To overcome the membrane permeability barrier and low nutrient availability in the environment, Gram-negative organisms have evolved many mechanisms dedicated to the intake of nutrients. One such mechanism is the long-chain fatty acid uptake pathway. This pathway involves machinery that transports fatty acids across the outer membrane and into the cell, where the lipid can be used for either nutrition or for remodeling the structure of the membrane. Interestingly, the fatty acid species that can be recognized by this machinery differ between organisms; the aquatic pathogen Vibrio cholerae demonstrates a much wider substrate recognition profile than other Gram-negative species. In this work we elaborate on the lipid nutrients accessible to V. cholerae, demonstrating that it can use lysophosphatidylcholine as both a carbon source as well as a source of fatty acids for remodeling its outer membrane. We identify the enzyme responsible for the breakdown of lysophosphatidylcholine, VolA (Vibrio outer membrane lysophospholipase A). VolA is conserved in many Vibrio species as well as other aquatic Gram-negatives, annotated as a putative lipase. We show VolA is co-expressed with the outer membrane fatty acid transporter FadL; FadL serves to transport across the outer membrane those long-chain fatty acids freed from lysophosphatidylcholine by VolA. VolA is expressed on the surface of the cell as a membrane anchored lipoprotein; this is novel as VolA is the first identified example of a surface-localized lipoprotein phospholipase. Biochemical characterization of VolA shows that it acts as a canonical lysophospholipase in vitro, suggesting that it works in tandem with the FadL transporter, freeing fatty acids from lysophosphatidylcholine at the surface of the cell to be brought in via the fatty acid uptake pathway. This work expands on the currently understood lipid uptake abilities of Vibrio cholerae, demonstrating a novel mechanism for utilizing a nutrient not previously thought to metabolized. VolA is an important to our understanding of the larger picture of lipid uptake and how it contributes to the survival of Gram-negative organisms. / text

Universiẗat, Diss., 2005--Würzburg.

Die opportunistische Hefe Candida albicans ist in der Lage durch ein koordiniertes Zusammenspiel bestimmter zellulärer Eigenschaften sich unterschiedlichen Umweltbedingungen anzupassen und unterschiedliche Nischen innerhalb des menschlichen Wirts zu kolonisieren. Die Sekretion hydrolytischer Enzyme, wie Proteinasen und Phospholipasen, stellt eine wichtige Eigenschaft des Pilzes dar, die als wesentlicher Faktor für die Aufrechterhaltung der Pathogenität von C. albicans angesehen wird. Ein Schwerpunkt der hier vorliegenden Studie ist die funktionale Charakterisierung des caPLB5-Gens, eines neuen Mitglieds der insgesamt 5 Mitglieder umfassenden Phospholipase-B-Genfamilie. Im Gegensatz zu den gut untersuchten sekretorischen PLBs caPlb1p and caPlb2p scheint das caPlb5-Protein GPI-verankert und letztlich zellwandgebunden zu sein. Mittels Northernexpressions-Studien ließen sich in verschiedenen C.-albicans-Stämmen und unterschiedlichen Wachstumsbedingungen caPLB5-spezifische Transkripte nachweisen. Während des Hefe-Hyphe-Wechsels in Lee’s Medium zeigte sich interessanterweise eine differentielle Regulation der Gene caPLB5, caPLB1 and caPLB2. Durch Sequenzanalyse einzelner caPLB5-Allele konnte die Anwesenheit zweier unterschiedlicher Allele in C. albicans bei verschiedenen Stämmen nachgewiesen werden. Die gezielte Geninaktivierung beider Allele in zwei verschiedenen Stämmen resultierte in einer attenuierten Virulenz, was sich im Mausmodell für systemische Infektion anhand des Kolonisationsgrads des Wirtsgewebes messen ließ. Die Phänotypen sowohl der Nullmutanten als auch der caPLB5-Revertanten belegen, dass die Phospholipase B caPlb5p für die vollständige Virulenz des Pathogens benötigt wird und dabei eine Rolle bei der in vivo Organbesiedlung spielt. Diese Arbeit präsentiert zudem die Isolierung und Charakterisierung des ATP-Binding-Cassette-(ABC)-Transporter-Gens caMLT1 aus C. albicans. CaMlt1p zählt zur MRP/CFTR-Unterfamilie ATP-bindender Transportproteine, eine Proteinkategorie, die in diesem Pilz bislang noch nicht beschrieben wurde. Energiebetriebene Transportproteine der ABC-Superfamilie schleusen eine Vielzahl unterschiedlicher Substrate aktiv durch biologische Membranen und erfüllen dabei wichtige Funktionen im zellulären Metabolismus und in der Entgiftung. Das caMlt1-Protein zeigt hohe sequenzielle und strukturelle Ähnlichkeiten zu den vakuolaren Efflux-Pumpen Ycf1p und Bpt1p von S. cerevisiae. Durch genomische Markierung mit dem grün fluoreszierenden GFP-Protein konnte caMlt1p in der vakuolaren Membran lokalisiert werden. Northernblothybridisierungen belegten die Induzierbarkeit der Gentranskripte durch die metabolischen Gifte Cadmium und CDNB, beides Substrate der scYcf1-Pumpe. Obwohl diese Untersuchungen darauf hindeuten, dass caMlt1p ein Ortholog von scYcf1p sein könnte, zeigte sich bei dem Komplementationsversuch einer scycf1-negativen S.