Thesis (MScAgric) -- University of Stellenbosch, 2002. / ENGLISH ABSTRACT: Fungi are an extremely diverse group of organisms and, by acting as pathogens, they can colonise various other organisms, including humans, plants and animals. The effect of this is usually detrimental, not only to agricultural crops and livestock, but also to human well-being. The extensive farming of crops and livestock requires persistent control of fungal populations, commonly through the use of chemical fungicides. However, the exclusive use of fungicides is no longer a sustainable practice, as a result of serious problems, such as increasing fungicide resistance in pathogen strains, the high costs of fungicides, as well as concern about the environment. The search by producers and scientists for alternative control measures is an ongoing process. The fungal cell wall consists of polysaccharides that not only playa role in protection of the fungi, but also in relaying signals for the invasion and infection of susceptible hosts. Chitin, a polysaccharide composed of N-acteylglucosamine (GleNAc) residues linked by P-1,4 glucosidic linkages, is one of the major components of the fungal cell wall, where it plays an important role in the apical growth of the vegetative hyphae. Chitinases (EC 3.2.1.14) are abundant proteins produced by a variety of microorganisms and plants and are necessary for the hydrolysis of the chitin polymer. During the invasion of many plant species by a pathogen, the production of a specific group of proteins, designated pathogenesis-related (PR) proteins that include chitinases, is induced as part of their defence response. Due to the facts that pathogenic fungi contain chitin in their cell walls and that plant chitinases are induced upon pathogen attack, chitinases have been confirmed as an integral and crucial part of the plant's natural defence response. Chitinases have increasingly been targeted to upregulate plants' endogenous disease resistance mechanisms through transgenic overexpression in a variety of hosts. Several species of fungi, including various Trichoderma spp., are potent biocontrol agents of plant pathogenic fungi and insects. The antagonistic activities of these biological control agents towards phytopathogens are based on the secretion of extracellular hydrolytic enzymes, such as cell wall-degrading chitinase enzymes. However, biological control is not restricted to naturally occurring biocontrol agents. Through the process of genetic transformation, other fungal or yeast species can be enhanced to produce their own chitinases or other antimicrobial substances more effectively in order to yield potent biocontrol agents. Various types of chitinases have been applied in the production of fungal resistant plants and some research has been done on the application of chitinases, from a variety of microorganisms, as biological control agents. In contrast, very little is known about the antifungal activity of the Saccharomyces cerevisiae chitinase enzyme, encoded by the CTS1-2 gene. The CTS1-2 gene was utilised in this study as a candidate for overexpression in both yeast and plant expression systems to analyse the ability of the encoding chitinase to inhibit fungal growth. The first objective of this study involved the high level expression and optimisation of the secretion of the CTS1-2 gene in S. cerevisiae to render recombinant yeast with enhanced antifungal abilities and with possible applications as a biocontrol agent to control plant pathogenic fungi. It was hypothesised that high-level expression and efficient secretion would be prerequisites in a biocontrol yeast strain. To this end, two strong promoters and terminators were included in the study and the secretion of the chitinase gene was evaluated by testing three different secretion signals. The secretion signals included: the native CTS1-2 secretion signal, the S. cerevisiae mating pheromone a-factor (MFa1) secretion signal, as well as the Trichoderma reesei f3-xylanase 2 (XYN2) secretion signal. The phosphoglycerate kinase 1 (PGK1) and alcohol dehydrogenase 2 (ADH2) promoters and terminators were employed to achieve high-level expression. The results obtained from the analysis of the recombinant yeasts showed that the PGK1 promoter-terminator constructs yielded high level CTS1-2-expressing and chitinase-producing strains of S. cerevisiae PRY488. The ability of the different secretion signals to efficiently secrete the overexpressed chitinase was analysed and it was found that the non-native secretion signals