centre of human nucleoside transporter hCNT1 implicated in the transport of antiviral/anticancer drugs through novel experimental approaches

1. Mutáns génbankokat hoztunk létre a humán hCNT1 cDNS-ből, valamint a 2. pontban létrehozott funkcióvesztéses mutáns E497Q-hCNT1 cDNS-ből (technikai kontroll), és funkciónyeréses mutánsok izolálásához elkezdtük a transzformációs kísérleteket. 2. Létrehoztuk az E497Q-hCNT1 funkcióvesztéses mutánst a hCNT1 cDNS molekula irányított pontmutációjával a mutáns génbank létrehozásának technikai ellenőrzése céljából. 3. Létrehoztuk az Aspergillus nidulans cntA::riboB pyrG89 pantoB100 riboB2 deléciós recipiens törzset a cntA::riboB pyrG89 pyroA4 biA1 riboB2 és pantoB100 riboB2 törzsek keresztezésével. A recipiens törzs a humán hCNT1 heterológ expressziós rendszerben történő funkcionális kifejeződésének ellenőrzését szolgálja, valamint az E497Q-hCNT1 funkcióvesztéses mutáns molekulából létrehozott mutáns génbank transzformációját szolgálja. 4. Létrehoztunk egy új, autonóm replikatív Aspergillus expressziós vektort. A 13,2 kb méretű pNNPanto vektort a pAnGFP integrálódó GFP fúziós kiindulási expressziós vektorból hoztuk létre 4 módosítást követően. 5. A hCNT1 és a 2. pontban leírt E497Q-hCNT1 funkcióvesztéses mutáns hCNT1 cDNS heterológ expressziós rendszerben történő kifejeztetésével igazoltuk, hogy a hCNT1 aktív uridin transzportert fejez ki A. nidulansban, valamint ellenőriztük, hogy a mutáns E497Q-hCNT1 nem képes uridin szállításra. 6. Létrehoztunk különböző „uptake” deficiens recipiens törzseket a mutáns génbankok transzformálására és a funkciónyeréses mutánsok screeneléséhez. | 1. Mutant gene banks from human hCNT1 cDNA and hCNT1 cDNA derived non-functional E497Q-hCNT1 molecule (for technical control purpose) were constructed and used for screening of gain-of-function mutants. 2. A non-functional E497Q-hCNT1 mutant hCNT1 cDNA molecule was constructed by directed point mutagenesis for the purpose of generation and using the E497Q-hCNT1 mutant gene bank to evaluate technically the process of mutant gene bank generation. 3. cntA::riboB pyrG89 pantoB100 riboB2 A. nidulans deletion mutant was developed by crossing cntA::riboB pyrG89 pyroA4 biA1 riboB2 with pantoB100 riboB2 for the purpose of test-transformation of (i) hCNT1 and E497Q-hCNT1 in the heterologue expression system; (ii) transformation with E497Q-hCNT1 mutant gene bank. 4. Novel autonomous expression vector of A. nidulans, pNNPanto was developed by engineering the existing pAnGFP integrative GFP fusion A. nidulans vector in four steps. 5. Heterologous expression of the human hCNT1 and E497Q-hCNT1 and the expression of A. nidulans CntA (AN5493.3) cDNAs cloned in pNNPanto autonomous expression vector were carried out in cntA::riboB pyrG89 pantoB100 riboB2 recipient strain. 6. Hypoxanthine/uric acid uptake deficient mutant uapA24 uapC201/401 azgA4 pantoB100 was developed by cross of uapA24 uapC201/401 azgA4 pabaA1 strain with pantoB100 riboB2 mutant for the purpose of direct screening for gain-of-function mutants