-cerevisiae-Mutante mit einer caMLT1-Genkopie keine Reversion des sensitiven Phänotyps gegenüber Cadmium oder CDNB. Auch wiesen die in dieser Arbeit konstruierten, camlt1-negativen Mutanten in C. albicans, die zur Identifizierung potentieller caMlt1p-Substrate eingesetzt wurden, keinen hypersensitiven Phänotyp gegenüber CdCl2, CDNB oder irgendeiner anderen getesteten inhibitorischen Substanz auf. CaMlt1p ist demzufolge kein funktionales Homolog von scYcf1p. Als vakuolar lokalisiertes Protein weist caMLT1 ein für diese Proteingruppe typisches Transkriptionsprofil auf. Die mRNA-Expression erfolgt dabei wachstumsphasenabhängig mit der höchsten Geninduktion während des Diauxic-Shifts, wenn ein Mangel an Glucose (und anderen Nährstoffen) im Medium entsteht. Eine generierte camlt1-Nullmutante war interessanterweise in einem murinen Peritonitismodell in ihrer Fähigkeit die Leber zu invadieren drastisch reduziert. Durch Reintegration einer funktionalen caMLT1-Genkopie konnte der Virulenzdefekt aufgehoben werden. CaMlt1p scheint in die Fähigkeit von C. albicans involviert zu sein an intestinale Organe adhärieren und Gewebebarrieren penetrieren zu können, möglicherweise durch Einbindung des Transporters in Stressantwort- und Detoxifikationsmechanismen. Beide Gene, caMLT1 und caPLB5, wurden auf zweierlei Weise inaktiviert: mittels einer klassischen Mutagenesemethode für C. albicans (dem URA3-Blaster-System im Ura--auxotrophen Stamm CAI4) und durch Entwicklung eines neuen dominanten Selektionssysstems. Die dominante Selektion basiert dabei auf der genomischen Insertion einer Einzelkopie eines mutierten caIMH3-Allels (MPAR), das Transformanten Resistenz gegenüber Mycophenolsäure (MPA) verleiht. Dieses System ermöglicht die genetische Manipulation von C. albicans Wildtypstämmen, wodurch die mühselige Konstruktion auxotropher und oft avirulenter Stämme nicht mehr nötig ist. / A coordinated interplay of certain traits enables the opportunistic yeast Candida albicans to adapt to different environmental conditions and to colonize different niches of the human host. Secretion of hydrolyzing enzymes, like proteinases and phospholipases, is an important characteristic of C. albicans which is considered to be integral to pathogenesis. This study focuses on the functional characterisation of the caPLB5 gene, a new member of the phospholipase B multigene family with five putative members. In contrast to the well characterized secretory PLBs caPlb1p and caPlb2p, the putative caPlb5-protein is likely to be GPI-anchored and ultimately bound to the cell wall. Northern expression studies showed caPLB5-specific transcripts in several strains of C. albicans under each growth condition tested. Interestingly, differential regulation of caPLB5, caPLB1 and caPLB2 could be detected during the yeast to hyphae transition in Lee’s medium. Sequence analysis of single caPLB5 allels resulted in the identification of two different alleles in several strains of C. albicans. The targeted gene disruption of both alleles in two different strains resulted in attenuated virulence as measured by host tissue colonization in a mouse model of systemic infection. The phenotypes expressed by null mutants and revertant strains of caPLB5 indicate that the phospholipase is required for complete virulence of this pathogen by playing a role for in vivo organ colonization. This study further presents the isolation and characterisation of the ABC-transporter gene caMLT1 in C. albicans belonging to the MRP/CFTR-subfamily of ATP-binding casette (ABC) transporters, a class of proteins so far not described in this fungus. Energy-driven transport proteins within the ABC-superfamily actively transport a wide variety of substances across biological membranes and fulfill important functions in cellular metabolism