The opportunistic fungal pathogen Cryptococcus neoformans causes lethal infections in immunocompromised patients. Macrophages are central to the host response to cryptococci; however, it is unclear how C. neoformans is recognised and phagocytosed by macrophages. Here we investigate the role of TLR4 in the non-opsonic phagocytosis of C. neoformans. We find that loss of TLR4 function unexpectedly increases phagocytosis of non-opsonised cryptococci by murine and human macrophages. The increased phagocytosis observed in Tlr4−/− cells was dampened by pre-treatment of macrophages with oxidised-LDL, a known ligand of scavenger receptors. The scavenger receptor, macrophage scavenger receptor 1 (MSR1) (also known as SR-A1 or CD204) was upregulated in Tlr4−/− macrophages. Genetic ablation of MSR1 resulted in a 75% decrease in phagocytosis of non-opsonised cryptococci, strongly suggesting that it is a key non-opsonic receptor for this pathogen. We go on to show that MSR1-mediated uptake likely involves the formation of a multimolecular signalling complex involving FcγR leading to SYK, PI3K, p38 and ERK1/2 activation to drive actin remodelling and phagocytosis. Altogether, our data indicate a hitherto unidentified role for TLR4/MSR1 crosstalk in the non-opsonic phagocytosis of C. neoformans.

The "Amoeboid Predator-Fungal Animal Virulence Hypothesis" posits that interactions with environmental phagocytes shape the evolution of virulence traits in fungal pathogens. In this hypothesis, selection to avoid predation by amoeba inadvertently selects for traits that contribute to fungal escape from phagocytic immune cells. Here, we investigate this hypothesis in the human fungal pathogens Cryptococcus neoformans and Cryptococcus deneoformans. Applying quantitative trait locus (QTL) mapping and comparative genomics, we discovered a cross-species QTL region that is responsible for variation in resistance to amoeba predation. In C. neoformans, this same QTL was found to have pleiotropic effects on melanization, an established virulence factor. Through fine mapping and population genomic comparisons, we identified the gene encoding the transcription factor BZP4 that underlies this pleiotropic QTL and we show that decreased expression of this gene reduces melanization and increases susceptibility to amoeba predation. Despite the joint effects of BZP4 on amoeba resistance and melanin production, we find no relationship between BZP4 genotype and escape from macrophages or virulence in murine models of disease. Our findings provide new perspectives on how microbial ecology shapes the genetic architecture of fungal virulence, and suggests the need for more nuanced models for the evolution of pathogenesis that account for the complexities of both microbe-microbe and microbe-host interactions.

Cryptococcus neoformans is an opportunistic fungal pathogen that can cause lethal cryptococcal meningitis in immunocompromised individuals such as those with HIV/AIDS. In addition, cryptococcal infections occasionally arise in immunocompetent individuals or those with previously undiagnosed immunodeficiencies. The course of cryptococcosis is highly variable in both patient groups, and there is rapidly growing evidence that genetic polymorphisms may have a significant impact on the trajectory of disease. Here, we review what is currently known about the nature of these polymorphisms and their impact on host response to C. neoformans infection.Thus far, polymorphisms in Fc gamma receptors, mannose-binding lectin, Dectin-2, Toll-like receptors and macrophage colony-stimulating factor have been associated with susceptibility to cryptococcal disease. Notably, however, in some cases the impact of these polymorphisms depends on the genetic background of the population; for example, the FCGR3A 158 F/V polymorphism was associated with an increased risk of cryptococcal disease in both HIV-positive and HIV-negative white populations, but not in Han Chinese patients. In most cases, the precise mechanism by which the identified polymorphisms influence disease progression remains unclear, although impaired fungal recognition and phagocytosis by innate immune cells appears to play a major role. Finally, we highlight outstanding questions in the field and emphasize the need for future research to include more diverse populations in their genetic association studies.

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Members of Cryptococcus gattii/neoformans species complex are the etiological agents of the potentially fatal human fungal infection cryptococcosis. C. gattii and its sister species cause disease in both immunocompetent and immunocompromised hosts, while the closely related species C. neoformans and C. deneoformans predominantly infect immunocompromised hosts. To date, most studies have focused on similarities in pathogenesis between these two groups, but over recent years, important differences have become apparent. In this review paper, we highlight some of the major phenotypic differences between the C. gattii and neoformans species complexes and justify the need to study the virulence and pathogenicity of the C. gattii species complex as a distinct cryptococcal group.