delivered significantly more protein to the extracellular environment. It was thus evident that the performance of the MFa1 and XYN2 secretion signals was superior to that of the native secretion signal. The antifungal activities of the recombinant chitinases produced by these constructs were tested in in vitro assays against Botrytis cinerea. The enzymes led to a significant reduction in hyphal development, caused by extreme structural damage to the hyphal tips, the hyphal cell walls as well as the ability of the fungus to form reproductive and survival structures, thereby confirming the antifungal abilities of this enzyme. The ADH2 promoter-terminator constructs yielded CTS1-2 transcripts, but no chitinase activity could be detected with any of these strains. The reasons for this still remain unclear. The second objective of this study was to assess the potential of the yeast chitinase gene to upregulate defence against fungal infection in planta. In order to elucidate this, the CTS1-2 gene was constitutively overexpressed in tobacco plants, targeting the chitinase both to the intra- and the extracellular environment. The results obtained showed that the transgenic tobacco lines regenerated in this study stably integrated the transgene, exhibiting transgene expression as well as the production of a biologically active yeast chitinase enzyme. The F, progeny were rigorously tested for resistance to B. cinerea, and both in vitro and in planta assays confirmed that the yeast chitinase increased the plant's tolerance to fungal infection; some of the lines showed disease resistance of 65 and 70%. The plants expressing an extracellularly targeted chitinase gene are still under evaluation. Interesting results are expected relating to the effect of the chitinase on the plant surface with regards to disease resistance to fungal pathogens. In conclusion, the combined set of results from both the yeast and plant overexpression studies has confirmed the strong antifungal effect of yeast chitinases. The yeast CTS1-2 chitinase could be instrumental in the development of a new generation of yeast strains with improved antifungal capabilities. This enzyme could also play an important role in genetic transformation technologies aimed at enhanced disease resistance. / AFRIKAANSE OPSOMMING: Swamme omsluit 'n uiterste diverse groep organismes wat mense, plante en diere deur patogeniese aksie kan koloniseer. Die uitkoms hiervan op landbougewasse, die veebedryf en menslike gesondheid is gewoonlik skadelik. Uitgebreide gewas- en veeboerderye benodig voortdurende beheer van fungiese populasies, tipies deur van chemiese swamdoders gebruik te maak. Die uitsluitlike gebruik van swamdoders is egter nie meer 'n lewensvatbare praktyk nie, hoofsaaklik as gevolg van probleme soos die opbou van weerstand van patogeniese rasse teen swamdoders, die hoë kostes van die middels, asook besorgheid oor die omgewing. Die soektog na alternatiewe beheermaatreëls deur produsente en wetenskaplikes bly 'n aaneenlopende proses. Die swamselwand bestaan uit polisakkariede wat nie net In rol in die beskerming van die swam speel nie, maar ook betrokke is in die oordrag van aanvals- en infeksieverwante seine in 'n vatbare gasheer. Chitien, 'n polisakkaried bestaande uit N-asetielglukosamien (GlcNAc) residu's gekoppel deur 13-1,4glukosidiese bindings, is een van die hoofkomponente van die swamselwand, waar dit 'n belangrike rol in die apikale groei van vegetatiewe hifes speel. Chitinases (EC 3.2.1.14) is proteïene wat oorvloedig deur 'n verskeidenheid van mikroërganismes en plante geproduseer word, waar hulle vir die hidrolise van die chitien polimeer noodsaaklik is. Tydens die infeksie van verskeie plantspesies deur In patogeen, word die produksie van 'n spesifieke groep proteïene, die sogenaamde patogeen-verwante (PR) proteïene wat chitinases insluit, as deel van die plant se verdedigingsreaksie geïnduseer. Die feit dat patogeniese swamselwande chitien bevat en dat plantchitinases tydens infeksie geïnduseer word, het daartoe gelei dat dit bevestig is dat chitinases In integrale en kritiese deel van die plant se natuurlike verdedigingsreaksie uitmaak. Chitinases word toenemend geteiken in pogings om die plant se intrinsieke siekteweerstandsmeganismes te verbeter deur transgeniese ooruitdrukking daarvan in 'n verskeidenheid van gashere. Verskeie swamspesies, insluitend verskillende Trichodenna-spesies, is kragtige bio-antagoniste van plantpatogeniese swamme. Die antagonistiese aksies van hierdie biologiese beheeragente teenoor fitopatogene