and detoxification. The protein encoded by the caMLT1 gene shows high similarities to the vacuolar efflux-pumps Ycf1p and Bpt1p of S. cerevisiae. Genomic tagging with the green fluorescent protein (GFP) revealed vacuolar membrane localization of caMlt1p. Northern hybridisation experiments documented the inducibility of gene transcripts by the metabolic poisons cadmium and CDNB, which are also substrates of the scYcf1-pump. While caMlt1p could be an orthologue of scYcf1p, complementation of a scycf1-negative S. cerevisiae mutant with a caMLT1-gene copy did not reverse the sensitive phenotype to these toxins. Moreover, the construction of camlt1-negative mutants in C. albicans allowed for screening of substrates putatively transported by caMlt1p. These null mutants showed no hypersensitive phenotype to neither CdCl2 nor CDNB or any other tested inhibitory substances, hence caMlt1p is not a functional homologue of scYcf1p. The caMLT1 mRNA expression pattern is typical for a vacuolar gene, showing an extensively growth phase dependent regulation with the highest gene induction during the diauxic transition when glucose (and other nutrients) becomes limited. Most interestingly, a generated mlt1 null mutant showed a dramatic reduction in liver invasion in a mouse peritonitis model. Reintegration of a functional caMLT1 gene copy reverted the virulence defect. CaMlt1p seems to be involved in the capability of C. albicans to adhere to the intestinal organs and penetrate tissue barriers putatively because of its involvement in mechanisms of stress response and detoxification. Both genes, caMLT1 and caPLB5, were inactivated by using a classical disruption method for C. albicans (the URA3-Blaster-system in Ura- auxotrophic strain CAI4) and by developing a new dominant selection system. Dominant selection is based on genomic insertion of a single copy of a mutated caIMH3 allel (MPAR) that renders transformants resistant to mycophenolic acid (MPA). Using this system, the cumbersome generation of auxotrophic strains, which are often avirulent, is obsolete, while C. albicans wild-type strains become amenable to genetic manipulation.

Submitted by Irene Nascimento (irene.kessia@ufpe.br) on 2017-03-08T16:58:52Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Juliana Gusmão de Araujo.pdf: 5234646 bytes, checksum: 87f79f1087a57c64407eda5226cb557f (MD5) / Made available in DSpace on 2017-03-08T16:58:52Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Juliana Gusmão de Araujo.pdf: 5234646 bytes, checksum: 87f79f1087a57c64407eda5226cb557f (MD5) Previous issue date: 2012-12-26 / O colesteatoma adquirido, mesmo com os conhecimentos acumulados desde sua primeira descrição, ainda se mantém como um problema de saúde pública distante de ser solucionado. O entendimento mais profundo da patogênese do colesteatoma é de extrema importância visto que a natureza desta lesão é destrutiva e causadora de complicações potencialmente graves. Apesar das teorias propostas e das várias proteínas terem sido identificados no colesteatoma, a verdadeira etiopatogenia da doença ainda carece de investigações. Objetivo: Identificar biomarcadores do colesteatoma adquirido utilizando a plataforma proteômica. Casuística e Métodos: Foram coletadas amostras de colesteatoma e também fragmento de pele da região retroauricular de 12 indivíduos submetidos a cirurgia para remoção do colesteatoma. As amostras foram armazenadas em solução salina e mantidas a -20ºC até pesagem tecidual e extração das proteínas. Eletroforese bidimensional foi realizada, os géis foram corados com nitrato de prata e suas imagens digitalizadas. Todas as análises foram realizadas em triplicatas. Os peptídeos extraídos após a digestão do spots foram levados à espectrometria de massa e os espectros obtidos foram analisados usando o algoritmo Mascot utilizando os bancos de dados de proteína do NCBI e SwissProt. Resultados: Dos 393 spots identificados na análise do extrato proteico de colesteatoma adquirido, apenas 10 estavam dentro dos parâmetros estatísticos aceitáveis pelo algoritmo Mascot. As principais proteínas detectadas no colesteatoma adquirido foram a cadeia beta do fibrinogênio, proteína da matriz extracelular 2, actina citoplasmática 1, heparan sulfato glucosamina 3-O-sulfotransferase 3A1, fator de necrose tumoral alfa induzido proteína 8-like 1, Stanniocalcina-2, lisofosfolipase eosinofílica e OFUT1.Conclusão: Foram identificadas proteínas envolvidas