Phospholipids trigger Cryptococcus neoformans capsular enlargement during interactions with amoebae and macrophages.

A remarkable aspect of the interaction of Cryptococcus neoformans with mammalian hosts is a consistent increase in capsule volume. Given that many aspects of the interaction of C. neoformans with macrophages are also observed with amoebae, we hypothesized that the capsule enlargement phenomenon also had a protozoan parallel. Incubation of C. neoformans with Acanthamoeba castellanii resulted in C. neoformans capsular enlargement. The phenomenon required contact between fungal and protozoan cells but did not require amoeba viability. Analysis of amoebae extracts showed that the likely stimuli for capsule enlargement were protozoan polar lipids. Extracts from macrophages and mammalian serum also triggered cryptococcal capsular enlargement. C. neoformans capsule enlargement required expression of fungal phospholipase B, but not phospholipase C. Purified phospholipids, in particular, phosphatidylcholine, and derived molecules triggered capsular enlargement with the subsequent formation of giant cells. These results implicate phospholipids as a trigger for both C. neoformans capsule enlargement in vivo and exopolysaccharide production. The observation that the incubation of C. neoformans with phospholipids led to the formation of giant cells provides the means to generate these enigmatic cells in vitro. Protozoan- or mammalian-derived polar lipids could represent a danger signal for C. neoformans that triggers capsular enlargement as a non-specific defense mechanism against potential predatory cells. Hence, phospholipids are the first host-derived molecules identified to trigger capsular enlargement. The parallels apparent in the capsular response of C. neoformans to both amoebae and macrophages provide additional support for the notion that certain aspects of cryptococcal virulence emerged as a consequence of environmental interactions with other microorganisms such as protists.

Phagocytosis of Cryptococcus neoformans by, and nonlytic exocytosis from, Acanthamoeba castellanii.

Cryptococcus neoformans, an encapsulated, pathogenic yeast, is endowed with a variety of virulence factors, including a polysaccharide capsule. During mammalian infection, the outcome of the interaction between C. neoformans and macrophages is central to determining the fate of the host. Previous studies have shown similarities between the interaction of C. neoformans with macrophages and with amoebae, resulting in the proposal that fungal virulence for mammals originated from selection by amoeboid predators. In this study, we investigated the interaction of C. neoformans with the soil amoeba Acanthamoeba castellanii. Comparison of phagocytic efficiency of the wild type, nonencapsulated mutants, and complemented strains showed that the capsule was antiphagocytic for amoebae. Capsular enlargement was associated with a significant reduction in phagocytosis, suggesting that this phenomenon protects against ingestion by phagocytic predators. C. neoformans var. neoformans cells were observed to exit amoebae several hours after ingestion, in a process similar to the recently described nonlytic exocytosis from macrophages. Cryptococcal exocytosis from amoebae was dependent on the strain and on actin and required fungal viability. Additionally, the presence of a capsule was inversely correlated with the likelihood of extrusion in certain strains. In summary, nonlytic exocytosis from amoebae provide another parallel to observations in fungus-macrophage interactions. These results provide additional support for the notion that some mechanisms of virulence observed during mammalian infection originated, and were selected for, by environmental interactions.

Paramecium species ingest and kill the cells of the human pathogenic fungus Cryptococcus neoformans.

A fundamental question in the field of medical mycology is the origin of virulence in those fungal pathogens acquired directly from the environment. In recent years, it was proposed that the virulence of certain environmental animal-pathogenic microbes, such as Cryptococcus neoformans, originated from selection pressures caused by species-specific predation. In this study, we analyzed the interaction of C. neoformans with three Paramecium spp., all of which are ciliated mobile protists. In contrast to the interaction with amoebae, some Paramecium spp. rapidly ingested C. neoformans and killed the fungus. This study establishes yet another type of protist-fungal interaction supporting the notion that animal-pathogenic fungi in the environment are under constant selection by predation.

Capsule enlargement in Cryptococcus neoformans confers resistance to oxidative stress suggesting a mechanism for intracellular survival.

Cryptococcus neoformans is a facultative intracellular pathogen. The most distinctive feature of C. neoformans is a polysaccharide capsule that enlarges depending on environmental stimuli. The mechanism by which C. neoformans avoids killing during phagocytosis is unknown. We hypothesized that capsule growth conferred resistance to microbicidal molecules produced by the host during infection, particularly during phagocytosis. We observed that capsule enlargement conferred resistance to reactive oxygen species produced by H(2)O(2) that was not associated with a higher catalase activity, suggesting a new function for the capsule as a scavenger of reactive oxidative intermediates. Soluble capsular polysaccharide protected C. neoformans and Saccharomyces cerevisiae from killing by H(2)O(2). Acapsular mutants had higher susceptibility to free radicals. Capsular polysaccharide acted as an antioxidant in the nitroblue tetrazolium (NBT) reduction coupled to beta-nicotinamide adenine dinucleotide (NADH)/phenazine methosulfate (PMS) assay. Capsule enlargement conferred resistance to antimicrobial peptides and the antifungal drug Amphotericin B. Interestingly, the capsule had no effect on susceptibility to azoles and increased susceptibility to fluconazole. Capsule enlargement reduced phagocytosis by environmental predators, although we also noticed that in this system, starvation of C. neoformans cells produced resistance to phagocytosis. Our results suggest that capsular enlargement is a mechanism that enhances C. neoformans survival when ingested by phagocytic cells.