is gebaseer op die uitskeiding van ekstrasellulêre hidrolitiese ensieme, soos die selwandverterende chitinase ensieme. Nietemin is biologiese beheer nie net tot bio-antagoniste wat natuurlik voorkom beperk nie. Deur die proses van genetiese transformasie kan ander swam- of gisspesies verbeter word om hul eie chitinases of ander antimikrobiese substanse meer effektief te produseer, wat aanleiding sal gee tot kragtige bio-antagoniste. Verskeie tipes chitinases is al in die produksie van swambestande plante ingespan en uitgebreide navorsing is gedoen op die toepassing van 'n reeks chitinases, afkomstig van 'n verskeidenheid van mikroërganismes, as biologiese beheeragente. In teenstelling is baie min bekend oor die antifungiese aktiwiteite van die Saccharomyces cerevisiae chitinase ensiem, wat deur die CTS1-2 geen ge-enkodeer word. Die CTS1-2-geen is in hierdie studie gebruik vir ooruitdrukking in beide gis- en plantuitdrukkingsisteme om die chitinase se vermoë om swamgroei te inhibeer, te ondersoek. Die eerste oorkoepelende oogmerk van hierdie studie het hoë-vlak uitdrukking en optimalisering van sekresie van die CTS1-2-geen in S. cerevisiae behels, met die toekomstige doelwit om 'n rekombinante gis met verbeterde antifungiese eienskappe en met moontlike toepassings as 'n bio-antagonis teen plantpatogeniese swamme te ontwikkel. Die hipotese was dat hoë-vlak uitdrukking en voldoende sekresie voorvereistes vir 'n bio-antagonisras is. Omdié rede is twee sterk promotors en termineerders by hierdie studie ingesluit en is die sekresie van die chitinase-geen geëvalueer deur drie verskillende sekresieseine te toets. Die sekresieseine sluit in: die wilde-tipe CTS1-2 sekresiesein, die S. cerevisiae paringsferomoon a-faktor (MFa1) sekresiesein, en die Trichoderma reesei p-xilanase (XYN2) sekresiesein. Die fosfogliseraat kinase 1 (PGK1) en alkohol dehidrogenase 2 (ADH2) promotors en termineerders is gebruik om hoë-vlak uitdrukking te dryf. Die resultate wat vanaf die analises van die rekombinante giste verkry is, het getoon dat die PGK1 promotor-termineerder konstrukte hoë-vlak CTS1-2-uitdrukkende en chitinase-produserende S. cerevisiae PRY488 rasse opgelewer het. Die vermoë van die verskillende sekresieseine om die ooruitgedrukte chitinase voldoende uit te skei, is geanaliseer, en daar is gevind dat die heteroloë sekresieseine aansienlik meer proteïene na die ekstrasellulêre omgewing geloods het. Dit was dus duidelik dat die MFa1 en XYN2 sekresieseine beter as die wilde-tipe sekresiesein presteer het. Die antifungiese aktiwiteit van die rekombinante chitinases wat deur hierdie konstrukte geproduseer is, is ook in in vitrotoetse teen Botryits cinerea getoets. Die teenwoordigheid van die ensieme het gelei tot 'n aansienlike afname in hife-ontwikkeling, veroorsaak deur ekstreme strukturele skade aan die hifepunte, die hifeselwande, asook die vermoë van die swam om voortplanting- en oorlewingstrukture te vorm. Die ADH2 promotor-termineerderkonstrukte het CTS1-2 transkripte vertoon, maar geen chitinase-aktiwiteite kon in hierdie konstrukte waargeneem word nie. Die redes hiervoor is tot op hede onbekend. Die tweede oogmerk van hierdie studie was om die potensiaal van die gischitinase om swaminfeksie in planta teë te werk, te ondersoek. Die CTS1-2-geen is konstitutief ooruitgedruk in tabakplante, waarin die chitinase na beide die intra- en ekstrasellulêre omgewing geteiken is. Resultate het getoon dat die geregenereerde transgeniese tabaklyne die transgeen stabiel geïntegreer het, transgeenuitdrukking vertoon en dat 'n biologies aktiewe chitinase-ensiem geproduseer is. 'n F1-generasie is aan strawwe toetse onderwerp om weerstand teen B. cinerea te ondersoek. Beide die in vitro en in planta toetse het bevestig dat die gischitinase die plant se verdraagsaamheid teenoor swaminfeksie verhoog het; sommige lyne het siekteweerstand van tussen 65 en 70% getoon. Die plante wat 'n ekstrasellulêre chitinase produseer, word steeds geëvalueer. Interessante resultate word verwag aangaande die effek van die chitinase op die plant se oppervlak met betrekking tot siekteweerstand teen swampatogene. Ten slotte, die gekombineerde stel resultate wat vanaf beide die gis- en plantuitdrukkingstudies verkry is, het die sterk antifungiese effek van gischitinases bevestig. Die gis CTS1-2 kan instrumenteel wees in die ontwikkeling van 'n nuwe generasie gisrasse met verbeterde antifungiese eienskappe. Die ensiem kan ook 'n belangrike rol in genetiese transformasietegnologieë, wat op verbeterde siekteweerstand gemik is, speel.

