Cryptococcus neoformans is the leading cause of fungal meningitis with limited treatment options, making early and accurate diagnosis critical for improved patient outcomes. Current diagnostic methods for cryptococcosis rely largely on capsule antigen detection and fungal culture, which are time-consuming and unable to identify mutation-driven antifungal resistance. In this study, we have developed a SuperSelective primer-based PCR (SSP-PCR) platform for the rapid and specific detection of azole resistance-associated single-nucleotide polymorphisms (SNPs). We demonstrate that SSP-PCR reliably detected a single copy of mutant allele in the presence of excess wild-type (WT) DNA, with sensitivity reaching a 1:10[4] mutant-to-WT ratio. Incorporating molecular beacon (MB) probes into our SSP-PCR platform further enhanced amplification specificity, enabling selective detection of the ERG11 Y145F (A434T) mutation, which is known to confer high azole resistance. Using genomic DNA from in vitro cultures and mouse lung tissues infected with either WT strain H99 or a fluconazole-hyper-resistant mrl1 clinical isolate that carries the ERG11(A434T) mutation, or both strains, we successfully detected the A434T mutant allele in both settings. Moreover, our SSP-PCR simultaneously identified ERG11(A434T) and the multi-azole resistance-associated ERG11(G1885A) mutant alleles in a single-tube duplex reaction. Collectively, this is the first study that utilizes the SSP-PCR platform to provide a robust and ultrasensitive molecular approach for the detection of azole resistance in C. neoformans, which provides a proof-of-concept methodology for its potential high-throughput clinical screening applications.

Cryptococcus neoformans is an opportunistic fungal pathogen that causes pulmonary infection in immunocompromised patients, which in severe cases leads to fatal meningoencephalitis. Cryptococcus exhibits unique glycobiology that plays important roles in pathogenesis. Unlike model yeast and other common fungal pathogens, Cryptococcus incorporates xylose, a five-carbon monosaccharide, into its glycans. One trimer motif, which consists of xylose in β-1,2 linkage to the reducing mannose of an α-1,3-mannose dimer, occurs in key cryptococcal glycoconjugates that include protein N- and O-linked glycans, glycosylinositol phosphorylceramides (GIPCs), and the capsule polysaccharides glucuronoxylomannan (GXM) and glucuronoxylomannogalactan (GXMGal). We previously identified cryptococcal β-1,2-xylosyltransferase 1 (Cxt1), which catalyzes the formation of this motif in GIPCs, GXM, and GXMGal. Here, we report the discovery of a second enzyme, cryptococcal β-1,2-xylosyltransferase 2 (Cxt2). Through characterization of cells that lack one or both corresponding genes (CXT1 and CXT2), we have dissected the biological roles of these enzymes, which are overlapping but not identical. Notably, Cxt1 and Cxt2 colocalize in the Golgi, influence the capsule in a strain-dependent manner, and together are responsible for all xylose addition to O-glycans. Overall, our work highlights the unique roles of these two enzymes and fills a gap in our understanding of cryptococcal glycan synthesis.IMPORTANCECryptococcus neoformans is an opportunistic fungal pathogen that causes almost 150,000 deaths each year worldwide. Cryptococcus synthesizes unique glycan structures that play important roles in its biology and pathogenesis. One abundant component of these structures is xylose, a five-carbon monosaccharide. Because xylose is not used by many fungal organisms, including model yeast, we have limited information about how cells add it to their glycans. Here we report a xylosyltransferase enzyme that performs this function, and we characterize specific biological roles of this protein and a closely related one we discovered earlier. We find that these proteins together perform all detectable xylose addition to an important class of protein-linked glycans (O-glycans). They also both participate in other synthetic processes, although this varies with the specific enzyme and strain background. These results contribute to our understanding of cryptococcal glycan synthesis and underscore the importance of testing multiple background strains.

Antibiotic resistance of microorganisms threatens global health and agriculture. Antimicrobial peptides can display broad-spectrum activity, making them candidate alternatives to antibiotics. Here, ten antimicrobial peptides were tested against fungal pathogens, including Candida albicans, Cryptococcus neoformans, Aspergillus niger, and Gram-negative (Escherichia coli and Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus) pathogens. Aurein 1.2, halictine-II, and macropin-I showed broad-spectrum activity. In particular, halictine-II showed high potency combined with low hemolytic activity, with the MIC values across species being 5 to 90-fold lower than the observed hemolytic concentration.