com a migração celular, regulação da apoptose, vias de sinalização, hiperproliferação celular, cicatrização e processos inflamatórios. Pudemos, desta maneira, traçar um perfil proteômico do colesteatoma adquirido. / The acquired cholesteatoma, even with all the knowledge accumulated since its first description, still remains a public health problem, far from being solved. A deeper understanding of its pathogenesis is extremely important since it is a destructive lesion that might cause potentially serious complications. Several proteins have been identified in cholesteatoma and a few theories were described, however the true etiology of the disease still needs investigation. Objective: Identify acquired cholesteatoma biomarkers using proteomics platform. Patients and methods: Cholesteatoma samples were collected and also a skin fragment of the surgical incision of twelve patients undergoing surgery for cholesteatoma removal. The samples were stored in saline solution and kept at -20 ° C until weighing tissue and proteins extraction. Two-dimensional electrophoresis was conducted, the gels were stained with silver nitrate and their images were digitized. All analyzes were performed in triplicate. The peptides extracted after spots digestion were taken to mass spectrometry and the spectra obtained were analyzed using the Mascot algorithm comparing databases of the NCBI and SwissProt protein. Results: Of the 393 spots identified in the analysis of protein extracts of acquired cholesteatoma, only 10 were within acceptable statistical parameters by Mascot algorithm. The proteins detected in acquired cholesteatoma were fibrinogen beta chain, extracellular matrix protein 2, actin cytoplasmic 1, heparan sulfate glucosamine 3-O-sulfotransferase 3A1, tumor necrosis factor alpha 8 induced proteinlike 1, stanniocalcin-2, eosinophil lysophospholipase and OFUT1. Conclusion: Proteins involved in cell migration, regulation of apoptosis, signaling pathways, cellular proliferation, wound healing and inflammatory processes were identified. We were able to draw a proteomic profile of acquired cholesteatoma.

The relationship among eosinophils, lysophospholipase activity and the immune response in animals infected with Trichinella spiralis was studied using in vivo and in vitro techniques. In an in vivo experiment, anti-thymocyte serum (ATS) was administered to mice infected with T. spiralis and its effects on intestinal lysophospholipase (EC 3.1.1.5.) activity, peripheral blood, bone marrow and intestinal eosinophilia were measured in the same experimental animal. The ATS caused a significant temporally related suppression of both the tissue lysophospholipase response and eosinophilia, in all three compartments. These findings support the hypothesis that parasite-induced eosinophilia is the cause of the increased lysophospholipase activity of parasitized tissue and that the responses are thymus cell-dependent. In vitro experiments demonstrated that the eosinophil was the primary inflammatory cell source of lysophospholipase among eosinophils, neutrophils macrophages and lymphocytes. The role of other cells and antigen in the production of the enzyme by the eosinophil was also investigated in vitro• Results demonstrated that eosinophils cultured with both T. spiralis antigen and other leukocytes yielded enzyme activities significantly greater than eosinophils cultured alone or with only antigen. More specific experiments showed that T-lymphocytes were the cells responsible for influencing the eosinophils' lysophospholipase activity in the presence of antigen, and that their influence was enhanced by the presence of macrophages. These results suggested that increased lysophospholipase activity present in parasitized tissue was not only due to increased numbers of eosinophils infiltrating parasitized tissue but was also due to each eosinophil synthesizing more of the enzyme. The necessity for antigen and other cells suggests a role for cell cooperation in the production of the enzyme, specifically T-lymphocytes and macrophage interaction with the eosinophil. A lymphocyte soluble factor collected from sensitized lymphocytes stimulated with specific antigen or concanavalin A was found to enhance the eosinophil lysophospholipase activity when added to cultures of eosinophils plus other peritoneal cells. The soluble factor did not stimulate the lysophospholipase activity of pure cultures of eosinophils.