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Contains fulltext : 235356.pdf (Publisher’s version ) (Open Access), Candida albicans is a major fungal pathogen of humans. It exists as a commensal in the oral cavity, gut or genital tract of most individuals, constrained by the local microbiota, epithelial barriers and immune defences. Their perturbation can lead to fungal outgrowth and the development of mucosal infections such as oropharyngeal or vulvovaginal candidiasis, and patients with compromised immunity are susceptible to life-threatening systemic infections. The importance of the interplay between fungus, host and microbiota in driving the transition from C. albicans commensalism to pathogenicity is widely appreciated. However, the complexity of these interactions, and the significant impact of fungal, host and microbiota variability upon disease severity and outcome, are less well understood. Therefore, we summarise the features of the fungus that promote infection, and how genetic variation between clinical isolates influences pathogenicity. We discuss antifungal immunity, how this differs between mucosae, and how individual variation influences a person's susceptibility to infection. Also, we describe factors that influence the composition of gut, oral and vaginal microbiotas, and how these affect fungal colonisation and antifungal immunity. We argue that a detailed understanding of these variables, which underlie fungal-host-microbiota interactions, will present opportunities for directed antifungal therapies that benefit vulnerable patients.

Contains fulltext : 235356.pdf (Publisher’s version ) (Open Access), Candida albicans is a major fungal pathogen of humans. It exists as a commensal in the oral cavity, gut or genital tract of most individuals, constrained by the local microbiota, epithelial barriers and immune defences. Their perturbation can lead to fungal outgrowth and the development of mucosal infections such as oropharyngeal or vulvovaginal candidiasis, and patients with compromised immunity are susceptible to life-threatening systemic infections. The importance of the interplay between fungus, host and microbiota in driving the transition from C. albicans commensalism to pathogenicity is widely appreciated. However, the complexity of these interactions, and the significant impact of fungal, host and microbiota variability upon disease severity and outcome, are less well understood. Therefore, we summarise the features of the fungus that promote infection, and how genetic variation between clinical isolates influences pathogenicity. We discuss antifungal immunity, how this differs between mucosae, and how individual variation influences a person's susceptibility to infection. Also, we describe factors that influence the composition of gut, oral and vaginal microbiotas, and how these affect fungal colonisation and antifungal immunity. We argue that a detailed understanding of these variables, which underlie fungal-host-microbiota interactions, will present opportunities for directed antifungal therapies that benefit vulnerable patients.

Contains fulltext : 235356.pdf (Publisher’s version ) (Open Access), Candida albicans is a major fungal pathogen of humans. It exists as a commensal in the oral cavity, gut or genital tract of most individuals, constrained by the local microbiota, epithelial barriers and immune defences. Their perturbation can lead to fungal outgrowth and the development of mucosal infections such as oropharyngeal or vulvovaginal candidiasis, and patients with compromised immunity are susceptible to life-threatening systemic infections. The importance of the interplay between fungus, host and microbiota in driving the transition from C. albicans commensalism to pathogenicity is widely appreciated. However, the complexity of these interactions, and the significant impact of fungal, host and microbiota variability upon disease severity and outcome, are less well understood. Therefore, we summarise the features of the fungus that promote infection, and how genetic variation between clinical isolates influences pathogenicity. We discuss antifungal immunity, how this differs between mucosae, and how individual variation influences a person's susceptibility to infection. Also, we describe factors that influence the composition of gut, oral and vaginal microbiotas, and how these affect fungal colonisation and antifungal immunity. We argue that a detailed understanding of these variables, which underlie fungal-host-microbiota interactions, will present opportunities for directed antifungal therapies that benefit vulnerable patients.

Contains fulltext : 200455.pdf (publisher's version ) (Open Access), Clearance of invading microbes requires phagocytes of the innate immune system. However, successful pathogens have evolved sophisticated strategies to evade immune killing. The opportunistic human fungal pathogen Candida albicans is efficiently phagocytosed by macrophages, but causes inflammasome activation, host cytolysis, and escapes after hypha formation. Previous studies suggest that macrophage lysis by C. albicans results from early inflammasome-dependent cell death (pyroptosis), late damage due to glucose depletion and membrane piercing by growing hyphae. Here we show that Candidalysin, a cytolytic peptide toxin encoded by the hypha-associated gene ECE1, is both a central trigger for NLRP3 inflammasome-dependent caspase-1 activation via potassium efflux and a key driver of inflammasome-independent cytolysis of macrophages and dendritic cells upon infection with C. albicans. This suggests that Candidalysin-induced cell damage is a third mechanism of C. albicans-mediated mononuclear phagocyte cell death in addition to damage caused by pyroptosis and the growth of glucose-consuming hyphae.

