Secreted phospholipase B (PLB) activity promotes the survival and replication of Cryptococcus neoformans in macrophages in vitro. We therefore investigated the role of mononuclear phagocytes and cryptococcal PLB in the dissemination of infection in a mouse model, using C. neoformans var. grubii wild-type strain H99, a PLB1 deletion mutant (⌬plb1), and a reconstituted strain (⌬plb1 rec ). PLB facilitated the entry of endotracheally administered cryptococci into lung IM. PLB was also required for lymphatic spread from the lung to regional lymph nodes and for entry into the blood. Langhans-type giant cells containing budding cryptococci were seen free in the lymphatic sinuses of hilar nodes of H99-and ⌬plb1 rec -infected mice, suggesting that they may have a role in the dissemination of cryptococcal infection. The transfer of infected lung macrophages to recipient mice by tail vein injections demonstrated that these cells can facilitate hematogenous dissemination of cryptococci to the brain, independent of cryptococcal PLB secretion. PLB activities of cryptococci isolated from lung macrophages or infected brains were not persistently increased. We conclude that mononuclear phagocytes are a vehicle for cryptococcal dissemination and that PLB activity is necessary for the initiation of interstitial pulmonary infections and for dissemination from the lung via the lymphatics and blood. PLB is not, however, essential for the establishment of neurological infections when cryptococci are presented within, or after passage through, mononuclear phagocytes.Cryptococcus neoformans is a common cause of potentially fatal fungal meningoencephalitis, especially in immunocompromised patients. Primary infections are acquired by inhalation of infectious propagules from environmental sources (1). However, mechanisms by which C. neoformans establishes pulmonary disease and disseminates to the central nervous system (CNS) are not understood.Recent studies using murine models and macrophage-like cell lines have implicated secreted phospholipase B (PLB), the protein produced by the PLB1 gene (5), in intracellular survival, growth, and replication of C. neoformans within macrophages (5,7,15). Furthermore, the persistence of cryptococcal infections has been correlated with the presence of viable cryptococci within macrophages (8, 10). Cryptococcal PLB also enhances pulmonary infections, possibly by inhibiting the development of a protective immune response in the lung, and is required for dissemination to pulmonary lymph nodes and the brain (15). It has been proposed that PLB initiates invasion of the lung interstitium by cryptococci since phospholipids in the pulmonary surfactant and the outer leaflet of mammalian cell membranes are preferred substrates of the enzyme (3, 17). The mechanisms by which cryptococcosis is established in the CNS are unknown, although it has been suggested that cryptococci cross the blood-brain barrier within monocytes or after the penetration of endothelial cells (4) and that CNS infection is associated with su...

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Cryptococci survive and replicate within macrophages and can use exogenous arachidonic acid for the production of eicosanoids. Phospholipase B1 (PLB1) has a putative, but uninvestigated, role in these processes. We have shown that uptake and esterification of radiolabeled arachidonic, palmitic, and oleic acids by the Cryptococcus neoformans var. grubii H99 wild-type strain and its PLB1 deletion mutant strain (the ⌬plb1 strain) are independent of PLB1, except under hyperosmolar stress. Similarly, PLB1 was required for metabolism of 1-palmitoyl lysophosphatidylcholine (LysoPC), which is toxic to eukaryotic cell membranes, under hyperosmolar conditions. During both logarithmic and stationary phases of growth, the physiologically relevant phospholipids, dipalmitoyl phosphatidylcholine (DPPC) and dioleoyl phosphatidylcholine, were taken up and metabolized via PLB1. Exogenous DPPC did not enhance growth in the presence of glucose as a carbon source but could support it for at least 24 h in glucose-free medium. Detoxification of LysoPC by reacylation occurred in both the H99 wild-type and the ⌬plb1 strains in the presence of glucose, but PLB1 was required when LysoPC was the sole carbon source. This indicates that both energy-independent (via PLB1) and energydependent transacylation pathways are active in cryptococci. Phospholipase A 1 activity was identified by PLB1-independent degradation of 1-palmitoyl-2-arachidonoyl phosphatidylcholine, but the arachidonoyl LysoPC formed was not detoxified by reacylation. Using the human macrophage-like cell line THP-1, we demonstrated the PLB1-dependent incorporation of macrophage-derived arachidonic acid into cryptococcal lipids during cryptococcus-phagocyte interaction. This pool of arachidonate can be sequestered for eicosanoid production by the fungus and/or suppression of host phagocytic activity, thus diminishing the immune response.