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Abstract Background The CryptoDex trial showed that dexamethasone caused poorer clinical outcomes and slowed fungal clearance in human immunodeficiency virus–associated cryptococcal meningitis. We analyzed cerebrospinal fluid (CSF) cytokine concentrations from participants over the first week of treatment to investigate mechanisms of harm and test 2 hypotheses: (1) dexamethasone reduced proinflammatory cytokine concentrations, leading to poorer outcomes and (2) leukotriene A4 hydrolase (LTA4H) genotype influenced the clinical impact of dexamethasone, as observed in tuberculous meningitis. Methods We included participants from Vietnam, Thailand, and Uganda. Using the Luminex system, we measured CSF concentrations of the following: interferon γ, tumor necrosis factor (TNF) α, granulocyte-macrophage colony-stimulating factor, monocyte chemoattractant 1, macrophage inflammatory protein 1α, and interleukin 6, 12p70, 8, 4, 10, and 17. We determined the LTA4H genotype based on the promoter region single-nucleotide polymorphism rs17525495. We assessed the impact of dexamethasone on cytokine concentration dynamics and the association between cytokine concentration dynamics and fungal clearance with mixed effect models. We measured the influence of LTA4H genotype on outcomes with Cox regression models. Results Dexamethasone increased the rate TNF-α concentration’s decline in (−0.13 log2pg/mL/d (95% confidence interval, −.22 to −.06 log2pg/mL/d; P = .03), which was associated with slower fungal clearance (correlation, −0.62; 95% confidence interval, −.83 to −.26). LTA4H genotype had no statistically significant impact on outcome or response to dexamethasone therapy. Better clinical outcomes were associated with higher baseline concentrations of interferon γ. Conclusions Dexamethasone may slow fungal clearance and worsen outcomes by increasing TNF-α concentration’s rate of decline. We investigated clinical and microbiological impacts of longitudinal cerebrospinal fluid cytokine concentrations in human immunodeficiency virus–associated cryptococcal meningitis, and interactions with dexamethasone therapy or leukotriene A4 hydrolase genotype. We found that faster declines in tumor necrosis factor α were associated with reduced fungal clearance.

Alternative transcription start site (TSS) usage regulation has been identified as a major means of gene expression regulation in metazoans. However, in fungi, its impact remains elusive as its study has thus far been restricted to model yeasts. Here, we first re-analyzed TSS-seq data to define genuine TSS clusters in 2 species of pathogenic Cryptococcu s. We identified 2 types of TSS clusters associated with specific DNA sequence motifs. Our analysis also revealed that alternative TSS usage regulation in response to environmental cues is widespread in Cryptococcus , altering gene expression and protein targeting. Importantly, we performed a forward genetic screen to identify a unique transcription factor (TF) named Tur1, which regulates alternative TSS (altTSS) usage genome-wide when cells switch from exponential phase to stationary phase. ChiP-Seq and DamID-Seq analyses suggest that at some loci, the role of Tur1 might be direct. Tur1 has been previously shown to be essential for virulence in C . neoformans . We demonstrated here that a tur1Δ mutant strain is more sensitive to superoxide stress and phagocytosed more efficiently by macrophages than the wild-type (WT) strain.

Dissemination from one organ system to another is common to many pathogens and often the key process separating simple illness from fatal infection. The pathogenic species offer a prime example. Cryptococcal infection is thought to begin in the lungs, as a mild or asymptomatic pneumonia. However, bloodborne dissemination from the lungs to the brain is responsible for the most devastating forms of infection. As with other disseminating infections, the transition likely depends on rare but crucial events, such as the crossing of a tissue barrier. By their nature, these events are difficult to study. Francis et al. (mBio 15:e03078-23, 2024, https://doi.org/10.1128/mbio.03078-23) have addressed this difficulty by developing a powerful imaging pipeline to scan through unprecedented volumes of tissue from mice infected with at multiple stages of infection. Their observations challenge some of our basic assumptions about cryptococcal pathogenesis, including when and how the organism reaches the bloodstream and the central nervous system.

Cryptococcus neoformans causes fatal fungal meningoencephalitis. Here, we study the roles played by fungal kinases and transcription factors (TFs) in blood-brain barrier (BBB) crossing and brain infection in mice. We use a brain infectivity assay to screen signature-tagged mutagenesis (STM)-based libraries of mutants defective in kinases and TFs, generated in the C. neoformans H99 strain. We also monitor in vivo transcription profiles of kinases and TFs during host infection using NanoString technology. These analyses identify signalling components involved in BBB adhesion and crossing, or survival in the brain parenchyma. The TFs Pdr802, Hob1, and Sre1 are required for infection under all the conditions tested here. Hob1 controls the expression of several factors involved in brain infection, including inositol transporters, a metalloprotease, PDR802 , and SRE1 . However, Hob1 is dispensable for most cellular functions in Cryptococcus deuterogattii R265, a strain that does not target the brain during infection. Our results indicate that Hob1 is a master regulator of brain infectivity in C. neoformans . Cryptococcus neoformans causes fatal fungal meningoencephalitis. Here, the authors identify fungal kinases and transcription factors involved in blood-brain barrier crossing and brain infection in mice.

