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ABSTRACT: We characterized Cryptococcus neoformans recombinant antiphagocytic protein 1 (rApp1) by SDS-PAGE, gel filtration chromatography, circular dichroism, and fluorescence spectroscopy. rApp1 produced by C. neoformans var. grubii contains an odd number of cysteines; therefore, it has the ability to form intermolecular disulfide bridges which can lead to the formation of amyloid fibrils in the absence of β-mercaptoethanol or DTT in vitro. Alternate approaches to over-expression of rApp1 in the Lepidopteran High Five(™) Insect cell line using pIZ/V5-His and in lentivirus were explored and are described. Finally, localization of App1 in vivo was examined in the presence and absence of the capsule.
Mycopathologia 10/2011; 173(5-6):395-405. · 1.65 Impact Factor
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ABSTRACT: The cryptococcus-specific protein antiphagocytic protein 1 (App1) regulates Cryptococcus neoformans virulence by controlling macrophage-driven fungal phagocytosis. This is accomplished through complement receptors (CR), specifically CR3. When inhaled, C. neoformans can cause a life-threatening meningoencephalitis in immunocompromised patients. Because glucose starvation can significantly change the gene expression and virulence of C. neoformans and because App1 is critical for phagocytosis in the lung-a low-glucose environment-we investigated the role of glucose in App1 expression. We found that App1 was upregulated dramatically under low-glucose conditions, and it was upregulated when C. neoformans cells were incubated in bronchoalveolar lavage (BAL) fluid, serum, and cerebrospinal fluid, which are low-glucose environments. Characterization of App1's regulation based on mammalian lung physiology revealed that App1 is upregulated via both increases in transcription and increases in mRNA stability. Our data provide new insights regarding C. neoformans adaptations to low-glucose environments.
Eukaryotic Cell 01/2011; 10(3):293-301. · 3.60 Impact Factor
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ABSTRACT: In previous studies, we showed that the pathogenic fungus Cryptococcus neoformans (Cn) produces a specific and unique protein called antiphagocytic protein 1 (App1), which inhibits phagocytosis of Cn by alveolar macrophages (AMs). Phagocytosis of Cn by AMs occurs mainly through a complement- or Ab-mediated mechanism. Among AM receptors, complement receptor 3 (CR3) and FcRgamma are the most common receptors involved in the phagocytic process. Because App1 inhibits phagocytosis of complement- but not Ab-coated erythrocytes, we investigated the role of CR3 in App1-macrophage interactions. We found that App1 binds to CR3 and if CR3 is absent from the surface of AMs, its antiphagocytic action is lost. When we investigated whether App1 would also bind to other complement receptor(s), we found that App1 does bind to complement receptor 2 (CR2) in a dose-dependent manner. In certain lymphoma cell lines, cellular proliferation is stimulated by complement through CR2, providing a potential use of App1 as a proliferation inhibitor of these cells. Initially discovered as an antiphagocytic protein regulating CR3-mediated innate immunity, App1 may also play a key role in the regulation of acquired immunity, because CR2 is mainly localized on B cells.
The Journal of Immunology 02/2009; 182(1):84-91. · 5.79 Impact Factor