Cryptococcosis often begins in the lungs and may later reach the central nervous system, where it can lead to severe or fatal disease. The course of infection is shaped by changing interactions between host cells, fungal adaptive states, and local tissue environments. Many virulence factors and immune responses have been studied in detail, but the evidence remains divided across organs, cell types, and experimental systems. As a result, pulmonary infection is better understood than later stages, including dissemination, crossing of the blood-brain barrier, CNS involvement, and long-term persistence. In this review, we use a stage-resolved phenomic framework to connect host cell states, fungal adaptive phenotypes, and tissue niches throughout the course of infection. Evidence from multi-omics studies, spatial analyses, cellular experiments, animal models, and clinical cohorts is brought together to show how phenotypic features differ between stages and how uneven the current evidence base remains. The strongest evidence remains concentrated in pulmonary infection, whereas dissemination, crossing of the blood-brain barrier, and CNS disease are supported by more model-dependent data. Evidence for persistence and reactivation is the most limited. By organizing these findings into a disease-course map, this review outlines testable questions for biomarker discovery, host-directed therapy, and risk stratification in cryptococcal infection.

Invasive fungal infections can be life-threatening, particularly in patients with impaired immunity, yet treatment remains limited to a few antifungal drug classes. During infection, fungal pathogens encounter changes in temperature, nutrient availability, immune pressure and drug exposure that challenge their growth and survival. tRNA modifications may contribute to adaptation by supporting tRNA stability, aminoacylation and codon decoding. Studies in Aspergillus fumigatus, Candida albicans and Cryptococcus neoformans have linked conserved tRNA-modification pathways to fungal development, host interaction, stress adaptation, virulence and the response to 5-fluorocytosine, with evidence ranging from genetic and infection phenotypes to biochemical characterization of modification reactions. In this Review, we examine how these studies connect modification chemistry and substrate tRNAs with translation, protein output and infection phenotypes. We also draw on mechanistic studies from model organisms and plant-pathogenic fungi to clarify translational links that remain unresolved in human fungal pathogens. We conclude by discussing when tRNA-modification pathways become functionally limiting under host-associated conditions and how they may alter antifungal responses.

Extensive clinical studies have convincingly demonstrated that fungal infections constitute a mounting global health concern. Candida albicans, C. auris, Aspergillus fumigatus, and Cryptococcus neoformans were classified by the WHO as critical priority fungi due to their widespread prevalence, high mortality rates associated with invasive mycoses, and the increasing spread of resistant strains. The currently available antifungal arsenal, comprising four major classes (azoles, polyenes, echinocandins, and flucytosine), is critically insufficient. Resistance to these agents is escalating worldwide, while the approval of new drugs remains alarmingly slow. This review provides a comprehensive overview of the molecular mechanisms underlying resistance to conventional and emerging antimycotics in critical priority fungi, including recently approved and investigational agents such as oteseconazole, rezafungin, ibrexafungerp, fosmanogepix, and olorofim. It examines current strategies to overcome resistance, including combination therapy, drug repurposing, targeted delivery, antibiofilm approaches, immunotherapy, and the application of natural antifungal compounds. Special emphasis is placed on antifungal peptides (AFPs) derived from plants, animals, bacteria, and fungi, along with their synthetic analogs, all of which have demonstrated efficacy against critical priority fungi. The review primarily focuses on the molecular targets of AFPs and on the development of fungal resistance under prolonged exposure, compared with conventional antifungals, in order to evaluate their potential as prototypes for next-generation antimycotics.

All pathogens rely on dynamic gene expression for host adaptation and disease causation. This is especially important for pathogens that normally are found in the environment, like Cryptococcus neoformans, given that environmental and host conditions are dramatically different. Epigenetic modifications, like those carried out by SET-domain histone lysine methyltransferases (HKMTs), are essential for this genome expression flexibility. While transcription factors and signaling cascades have been well-studied in C. neoformans's host adaptation, the role of HKMTs in this process has not been explored. Here, we characterize the gene SET202, previously reported as a putative HKMT important for growth in host lungs. We show that set202Δ mutants have 50% less phagosomal permeabilization than the wild-type fungi, have phagosomal maturation defects, and poor intracellular survival. Additionally, they are defective in traits promoting virulence, including significantly smaller capsules, less melanin, and poor growth under host-like conditions. These phenotypes contributed to reduced virulence in a murine model of infection but, notably, this defect was host-temperature-dependent. In order to explain mechanistically how SET202 deletion could affect such a wide range of processes, we assessed and demonstrated, for the first time, that Set202 is a true histone-3-lysine 36 methyltransferase, supporting its role in epigenetically modifying the fungal genome to enable adaptation to the hostile host conditions. Consequently, our findings support the study of the biological function of Set202, as it can serve as a potential future therapeutic target for the treatment of infections caused by C. neoformans, and possibly other pathogenic fungi.