Anaplasma phagocytophilum and A. marginale are the etiologic agents of bovine anaplasmosis and human granulocytic anaplasmosis, respectively. As obligate intracellular pathogens, binding and entry of host cells is a prerequisite for survival. The molecular events associated with these processes are poorly understood. Identifying the adhesins mediating binding, delineating their key functional domains, and determining the molecular determinants to which they bind not only benefits better understanding of Anaplasma spp. pathobiology, but could also benefit the development of novel approaches for protecting against infection. We previously demonstrated that A. phagocytophilum outer membrane protein A (ApOmpA) is critical for bacterial binding and entry host through recognition of α2,3-sialic acid and α1,3-fucose of its receptors, including 6-sulfo-sLex. In this study, we determined that two amino acids, G61 and K64, within its binding domain (ApOmpA59-74), are essential for ApOmpA function. We also confirmed the ability of ApOmpA to act as an adhesin and invasin as it conferred adhesiveness and invasiveness to inert beads. We next extended our studies to A. marginale as it also expresses OmpA (AmOmpA) and its role in infection has not been studied. Molecular models of ApOmpA and AmOmpA were nearly identical, especially in the ApOmpA binding domain and its counterpart in AmOmpA. Antisera raised against AmOmpA or its putative binding domain inhibit A. marginale infection. AmOmpA G55 and K58 are contributory and K59 is essential for AmOmpA to bind to host cells. AmOmpA binding is dependent on α2,3-sialic acid and α1,3-fucose. Coating inert beads with AmOmpA conferred the ability to bind to and be taken up by host cells, confirming that it acts as an adhesin and invasin. 6-sulfo-sLex is dispensable for AmOmpA binding and A. marginale infection. ApOmpA works cooperatively with Asp14 (14-kDa A. phagocytophilum surface protein) to promote optimal infection of host cells. We found that Asp14 is conserved across A. phagocytophilum strains and in A. marginale and confirmed the ability of Asp14 to act as an adhesin and invasin as it conferred adhesiveness and invasiveness to inert beads. Collectively, this work advances our understanding of A. phagocytophilum and A. marginale adhesion and invasion of host cells.

Periodontal disease (PD) is a polymicrobial infection characterized by inflammation of the gingiva, alveolar bone resorption, and tooth loss (edentulism). Treponema denticola along with Porphyromonas gingivalis and Tannerella forsythia are among the “Red Complex” and are main etiological agents in PD. Treponemes are a member of the Spirochaeta phylum and are obligate anaerobes, that express pyruvate ferredoxin oxidoreductase (PFOR). The enzyme catalyzes the oxidation of pyruvate to acetyl-CoA and reduced ferredoxin. Amixicile is a novel bacteriostatic derivative of nitazoxanide and an inhibitor of PFOR. In light of the fact that Treponemes express PFOR, this study was conducted to investigate the susceptibility of oral Treponemes to AMX. All oral Treponemes tested were susceptible to AMX and the MIC values were determined ranging of 1.5-4.5 μg mL-1 for an initial starting cell concentration of 1.9x106 cells mL-1. Other potentially therapeutic effects for AMX for T. denticola were investigated: motility, hydrogen sulfide production, and serum sensitivity. AMX reduced overall spirochete motility by 50% at sub-MIC concentrations. There was a dose dependent decrease in H2S production in T. denticola at sub-MIC and MIC values. Furthermore, prior exposure of AMX led to increases in serum sensitivity. Taking into account the fact that other periodontal red complex bacteria express PFOR, AMX could serve as a new selective adjunctive treatment for periodontal disease.