Contains fulltext : 200455.pdf (publisher's version ) (Open Access), Clearance of invading microbes requires phagocytes of the innate immune system. However, successful pathogens have evolved sophisticated strategies to evade immune killing. The opportunistic human fungal pathogen Candida albicans is efficiently phagocytosed by macrophages, but causes inflammasome activation, host cytolysis, and escapes after hypha formation. Previous studies suggest that macrophage lysis by C. albicans results from early inflammasome-dependent cell death (pyroptosis), late damage due to glucose depletion and membrane piercing by growing hyphae. Here we show that Candidalysin, a cytolytic peptide toxin encoded by the hypha-associated gene ECE1, is both a central trigger for NLRP3 inflammasome-dependent caspase-1 activation via potassium efflux and a key driver of inflammasome-independent cytolysis of macrophages and dendritic cells upon infection with C. albicans. This suggests that Candidalysin-induced cell damage is a third mechanism of C. albicans-mediated mononuclear phagocyte cell death in addition to damage caused by pyroptosis and the growth of glucose-consuming hyphae.

Contains fulltext : 200455.pdf (publisher's version ) (Open Access), Clearance of invading microbes requires phagocytes of the innate immune system. However, successful pathogens have evolved sophisticated strategies to evade immune killing. The opportunistic human fungal pathogen Candida albicans is efficiently phagocytosed by macrophages, but causes inflammasome activation, host cytolysis, and escapes after hypha formation. Previous studies suggest that macrophage lysis by C. albicans results from early inflammasome-dependent cell death (pyroptosis), late damage due to glucose depletion and membrane piercing by growing hyphae. Here we show that Candidalysin, a cytolytic peptide toxin encoded by the hypha-associated gene ECE1, is both a central trigger for NLRP3 inflammasome-dependent caspase-1 activation via potassium efflux and a key driver of inflammasome-independent cytolysis of macrophages and dendritic cells upon infection with C. albicans. This suggests that Candidalysin-induced cell damage is a third mechanism of C. albicans-mediated mononuclear phagocyte cell death in addition to damage caused by pyroptosis and the growth of glucose-consuming hyphae.

Contains fulltext : 200455.pdf (publisher's version ) (Open Access), Clearance of invading microbes requires phagocytes of the innate immune system. However, successful pathogens have evolved sophisticated strategies to evade immune killing. The opportunistic human fungal pathogen Candida albicans is efficiently phagocytosed by macrophages, but causes inflammasome activation, host cytolysis, and escapes after hypha formation. Previous studies suggest that macrophage lysis by C. albicans results from early inflammasome-dependent cell death (pyroptosis), late damage due to glucose depletion and membrane piercing by growing hyphae. Here we show that Candidalysin, a cytolytic peptide toxin encoded by the hypha-associated gene ECE1, is both a central trigger for NLRP3 inflammasome-dependent caspase-1 activation via potassium efflux and a key driver of inflammasome-independent cytolysis of macrophages and dendritic cells upon infection with C. albicans. This suggests that Candidalysin-induced cell damage is a third mechanism of C. albicans-mediated mononuclear phagocyte cell death in addition to damage caused by pyroptosis and the growth of glucose-consuming hyphae.