Multipurpose prevention technologies for sexual and reproductive health: mapping global needs for introduction of new preventive products.

OBJECTIVES: Worldwide, women face sexual and reproductive health (SRH) risks including unintended pregnancy and sexually transmitted infections (STIs) including HIV. Multipurpose prevention technologies (MPTs) combine protection against two or more SRH risks into one product. Male and female condoms are the only currently available MPT products, but several other forms of MPTs are in development. We examined the global distribution of selected SRH issues to determine where various risks have the greatest geographical overlap. STUDY DESIGN: We examined four indicators relevant to MPTs in development: HIV prevalence, herpes simplex virus type 2 prevalence (HSV-2), human papillomavirus prevalence (HPV) and the proportion of women with unmet need for modern contraception. Using ArcGIS Desktop, we mapped these indicators individually and in combination on choropleth and graduated symbol maps. We conducted a principal components analysis to reduce data and enable visual mapping of all four indicators on one graphic to identify overlap. RESULTS: Our findings document the greatest overlapping risks in Sub-Saharan Africa, and we specify countries in greatest need by specific MPT indication. CONCLUSIONS: These results can inform strategic planning for MPT introduction, market segmentation and demand generation; data limitations also highlight the need for improved (non-HIV) STI surveillance globally. IMPLICATIONS: MPTs are products in development with the potential to empower women to prevent two or more SRH risks. Geographic analysis of overlapping SRH risks demonstrates particularly high need in Sub-Saharan Africa. This study can help to inform strategic planning for MPT introduction, market segmentation and demand generation.

Impaired synthesis of prostaglandin E2 by lung fibroblasts and alveolar epithelial cells from GM-CSF-/- mice: implications for fibroproliferation.

Prostaglandin E(2) (PGE(2)) is a potent suppressor of fibroblast activity. We previously reported that bleomycin-induced pulmonary fibrosis was exaggerated in granulocyte-macrophage colony-stimulating factor knockout (GM-CSF(-/-)) mice compared with wild-type (GM-CSF(+/+)) mice and that increased fibrosis was associated with decreased PGE(2) levels in lung homogenates and alveolar macrophage cultures. Pulmonary fibroblasts and alveolar epithelial cells (AECs) represent additional cellular sources of PGE(2) within the lung. Therefore, we examined fibroblasts and AECs from GM-CSF(-/-) mice, and we found that they elaborated significantly less PGE(2) than did cells from GM-CSF(+/+) mice. This defect was associated with reduced expression of cyclooxygenase-1 and -2 (COX-1 and COX-2), key enzymes in the biosynthesis of PGE(2). Additionally, proliferation of GM-CSF(-/-) fibroblasts was greater than that of GM-CSF(+/+) fibroblasts, and GM-CSF(-/-) AECs were impaired in their ability to inhibit fibroblast proliferation in coculture. The addition of GM-CSF to fibroblasts from GM-CSF(-/-) mice increased PGE(2) production and decreased proliferation. Similarly, AECs isolated from GM-CSF(-/-) mice with transgenic expression of GM-CSF under the surfactant protein C promoter (SpC-GM mice) produced more PGE(2) than did AEC from control mice. Finally, SpC-GM mice were protected from fluorescein isothiocyanate-induced pulmonary fibrosis. In conclusion, these data demonstrate that GM-CSF regulates PGE(2) production in pulmonary fibroblasts and AECs and thus plays an important role in limiting fibroproliferation.

Protection from fluorescein isothiocyanate-induced fibrosis in IL-13-deficient, but not IL-4-deficient, mice results from impaired collagen synthesis by fibroblasts.

Intratracheal injection of FITC results in acute lung injury and progresses to fibrosis by day 21 postchallenge. In response to FITC, BALB/c mice produce IL-4 and IL-13 in the lung. To investigate whether IL-4 and/or IL-13 were important profibrotic mediators in this model, we examined the fibrotic response to FITC in mice that were genetically deficient in IL-4 (IL-4(-/-)), IL-13 (IL-13(-/-)), or IL-4 and IL-13 combined (IL-4/13(-/-)). Baseline levels of collagen were similar in all mice. In response to FITC, both BALB/c and IL-4(-/-) mice developed fibrosis, whereas the IL-13(-/-) and IL-4/13(-/-) mice were significantly protected, as measured by total lung collagen levels and histology. Total leukocyte recruitment to the lung was similar in all four strains of mice when measured on days 7, 14, and 21 post-FITC. BALB/c mice showed prominent eosinophilia on day 7 that was absent in IL-4(-/-), IL-13(-/-), and IL-4/13(-/-) mice, suggesting that eosinophilia is not necessary for development of a fibrotic response. There were no significant differences in the percentages of any other leukocytes analyzed between the genotypes. Similarly, protection in IL-13(-/-) mice was not associated with alterations in cytokine or eicosanoid profiles. Interestingly, TGF-beta1 production was not reduced in IL-13(-/-) mice. Analyses of fibroblasts isolated from the four genotypes demonstrated that although there were similar numbers of fibroblasts present in cultures of lung minces, fibroblasts from IL-13-deficient strains have reduced basal and stimulated levels of collagen production. IL-13Ralpha1 expression increases on fibroblasts during fibrotic responses in vivo, and IL-13 increases collagen synthesis in fibroblasts. Thus, IL-13 mediates its profibrotic actions through direct effects on fibroblast production of extracellular matrix.