Fungal-Induced Cell Cycle Impairment, Chromosome Instability and Apoptosis via Differential Activation of NF-κB

Abstract

Microbial pathogens have developed efficient strategies to compromise host immune responses. Cryptococcus neoformans is a facultative intracellular pathogen, recognised as the most common cause of systemic fungal infections leading to severe meningoencephalitis, mainly in immunocompromised patients. This yeast is characterized by a polysaccharide capsule, which inhibits its phagocytosis. Whereas phagocytosis escape and macrophage intracellular survival have been intensively studied, extracellular survival of this yeast and restraint of host innate immune response are still poorly understood. In this study, we have investigated whether C. neoformans affected macrophage cell viability and whether NF-κB (nuclear factor-κB), a key regulator of cell growth, apoptosis and inflammation, was involved. Using wild-type (WT) as well as mutant strains of C. neoformans for the pathogen side, and WT and mutant cell lines with altered NF-κB activity or signalling as well as primary macrophages for the host side, we show that C. neoformans manipulated NF-κB-mediated signalling in a unique way to regulate macrophage cell fate and viability. On the one hand, serotype A strains reduced macrophage proliferation in a capsule-independent fashion. This growth decrease, which required a critical dosage of NF-κB activity, was caused by cell cycle disruption and aneuploidy, relying on fungal-induced modification of expression of several cell cycle checkpoint regulators in S and G2/M phases. On the other hand, C. neoformans infection induced macrophage apoptosis in a capsule-dependent manner with a differential requirement of the classical and alternative NF-κB signalling pathways, the latter one being essential. Together, these findings shed new light on fungal strategies to subvert host response through uncoupling of NF-κB activity in pathogen-controlled apoptosis and impairment of cell cycle progression. They also provide the first demonstration of induction of aneuploidy by a fungal pathogen, which may have wider implications for human health as aneuploidy is proposed to promote tumourigenesis.

Author Summary

Cryptococcus neoformans, the only encapsulated pathogenic yeast, is responsible for severe opportunistic meningoencephalitis mostly in immunocompromised patients. It is a facultative intracellular pathogen and, as such, has the ability to survive intra- and extracellularly. Whereas interactions of C. neoformans with macrophages, especially its phagocytosis escape and intracellular survival, have been intensively studied, little is known about other schemes allowing extracellular survival of this yeast and restraint of host innate immune response. Here, we report that Cryptococcus neoformans compromised macrophage viability in two ways. Firstly, fungal infection elicited a strong decrease in macrophage proliferation in a capsule-independent fashion. This inhibition was subsequent to fungal-induced cell cycle disruption and chromosome aberrations (aneuploidy), a phenomenon commonly triggered by bacteria or viruses but for the first time described for a fungus. Secondly, this pathogen promoted apoptosis in a capsule-dependent manner. Our findings unravel a new process by which a fungal pathogen dampens the immune response using uncoupled activity of NF-κB (a key regulator of cell growth, apoptosis and inflammation) in fungal-induced apoptosis and inhibition of cell proliferation. This may have larger implications for human health, as animal models suggest that aneuploidy promotes tumourigenesis.