Invasive Pilzinfektionen stellen ein großes Problem bei Transplantatempfängern dar. Candida und Aspergillus spp. sind die häufigsten pathogenen Pilze bei Patienten nach KMT und Organtransplantationen. Diese Infektionen sind durch eine hohe Morbidität und Mortalität gekennzeichnet, insbesondere bei Patienten mit persistierender Granulozytopenie und damit jene nach allogener KMT. Die Mortalitätsrate kann durch eine Frühdiagnostik und durch Gabe einer geeigneten Therapie wesentlich reduziert werden. Die Symptome und Zeichen einer systemischen Pilzinfektion sind bei Transplantatempfängern untypisch. Die serologische Diagnostik von invasiven Candidosen oder Aspergillosen stellt eine zusätzliche Möglichkeit zur klinischen Untersuchung und anderen diagnostischen Maßnahmen dar. Diese Untersuchung umfasste 252 Patienten (17 Patienten nach KMT) und 235 Patienten von Intensivtherapiestationen (z. B. nach Organtransplantationen) in den Jahren 1991-1994 von der Humboldt-Universität zu Berlin (Charite). Die Patientenseren wurden routinemäßig auf das Vorkommen einer Candida- und Aspergillus Antigenämie geprüft. Zum Nachweis von zirkulierendem Galactomannan wurde ein Latex Agglutinationstest- Pastorex Candida und Aspergillus, Sanofi Diagnostics Pasteur, genutzt. Invasive Aspergillus-Pilzinfektionen wurden bei acht von 235 Patienten unter Intensivtherapie gefunden. Alle acht Patienten mit invasiver Aspergillose hatten einen positiven Aspergillus-Antigen-Test. Der direkte Nachweis von Antigenbestandteilen von Candida oder Aspergillus spp. erwies sich als vielversprechender frühdiagnostischer Test bei kritisch kranken und immunsupprimierten Patienten. / Invasive fungal infections are a major problem in transplant recipients. Candida and Aspergillus spp. are the most common fungal pathogens causing infection in patients undergoing BMT or solid organ transplantation. These infections are characterised by high morbidity and mortality, especially in patients with persistent granulocytopenia and in these receiving allogeneic bone marrow transplant. The mortality rate can be substantially reduced if an early diagnosis is made and the proper therapy given. The symptoms and signs of deep fungal infection in the transplant recipients are unreliable and often absent regardless of the type of organism or the site of infection. Laboratory tests are essential to establish the diagnosis of invasive fungal infection. The serological diagnosis of invasive candidosis or aspergillosis is at best an adjunct to clinical evaluation and other diagnostic procedures. The study comprises 252 patients undergoing allogeneic bone marrow transplantation (17 patients) and 235 patients from intensive care units (for instance after solid organ transplantation) in the years 1991-1994 at the Humboldt-University of Berlin (Charité). The serum of the patients were routinely screened for the occurrence of Candida and Aspergillus antigenemia (circulating galactomannan was detected using a latex agglutination test-Pastorex Candida and Aspergillus, Sanofi Diagnostics Pasteur). Invasive Aspergillus fungal infection was found in eight of the 235 intensive care patients. All these eight patients with invasive aspergillosis had an positive Aspergillus antigen test. The direct detection of antigenic components of Aspergillus and Candida spp. in serum appears promising as an early diagnostic test in critical ill and immunocompromised patients.

Made available in DSpace on 2016-03-22T17:27:02Z (GMT). No. of bitstreams: 1 is2.pdf: 1552188 bytes, checksum: 842b31ea5e16c999c9c802e21d14ccce (MD5) Previous issue date: 2013-07-22 / Tinea nigra is a rare superficial mycosis caused by Hortaea werneckii. This infection presents as asymptomatic brown to black macule mostly in palmo-plantar regions. This study presents dermatoscopy examination of a lesion, which demonstrated a homogeneous nonmelanocytic pigmented pattern with spicules in the macula; scanning electron microscopy (SEM) examination of the fungal culture, showing sympodial conidiogenesis; and SEM examination of a sample of the lesion, that revealed the epidermis with keratinocytes, elimination of fungal filaments and important aggregation of hyphae. SEM s findings correlated with those of dermatoscopic examination and allowed also documenting the mode of dissemination of tinea nigra, showing how hyphae are eliminated on lesion s surface / Tinea nigra é uma rara micose superficial causada pelo fungo Hortaea werneckii. Esta infecção apresenta-se como mancha assintomática acastanhada ou enegrecida, mais frequentemente na região palmo-plantar. Foram realizados neste estudo: dermatoscopia de uma lesão, a qual demonstrou um padrão homogêneo de pigmentação nãomelanocítica com espículas na mácula; microscopia eletrônica de varredura (MEV) da cultura fúngica, mostrando conidiogênese simpodial; e MEV de uma amostra da lesão, a qual revelou epiderme com queratinócitos, eliminação de filamentos fúngicos e importante agregação de hifas. Os achados de MEV se correlacionaram com aqueles do exame dermatoscópico e ainda permitiram documentar o modo de disseminação da tinea nigra, demonstrando como as hifas são eliminadas na superfície da lesão

