Synergistic effect of pedalitin and amphotericin B against Cryptococcus neoformans by in vitro and in vivo evaluation.

Cryptococcosis is an opportunistic fungal infection responsible for high morbidity and mortality in immunocompromised patients. Combination of antifungal substances is a promising way to increase the percentage of successful treatment. Pedalitin (PED) is a natural substance obtained from Pterogyne nitens. The aim of this study was to verify the efficacy of PED alone and in combination with amphotericin B (AmB) in vitro and in vivo against Cryptococcus spp. In the in vitro assay, minimum inhibitory concentrations (MICs) of 0.125 mg/L for AmB and 3.9 mg/L for PED were found when the substances were tested alone, whilst in the combination treatment the active concentration of both decreased, with MICs of 0.03 mg/L for AmB and 1 mg/L for PED. In the survival assay, fungal burden study and histopathological assays it was possible to study the efficacy of the substances alone and in combination. The efficacy of combination therapy was considered better than monotherapy as evaluated in a Galleria mellonella model and a murine model. Thus, the combination of PED and AmB is an interesting alternative for anticryptococcal fungal treatment. Moreover, a correlation was observed between the invertebrate and murine models for this antifungal treatment combination.

Alterations of protein expression in conditions of copper-deprivation for Paracoccidioides lutzii in the presence of extracellular matrix components.

BACKGROUND: Paracoccidioides spp is a fungi genus and the agent of paracoccidioidomycosis. The strategies of infection used by these pathogens involve the expression of proteins related to adaptation to the host, particularly regarding the uptake of micronutrients. This study analyzed the adhesion of Paracoccidioides lutzii during conditions of copper (Cu) and iron (Fe) deprivation, while also evaluating the proteins expressed in conditions of Cu depletion in the presence of four extracellular matrix (ECM) components (laminin, fibronectin and types I and IV collagen). RESULTS: We cultured the P. lutzii in a chemically defined media without Cu and Fe. The fungus was then placed in contact with different ECM components and adhesion was evaluated. A significant increase in binding to all ECM components was observed when the fungus was cultured without Cu; which might be related to some adhesins expression. A proteomic assay was developed and revealed 39 proteins expressed that are involved in processes such as virulence, protein synthesis, metabolism, energy, transcription, transport, stress response and the cell cycle when the fungus was interacting with the ECM components. The up-regulated expression of two important adhesins, enolase and 14-3-3, was observed at the fungal cell wall during the interaction with the ECM components, indicating the role of these proteins in the Paracoccidioides-host interaction. CONCLUSIONS: This study is important for determining prospective proteins that may be involved in the interaction of Paracoccidioides with a host. Understanding the adaptive response to different growth conditions, elucidating the processes of adhesion and cell invasion, and identifying the proteins that are differentially expressed during the fungus-host interaction may help elucidate mechanisms used for survival and growth of Paracoccidioides in various human tissues.

Surface-expressed enolase contributes to the adhesion of Paracoccidioides brasiliensis to host cells.

Paracoccidioidomycosis is a systemic mycosis caused by the dimorphic fungus Paracoccidioides brasiliensis. Understanding the interactions between P. brasiliensis and the host tissue depends on the study of the different steps of the process of colonization, especially adhesion, in which the pathogen recognizes ligands on the surface of host cells. This study aimed to verify the role of enolase in the host cell-fungus interaction and the ability of enolase to bind to extracellular matrix components, to determine its subcellular localization, and to study the P. brasiliensis enolase amino acid sequence. The data revealed that fibronectin is the major ligand of enolase. Evaluation of the location of enolase at an ultrastructural level revealed that it is distributed in various cellular compartments, but at a high level in the cell wall. The analysis of the amino acid sequence revealed an internal plasminogen-binding motif ((254)FYKADEKKY(262)), which is conserved in most organisms and described as an important interaction site of the enolase with the host cell surface. This suggests that enolase performs additional functions related to the glycolytic pathway and also plays a role of adhesion in P. brasiliensis. Therefore, this study increases the knowledge about the characteristics of enolase and its influence on the binding process of P. brasiliensis.