Extrachromosomal genetic elements in fungi

All fungi like eukaryotes possess mitochondria, which are the sites of the oxydative phosphorylation. As eukaryote evolution depends on oxygenic atmosphere, these organisms are primarily aerobic. Except a small group of strict anaerobes (those which lost the capacity of oxydative pathways living in special milieu in association with rumen of grass-eating animals) all fungi can utilize various compounds as carbon sources via oxidative phosphorylation pathways resulting in high energy yield. Certain groups of fungi – i.e. most of the yeasts – under anaerobe conditions, are able to supply themselves with lower levels of fermentation energy, too exhibiting a slow growing capacity utilizing the same amount of carbon source. The mutation of mitochondrial genome or mitochondrial functions encoded by nuclear genes of these fungi might result in a so-calledpetitephenotype producing small colonies on solid media due to their slow growing capacity. These mutants can utilize only fermentable carbon sources. Filamentous fungi have only limited possibilities to produce such phenotypes. ExceptZygomycetes(where the shortage of oxygen induces dimorphic transitions) filamentous fungi can grow and develop their vegetative and sexual reproductive structures only in aerobe milieu. However amongNeurosporaspecies there are several mitochondrial mutations resulting in morphological phenotypes. These are due to the lower energy level provided by the reduced capacity of cytochrome-oxidase enzymes. These mutants (e.g.pokystopper) can be considered aspetiteanalogues. The complete loss of mitochondrial functions – such asrho zerocharacter in yeast – cannot be survived by filamentous fungi. Podospora anserinaand some of its close relatives exhibit a so-calledsenescencephenotype, which means that the growing hyphae in the youngest part of the colonies stop growing and start to die within a short period of time. This phenomenon – discussed below – is also connected to reduced function of mitochondria.The first part of this paper gives a short overview of the genetic organization of mitochondria of fungi, based on the most recent data of three filamentous fungi:Aspergillus nidulansNeurospora crassaand Podospora anserina. Their data are compared to those of the well-characterizedSaccharomyces cerevisiae. In the second part we summarize what we know about other extrachrosomal elements, such as DNA plasmids of various origins and structures, and dsRNAs or virus like particles (VLP). Also discussed are their roles and/or putative functions in the life of the fungi

In vitro interactions of Candida parapsilosis wild type and lipase deficient mutants with human monocyte derived dendritic cells

<p>Abstract</p> <p>Background</p> <p><it>Candida parapsilosis </it>typically is a commensal of human skin. However, when host immune defense is compromised or the normal microflora balance is disrupted, <it>C. parapsilosis </it>transforms itself into an opportunistic pathogen. <it>Candida</it>-derived lipase has been identified as potential virulence factor. Even though cellular components of the innate immune response, such as dendritic cells, represent the first line of defense against invading pathogens, little is known about the interaction of these cells with invading <it>C. parapsilosis</it>. Thus, the aim of our study was to assess the function of dendritic cells in fighting <it>C. parapsilosis </it>and to determine the role that <it>C. parapsilosis</it>-derived lipase plays in the interaction with dendritic cells.</p> <p>Results</p> <p>Monocyte-derived immature and mature dendritic cells (iDCs and mDCs, respectively) co-cultured with live wild type or lipase deficient <it>C. parapsilosis </it>strains were studied to determine the phagocytic capacity and killing efficiency of host cells. We determined that both iDCs and mDCs efficiently phagocytosed and killed <it>C. parapsilosis</it>, furthermore our results show that the phagocytic and fungicidal activities of both iDCs and mDCs are more potent for lipase deficient compared to wild type yeast cells. In addition, the lipase deficient <it>C. parapsilosis </it>cells induce higher gene expression and protein secretion of proinflammatory cytokines and chemokines in both DC types relative to the effect of co-culture with wild type yeast cells.</p> <p>Conclusions</p> <p>Our results show that DCs are activated by exposure to <it>C. parapsilosis</it>, as shown by increased phagocytosis, killing and proinflammatory protein secretion. Moreover, these data strongly suggest that <it>C. parapsilosis </it>derived lipase has a protective role during yeast:DC interactions, since lipase production in wt yeast cells decreased the phagocytic capacity and killing efficiency of host cells and downregulated the expression of host effector molecules.</p

The role of the Aspergillus nidulans high mobility group B protein HmbA, the orthologue of Saccharomyces cerevisiae Nhp6p

The mammalian HMGB1 is a high-mobility-group B protein, which is both an architectural and functional element of chromatin. Nhp6p, the extensively studied fungal homologue of HMGB1 in Saccharomyces cerevisiae has pleiotropic physiological functions. Despite the existence of Nhp6p orthologues in filamentous ascomycetes, little is known about their physiological roles besides their contribution to sexual development. Here we study the function of HmbA, the Aspergillus nidulans orthologue of Nhp6p. We show that HmbA influences the utilization of various carbon- and nitrogen sources, stress tolerance, secondary metabolism, hyphae elongation and maintenance of polarized growth. Additionally, by conducting heterologous expression studies, we demonstrate that HmbA and Nhp6p are partially interchangeable. HmbA restores SNR6 transcription and fitness of nhp6AΔBΔ mutant and reverses its heat sensitivity. Nhp6Ap complements several phenotypes of hmbAΔ , including ascospore formation, utilization of various carbon- and nitrogen-sources, radial growth rate, hypha elongation by polarized growth. However, Nhp6Ap does not complement sterigmatocystin production in a hmbAΔ strain. Finally, we also show that HmbA is necessary for the normal expression of the endochitinase chiA , a cell wall re-modeller that is pivotal for the normal mode of maintenance of polar growth