Introdução: Fungos são alérgenos cuja importância nas doenças alérgicas respiratórias é subestimada por falta de extratos alergênicos adequados para diagnóstico, quer através de testes cutâneos, quer através de dosagem de IgE específica. Botucatu possui características próprias por se localizar numa \"cuesta\" e possuir vegetação variada que cobre grande parte da cidade. A partir das plantas os fungos ganham o ar através da esporulação podendo ser inalados e sensibilizar pacientes com alergias respiratórias. Este trabalho se propõe a estudar quais os fungos mais prevalentes em Botucatu e a taxa de sensibilização dos principais gêneros entre os portadores de asma e rinite procedentes dessa região. Casuística e Métodos: Foi desenvolvido um aparelho caça esporos para a captura dos mesmos em quatro pontos da cidade. Foram feitas cinco coletas/ponto/semana durante um ano para contagem e identificação das UFC (unidades formadoras de colônias). Foram selecionados pacientes de Botucatu e região com asma e rinite, nos quais se aplicou testes de puntura para os principais alérgenos, incluindo onze diferentes fungos. Foram também realizados testes intradérmicos com extratos de fungos nos pacientes cujo teste de puntura ao fungo em questão tenha sido negativo. Resultados: Foram identificados 67 gêneros sendo os mais prevalentes: Cladosporium, Epicoccum, Levedura, Aspergillus, Helminthosporium, Nigrospora, Monilia, Fusarium, Hyalodendrum, Penicillium, Curvularia, Humicola, Alternaria, Trichoderma e Sphaerosporium. Foram selecionados 207 pacientes dos quais 118 (57%) apresentaram pelo menos um teste positivo a fungo. O teste intradérmico foi responsável por 88% do diagnóstico de sensibilização A dosagem de IgE específica mostrou baixa sensibilidade (4%). Os fungos mais sensibilizantes foram: Aspergillus, Neurospora, Candida, Penicillium e Cladosporium. Observou-se maior positividade a fungos no grupo de asma grave, sendo que Aspergillus, Neurospora e Candida foram mais prevalentes nesse grupo. Encontrou-se associação positiva entre pacientes com asma atendidos em Pronto-Socorros durante o ano estudado e o total de UFC nesse período. Conclusões: Os fungos mais prevalentes no planeta são também encontrados em Botucatu, embora haja fungos característicos dessa região; a sensibilização a fungos é expressiva na população estudada e guarda relação com maior gravidade da asma; o teste intradérmico foi o melhor meio diagnóstico para sensibilização a fungos / Purpose: Fungi are allergens whose importance has been underestimated due to the absence of reliable extracts for the diagnosis of sensitization in vivo or in vitro. Botucatu has unique characteristics because of its location on a \"cuesta\" and its varied vegetation which cover a large area of the city. The fungi reach the air from the plants through sporulation and they can be inhaled and sensitize patients with allergic diseases. Here we studied the most prevalent fungi in the air of Botucatu and the rate of sensitization to the main fungi genus among patients with asthma and rhinitis. Methods: We developed a spore trap to catch the spores from the air in four points in the city. Five collections were taken weekly during a whole year. Colony forming units (CFU) were counted and the fungi on Petri dishes were classified. Patients with asthma and rhinitis have been selected and submitted to prick tests with the main allergens including eleven different types of fungi. Intradermal tests with fungi extract were also performed in the patients whose prick test to these fungi had been negative. Results: Sixty-seven genus of fungi have been identified and the more prevalent were: Cladosporium, Epicoccum, yeast, Aspergillus, Helminthosporium, Nigrospora, Monilia, Fusarium, Hyalodendrum, Penicillium, Curvularia, Humicola, Alternaria, Trichoderma and Sphaerosporium. Two hundred and seven patients have been selected and 118 (57%) have been at least one positive test to fungus. Intradermal test made the diagnosis in 88% of all diagnostics of sensitization, whereas \"in vitro\" specific IgE had low sensibility (4%). The more sensitizer fungi were: Aspergillus, Neurospora, Candida, Penicillium and Cladosporium. There was a significant correlation between severe asthma and fungi sensitization and the fungi more prevalent in that group of patient were: Aspergillus, Neurospora and Candida. Moreover, there was a positive correlation between the number of asthmatic patients attended in Emergencies and the total of CFU in the study period. Conclusion: We recognize in the air fungi specific to Botucatu although the most prevalent fungi are the same found in other places in the world; the fungi sensitization among patients with allergic respiratory diseases is meaningful and correlated with asthma severity; the intradermal test was the best mean to diagnose fungi sensitization

