| Background and PurposeC. neoformans is an environmental pathogen found primarily in pigeon droppings and soil contaminated with avian guanos throughout the world. It is a heterothallic, basidiomycetous, pathogenic fungus that infects both immunocompetent and immunocompromised individuals. The prevalence of disease caused by this organism has increased dramatically as a result of human immunodeficiency virus (HIV) infection, organ transplantation, cytotoxic chemotherapy and corticosteroid use. And the infection caused by C. neoformans can result in chronic dormant infection with the potential for acute outbreaks. C. neoformans has several well established virulence factors:capsule, melanin, expression of urease and phospholipase, and the ability to grow at human body temperature.C. neoformans is strictly an obligate aerobic pathogenic yeast. In laboratory conditions, atmospheric levels of oxygen (21%) are required for optimal growth of C. neoformans and lower oxygen concentrations lead to a significant reduction in cell growth. The inhaled C. neoformans cells reach the central nervous system by haematogenous dissemination and cause life-threatening meningoencephalitis. It is well known that oxygen concentrations in the human brain are reported to be drastically lower than in the atmosphere and vary significantly among anatomical sites. Moreover, inflammation, thrombosis, and necrosis associated with infection are thought to lead to increased degrees of hypoxia. Thus, in order to establish infection in the host, C. neoformans cells must sense and adapt to rapidly changing oxygen level. Recent studies have demonstrated the obligate aerobic pathogenic yeast C. neoformans can adapt to and survive in hypoxic conditions, and indicated that the central strategy of C. neoformans lies in adjustment of its proliferation rate to the available oxygen levels in CNS, Sterol-response element binding protein (SREBP) and Tco pathways seem to be two sensing systems involved in hypoxia sensing in C. neoformans, while there is still a lack of information on the effect of hypoxia on virulence of C. neoformans. We found that C. neoformans did not form apparent capsule under hypoxia conditions. Hence we propose a hypothesis that cryptococcal response to hypoxia might be the driving force for developing a state of dormant infection which is characterized by extensive changes in phenotype and virulence factors.This study will focus on the effect of different oxygen concentrations caused by AnaeroPack Series on virulence of C. neoformans, in order to provide new ideas for the role of hypoxia in chronic dormant infection caused by C. neoformans.MethodsC. neoformans type strains (BLS71, BLS63, ATCC32609, ATCC34874and YD53, serotype A, B, C, D and AD, respectively) and clinical isolates were grown in YPD at37℃. The cultures were then spotted on to YPD, YES, DOPA (dihydroxyphenylalanine) and caffeic acid agars, urea agar and egg yolk agar under various oxygen levels caused by AnaeroPack Series. The colony diameter, capsule size, pigmentation, urease and phospholipase activity of strains were observed and the effect of different oxygen concentrations on virulence factors of C. neoformans was investigated.Results1. Morphology observationThe test strains can grow under different oxygen concentrations and grow better under normoxic conditions than other conditions. C. neoformans slows its growth as the oxygen concentration declines. All test strains did not form visible yeast-like colony under anaerobic condition and colony diameters were significantly smaller than other three groups. No differences of colony diameters of all strains were found under the same oxygen level culture conditions. No differences of growth rates and colony sizes between the YPD and YES media were found.2. Melanin productionWith exception of anaerobic condition, all species were able to produce melanin on DOPA media after3and7days under other three culture conditions. Conversely, most strains showed no evidence of pigmentation on caffeic acid agar under different oxygen concentrations except normoxic conditions.3. CapsuleWith exception of YD53, all strains form polysaccharide capsules under any of oxygen levels. In capsule non-inducing conditions, there was significant difference between the four oxygen concentrations in BLS71、BLS63、ATCC32609and clinical isolates. BLS71、BLS63and clinical isolates have a smaller capsule under anaerobic condition. BLS71and clinical isolates have a greater capsule in capsule-inducing conditions than non-inducing conditions under any of the four oxygen concentrations.4. Urease and Phospholipase activityAll strains except ATCC32609display pink colour intensity after incubation in atmospheric conditions for7days. All tested strains showed no urease activity under anaerobic condition after incubation for14days. With exception of anaerobic condition, all C. neoformans in this study expressed phospholipase activity after7days. The PZs of BLS71, ATCC32609and ATCC34874in normoxic conditions were smaller than in other conditions. The PZs of the same strains increased as the oxygen concentration decline. There was no significant correlation of phospholipase activity with different strains in the same culture condition.ConclusionC. neoformans can grow under anaerobic condition. Hypoxia may have some inhibiting effect on capsule formation, melanin production and the activities of phospholipase and urease of C. neoformans. The strains tested may have different susceptibility to oxygen. This study suggested that serotypes may affect the susceptibility of C. neoformans to various oxygen concentrations. |
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