Mikroszkópikus gombák (Aspergillus, Fusarium, Cryptococcus) mitokondriális genomszerveződésének összehasonlító elemzése = Study on the mitochondrial genome organisation of some microscopic fungi

Munkánk során meghatároztuk egy Aspergillus niger (1a típus, 31103 bp) és egy A. tübingensis (2b típus, 33656 bp) törzs teljes mitokondriális DNS szekvenciáját. A két genom géntartalma és a gének sorrendje megegyezik, különbséget mindössze a restrikciós enzimek hasítási mintázatában tapasztaltunk. Megállapítottuk, hogy a méretbeli eltérésekért a cox1, atp9 és a ndh4L gének intron-tartalma felelős. Elkészítettük a korábban már RFLP mintázat alapján elkülönített hat A. tübingensis mtDNS fizikai és funkcionális térképét. Eredményeink bizonyították, hogy a tapasztalt intraspecifikus polimorfizmus intron-szerzéssel illetve restrikciós hasítóhelyeket érintő pontmutációkkal magyarázható. Növény-patogén Fusarium törzsek biodiverzitását a mtDNS RFLP mintázata alapján tanulmányoztuk. Több haplotípust sikerült elkülönítenünk a vizsgált 3 fajban. Meghatároztuk e haplotípusok gazdanövény szerinti eloszlását. F. proliferatum lineáris, mitokondriumban lokalizált DNS plazmidjának teljes szekvenálását elvégeztük. A plazmid funkciójára jelenleg nincs bizonyítékunk. Cryptococcus neoformans két varietas-ának mtDNS szerveződését hasonlítottuk össze. Megállapítottuk, hogy a tapasztalt méretbeli különbséget a cox1, cob és LRNS gének eltérő intron-tartalma okozza. Egy másik vizsgált faj, a Trichosporon pullulans 18 kb méretű mtDNS-e a legkisebb, NADH géneket is tartalmazó mtDNS-nek bizonyult élesztőgombák között. | In the present work the complete mitochondrial DNA (mtDNA) of Aspergillus niger mtDNA type 1a (31103 bp) was sequenced and compared to the Aspergillus tubingensis type 2b (33656 bp) mtDNA. The patterns of restriction sites were similar, the gene content and order was identical. The size difference was principally attributed to the intron content of their cox1, atp9 and ndh4L genes. A. tubingensis isolates were earlier classified into six groups on the basis of the mtDNA RFLP pattern. The reason of the intraspecific mtDNA variability was investigated and proved that polymorphism due to intron acquisition and also sporadic point mutations affecting the recognition motifs of the restriction enzymes. Biodiversity of plant-pathogenic Fusarium isolates belonging to 3 different species were studied. On the basis of the RFLP pattern of their mtDNA several haplotypes were identified. The distribution of these haplotypes among host species was determined. The complete nucleotide sequence of a 10 kb linear DNA plasmid was identified, however, its function is still unknown. The functional map of the mtDNAs of two Cryptococcus neoformans varities was constructed. Characteristic sequences were cloned, sequenced and verified that the intron-content of coxI, cob and LRNA genes are responsible for the size differences of the two strains. The study of the organisation of Trichosporon pullulans mtDNA revealed that it is smallest known mtDNA among yeast carrying NADH dehidrogenase genes

Patogén Crytococcusok ellen hatásos killer toxin izolálása, jellemzése és a kódoló gén lokalizálása, klónozása Filobasidium capsulogenumban = Isolation and characterization of killer toxin active against pathogen Cryptococcus, localization and cloning of the toxin coding gene(s) in Filobasidium capsulogenum.