UNLABELLED: The genus Malassezia includes 14 species that are found on the skin of humans and animals and are associated with a number of diseases. Recent genome sequencing projects have defined the gene content of all 14 species; however, to date, genetic manipulation has not been possible for any species within this genus. Here, we develop and then optimize molecular tools for the transformation of Malassezia furfur and Malassezia sympodialis using Agrobacterium tumefaciens delivery of transfer DNA (T-DNA) molecules. These T-DNAs can insert randomly into the genome. In the case of M. furfur, targeted gene replacements were also achieved via homologous recombination, enabling deletion of the ADE2 gene for purine biosynthesis and of the LAC2 gene predicted to be involved in melanin biosynthesis. Hence, the introduction of exogenous DNA and direct gene manipulation are feasible in Malassezia species. IMPORTANCE: Species in the genus Malassezia are a defining component of the microbiome of the surface of mammals. They are also associated with a wide range of skin disease symptoms. Many species are difficult to culture in vitro, and although genome sequences are available for the species in this genus, it has not been possible to assess gene function to date. In this study, we pursued a series of possible transformation methods and identified one that allows the introduction of DNA into two species of Malassezia, including the ability to make targeted integrations into the genome such that genes can be deleted. This research opens a new direction in terms of now being able to analyze gene functions in this little understood genus. These tools will contribute to define the mechanisms that lead to the commensalism and pathogenicity in this group of obligate fungi that are predominant on the skin of mammals.

UNLABELLED: Fungal diseases represent a major burden to health care globally. As with other pathogenic microbes, there is a limited number of agents suitable for use in treating fungal diseases, and resistance to these agents can develop rapidly. Cryptococcus neoformans is a basidiomycete fungus that causes cryptococcosis worldwide in both immunocompromised and healthy individuals. As a basidiomycete, it diverged from other common pathogenic or model ascomycete fungi more than 500 million years ago. Here, we report C. neoformans genes that are essential for viability as identified through forward and reverse genetic approaches, using an engineered diploid strain and genetic segregation after meiosis. The forward genetic approach generated random insertional mutants in the diploid strain, the induction of meiosis and sporulation, and selection for haploid cells with counterselection of the insertion event. More than 2,500 mutants were analyzed, and transfer DNA (T-DNA) insertions in several genes required for viability were identified. The genes include those encoding the thioredoxin reductase (Trr1), a ribosome assembly factor (Rsa4), an mRNA-capping component (Cet1), and others. For targeted gene replacement, the C. neoformans homologs of 35 genes required for viability in ascomycete fungi were disrupted, meiosis and sporulation were induced, and haploid progeny were evaluated for their ability to grow on selective media. Twenty-one (60%) were found to be required for viability in C. neoformans. These genes are involved in mitochondrial translation, ergosterol biosynthesis, and RNA-related functions. The heterozygous diploid mutants were evaluated for haploinsufficiency on a number of perturbing agents and drugs, revealing phenotypes due to the loss of one copy of an essential gene in C. neoformans. This study expands the knowledge of the essential genes in fungi using a basidiomycete as a model organism. Genes that have no mammalian homologs and are essential in b

Item does not contain fulltext, Little is known regarding the role of NK cells during primary and secondary disseminated Candida albicans infection. We assessed the role of NK cells for host defense against candidiasis in immunocompetent, as well as immunodeficient, hosts. Surprisingly, depletion of NK cells in immunocompetent WT mice did not increase susceptibility to systemic candidiasis, suggesting that NK cells are redundant for antifungal defense in otherwise immunocompetent hosts. NK-cell-depleted mice were found to be protected as a consequence of attenuation of systemic inflammation. In contrast, the absence of NK cells in T/B/NK-cell-deficient NSG (NOD SCID gamma) mice led to an increased susceptibility to both primary and secondary systemic C. albicans infections compared with T/B-cell-deficient SCID mice. In conclusion, this study demonstrates that NK cells are an essential and nonredundant component of anti-C. albicans host defense in immunosuppressed hosts with defective T/B-lymphocyte immunity, while contributing to hyperinflammation in immunocompetent hosts. The discovery of the importance of NK cells in hosts with severe defects of adaptive immunity might have important consequences for the design of adjunctive immunotherapeutic approaches in systemic C. albicans infections targeting NK-cell function.

