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.We thank Dr J.D. Nosanchuk for the use of defensins and Dr Steinman for the kind gift of A. castellanii strains. We thank Dr J.C. Arguelles and Pilar González (Universidad de Murcia, Spain) for providing protocols to measure catalase activity, and Drs Carlos and Juana Maria Gancedo (CSIC, Spain) for the permission to use their technical resources and for their helpful discussions. We are indebted to Dr F. Usera and Rosa Hidalgo for their collaboration, help and technical support in the use of the γ-irradiator from the animal facility from the National Center for Biotechnology (CSIC, Spain). We warmly thank Josefa Casas for her technical support, and all the members from the Mycology Service from the National Center for Microbiology (Instituto de Salud Carlos III) for their helpful discussions. M.V.C. is funded by a research contract from the Agencia Española de Cooperación Internacional (AECI). O.Z. is a ‘Ramón y Cajal’ fellow from the Ministerio Español de Educación y Ciencia (MEC) and is funded by Grants MPY1025/06 from the MEC and 1181/06 from el Instituto de Salud Carlos III.S

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

Socializing One Health: an innovative strategy to investigate social and behavioral risks of emerging viral threats

In an effort to strengthen global capacity to prevent, detect, and control infectious diseases in animals and people, the United States Agency for International Development’s (USAID) Emerging Pandemic Threats (EPT) PREDICT project funded development of regional, national, and local One Health capacities for early disease detection, rapid response, disease control, and risk reduction. From the outset, the EPT approach was inclusive of social science research methods designed to understand the contexts and behaviors of communities living and working at human-animal-environment interfaces considered high-risk for virus emergence. Using qualitative and quantitative approaches, PREDICT behavioral research aimed to identify and assess a range of socio-cultural behaviors that could be influential in zoonotic disease emergence, amplification, and transmission. This broad approach to behavioral risk characterization enabled us to identify and characterize human activities that could be linked to the transmission dynamics of new and emerging viruses. This paper provides a discussion of implementation of a social science approach within a zoonotic surveillance framework. We conducted in-depth ethnographic interviews and focus groups to better understand the individual- and community-level knowledge, attitudes, and practices that potentially put participants at risk for zoonotic disease transmission from the animals they live and work with, across 6 interface domains. When we asked highly-exposed individuals (ie. bushmeat hunters, wildlife or guano farmers) about the risk they perceived in their occupational activities, most did not perceive it to be risky, whether because it was normalized by years (or generations) of doing such an activity, or due to lack of information about potential risks. Integrating the social sciences allows investigations of the specific human activities that are hypothesized to drive disease emergence, amplification, and transmission, in order to better substantiate behavioral disease drivers, along with the social dimensions of infection and transmission dynamics. Understanding these dynamics is critical to achieving health security--the protection from threats to health-- which requires investments in both collective and individual health security. Involving behavioral sciences into zoonotic disease surveillance allowed us to push toward fuller community integration and engagement and toward dialogue and implementation of recommendations for disease prevention and improved health security

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

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

AbstractObjectivesWorldwide, 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 designWe 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.ResultsOur findings document the greatest overlapping risks in Sub-Saharan Africa, and we specify countries in greatest need by specific MPT indication.ConclusionsThese 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.ImplicationsMPTs 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