RNA interference (RNAi) is a widely conserved genome-defense mechanism that protects eukaryotes against viruses and transposable elements. We previously showed that RNAi loss can lead to hypermutation and antifungal drug resistance in the human fungal pathogen Cryptococcus neoformans, illuminating the potential clinical relevance of this pathway. In this study, we identified another function of RNAi in C. neoformans: mycovirus restriction. By screening known RNAi-deficient C. neoformans isolates, we identified a novel dsRNA mycovirus of the Orthototiviridae family, which we named CnTV1. We subsequently detected CnTV1 in three additional RNAi-deficient isolates. All CnTV1-positive isolates shared an ancestral nuclear mitochondrial DNA segment (NUMT) insertion that disrupts the gene encoding Argonaute (Ago1). Restoration of RNAi in sexually produced zygotes efficiently eliminated CnTV1. RNAi rescue by CRISPR-Cas9-mediated allele exchange eliminated CnTV1 during vegetative growth, further demonstrating RNAi is sufficient for mycoviral control. Loss of the RNA helicase Ski2 or the exoribonuclease Xrn1 increased CnTV1 abundance, revealing RNAi-independent restriction of the virus. Restoration of RNAi cleared the virus even in the absence of Ski2 or Xrn1, indicating a dominant role for RNAi in antiviral defense. To investigate the biological implications of CnTV1 infection, we developed a cytoplasmic-mixing approach and generated isogenic strain pairs in an RNAi-deficient background that differ only in viral infection status. Leveraging these strains, we show that CnTV1 infection leads to coordinated, low-magnitude transcriptomic changes and that strains lacking the mycovirus were moderately less virulent in a murine infection model. Our findings reveal that RNAi serves as a dominant antiviral defense system in C. neoformans and suggest that naturally occurring RNAi deficiency may be more prevalent than previously appreciated. This work highlights mycovirus persistence as an important consequence of RNAi loss in this WHO-designated critical priority fungal pathogen.

Infections due to Cryptococcus species are estimated to cause over 100,000 deaths a year. No cryptococcal vaccines are approved for human use. We have shown that vaccine formulations consisting of C. neoformans chitin deacetylase (Cda)2 and Cda1 adjuvanted with Cationic Adjuvant Formulation 01 (CAF01) protect mice against experimental cryptococcosis by a mechanism dependent upon CD4 [+] T cells. Here, using overlapping peptide libraries, we mapped epitopes in Cda2 and Cda1 that stimulated interferon-gamma (IFNγ) release from splenocytes of C57BL/6 and DR4 mice immunized with these proteins. DR4 mice lack mouse major histocompatibility class II (MHC-II) proteins and express chimeric MHC-II proteins with the specificity of the human HLA-DR allele, HLA-DRB1*04:01. Experimental results were then compared with MHC-II binding predictions using the Immune Epitope Database (IEDB) NetMHCIIpan 4.1 BA MHC-II epitope prediction tool. Unique epitopes in Cda2 and Cda1 were discovered for each mouse strain, some of which were not predicted to bind well to MHC-II. CD4 [+] T cells were responsible for cytokine release as IFNγ production was lost if CD4 [+] T cells were depleted from the splenocyte population. Finally, we tested a hybrid recombinant protein that consisted of portions of Cda2 and Cda1 containing the MHC-II/CD4 [+] T cell epitopes which induce CD4 [+] T cells in DR4 mice. When administered as a CAF01-adjuvanted vaccine, the hybrid protein protected DR4 mice from an otherwise lethal C. neoformans pulmonary challenge. These results provide a proof of principle regarding the utility of MHC-II/CD4 [+] T cell epitope mapping in cryptococcal vaccine development.

Mononuclear phagocytes are widely distributed throughout the body and interact with other cellular systems to preserve homeostasis and health. Recent studies have revealed the remarkable plasticity and adaptability of their cellular recognition and trophic responses beyond phagocytosis and immunity. We invited several early-career investigators to review the nature and interactions of selected Mononuclear Phagocyte populations within established organ systems, apart from lympho-haematopoietic tissues. This special issue indicates that the Mononuclear Phagocyte System (MPS) can be considered on a par with Neuro-Endocrine and other physiologic organ systems in the mouse and human. As a guide to the context of individual reviews, we provide a concise overview of the field and suggestions for further reading and research.