Item does not contain fulltext, Little is known regarding the role of NK cells during primary and secondary disseminated Candida albicans infection. We assessed the role of NK cells for host defense against candidiasis in immunocompetent, as well as immunodeficient, hosts. Surprisingly, depletion of NK cells in immunocompetent WT mice did not increase susceptibility to systemic candidiasis, suggesting that NK cells are redundant for antifungal defense in otherwise immunocompetent hosts. NK-cell-depleted mice were found to be protected as a consequence of attenuation of systemic inflammation. In contrast, the absence of NK cells in T/B/NK-cell-deficient NSG (NOD SCID gamma) mice led to an increased susceptibility to both primary and secondary systemic C. albicans infections compared with T/B-cell-deficient SCID mice. In conclusion, this study demonstrates that NK cells are an essential and nonredundant component of anti-C. albicans host defense in immunosuppressed hosts with defective T/B-lymphocyte immunity, while contributing to hyperinflammation in immunocompetent hosts. The discovery of the importance of NK cells in hosts with severe defects of adaptive immunity might have important consequences for the design of adjunctive immunotherapeutic approaches in systemic C. albicans infections targeting NK-cell function.

Item does not contain fulltext, Little is known regarding the role of NK cells during primary and secondary disseminated Candida albicans infection. We assessed the role of NK cells for host defense against candidiasis in immunocompetent, as well as immunodeficient, hosts. Surprisingly, depletion of NK cells in immunocompetent WT mice did not increase susceptibility to systemic candidiasis, suggesting that NK cells are redundant for antifungal defense in otherwise immunocompetent hosts. NK-cell-depleted mice were found to be protected as a consequence of attenuation of systemic inflammation. In contrast, the absence of NK cells in T/B/NK-cell-deficient NSG (NOD SCID gamma) mice led to an increased susceptibility to both primary and secondary systemic C. albicans infections compared with T/B-cell-deficient SCID mice. In conclusion, this study demonstrates that NK cells are an essential and nonredundant component of anti-C. albicans host defense in immunosuppressed hosts with defective T/B-lymphocyte immunity, while contributing to hyperinflammation in immunocompetent hosts. The discovery of the importance of NK cells in hosts with severe defects of adaptive immunity might have important consequences for the design of adjunctive immunotherapeutic approaches in systemic C. albicans infections targeting NK-cell function.

1 april 2012, Contains fulltext : 110647.pdf (publisher's version ) (Closed access), In experimental studies, the role of complement in antifungal host defense has been attributed to its opsonizing capability. In this study, we report that in humans an activated complement system mainly augments Candida albicans-induced host proinflammatory cytokine production via C5a-C5aR signaling, while phagocytosis and intracellular killing of Candida are not influenced. By blocking the C5a-C5aR signaling pathway, either with anti-C5a antagonist antibodies or with the C5aR antagonist W-54001, C. albicans-induced IL-6 and IL-1beta levels were significantly reduced. Recombinant C5a augmented cytokine production. In addition, using serum from patients with various complement deficiencies, we demonstrated a crucial role of C5, but not C6 or the membrane attack complex, in C. albicans-induced IL-6 and IL-1beta production in monocytes. These findings reveal a central role of anaphylatoxin C5a in augmenting host proinflammatory cytokine production upon contact with C. albicans, and define the role of the complement system in anti-Candida host defense in humans.

01 april 2012, Contains fulltext : 110647.pdf (publisher's version ) (Closed access), In experimental studies, the role of complement in antifungal host defense has been attributed to its opsonizing capability. In this study, we report that in humans an activated complement system mainly augments Candida albicans-induced host proinflammatory cytokine production via C5a-C5aR signaling, while phagocytosis and intracellular killing of Candida are not influenced. By blocking the C5a-C5aR signaling pathway, either with anti-C5a antagonist antibodies or with the C5aR antagonist W-54001, C. albicans-induced IL-6 and IL-1beta levels were significantly reduced. Recombinant C5a augmented cytokine production. In addition, using serum from patients with various complement deficiencies, we demonstrated a crucial role of C5, but not C6 or the membrane attack complex, in C. albicans-induced IL-6 and IL-1beta production in monocytes. These findings reveal a central role of anaphylatoxin C5a in augmenting host proinflammatory cytokine production upon contact with C. albicans, and define the role of the complement system in anti-Candida host defense in humans.

