Intento de Aislamiento del Paracoccidioides brasiliensis del suelo

Resumen Cuarenta y cinco muestras de tierra, preferentemente de cafetales a más de 500 m de altitud sobre el nivel del mar, de varias regiones de Venezuela fueron inoculadas por vía endovenosa en ratones.No pudo aislarse elParacoccidioides brasiliensis.Se discuten brevemente las posibles causas de los resultados negativos y se proponen otras téchnicas para seguir intentando la determinación del habitat deParacoccidioides brasiliensis.

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Summary Forty five soil samples, most of them from coffee plantations at more than 500 m altitude, from several parts from Venezuela were inoculated intravenously into mice. Paracoccidioides brasilicnsis was not isolated.The authors discuss the possible reasons of the negative results and propose other techniques to apply in the future in order to isolateParacoccidioides brasilicnsis from nature.

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Résumé Quarante cinq échantillons de terre furent inoculés par voie intravéneuse — des souris. Les échantillons provenaient principalement de plantations de café — plus de 500 m d'altitude de plusieurs régions du Venezuela.LeParacoccidioides brasiliensis n'a pu etre isolé.Les auteurs discutent les causes éventuelles de cet échec et proposent des méthodes différentes pour reprendre la recherche duParacoccidioides brasiliensis dans la nature.

     

Factores Determinantes de la Fase Levaduriforme del Paracoccidioides brasiliensis

Resumen Se cultivaron 4 cepas autóctonas deP. brasiliensis en los medios líquidos siguientes: 1) medio con neopetona Difco dialisado deEvans &Kessel; 2) caldo infusión de carne glucosado al 1 %; 3) caldo extracto de carne glucosado al 1 % y 4) medio de Sabouraud glucosado al 4 %, distribuidos en frascos de Erlenmeyer de 300 cc a razón de 150 ml cada uno e incubados a 28°C en un agitador mecánico de vaivén con un desplazamiento de 5 cm y a 90 ciclos por minuto. En estas condiciones se obtuvo, en todas las cepas, la transformación de la fase filamentosa en levaduriforme o parasitaria con producción de abundantes microformas de 1 µ, aproximadamente, de diámetro.

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Summary We have cultured 4 autochthonous strains ofP. brasiliensis in the following liquid media: 1) media with dialysed Difco Neopeptone ofEvans &Kessel; 2) 1 % glucose meat infusion broth; 3) 1 % glucose meat extract broth and 4) 4 % glucose Sabouraud media, distributed in 300 cc Erlenmeyer flasks containing 150 ml each, incubated at 28°C in an electric shaker with a speed range of 90 strokes per minute and a wide degree of agitation of 2'.We obtained under these conditions the conversion of the filamentous phase of the four strains into the yeast phase producing abundant microforms of about 1 µ of diameter.

     

Aleuriospores of Paracoccidioides brasiliensis

In soil extract agar and in Bennett medium abundant aleuriospores ofParacoccidioides brasiliensis have been observed. The possibility that these spores could be the infecting elements for man is discussed.

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Resumen Se han observado abundantes aleuriosporas deParacoccidioides brasiliensis en agar extracto de tierra y en medio de Bennett. Se discute la posibilidad de que éstas esporas puedan ser los elementos que infectan al hombre.

     

Ornithine decarboxylase in Paracoccidioides brasiliensis

Ornithine decarboxylase in Paracoccidioides brasiliensis, a dimorphic human pathogenic fungus, was more active at 37° C in the yeast phase and at 30° C in the mycelial phase. In contrast to other fungal systems, yeast growth and mycelium-to-yeast transition in P. brasiliensis were accompanied by a high activity of ornithine decarboxylase at the onset of the budding process, the activity of which was inhibited by 1,4-diamino-2-butanone. The activity of ornithine decarboxylase remained at a basal level during vegetative growth of both the mycelial phase and the late stage of yeast phase, and also through the yeast-to-mycelium transition. Received: 18 December 1995 / Accepted: 8 March 1996     

The pathobiology of Paracoccidioides brasiliensis

Paracoccidioides brasiliensis causes one of the most prevalent systemic mycoses in Latin America--paracoccidioidomycosis. It is a dimorphic fungus that undergoes a complex transformation in vivo, with mycelia in the environment producing conidia, which probably act as infectious propagules upon inhalation into the lungs, where they transform to the pathogenic yeast form. This transition is readily induced in vitro by temperature changes, resulting in modulation of the composition of the cell wall. Notably, the polymer linkages change from beta-glucan to alpha-glucan, possibly to avoid beta-glucan triggering the inflammatory response. Mammalian oestrogens inhibit this transition, giving rise to a higher incidence of disease in males. Furthermore, the susceptibility of individuals to paracoccidioidomycosis has a genetic basis, which results in a depressed cellular immune response in susceptible patients; resistance is conferred by cytokine-stimulated granuloma formation and nitric oxide production. The latency period and persistence of the disease and the apparent lack of efficacy of humoral immunity are consistent with P. brasiliensis existing as a facultative intracellular pathogen.     