A criptococose é uma infecção fúngica causada pela levedura encapsulada Cryptococcus neoformans que acomete freqüentemente o meningoencéfalo de pacientes imunocomprometidos. No Brasil, cerca de 6% dos pacientes com AIDS desenvolvem a criptococose. A construção da resposta imune na criptococose envolve fagócitos e linfócitos T e B presentes no granuloma. Atualmente, são descritos três subtipos de linfócitos B: linfócitos B-1 a, B-1 b e B-2, o último conhecido como linfócito B \"convencional\". Os linfócitos B-1 (B-1a e B-1b) expressam concomitantemente imunofenótipo de macrófagos, células B e células T. Estes linfócitos receberam o nome de fagócitos mononucleares derivados de linfócitos B-1 e possuem a capacidade de migrar do peritônio até um foco inflamatório não específico, fagocitarem, expressarem antígeno e secretarem anticorpos e citocinas. Portanto, avaliamos a participação destes linfócitos B-1 na criptococose através de ensaios in vitro e in vivo com duas cepas diferentes de Cryptococcus neoformans variedade neoformans sorotipo A. Os linfócitos B-1 fagocitaram e destruiram as leveduras de Cryptococcus neoformans, principalmente na presença de complemento. Altas produções de NO encontradas no sobrenadante de cultura do ensaio de fagocitose associada à alta atividade peroxidásica e capacidade destes linfócitos em gerar EROs, poderiam justificar a alta atividade de \"killing\" encontrada nestes linfócitos B-1. Ainda, quando estimuladas com o fungo, os linfócitos B-1 secretaram principalmente IL-12 e IFN-γ, enquanto a IL-10 diminuiu significativamente. Na infecção experimental os camundongos BALB/xid, desprovidos de linfócitos B-1, apresentaram maior susceptibilidade à criptococose, com ambas as cepas, quando comparados aos camundongos BALB controle (BALB/c). Estes animais BALB/xid apresentaram maior carga fúngica nos órgãos baço, fígado e pulmão e morreram precocemente. A concentração de IL-10 foi maior nos órgãos dos camundongos BALB/xid, enquanto a IL-4 aumentou nos camundongos BALB/c. A concentração das citocinas de padrão Th1 (IL-12, IFN-γ e IL-2). consideradas protetoras na criptococose foi maior nos camundongos BALB/c. Além· da produção de citocinas protetoras, os órgãos dos camundongos BALB/c infectados com a cepa de menor cápsula apresentaram um maior população de linfócitos B-1. Estes linfócitos provavelmente estavam participando do processo inflamatório e contribuindo para a cura da infecção. Embora outras investigações sejam necessárias para melhor compreendermos a participação de linfócitos B-1 na criptococose, podemos considerar que estas células interagem com leveduras de Cryptococcus neoformans e podem ser decisivas para uma resposta protetora na criptococose experimental. / Abstract not available.

A criptococose é uma infecção fúngica causada pela levedura encapsulada Cryptococcus neoformans que acomete freqüentemente o meningoencéfalo de pacientes imunocomprometidos. No Brasil, cerca de 6% dos pacientes com AIDS desenvolvem a criptococose. A construção da resposta imune na criptococose envolve fagócitos e linfócitos T e B presentes no granuloma. Atualmente, são descritos três subtipos de linfócitos B: linfócitos B-1 a, B-1 b e B-2, o último conhecido como linfócito B \"convencional\". Os linfócitos B-1 (B-1a e B-1b) expressam concomitantemente imunofenótipo de macrófagos, células B e células T. Estes linfócitos receberam o nome de fagócitos mononucleares derivados de linfócitos B-1 e possuem a capacidade de migrar do peritônio até um foco inflamatório não específico, fagocitarem, expressarem antígeno e secretarem anticorpos e citocinas. Portanto, avaliamos a participação destes linfócitos B-1 na criptococose através de ensaios in vitro e in vivo com duas cepas diferentes de Cryptococcus neoformans variedade neoformans sorotipo A. Os linfócitos B-1 fagocitaram e destruiram as leveduras de Cryptococcus neoformans, principalmente na presença de complemento. Altas produções de NO encontradas no sobrenadante de cultura do ensaio de fagocitose associada à alta atividade peroxidásica e capacidade destes linfócitos em gerar EROs, poderiam justificar a alta atividade de \"killing\" encontrada nestes linfócitos B-1. Ainda, quando estimuladas com o fungo, os linfócitos B-1 secretaram principalmente IL-12 e IFN-γ, enquanto a IL-10 diminuiu significativamente. Na infecção experimental os camundongos BALB/xid, desprovidos de linfócitos B-1, apresentaram maior susceptibilidade à criptococose, com ambas as cepas, quando comparados aos camundongos BALB controle (BALB/c). Estes animais BALB/xid apresentaram maior carga fúngica nos órgãos baço, fígado e pulmão e morreram precocemente. A concentração de IL-10 foi maior nos órgãos dos camundongos BALB/xid, enquanto a IL-4 aumentou nos camundongos BALB/c. A concentração das citocinas de padrão Th1 (IL-12, IFN-γ e IL-2). consideradas protetoras na criptococose foi maior nos camundongos BALB/c. Além· da produção de citocinas protetoras, os órgãos dos camundongos BALB/c infectados com a cepa de menor cápsula apresentaram um maior população de linfócitos B-1. Estes linfócitos provavelmente estavam participando do processo inflamatório e contribuindo para a cura da infecção. Embora outras investigações sejam necessárias para melhor compreendermos a participação de linfócitos B-1 na criptococose, podemos considerar que estas células interagem com leveduras de Cryptococcus neoformans e podem ser decisivas para uma resposta protetora na criptococose experimental. / Abstract not available.

