Cryptococcus neoformans is a facultative intracellular pathogen and its interaction with macrophages is a key event determining the outcome of infection. Urease is a major virulence factor in C. neoformans but its role during macrophage interaction has not been characterized. Consequently, we analyzed the effect of urease on fungal-macrophage interaction using wild-type, urease-deficient and urease-complemented strains of C. neoformans. The frequency of non-lytic exocytosis events was reduced in the absence of urease. Urease-positive C. neoformans manifested reduced and delayed intracellular replication with fewer macrophages displaying phagolysosomal membrane permeabilization. The production of urease was associated with increased phagolysosomal pH, which in turn reduced growth of urease-positive C. neoformans inside macrophages. Interestingly, the ure1 mutant strain grew slower in fungal growth medium which was buffered to neutral pH (pH 7.4). Mice inoculated with macrophages carrying urease-deficient C. neoformans had lower fungal burden in the brain than mice infected with macrophages carrying wild-type strain. In contrast, the absence of urease did not affect survival of yeast when interacting with amoebae. Because of the inability of the urease deletion mutant to grow on urea as a sole nitrogen source, we hypothesize urease plays a nutritional role involved in nitrogen acquisition in the environment. Taken together, our data demonstrate that urease affects fitness within the mammalian phagosome, promoting non-lytic exocytosis while delaying intracellular replication and thus reducing phagolysosomal membrane damage, events that could facilitate cryptococcal dissemination when transported inside macrophages. This system provides an example where an enzyme involved in nutrient acquisition modulates virulence during mammalian infection. Cryptococcus neoformans is a relatively frequent cause of life-threatening infection in severely immunocompromised patients, especially those with AIDS. Persistence of infection involves residence within macrophages, where C. neoformans can survive and replicate while residing in the phagolysosome. New treatments may be developed from a better understanding of how this pathogen resists clearance from and adapts for persistence in host phagocytic cells. In this study, we demonstrate a novel role for urease, a major virulence factor of C. neoformans, in its interaction with macrophages. This enzyme is able to break down urea into ammonia, which is a base, thus raising the surrounding pH. In the context of a mammalian infection, we show that cryptococcal urease increases the phagolysosomal pH which delays yeast replication, therefore causing less damage to macrophages and prolongs intracellular residence. Moreover, urease promotes C. neoformans exit from macrophages without killing the host cells. Overall, our data implies that urease also contributes to virulence by allowing the pathogen to persist and disseminate in macrophages.

Cryptococcus neoformans and Cryptococcus gattii are two species complexes in the large fungal genus Cryptococcus and are responsible for potentially lethal disseminated infections. These two complexes share several phenotypic traits, such as production of the protective compound melanin. In C. neoformans, the pigment associates with key cellular constituents that are essential for melanin deposition within the cell wall. Consequently, melanization is modulated by changes in cell-wall composition or ultrastructure. However, whether similar factors influence melanization in C. gattii is unknown. Herein, we used transmission EM, biochemical assays, and solid-state NMR spectroscopy of representative isolates and "leaky melanin" mutant strains from each species complex to examine the compositional and structural factors governing cell-wall pigment deposition in C. neoformans and C. gattii. The principal findings were the following. 1) C. gattii R265 had an exceptionally high chitosan content compared with C. neoformans H99; a rich chitosan composition promoted homogeneous melanin distribution throughout the cell wall but did not increase the propensity of pigment deposition. 2) Strains from both species manifesting the leaky melanin phenotype had reduced chitosan content, which was compensated for by the production of lipids and other nonpolysaccharide constituents that depended on the species or mutation. 3) Changes in the relative rigidity of cell-wall chitin were associated with aberrant pigment retention, implicating cell-wall flexibility as an independent variable in cryptococcal melanin assembly. Overall, our results indicate that cell-wall composition and molecular architecture are critical factors for the anchoring and arrangement of melanin pigments in both C. neoformans and C. gattii species complexes.

Cryptococcus neoformans is a fungal pathogen with worldwide distribution. C. neoformans resides within mature phagolysosomes where it often evades killing and replicates. C. neoformans induces phagolysosomal membrane permeabilization (PMP), but the mechanism for this phenomenon and its consequences for macrophage viability are unknown. In this study, we used flow cytometry methodology in combination with cell viability markers and LysoTracker to measure PMP in J774.16 and murine bone marrow–derived macrophages infected with C. neoformans. Our results showed that cells manifesting PMP were positive for apoptotic markers, indicating an association between PMP and apoptosis. We investigated the role of phospholipase B1 in C. neoformans induction of PMP. Macrophages infected with a C. neoformans Δplb1 mutant had reduced PMP compared with those infected with wild-type and phospholipase B1–complemented strains, suggesting a mechanism of action for this virulence factor. Capsular enlargement inside macrophages was identified as an additional likely mechanism for phagolysosomal membrane damage. Macrophages undergoing apoptosis did not maintain an acidic phagolysosomal pH. Induction of PMP with ciprofloxacin enhanced macrophages to trigger lytic exocytosis whereas nonlytic exocytosis was common in those without PMP. Our results suggest that modulation of PMP is a critical event in determining the outcome of C. neoformans–macrophage interaction.