Intrayeast hyphae in Paracoccidioides brasiliensis

The occurrence of a live hypha inside a dead yeast during the transformation from yeast to mycelium of Paracoccidioides brasiliensis is described.     

Cytosolic Neutral Proteinases of Paracoccidioides brasiliensis

Cytosolic proteinases were assayed in both morphological phases of Paracoccidioides brasiliensis. Preparations from the mycelial phase were more active in vitro than those from the yeast cells. Optimal proteinase activities for both phases occurred at pH's between 6.0 and 9.0, and at 45°C. Gelatin-SDS-PAGE electrophoresis separated several bands (58–112 kDa) in mycelial preparations; a single band (70 kDa) was seen in yeast preparations. Enzymatic activities were inhibited by antipain, phenyl methyl sulfonyl fluoride (PMSF), and chymostatin, suggestive of serine proteinases. Partial inhibition of the mycelial enzymes by ethylene diamine tetraacetic acid (EDTA), 1,10-phenanthroline, and iodoacetamide, also suggested the presence of cysteine- and metallo-proteinases. The enzymatic activity increased in preparations extracted from yeast cells transforming to mycelia, and decreased in preparations obtained from the reverse process. Received: 29 September 1997 / Accepted: 19 February 1998     

Innate immunity to Paracoccidioides brasiliensis infection

Innate immunity is based in pre-existing elements of the immune system that directly interact with all types of microbes leading to their destruction or growth inhibition. Several elements of this early defense mechanism act in concert to control initial pathogen growth and have profound effect on the adaptative immune response that further develops. Although most studies in paracoccidioidomycosis have been dedicated to understand cellular and humoral immune responses, innate immunity remains poorly defined. Hence, the main purpose of this review is to present and discuss some mechanisms of innate immunity developed by resistant and susceptible mice to Paracoccidioides brasiliensis infection, trying to understand how this initial host-pathogen interface interferes with the protective or deleterious adaptative immune response that will dictate disease outcome. An analysis of some mechanisms and mediators of innate immunity such as the activation of complement proteins, the microbicidal activity of natural killer cells and phagocytes, the production of inflammatory eicosanoids, cytokines, and chemokines among others, is presented trying to show the important role played by innate immunity in the host response to P. brasiliensis infection.     

Characteristics of 17Paracoccidioides brasiliensis isolates

We have studied the physiological and morphological features of 17 isolates ofParacoccidioides brasiliensis in order to define their phenotypes. The isolates were cultured at room temperature on potato dextrose agar (PDA, Difco) slants for mycelial growth and in 1% dextrose brain heart infusion agar (BHIA, Difco) at 37°C for the study of yeast forms. Most mycelial and yeast forms grew well between pH 5.6–9.4. In their response to osmotic pressure the isolates were separated in three groups: intolerant, intermediate and tolerant. They also varied in carbohydrate assimilation tests, which indicated important metabolic variation. No clear differences were observed in phenol oxidase tests, KNO₃, starch, casein and arbutin assimilation tests. Only 1 of the isolates, Bt-19, had gelatinase activity. No correlation was observed between the above differences and virulence. Two patterns of growth were observed in the mycelial cultures, glabrous and cottonous, the latter being correlated with increased virulence for ddY mice. Most yeast forms grew as cerebriform colonies, but Pb-HC and Bt-19 colonies had a cobblestone-like surface.     

Ultrastructure of Dimorphic Transformation in Paracoccidioides brasiliensis

The fine structure of Paracoccidioides brasiliensis undergoing temperature-dependent transformation from mycelium to yeast and vice versa (M right harpoon over left harpoon Y) was studied. The transitional form to mycelium from the yeast appears as an elongated bud that extends from the yeast and which has a mixture of characteristics from both the yeast and the mycelium. The transitional form to yeast from the mycelium starts with enlargement of the interseptal spaces and cracking of the outer electron-dense layer of the cell wall of the hypha. Later the interseptal spaces tend to become round and separate. In M --> Y only few interseptal spaces seem to transform. The yeast is produced by self-transformation of the hypha. In Y --> M a new structure is formed and the yeast dies. Intrahyphal hyphae are observed during the transformation from M --> Y, and intrayeast hyphae during the Y --> M. Due to the high mortality and breakage observed in both types of transformations, we believe that wound of the yeast or the mycelium could elicit this phenomenon.