01 april 2012, Contains fulltext : 110647.pdf (publisher's version ) (Closed access), In experimental studies, the role of complement in antifungal host defense has been attributed to its opsonizing capability. In this study, we report that in humans an activated complement system mainly augments Candida albicans-induced host proinflammatory cytokine production via C5a-C5aR signaling, while phagocytosis and intracellular killing of Candida are not influenced. By blocking the C5a-C5aR signaling pathway, either with anti-C5a antagonist antibodies or with the C5aR antagonist W-54001, C. albicans-induced IL-6 and IL-1beta levels were significantly reduced. Recombinant C5a augmented cytokine production. In addition, using serum from patients with various complement deficiencies, we demonstrated a crucial role of C5, but not C6 or the membrane attack complex, in C. albicans-induced IL-6 and IL-1beta production in monocytes. These findings reveal a central role of anaphylatoxin C5a in augmenting host proinflammatory cytokine production upon contact with C. albicans, and define the role of the complement system in anti-Candida host defense in humans.

1 november 2010, Contains fulltext : 88980.pdf (publisher's version ) (Open Access), The secretion of aspartic proteases (Saps) has long been recognized as a virulence-associated trait of the pathogenic yeast Candida albicans. In this study, we report that different recombinant Saps, including Sap1, Sap2, Sap3, and Sap6, have differing abilities to induce secretion of proinflammatory cytokines by human monocytes. In particular Sap1, Sap2, and Sap6 significantly induced interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNF-alpha), and IL-6 production. Sap3 was able to stimulate the secretion of IL-1beta and TNF-alpha. All Saps tested were able to induce Ca(2+) influx in monocytes. Treatment of these Saps with pepstatin A did not have any effect on cytokine secretion, indicating that their stimulatory potential was independent from their proteolytic activity. The capacity of Saps to induce inflammatory cytokine production was also independent from protease-activated receptor (PAR) activation and from the optimal pH for individual Sap activity. The interaction of Saps with monocytes induced Akt activation and phosphorylation of IkappaBalpha, which mediates translocation of NF-kappaB into the nucleus. Overall, these results suggest that individual Sap proteins can induce an inflammatory response and that this phenomenon is independent from the pH of a specific host niche and from Sap enzymatic activity. The inflammatory response is partially dependent on Sap denaturation and is triggered by the Akt/NF-kappaB activation pathway. Our data suggest a novel, activity-independent aspect of Saps during interactions of C. albicans with the host.

1 januari 2010, Contains fulltext : 88208.pdf (publisher's version ) (Open Access), The innate immune system recognizes pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRR) and transduces downstream signaling to activate the host defense. Here we report that in addition to direct PAMP-PRR interactions, live Candida albicans cells can release soluble factors to actively potentiate interleukin-6 (IL-6) and IL-8 production induced in human mononuclear cells by the fungi. Although protease-activated receptor 1 (PAR1) and PAR2 ligation can moderately upregulate Toll-like receptor 4 (TLR4)-mediated IL-8 production, no effect on the C. albicans-induced cytokine was apparent. Similarly, the blockade of PAR signaling did not reverse the potentiation of cytokine production induced by soluble factors released by C. albicans. In conclusion, C. albicans releases soluble factors that potentiate cytokine release in a PAR1/2-independent manner. Thus, human PAR1 and PAR2 have a redundant role in the activation of human cells by C. albicans.

01 november 2010, Contains fulltext : 88980.pdf (publisher's version ) (Open Access), The secretion of aspartic proteases (Saps) has long been recognized as a virulence-associated trait of the pathogenic yeast Candida albicans. In this study, we report that different recombinant Saps, including Sap1, Sap2, Sap3, and Sap6, have differing abilities to induce secretion of proinflammatory cytokines by human monocytes. In particular Sap1, Sap2, and Sap6 significantly induced interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNF-alpha), and IL-6 production. Sap3 was able to stimulate the secretion of IL-1beta and TNF-alpha. All Saps tested were able to induce Ca(2+) influx in monocytes. Treatment of these Saps with pepstatin A did not have any effect on cytokine secretion, indicating that their stimulatory potential was independent from their proteolytic activity. The capacity of Saps to induce inflammatory cytokine production was also independent from protease-activated receptor (PAR) activation and from the optimal pH for individual Sap activity. The interaction of Saps with monocytes induced Akt activation and phosphorylation of IkappaBalpha, which mediates translocation of NF-kappaB into the nucleus. Overall, these results suggest that individual Sap proteins can induce an inflammatory response and that this phenomenon is independent from the pH of a specific host niche and from Sap enzymatic activity. The inflammatory response is partially dependent on Sap denaturation and is triggered by the Akt/NF-kappaB activation pathway. Our data suggest a novel, activity-independent aspect of Saps during interactions of C. albicans with the host.