Courtship is pivotal for successful mating. However, courtship is challenging for the Cryptococcus neoformans species complex, comprised of opportunistic fungal pathogens, as the majority of isolates are α mating type. In the absence of mating partners of the opposite mating type, C. deneoformans can undergo unisexual reproduction, during which a yeast-to-hyphal morphological transition occurs. Hyphal growth during unisexual reproduction is a quantitative trait, which reflects a strain's ability to undergo unisexual reproduction. In this study, we determined whether unisexual reproduction confers an ecological benefit by promoting foraging for mating partners. Through competitive mating assays using strains with different abilities to produce hyphae, we showed that unisexual reproduction potential did not enhance competition for mating partners of the same mating type, but when cells of the opposite mating type were present, cells with enhanced hyphal growth were more competitive for mating partners of either the same or opposite mating type. Enhanced mating competition was also observed in a strain with increased hyphal production that lacks the mating repressor gene GPA3, which contributes to the pheromone response. Hyphal growth in unisexual strains also enables contact between adjacent colonies and enhances mating efficiency during mating confrontation assays. The pheromone response pathway activation positively correlated with unisexual reproduction hyphal growth during bisexual mating and exogenous pheromone promoted bisexual cell fusion. Despite the benefit in competing for mating partners, unisexual reproduction conferred a fitness cost. Taken together, these findings suggest C. deneoformans employs hyphal growth to facilitate contact between colonies at long distances and utilizes pheromone sensing to enhance mating competition.

Summary Half a century after the introduction of Amphotericin B the management of cryptococcosis remains unsatisfactory. The disease, caused primarily by the two fungal species Cryptococcus neoformans and Cryptococcus gattii, remains responsible for considerable morbidity and mortality despite standard medical care. Current therapeutic options are limited to Amphotericin B, azoles and 5‐flucytosine. However, this organism has numerous well‐characterized virulence mechanisms that are amenable to pharmacological interference and are thus potential therapeutic targets. Here, we discuss existing approved antifungal drugs, resistance mechanisms to these drugs and non‐standard antifungal drugs that have potential in treatment of cryptococcosis, including immunomodulatory strategies that synergize with antifungal drugs, such as cytokine administration or monoclonal antibodies. Finally, we summarize attempts to target well‐described virulence factors of Cryptococcus, the capsule or fungal melanin. This review emphasizes the pressing need for new therapeutic alternatives for cryptococcosis.

Cryptococcosis is a major opportunistic infection and is one of the leading causes of death in adults living with HIV in sub-Saharan Africa. Recent estimates indicate that more than 130,000 people may die annually of cryptococcal meningitis in this region. Although complete diagnostic autopsy (CDA) is considered the gold standard for determining the cause of death, it is seldom performed in low income settings. In this study, a CDA was performed in 284 deceased patients from Mozambique (n = 223) and Brazil (n = 61). In depth histopathological and microbiological analyses were carried out in all cases dying of cryptococcosis. We determined the cryptococcal species, the molecular and sero-mating types and antifungal susceptibility. We also described the organs affected and reviewed the clinical presentation and patient management. Among the 284 cases included, 17 fatal cryptococcal infections were diagnosed. Cryptococcus was responsible for 16 deaths among the 163 HIV-positive patients (10%; 95%CI: 6–15%), including four maternal deaths. One third of the cases corresponded to C. gattii (VGI and VGIV molecular types, Bα and Cα strains) and the remaining infections typed were caused by C. neoformans var. Grubii (all VNI and Aα strains). The level of pre-mortem clinical suspicion was low (7/17, 41%), and 7/17 patients (41%) died within the first 72 hours of admission. Cryptococcosis was responsible for a significant proportion of AIDS-related mortality. The clinical diagnosis and patient management were inadequate, supporting the need for cryptococcal screening for early detection of the disease. This is the first report of the presence of C. gattii infection in Mozambique.

