Keigo Ueno

The fungal pathogen Cryptococcus neoformans contains about 200 µg of β-1,3-glucan (1,3BG) per 1 mg of dry cell weight when grown under standard culture conditions (YPD medium at 30 °C under aerobic conditions). However, 1,3BG exposure is tightly suppressed, even in capsule-deficient strains, allowing the fungus to evade recognition by the immune receptor dectin-1 and anti-1,3BG antibodies. Although other pathogenic fungi mask 1,3BG with α-1,3-glucan (1,3AG) to evade dectin-1 recognition, the factors responsible for 1,3BG masking and dectin-1 evasion in C. neoformans remain incompletely understood. To identify capsule-independent 1,3BG masking and dectin-1-evasion factors, we generated a series of cell wall-related gene deletion strains in the capsule-deficient strain cap59 Δ by using CRISPR-Cas9 and screened for mutants that failed to evade dectin-1 binding. We found eight deletants ( cap59 Δ/ mpk1 Δ, cap59 Δ/ chs3 Δ, cap59 Δ/ kre5 Δ, cap59 Δ/ crz1 Δ, cap59 Δ/ kre6 Δ/ skn1 Δ, cap59 Δ/ hxl1 Δ, cap59 Δ/ uge1 Δ, and cap59 Δ/ ugt1 Δ) that exhibited increased binding to dectin-1 or anti-1,3BG antibody, or both. As a similar phenotype was not observed in cap59 Δ/ ags1 Δ, 1,3AG-mediated masking of 1,3BG appears to play a limited role in C. neoformans . These eight deletants induced significantly greater secretion of IL-6 and IL-1β from dendritic cells (DCs) than did cap59 Δ. This enhanced inflammatory response was markedly attenuated in dectin-1-deficient DCs, indicating that the increased immunogenicity was driven by 1,3BG exposure and subsequent dectin-1 recognition. Collectively, these findings demonstrate that multiple genes involved in maintaining cell wall integrity—including those involved in β-1,6-glucan and chitosan biosynthesis—are essential for regulating 1,3BG exposure and enabling C. neoformans to evade dectin-1-mediated immune recognition. Highlights: We identified new capsule-independent β-1,3-glucan-masking genes in C. neoformans Gene deletants had higher dectin-1 deposition than parental acapsular cap59 Δ Deletion of these genes enhanced IL-6 and IL-1β secretion by dendritic cells The enhanced cytokine response was suppressed in dendritic cells lacking dectin-1. Deletant strains may serve as new whole-cell antigens for cryptococcal vaccines

Cryptococcus neoformans is the primary causative agent of cryptococcosis. Since C. neoformans thrives in environments and its optimal growth temperature is 25–30°C, it needs to adapt to heat stress in order to cause infection in mammalian hosts. In this study, we aimed to investigate the role of an uncharacterized gene, CNAG_03308. Although the CNAG_03308 deletion strain grew as well as the parent strain KN99, it produced yeast cells with abnormal morphology at 37°C and failed to propagate at 39°C. Furthermore, the deletion strain exhibited slower growth at 37°C in the presence of congo red, which is a cell wall stressor. When cultured at 39°C, the deletion strain showed strong staining with fluorescent probes for cell wall chitin and chitosan, including FITC-labeled wheat germ agglutinin, Eosin Y, and calcofluor white. The transmission electron microscopy of the deletion strain revealed a thickened inner layer of the cell wall containing chitin and chitosan under heat stress. This cell-surface altered deletion strain induced dendritic cells to secrete more interleukin (IL)-6 and IL-23 than the control strains under heat stress. In a murine infection study, C57BL/6 mice infected with the deletion strain exhibited lower mortality and lower fungal burden in the lungs and brain compared to those infected with the control strains. Based on these findings, we concluded that CNAG_03308 gene is necessary for C. neoformans to adapt to heat stress both in vitro and in the host environment. Therefore, we designated the CNAG_03308 gene as TVF1, which stands for thermotolerance and virulence-related factor 1. Cryptococcus neoformans is a fungal pathogen causing cryptococcosis, which requires thermotolerance to proliferate in the host environment. In the present study, we identified a novel gene, TVF1 (CNAG_03308), required for thermotolerance and virulence by reverse genetics approach.

Cryptococcosis is a mycosis caused by Cryptococcus neoformans and C. gattii species complexes. Although this infection is potentially lethal, no prophylactic vaccine is yet commercially available, and the immune memory that enables prevention is still under investigation. These pathogens have a capsule layer for immune evasion and a sophisticated mechanism to advance the infection, and it is expected that these characteristics will make it difficult to develop prophylactic vaccines and to decipher the protective immunity. The current vaccine studies are focused on subunit, mRNA, DNA, and viral vector vaccines, with whole‐cell vaccines also proving successful against cryptococcal infections. Cryptococcal whole‐cell vaccines have been composed of highly immunostimulating strains with low‐pathogenicity that are modified by genetic recombination technology. Examples include the whole‐cell vaccines H99γ, sgl1∆, fbp1∆, znf2^oe, cda1/2/3∆, cap59∆, and cap60∆. Some of these whole‐cell vaccines were found to be highly effective in prolonging life and suppressing the fungal burden after an infection challenge in mice, and to be cross‐reactive to C. neoformans, C. gattii, and other fungal pathogens. Furthermore, for some vaccines, the protective effect can be retained even in an immunocompromised host depleted of CD4⁺ T cells. These findings have provided new insights into protective immunity that should aid in vaccine development. In this review, we highlight the upsides and downsides of whole‐cell vaccines against cryptococcosis.

Cryptococcus gattii is a capsular pathogenic fungus causing life‐threatening cryptococcosis. Although the capsular polysaccharides (CPs) of C. gattii are considered as virulence factors, the physiological significance of CP biosynthesis and of CPs themselves is not fully understood, with many conflicting data reported. First, we demonstrated that CAP gene deletant of C. gattii completely lacked capsule layer and its virulence, and that the strain was susceptible to host‐related factors including oxidizing, hypoxic, and hypotrophic conditions in vitro. Extracellular CPs recovered from culture supernatant bound specifically to C. gattii acapsular strains, not to other fungi and immune cells, and rendered them the immune escape effects. In fact, dendritic cells (DCs) did not efficiently uptake the CP‐treated acapsular strains, which possessed no visible capsule layer, and a decreased amount of phosphorylated proteins and cytokine levels after the stimulation. DCs recognized C. gattii acapuslar cells via an immune receptor CD11b‐ and Syk‐related pathway; however, CD11b did not bind to CP‐treated acapsular cells. These results suggested that CPs support immune evasion by coating antigens on C. gattii and blocking the interaction between CD11b and C. gattii cells. Here, we describe the importance of CPs in pathogenicity and immune evasion mechanisms of C. gattii.

Cryptococcosis is a potentially lethal disease caused by fungal pathogens including Cryptococcus neoformans and Cryptococcus gattii species complex. These fungal pathogens live in the environment and are associated with certain tree species and bird droppings. This infectious disease is not contagious, and healthy individuals may contract cryptococcal infections by inhaling the airborne pathogens from the environment. Although cleaning a contaminated environment is a feasible approach to control environmental fungal pathogens, prophylactic immunization is also considered a promising method to regulate cryptococcal infections. We review the history of the development of cryptococcal vaccines, vaccine components, and the various forms of immune memory induced by cryptococcal vaccines.