RESUMO
Transmission electron microscopy was used to study ultrastructures in Paracoccidioides brasiliensis yeast cells after ingestion by nonactivated or cytokine-activated murine peritoneal macrophages. Yeast cells ingested by nonactivated macrophages had typical bi- and trilayered cell walls, plasma membranes, mitochondria, nuclei, vacuoles, etc., which remained intact for 24 h of coculture. In contrast, yeast cells ingested by activated macrophages exhibited abnormal mitochondrial ultrastructures within 4 h of interaction. Subsequent events that occurred were the formation of several clear vacuoles per cell, disintegration of the cytoplasm, and development of empty cells with intact walls. These findings provide, for the first time, insights into stepwise damage to fungal cells by activated macrophages (of particular interest in this instance because of prior evidence that the damage is due to nonoxidative mechanisms) and give possible clues regarding fungicidal mechanisms.
Assuntos
Ativação de Macrófagos , Macrófagos/microbiologia , Fungos Mitospóricos/ultraestrutura , Paracoccidioides/ultraestrutura , Animais , Parede Celular/ultraestrutura , Macrófagos/ultraestrutura , Camundongos , Camundongos Endogâmicos BALB C , Mitocôndrias/ultraestrutura , Cavidade Peritoneal/citologia , Fagocitose , Baço/citologia , Vacúolos/ultraestruturaRESUMO
The plating efficiency of Paracoccidioides brasiliensis on standard mycological media is poor, impairing its isolation and recovery from various sources, particularly infected tissues. We describe a medium that markedly improves P. brasiliensis plating efficiency. It consists of a synthetic medium (modified McVeigh-Morton) supplemented with 4% (v:v) horse serum and 5% (v:v) culture filtrate from stationary phase P. brasiliensis cultures. A commercially available medium (brain-heart infusion), ordinarily inferior to unsupplemented McVeigh-Morton medium, is at least as efficacious as supplemented McVeigh-Morton medium when supplemented in this manner. We show that plating efficiency varies among P. brasiliensis isolates and can even vary with the isolate's history of passage in culture. In contrast, all isolates studied could produce the growth enhancing factors present in culture filtrate. Some siderophores produced by other fungi can be substituted for the culture filtrate, whereas others can be substituted for both the filtrate and serum. The enhancing effect of filtrate and/or serum could be removed by chelating iron. P. brasiliensis-produced siderophores are likely to be the growth enhancing moiety in culture filtrates.