01 januari 2010, Contains fulltext : 88208.pdf (publisher's version ) (Open Access), The innate immune system recognizes pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRR) and transduces downstream signaling to activate the host defense. Here we report that in addition to direct PAMP-PRR interactions, live Candida albicans cells can release soluble factors to actively potentiate interleukin-6 (IL-6) and IL-8 production induced in human mononuclear cells by the fungi. Although protease-activated receptor 1 (PAR1) and PAR2 ligation can moderately upregulate Toll-like receptor 4 (TLR4)-mediated IL-8 production, no effect on the C. albicans-induced cytokine was apparent. Similarly, the blockade of PAR signaling did not reverse the potentiation of cytokine production induced by soluble factors released by C. albicans. In conclusion, C. albicans releases soluble factors that potentiate cytokine release in a PAR1/2-independent manner. Thus, human PAR1 and PAR2 have a redundant role in the activation of human cells by C. albicans.

01 januari 2010, Contains fulltext : 88208.pdf (publisher's version ) (Open Access), The innate immune system recognizes pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRR) and transduces downstream signaling to activate the host defense. Here we report that in addition to direct PAMP-PRR interactions, live Candida albicans cells can release soluble factors to actively potentiate interleukin-6 (IL-6) and IL-8 production induced in human mononuclear cells by the fungi. Although protease-activated receptor 1 (PAR1) and PAR2 ligation can moderately upregulate Toll-like receptor 4 (TLR4)-mediated IL-8 production, no effect on the C. albicans-induced cytokine was apparent. Similarly, the blockade of PAR signaling did not reverse the potentiation of cytokine production induced by soluble factors released by C. albicans. In conclusion, C. albicans releases soluble factors that potentiate cytokine release in a PAR1/2-independent manner. Thus, human PAR1 and PAR2 have a redundant role in the activation of human cells by C. albicans.

