Interactions of Paracoccidioides brasiliensis with host cells: recent advances.

Host-fungal interactions are inherently complex and dynamic. In order to identify new microbial targets and develop more effective antifungal therapies, it is important to understand the cellular and molecular mechanisms of disease. Paracoccidioidomycosis provokes a variety of clinical symptoms, and Paracoccidioides brasiliensis can reach many tissues, but primarily attacks the lungs. The ability of the pathogen to interact with the host surface structures is essential to further colonization, invasion, and growth. Epithelial cells may represent the first host barrier or the preferential site of entry of the fungus. For this reason, interactions between P. brasiliensis and Vero/A549 epithelial cells were evaluated, with an emphasis on the adherence, induction of cytoskeletal alterations, and differential signaling activity of the various surface molecules. The adhesion to and invasion of epithelial cells by P. brasiliensis may represent strategies employed to thwart the initial host immune response, and may help in the subsequent dissemination of the pathogen throughout the body.

Identification of a gene encoding adaptin-like protein in the Paracoccidioides brasiliensis genome by random amplified polymorphic DNA analysis.

Paracoccidioides brasiliensis isolates are not homogeneous in their patterns of pathogenicity in animals and adhesion to epithelial cells. During this investigation, genotypic differences were observed between two samples of P. brasiliensis strain 18 yeast phase (Pb18) previously cultured many times, one taken before (Pb18a) and the other after (Pb18b) animal inoculation. Random amplified polymorphic DNA analysis using the primer OPJ4 distinguished Pb18b from Pb18a by one 308 bp DNA fragment, which after cloning and sequencing was shown to encode a polypeptide sequence homologous to the protein beta-adaptin. It is suggested, by comparison to other micro-organisms, that this protein might play an important role in the virulence of P. brasiliensis. This result demonstrates the influence of in vitro subculturing on the genotype of this organism.

Isolation and partial characterization of a 30 kDa adhesin from Paracoccidioides brasiliensis.

The virulence of Paracoccidioides brasiliensis can be attenuated or lost after long periods of repeated subculturing and reestablished after animal inoculation. Only one adhesin (gp43) has been described until now, among the various identified components of P. brasiliensis, and gp43 shows adhesion to laminin. Thus, the present study was designed to isolate and characterize factors putatively related to the capacity of this fungus to adhere to the host by comparing P. brasiliensis samples, taken before and after animal inoculation. The two samples differed in their pattern of adhesion and invasion. The sample recently isolated from animals (Pb18b) demonstrated a greater capacity to adhere and to invade the Vero cells than the one subcultured in vitro (Pb18a). Extract from Pb18b also showed higher levels of protein expression than that from Pb18a, when two-dimensional electrophoresis gels were compared. A protein species of 30 kDa, pI 4.9, was more evident in the Pb18b extract and had properties of adhesin. Laminin, but none of the other extracellular matrix (ECM) components, such as fibronectin, collagen I and IV, bound specifically to the P. brasiliensis 30 kDa protein. The roles of 30 kDa and gp43 in cellular interactions were investigated and the adhesion of P. brasiliensis yeast cells was intensively inhibited by pre-treatment of epithelial cells with 30 kDa protein and gp43. Thus, this study presents evidence that adhesion capacity could be related to virulence, and that a 30 kDa adhesin accumulated differentially in samples with different levels of pathogenicity. This protein and its adhesion characteristics are being published for the first time and may be related to the virulence of P. brasiliensis.

Adesäo e invasäo de Paracoccidioides brasiliensis em cultura de células vero / Adherence and invasion of Paraccidioides brasiliensis (isolate 18) to Vero cells

A capacidade de aderir e invadir tecidos do hospedeiro tem sido imputada como importante mecanismo de patogenicidade. Neste trabalho foi estudado o processo de adesäo e invasäo de P. brasiliensis amostra 18, fase L, as culturas celulares da linhagem Vero, usando várias técnicas de microscopia. P. brasiliensis aderiu às células Vero após 30 minutos e formas intracitoplasmáticas apareceram após 5 horas de infecçäo. As células Vero apresentaram modificaçöes na presença do fungo, comportando-se como célula fagocítica, formando protusöes e projeçöes citoplasmáticas em dedo de luva, na tentativa de interiorizar o fungo. O fungo aderido às células exibiu padräo de reconhecimento semelhante ao de culturas com os soros anti-"cell-free" e anti-gp 43. Ambos os antígenos distribuíram-se regularmente na parede fúngica, de maneira específica, e a gp 43 foi mais evidente na conexäo entre as células mäe e filha e também nas proximidades da interaçäo fungo-célula. Células de P. brasiliensis näo foram observadas intracelularmente após tratamento com citocalasina D, sugerindo que um dos mecanismos que o fungo utiliza durante o processo de invasäo e via microfilamentos de actina. Por meio deste trabalho, informaçöes relevantes sobre os processos de adesäo e p emvolvimento do citoesqueleto durante a invasäo puderam ser demonstradas, podendo auxiliar no entendimento da patogênese de P. brasiliensis.