Expression and Characterization of Cryptococcus neoformans Recombinant App1

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.

Cell Wall-Associated Virulence Factors Contribute to Increased Resilience of Old Cryptococcus neoformans Cells

“…In addition to the role of App1 in inhibiting phagocytosis and macrophage-mediated killing, Qureshi et al (2012) hypothesized that App1 may also play a role in melanization as it has amyloid properties and amyloids have been shown to play a role in melanin biosynthesis in other species. Interestingly, we found that young app1 Δ cells melanized to the same extent as wildtype cells, whereas old app1 Δ cells exhibited lower levels of melanization compared to old wildtype cells.…”

“…Furthermore, though melanization enhances app1 Δ cells resistance against AMB killing, there is still no significant difference between young and old melanized app1 Δ cells. Future studies are required to determine whether loss of App1 alters cell wall structure as App1 is located in the cell wall (Qureshi et al, 2012). If true, it would be interesting to further explore if such changes in the cell wall’s composition may also have an effect on the cell membrane, which is below the cell wall.…”

“…Antiphagocytic protein 1 (App1) is a virulence factor that is unique to C. neoformans (Luberto et al, 2003) and located in the cell wall of C. neoformans (Qureshi et al, 2012). This protein is also secreted into the supernatant of cultures and detectable in bronchoalveolar lavage fluid, serum, and cerebral spinal fluid of patients (Luberto et al, 2003; Stano et al, 2009; Williams and Del Poeta, 2011).…”

“…Knockout mutants lacking APP1 are less virulent in mice, indicating this virulence factor plays an important role in establishing infection. Interestingly, Qureshi et al (2012) found App1 to have amyloid properties and argue it may also play additional roles in pathogenesis. For example, amyloids have been shown to help evade the immune system by producing a protective coating around the cell wall in various other microbes (Gebbink et al, 2005; Qureshi et al, 2012).…”

“…Interestingly, Qureshi et al (2012) found App1 to have amyloid properties and argue it may also play additional roles in pathogenesis. For example, amyloids have been shown to help evade the immune system by producing a protective coating around the cell wall in various other microbes (Gebbink et al, 2005; Qureshi et al, 2012). Furthermore, different amyloids have been shown to be important for melanin biosynthesis (Qureshi et al, 2012).…”