Legionella pneumophila ist ein fakultativ intrazellulär replizierendes Bakterium und der Erreger der Legionärskrankheit, einer schweren Pneumonie. Das Typ IVB Dot/Icm Proteinsekretionssystem und dessen Effektoren sind wesentlich an der Virulenz des Bakteriums beteiligt. Das Ziel der vorliegenden Arbeit war die Charakterisierung der Patatin-ähnlichen Proteine von L. pneumophila - insbesondere von PatA, das vom Dot/Icm Sekretionssystem in Wirtszellen eingeschleußt wird. Im Rahmen dieser Arbeit wurden folgende Ergebnisse erzielt: Die 11 Patatin-ähnlichen Proteine von L. pneumophila zeigen hauptsächlich Lysophospholipase A-Aktivität. PatA besitzt außerdem Phosphatidylglyzerol-spezifische Phospholipase A-Aktivität. Serin-72, welches in ein G-X-S-X-G Lipasemotiv eingebettet ist, ist für die Aktivität des Proteins essentiell. PatA ist nach Expression in A549 Epithelzellen in der Zytoplasmamembran oder einer damit eng assoziierten Struktur lokalisiert, die lipolytische Aktivität ist hierfür nicht entscheidend. Die Deletion einer C-terminalen Proteinregion führt zum Verlust der membranständigen Lokalisation. Virulenzattenuierte L. pneumophila Mutanten bilden unter Präsenz von Amöben - im Gegensatz zu Wildtypstämmen - eine Koloniemorphologie aus, die Scattermorphologie genannt wurde. Auf Basis der Scattermorphologie wurde eine Transposon-mutagenisierte Legionella Klonbank auf Wirtszellkolonisationsdefekte überprüft. Dabei wurden 119 kolonisationsdefekte Mutanten isoliert und 70 neue putative Wirtszellkolonisationsgene, darunter zwei Gene Patatin-ähnliche Proteine (patD, patF), identifiziert. patD befindet sich in einem Operon mit bdhA, dem Gen einer putativen 2-Hydroxybutyrat-Dehydrogenase. Das Operon spielt eine Rolle im Poly-Beta-Hydroxybutyrat (PHB) Stoffwechsel des Bakteriums und wird für die Replikation in Wirtszellen benötigt. Die Studie liefert die ersten experimentell fundierten Ergebnisse, die die Bedeutung des PHB-Metabolismus für die Virulenz des Bakteriums belegen, Legionella pneumophila is the causative agent of Legionnaires’ disease, a potentially fatal pneumonia. One mayor virulence determinant is the Dot/Icm Type IVB secretion system and its effector proteins. Aim of the present work was the characterization of patatin-like proteins of L. pneumophila, and in particular PatA, which is carried by the Dot/Icm secretion system into host cells. Within this work following results were obtained: The 11 patatin-like proteins of L. pneumophila possess majorly lysophospholipase A activity. L. pneumophila PatA additionally shows Phosphatidylglycerole-specific phospholipase A activity. Serin-72, which is embedded in an G-X-S-X-G lipase motiv is essential for the lipolytic activity of PatA. PatA locates to, or close to, the cytoplasmic membrane when expressed in A549 epithelial cells. The lipolytic activity of PatA is not required for membrane targeting and deletion of a C-terminal region abolishes proper targeting. Virulence attenuated L. pneumophila mutants, develop an easy recognizable phenotype during co-culture with A. castellanii on agar plates that was named „scatterphenotype“. On the basis of the scatterphenotype, a new assay was developed allowing screening of huge clone banks with respect to amoebae sensitivity, a marker for reduced virulence. Here, a collection of several thousand transposon mutagenized L. pneumophila clones was screened and a total of 119 amoebae sensitive mutants was isolated. Among those, 70 novel putative host cell colonization and virulence genes were identified including two members of the patatin-like protein family (patD, PatF). patD is cotranscribed with bdhA, therefore forming an operon. bdhA encodes a putative 3-hydroxybutyrate dehydrogenase. The operon is involved in the poly-beta-hydroxybutyrate (PHB) metabolism of L. pneumophila and is needed for replication in host cells. The study provides the first experimentally funded data showing the linkage of PHB metabolism and virulence of L. pneumophi

Candida species are the most common cause of opportunistic fungal infection worldwide. Here we report the genome sequences of six Candida species and compare these and related pathogens and non-pathogens. There are significant expansions of cell wall, secreted and transporter gene families in pathogenic species, suggesting adaptations associated with virulence. Large genomic tracts are homozygous in three diploid species, possibly resulting from recent recombination events. Surprisingly, key components of the mating and meiosis pathways are missing from several species. These include major differences at the mating-type loci (MTL); Lodderomyces elongisporus lacks MTL, and components of the a1/2 cell identity determinant were lost in other species, raising questions about how mating and cell types are controlled. Analysis of the CUG leucine-to-serine genetic-code change reveals that 99% of ancestral CUG codons were erased and new ones arose elsewhere. Lastly, we revise the Candida albicans gene catalogue, identifying many new genes.