Cryptococcus neoformans is a pathogenic yeast capable of a unique and intriguing form of cell-to-cell transfer between macrophage cells. The mechanism for cell-to-cell transfer is not understood. In this study, we imaged mouse macrophages with CellTracker Green 5-chloromethylfluorescein diacetate–labeled cytosol to ascertain whether cytosol was shared between donor and acceptor macrophages. Analysis of several transfer events detected no transfer of cytosol from donor-to-acceptor mouse macrophages. However, blocking Fc and complement receptors resulted in a major diminution of cell-to-cell transfer events. The timing of cell-to-cell transfer (11.17 min) closely approximated the sum of phagocytosis (4.18 min) and exocytosis (6.71 min) times. We propose that macrophage cell-to-cell transfer represents a nonlytic exocytosis event, followed by phagocytosis into a macrophage that is in close proximity, and name this process Dragotcytosis (“Dragot” is a Greek surname meaning “sentinel”), as it represents sharing of a microbe between two sentinel cells of the innate immune system. Key Points C. neoformans transfers between macrophages in coordinated exocytosis/phagocytosis. Opsonin receptors are used for the transfer process. C. neoformans must be viable for the transfer process to occur. Visual Abstract

A pet cockatoo was the suspected source of Cryptococcus neoformans recovered from an immunocompromised patient with cryptococcosis based on molecular analyses available in 2000. Here, we report whole genome sequence analysis of the clinical and cockatoo strains. Both are closely related MATα strains belonging to the VNII lineage, confirming that the human infection likely originated from pet bird exposure. The two strains differ by 61 single nucleotide polymorphisms, including eight nonsynonymous changes involving seven genes. To ascertain whether changes in these genes are selected for during mammalian infection, we passaged the cockatoo strain in mice. Remarkably, isolates obtained from mouse tissue possess a frameshift mutation in one of the seven genes altered in the human sample (LQVO5_000317), a gene predicted to encode an SWI–SNF chromatin-remodeling complex protein. In addition, both cockatoo and patient strains as well as mouse-passaged isolates obtained from brain tissue had a premature stop codon in a homologue of ZFC3 (LQVO5_004463), a predicted single-zinc finger containing protein, which is associated with larger capsules when deleted and reverted to a full-length protein in the mouse-passaged isolates obtained from lung tissue. The patient strain and mouse-passaged isolates show variability in virulence factors, with differences in capsule size, melanization, rates of nonlytic expulsion from macrophages, and amoeba predation resistance. Our results establish that environmental strains undergo genomic and phenotypic changes during mammalian passage, suggesting that animal virulence can be a mechanism for genetic change and that the genomes of clinical isolates may provide a readout of mutations acquired during infection.

Among the best-studied interactions between soil phagocytic predators and a human-pathogenic fungus is that of Acanthamoeba castellanii and Cryptococcus neoformans . The experimental conditions used in amoeba-fungus confrontation assays can have major effects on whether the fungus or the protozoan is ascendant in the interaction. In the presence of Mg ²⁺ and Ca ²⁺ in phosphate-buffered saline (PBS), C. neoformans was consistently killed when incubated with A. castellanii . A. castellanii survived better in the presence of Mg ²⁺ and Ca ²⁺ , even when incubated with C. neoformans . In the absence of Mg ²⁺ and Ca ²⁺ , C. neoformans survived when incubated with A. castellanii , and the percentage of dead amoebae was higher than when incubated without yeast cells. These results show that the presence of Mg ²⁺ and Ca ²⁺ can make a decisive contribution toward tilting the outcome of the interaction in favor of the amoeba. Of the two metals, Mg ²⁺ had a stronger effect than Ca ²⁺ . The cations enhanced A. castellanii activity against C. neoformans via enhanced phagocytosis, which is the major mechanism by which amoebae kill fungal cells. We found no evidence that amoebae use extracellular killing mechanisms in their interactions with C. neoformans . In summary, the presence of Mg ²⁺ and Ca ²⁺ enhanced the cell adhesion on the surfaces and the motility of the amoeba, thus increasing the chance for contact with C. neoformans and the frequency of phagocytosis. Our findings imply that the divalent cation concentration in soils could be an important variable for whether amoebae can control C. neoformans in the environment. IMPORTANCE The grazing of soil organisms by phagocytic predators such as amoebae is thought to select for traits that enable some of them to acquire the capacity for virulence in animals. Consequently, knowledge about the interactions between amoebae and soil microbes, such as pathogenic fungi, is important for understanding how virulence can emerge. We show that the interaction between an amoeba and the pathogenic fungus C. neoformans is influenced by the presence in the assay of magnesium and calcium, which potentiate amoebae. The results may also have practical applications, since enriching soils with divalent cations may reduce C. neoformans numbers in contaminated soils.