As a foodborne pathogen, Listeria monocytogenes (Lm) encounters many barriers to invasion and dissemination in the host that may change the nature of host response. Lm has been most commonly studied using intravenous (i.v.) inoculation, however, a method that delivers a bolus of bacteria directly to the bloodstream. Thus, little is known about what systemic and local mediators are triggered during the natural course of infection and how these may impact susceptibility. Our laboratory used foodborne transmission of Lm in mice to assess whether the method of transmission and the specific organ microenvironment could affect infection-induced secretion of type I interferon or prostaglandin E2. Type I interferon is a pro-inflammatory effector secreted in response to viruses that has been proposed to paradoxically down-regulate innate immunity to intracellular bacteria. In contrast to i.v. infection, type I interferon was not detrimental to the immune response when Lm were acquired orally. In fact, most of the anti-inflammatory effects of type I interferon in the spleen were attributable to i.v. but not foodborne infection. Importantly however, downregulation of the receptor for interferon gamma (IFNGR1), previously ascribed to the type I interferon response, was found to be a consequence of infection and unrelated to type I interferon. In the liver, robust recruitment and activation of neutrophils (PMN) is thought to be required for initiation of Lm immunity. Prostaglandin E2 (PGE2) is a lipid mediator most commonly associated with pain and fever that has also been demonstrated to have anti-inflammatory or tolerogenic effects. It is unknown, however, whether foodborne infection induces PGE2 in the liver and if PGE2 then down-regulates PMN activities. Recruitment of PMN to the liver following foodborne infection was robust in both susceptible and resistant animals. Bone marrow PMN from each killed Lm ex vivo with similar efficiency, thus suggesting that if PMN were dysfunctional during the course of natural infection, they were responding to cues in the microenvironment. Accordingly, significantly more PGE2 was made ex vivo by cells from the livers of susceptible animals than from resistant animals. When PGE2 was applied to naïve PMN prior to exposure to Lm, it consistently dampened the killing efficiency of these cells, suggesting that this lipid better known for its pro-inflammatory roles might have anti-inflammatory effects during Lm infection. Overall, these studies indicate that mediators produced as a result of infection may have very different roles dependent on route of inoculation, timing, and the specific organ examined.

Porphyromonas gingivalis, a Gram negative anaerobe implicated in the progression of periodontal disease, is capable of surviving and causing infection despite high levels of reactive nitrogen species found in the oral cavity due to its efficient nitrosative stress response. HcpR is an important sensor-regulator that plays a vital step in the initiation of the nitrosative stress response in many Gram negative anaerobic bacteria. We employ a combination of X-ray crystallography, SAXS, resonance Raman spectroscopy, UV-Vis spectroscopy, and molecular biology techniques to better understand this key regulator. Knockout of the hcpR gene in W83 P. gingivalis results in the inability of the bacteria to grow in physiological concentrations of nitrite and complementation of hcpR using the novel plasmid Pg108 rescues this phenotype. HcpR causes a drastic, dose dependent upregulation of PG0893, a gene coding for a putative NO reductase, when exposed to nitrite or nitric oxide. Full transcriptome sequencing reveals that hcp is the only significantly upregulated gene when P. gingivalis is exposed to nitrite and knockout of hcp resulted in a phenotype that is similar to that of the hcpR deficient strain. HcpR directly regulates the expression of hcp via direct binding to an inverted repeat sequence in the promoter region of the hcp gene. We present a 2.6 Å crystal structure of the N-terminal sensing domain of HcpR and show that it is FNR-CRP regulator. A putative hydrophobic heme binding pocket was identified in the junction between the N-terminal domain and the dimerization helix. Mutation of two methionine residues (Met68 and Met145) in this pocket abrogates activation of HcpR thus verifying the binding site. Heme bound to HcpR exhibits heme iron as a hexa-coordinate system in the absence of nitric oxide (NO) and upon nitrosylation transitions to a penta-coordinated system. Finally, Small Angle X-ray Scattering experiments of the full length HcpR reveal that the C-terminal DNA binding domain of HcpR has a high degree of interdomain flexibility.