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

Abstract

As Cryptococcus neoformans mother cells generationally age, their cell walls become thicker and cell-wall associated virulence factors are upregulated. Antiphagocytic protein 1 (App1), and laccase enzymes (Lac1 and Lac2) are virulence factors known to contribute to virulence of C. neoformans during infection through inhibition of phagocytic uptake and melanization. Here we show that these cell-wall-associated proteins are not only significantly upregulated in old C. neoformans cells, but also that their upregulation likely contributes to the increased resistance to antifungal and host-mediated killing during infection and to the subsequent accumulation of old cells. We found that old cells melanize to a greater extent than younger cells and as a consequence, old melanized cells are more resistant to killing by amphotericin B compared to young melanized cells. A decrease in melanization of old lacΔ mutants lead to a decrease in old-cell resilience, indicating that age-related melanization is contributing to the overall resilience of older cells and is being mediated by laccase genes. Additionally, we found that older cells are more resistant to macrophage phagocytosis, but this resistance is lost when APP1 is knocked out, indicating that upregulation of APP1 in older cells is in part responsible for their increased resistance to phagocytosis by macrophages. Finally, infections with old cells in the Galleria mellonella model support our conclusions, as loss of the APP1, LAC1, and LAC2 gene ablates the enhanced virulence of old cells, indicating their importance in age-dependent resilience.

Replicative Aging in Pathogenic Fungi

“…In older C. neoformans cells, remodeling of the cell wall with more abundant beta-glucans has been described [ 47 , 48 ]. In 10-generation-old C. neoformans cells, accumulation of antiphagocytic protein (App1), which is a key virulence factor [ 29 ] located in the cell wall, is observed.…”

“…Notably, melanin variants enriched in select lipids (sterol esters and poly-isoprenoids) were recently suggested to be linked to cell aging [ 68 ]. The disproportionate inheritance is supported by upregulated levels of LAC1 and LAC2 , which contribute to melanin formation, in 10-generation-old C. neoformans cells [ 29 ]. LAC1 is widely conserved in Candida and its role in age-related phenotypic variation should be examined further in Candida species, especially in the emergent C. auris .…”

“…Interestingly, despite slower doubling time in older mother cells, increased virulence was observed in older mother cells from C. neoformans , C. glabrata, and C. auris [ 17 , 18 , 29 ]. When older cells from these three pathogens were injected in Galleria mellonella (waxworm) larvae, the survival rates of the larvae were significantly lower than the survival rates of the larvae infected with the young daughter cells [ 17 , 18 , 29 ]. This is consistent with the observation that older cells from these pathogenic fungi were phagocytosed at a significantly lower rate by macrophages when compared with their respective daughter cells.…”

“…This is consistent with the observation that older cells from these pathogenic fungi were phagocytosed at a significantly lower rate by macrophages when compared with their respective daughter cells. Macrophage- and neutrophil-mediated killings were also lower in the mother cells isolated from these pathogens [ 17 , 18 , 29 ].…”

“…Again, this is consistent with gene expression analysis of older C. neoformans cells that indicated increased expression of three important established virulence factors. These factors include antiphagocytic protein (App1p) and laccase proteins (Lac1p and Lac2p) [ 29 ]. Murine experiments with C. glabrata have also suggested that older C. glabrata cells accumulate during chronic infection as evidenced by a higher bud scar count in yeast cells isolated from infected mice.…”