Legionella pneumophila ist ein fakultativ intrazellulär replizierendes Bakterium und der Erreger der Legionärskrankheit, einer schweren Pneumonie. Das Typ IVB Dot/Icm Proteinsekretionssystem und dessen Effektoren sind wesentlich an der Virulenz des Bakteriums beteiligt. Das Ziel der vorliegenden Arbeit war die Charakterisierung der Patatin-ähnlichen Proteine von L. pneumophila - insbesondere von PatA, das vom Dot/Icm Sekretionssystem in Wirtszellen eingeschleußt wird. Im Rahmen dieser Arbeit wurden folgende Ergebnisse erzielt: Die 11 Patatin-ähnlichen Proteine von L. pneumophila zeigen hauptsächlich Lysophospholipase A-Aktivität. PatA besitzt außerdem Phosphatidylglyzerol-spezifische Phospholipase A-Aktivität. Serin-72, welches in ein G-X-S-X-G Lipasemotiv eingebettet ist, ist für die Aktivität des Proteins essentiell. PatA ist nach Expression in A549 Epithelzellen in der Zytoplasmamembran oder einer damit eng assoziierten Struktur lokalisiert, die lipolytische Aktivität ist hierfür nicht entscheidend. Die Deletion einer C-terminalen Proteinregion führt zum Verlust der membranständigen Lokalisation. Virulenzattenuierte L. pneumophila Mutanten bilden unter Präsenz von Amöben - im Gegensatz zu Wildtypstämmen - eine Koloniemorphologie aus, die Scattermorphologie genannt wurde. Auf Basis der Scattermorphologie wurde eine Transposon-mutagenisierte Legionella Klonbank auf Wirtszellkolonisationsdefekte überprüft. Dabei wurden 119 kolonisationsdefekte Mutanten isoliert und 70 neue putative Wirtszellkolonisationsgene, darunter zwei Gene Patatin-ähnliche Proteine (patD, patF), identifiziert. patD befindet sich in einem Operon mit bdhA, dem Gen einer putativen 2-Hydroxybutyrat-Dehydrogenase. Das Operon spielt eine Rolle im Poly-Beta-Hydroxybutyrat (PHB) Stoffwechsel des Bakteriums und wird für die Replikation in Wirtszellen benötigt. Die Studie liefert die ersten experimentell fundierten Ergebnisse, die die Bedeutung des PHB-Metabolismus für die Virulenz des Bakteriums belegen, Legionella pneumophila is the causative agent of Legionnaires’ disease, a potentially fatal pneumonia. One mayor virulence determinant is the Dot/Icm Type IVB secretion system and its effector proteins. Aim of the present work was the characterization of patatin-like proteins of L. pneumophila, and in particular PatA, which is carried by the Dot/Icm secretion system into host cells. Within this work following results were obtained: The 11 patatin-like proteins of L. pneumophila possess majorly lysophospholipase A activity. L. pneumophila PatA additionally shows Phosphatidylglycerole-specific phospholipase A activity. Serin-72, which is embedded in an G-X-S-X-G lipase motiv is essential for the lipolytic activity of PatA. PatA locates to, or close to, the cytoplasmic membrane when expressed in A549 epithelial cells. The lipolytic activity of PatA is not required for membrane targeting and deletion of a C-terminal region abolishes proper targeting. Virulence attenuated L. pneumophila mutants, develop an easy recognizable phenotype during co-culture with A. castellanii on agar plates that was named „scatterphenotype“. On the basis of the scatterphenotype, a new assay was developed allowing screening of huge clone banks with respect to amoebae sensitivity, a marker for reduced virulence. Here, a collection of several thousand transposon mutagenized L. pneumophila clones was screened and a total of 119 amoebae sensitive mutants was isolated. Among those, 70 novel putative host cell colonization and virulence genes were identified including two members of the patatin-like protein family (patD, PatF). patD is cotranscribed with bdhA, therefore forming an operon. bdhA encodes a putative 3-hydroxybutyrate dehydrogenase. The operon is involved in the poly-beta-hydroxybutyrate (PHB) metabolism of L. pneumophila and is needed for replication in host cells. The study provides the first experimentally funded data showing the linkage of PHB metabolism and virulence of L. pneumophi

CandidaDB is a database dedicated to the genome of the most prevalent systemic fungal pathogen of humans, Candida albicans. CandidaDB is based on an annotation of the Stanford Genome Technology Center C.albicans genome sequence data by the European Galar Fungail Consortium. CandidaDB Release 2.0 (June 2004) contains information pertaining to Assembly 19 of the genome of C.albicans strain SC5314. The current release contains 6244 annotated entries corresponding to 130 tRNA genes and 5917 protein-coding genes. For these, it provides tentative functional assignments along with numerous pre-run analyses that can assist the researcher in the evaluation of gene function for the purpose of specific or large-scale analysis. CandidaDB is based on GenoList, a generic relational data schema and a World Wide Web interface that has been adapted to the handling of eukaryotic genomes. The interface allows users to browse easily through genome data and retrieve information. CandidaDB also provides more elaborate tools, such as pattern searching, that are tightly connected to the overall browsing system. As the C.albicans genome is diploid and still incompletely assembled, CandidaDB provides tools to browse the genome by individual supercontigs and to examine information about allelic sequences obtained from complementary contigs. CandidaDB is accessible at http://genolist.pasteur.fr/CandidaDB.