A basidiomycota Filobasidium capsuligenum egy olyan fehérjét szekretál, amely elpusztítja a patogén Cryptococcus neoformanst. Pályázatunkban ezen killer toxin genetikai determinánsát derítettük fel, a toxin fehérje izolálását, jellemzését valósítottuk meg, ill a toxin hatásmechanizmusát is feltártuk. Az érzékenységi tesztek alapján az FC-1 toxin specifikusan, csak a C. neoformans-ra hatott. Bebizonyítottuk, hogy e hatás citocidikus. A toxin protein természetű: hő hatására elbomlik, aktivitásának optimuma pH 4-6. A toxin receptoraként az érzékeny sejtek sejtfalának béta-1,6-glükánját határoztuk meg. E specificitást arra használtuk fel, hogy a toxint affinitáskromatográfiával tisztítsuk. A pusztulán-Sepharose 6B oszlop legaktívabb frakciójával SDS-PAGE-t végeztünk, és 3 protein sávot találtunk a 19 - 51 kDa tartományban. További kísérletek szükségesek a killer aktivitású protein azonosítására. A F. capsuligenum sejtek minilizátainak agaróz gélelektroforézisével kiderítettük, hogy az FC-1 proteint kromoszómális gén kódolja. A toxin hatásmódjának tanulmányozására a celluláris DNS mennyiségének változását a sejtciklus függvényében (DNS szintézis gátlás?) ill. a toxin kezelt sejtek FITC festődését (sejtfal bioszintézis gátlás?) vizsgáltuk. Eredményeink szerint a killing mechanizmus sem a sejtciklustól, sem pedig a sejtfal bioszintézistől nem függ. Sokkal inkább ionofor proteinként hat, amely a citoplazma membránfunkcióját rombolja. | The basidiomycetous yeast Filobasidium capsuligenum produces a killer toxin (FC-1) which is highly effective against the pathogen Cryptococcus neoformans. The goal of this project was to characterize the toxin, determine the genetic trait coding for it, and study its effect on C. neoformans cells. It was demonstrated that FC-1 toxin was specific for C. neoformans. The toxin had a cytocidal effect. FC-1 proved to be a protein: sensitive for heat and proteolytic enzymes. The optimal pH for the activity was within the range of pH 4-6. As receptor site for the killer protein beta-1,6-glucan in the cell wall of sensitive cells was determined. The specificity was used for purification of the toxin through affinity chromatography. The most active fraction from a pustulan-Sepharose 6B column was subjected to SDS-PAGE, and three protein bands, in molecular mass of 19 - 51 kDa, were detected. Additional investigations are necessary to identify the protein with killer property. Agarose gel electrophoresis of F. capsuligenum cell minilysate revealed that FC-1 protein is coded by the chromosomal DNA. Analysis of cellular DNA (inhibition of DNA synthesis?) by laser scanning cytometry and FITC staining (inhibition of cell wall biosynthesis?) of the toxin-treated cells revealed that the killing mechanism of FC-1 is neither cell cycle- nor cell wall biosynthesis-dependent; rather it might act as an ionophoric protein that disrupts the cytoplasmic membrane function

The role of the Aspergillus nidulans high mobility group B protein HmbA, the orthologue of Saccharomyces cerevisiae Nhp6p

The mammalian HMGB1 is a high-mobility-group B protein, which is both an architectural and functional element of chromatin. Nhp6p, the extensively studied fungal homologue of HMGB1 in Saccharomyces cerevisiae has pleiotropic physiological functions. Despite the existence of Nhp6p orthologues in filamentous ascomycetes, little is known about their physiological roles besides their contribution to sexual development. Here we study the function of HmbA, the Aspergillus nidulans orthologue of Nhp6p. We show that HmbA influences the utilization of various carbon- and nitrogen sources, stress tolerance, secondary metabolism, hyphae elongation and maintenance of polarized growth. Additionally, by conducting heterologous expression studies, we demonstrate that HmbA and Nhp6p are partially interchangeable. HmbA restores SNR6 transcription and fitness of nhp6AΔBΔ mutant and reverses its heat sensitivity. Nhp6Ap complements several phenotypes of hmbAΔ , including ascospore formation, utilization of various carbon- and nitrogen-sources, radial growth rate, hypha elongation by polarized growth. However, Nhp6Ap does not complement sterigmatocystin production in a hmbAΔ strain. Finally, we also show that HmbA is necessary for the normal expression of the endochitinase chiA , a cell wall re-modeller that is pivotal for the normal mode of maintenance of polar growth

A complete nicotinate degradation pathway in the microbial eukaryote Aspergillus nidulans

Several strikingly different aerobic and anaerobic pathways of nicotinate breakdown are extant in bacteria. Here, through reverse genetics and analytical techniques we elucidated in Aspergillus nidulans, a complete eukaryotic nicotinate utilization pathway. The pathway extant in this fungus and other ascomycetes, is quite different from bacterial ones. All intermediate metabolites were identified. The cognate proteins, encoded by eleven genes (hxn) mapping in three clusters are co-regulated by a specific transcription factor. Several enzymatic steps have no prokaryotic equivalent and two metabolites, 3-hydroxypiperidine-2,6-dione and 5,6-dihydroxypiperidine-2-one, have not been identified previously in any organism, the latter being a novel chemical compound. Hydrolytic ring opening results in α-hydroxyglutaramate, a compound not detected in analogous prokaryotic pathways. Our earlier phylogenetic analysis of Hxn proteins together with this complete biochemical pathway illustrates convergent evolution of catabolic pathways between fungi and bacteria