Key Points Cryptococcosis is a widespread opportunistic fungal infection of humans and other animals. Cryptococcus species that infect humans likely evolved as accidental pathogens in response to environmental selective pressure. Recent genomic analyses have highlighted the evolutionary history of Cryptococcus spp. and narrowed down the geographical origin of an unusual, hypervirulent outbreak. Despite being accidental pathogens, cryptococci display a remarkable ability to manipulate the human immune response to facilitate disease establishment and spread. Detailed in vivo and in vitro characterization of Cryptococcus spp. has started to elucidate the details of multiple mechanisms of pathogenesis that probably have important roles in disease severity. These include changes in fungal morphology, interactions with host phagocytes and mechanisms that allow Cryptococcus spp. to disseminate from the lung to the central nervous system. Renewed efforts to develop improved therapeutic approaches have highlighted potential new drugs and new uses for old drugs in the fight against cryptococcal disease. Recent studies have elucidated multiple virulence mechanisms used by Cryptococcus spp. to infect, disseminate within and ultimately kill their human host. In this Review, May et al . describe these recent developments in understanding host–fungal interactions, discuss how they affect disease severity and debate current and future therapeutic interventions against cryptococcosis. Cryptococcosis is a globally distributed invasive fungal infection that is caused by species within the genus Cryptococcus which presents substantial therapeutic challenges. Although natural human-to-human transmission has never been observed, recent work has identified multiple virulence mechanisms that enable cryptococci to infect, disseminate within and ultimately kill their human host. In this Review, we describe these recent discoveries that illustrate the intricacy of host–pathogen interactions and reveal new details about the host immune responses that either help to protect against disease or increase host susceptibility. In addition, we discuss how this improved understanding of both the host and the pathogen informs potential new avenues for therapeutic development.

In humans, and species complexes are the leading cause of fungal meningitis globally. To establish CNS infection, must breach the BBB, primarily comprised of specialized brain microvascular endothelial cells (BMECs), which is the prerequisite for cryptococci to invade the brain. Despite its clinical impact, the mechanisms underlying host-pathogen interaction at the BBB particularly involving environmental isolates remain under-characterized. This study aimed to investigate the cyto-morphological and transcriptomic responses of HBMECs to infection by clinical and environmental isolates using a dual approach-ultrastructural electron microscopy and high-throughput dual RNA-Seq. HBMECs were infected with molecularly typed clinical and environmental isolates of and at two infection time points (4 hpi and 18 hpi). Transmission electron microscopy was used to visualize host cell ultrastructural alterations, while dual RNA-Seq was performed to assess differential gene expression in both host and pathogen. TEM revealed extensive ultrastructural changes in infected HBMECs, including membrane ruffling, increased microvilli, mitochondrial alterations, ER dilation, Golgi fragmentation, nuclear deformation, and autophagosome formation. Transcriptomic profiling demonstrated functional enrichment of several critical cryptococcal virulence-associated genes linked to immune evasion and stress adaptation including various immune signaling pathways elicited by the HBMECs as a counter measure to the cryptococcal invasion. Clinical and environmental isolates exhibit comparable invasive potential and elicit similar host endothelial responses with consistent effects observed across all isolates and time points. This integrative study combining ultrastructural and transcriptomic analyses highlights conserved host-pathogen interactions at the BBB, identifies potential molecular targets for antifungal therapy and underscores the pathogenic relevance of environmental reservoirs in cryptococcal meningitis. cryptococcal meningitis, blood-brain barrier, invasion, transmission electron microscopy, Dual RNASeq, differential gene expression, host-pathogen interaction, HBMECs, ultrastructural alterations.

Cryptococcosis has become a major global health problem since the advent of the HIV pandemic in 1980s. Although its molecular epidemiology is well-defined, using isolates recovered since then, no pre-HIV-pandemic era epidemiological data exist. We conducted a molecular epidemiological study using 228 isolates of the C. neoformans/C. gattii species complexes isolated before 1975. Genotypes were determined by URA5 restriction fragment length polymorphism analysis and multi-locus sequence typing. Population genetics were defined by nucleotide diversity measurements, neutrality tests, and recombination analysis. Growth at 37°C, melanin synthesis, capsule production, and urease activity as virulence factors were quantified. The pre-HIV-pandemic isolates consisted of 186 (81.5%) clinical, 35 (15.4%) environmental, and 7 (3.1%) veterinary isolates. Of those, 204 (89.5%) belonged to C. neoformans VNI (64.0%), VNII (14.9%) and VNIV (10.5%) while 24 (10.5%) belonged to C. gattii VGIII (7.5%), VGI (2.6%) and VGII (0.5%). Among the 47 sequence types (STs) identified, one of VNII and 8 of VNIV were novel. ST5/VNI (23.0%) in C. neoformans and ST75/VGIII (25.0%) in C. gattii were the most common STs in both species complexes. Among C. neoformans, VNIV had the highest genetic diversity (Hd = 0.926) and the minimum recombination events (Rm = 10), and clinical isolates had less genetic diversity (Hd = 0.866) than environmental (Hd = 0.889) and veterinary isolates (Hd = 0.900). Among C. gattii, VGI had a higher nucleotide diversity (π = 0.01436) than in VGIII (π = 0.00328). The high-virulence genotypes (ST5/VNI and VGIIIa/serotype B) did not produce higher virulence factors levels than other genotypes. Overall, high genetic variability and recombination rates were found for the pre-HIV-pandemic era among strains of the C. neoformans/C. gattii species complexes. Whole genome analysis and in vivo virulence studies would clarify the evolution of the genetic diversity and/or virulence of isolates of the C. neoformans/C. gattii species complexes during the pre- and post-HIV-pandemic eras.