Cryptococcosis is a severe systemic mycosis, and relapse may be influenced by fungal subpopulations that survive fluconazole exposure. This retrospective cohort study investigated whether fluconazole minimum inhibitory concentration (MIC) and the HetR phenotype of the first available diagnostic cerebrospinal-fluid isolate were associated with recorded relapse within six months in 70 patients with neurocryptococcosis from Amazonas, Brazil. One clinical isolate per patient, comprising 64 Cryptococcus neoformans VNI and six Cryptococcus gattii VGII isolates, was evaluated by broth microdilution and endpoint-based HFL testing. Using an operational criterion of HFL/MIC ≥8, corresponding to visible growth at least three twofold dilutions above the MIC, 43 of 70 isolates (61.4%) were classified as displaying a HetR phenotype. Six-month follow-up was available for 69 patients. Recorded relapse occurred in 12 of 42 patients infected with HetR isolates (28.6%) and in 4 of 27 patients infected with non-HetR isolates (14.8%). Although relapse was numerically more frequent in the HetR group, the association was not statistically significant (conditional odds ratio, 2.27; exact 95% confidence interval, 0.59-10.96; two-sided Fisher's exact test, p=0.248). The HFL/MIC ratio also did not differ significantly according to recorded relapse status (p=0.165). The HetR phenotype of the first available diagnostic isolate, defined operationally in this study as HFL/MIC ≥8, was frequent but was not significantly associated with recorded relapse within six months in this cohort.

Hyaluronic acid (HA) is a biologically versatile polysaccharide synthesized by vertebrates and several microbial pathogens. To date, Cryptococcus neoformans CPS1p is the only reported bona fide hyaluronic acid synthase (HAS) in fungi, which is functionally related to bacterial HASs. Considering the phylogenetic and biochemical connection between HASs and chitin synthases (CHSs), essential for fungal cell wall synthesis, it is reasonable to hypothesize the former might be more common in fungi than expected. In this work, a comprehensive in silico survey of putative HASs in the Fungal Tree of Life was carried out. 68 putative HASs, mainly in Basidiomycota, were found, although other artificial intelligence-inferred putative HASs were found among Ascomycota. Global fold and arrangement of essential amino acids were shared by all kingdoms' HASs; however, C. neoformas CPS1p and additional putative fungal HASs (fungal HASs) showed exclusive conserved sequence signatures. Moreover, fungal HASs bore an only 3-helices transmembrane pore and their gating loop, which regulates the entrance of substrates to the catalytic site, was directly connected to an also exclusive intrinsically disordered C-terminus. Phylogenetically, fungal HASs were found in a clade different to that of bacterial, animal and viral HASs, and might share a common ancestor with Class I, III or VI CHSs. The atypical features of fungal HASs could influence the size and biological role of the HA they potentially synthesize and also highlight regulatory differences among HASs at the level of the gating loop configuration.

During human infection, Cryptococcus neoformans can disseminate to the central nervous system, causing often-fatal cryptococcal meningitis in individuals with compromised immunity. To acutely adapt to host micro-environments, C. neoformans employs mechanisms to reprogram its translatome to provide cells with tools to survive the stressors of the human host. The identities and functions of ribosome-associated factors that may modulate this process in C. neoformans remain unknown. We hypothesized that following host-related stressors, specific proteins associate with ribosomes and influence stress adaptation in C. neoformans. Utilizing a proteomic approach, termed RiboPROT, we analyzed the protein composition of mRNA-associated ribosomes from cells grown under unstressed (30°C), thermally stressed (shifted to 37°C), and oxidatively stressed (shifted to 2 mM H2O2) conditions. We identified unique factors enriched with actively translating ribosomes during host-related stressors. Phenotypic analysis of deletion mutants in selected ribosome-associated factors revealed that the J-domain protein Sis1 is important for growth at elevated temperature, Hsp70 and Hsp90 function, and virulence. The sis1Δ mutant also exhibited sensitivity to multiple drugs that cause mitochondrial perturbation, and growth on acetate as a sole carbon source suppressed the thermal growth defect at 38°C, suggesting that Sis1 supports mitochondrial homeostasis during thermal stress. Additionally, the sis1Δ mutant displayed transient hyper-repression of translation initiation during a shift to acute thermal stress at human body temperature but not at human febrile temperature. Global features of stress-responsive translatome reprogramming remained intact in the absence of Sis1, suggesting its role in thermotolerance and virulence is likely linked to regulation of specific targets involved in mitochondrial homeostasis in C. neoformans.IMPORTANCEFungal pathogens are an underrepresented and emerging global public health threat. In 2022, the World Health Organization recognized Cryptococcus neoformans as the top fungal high-priority pathogen, signifying the urgency for increased research and new therapeutics. The mechanisms by which C. neoformans adapts to the insults of the human body and causes systemic disease remain incompletely understood. Although the ribosome is essential to fungal viability and is targetable by small molecules, the conservation of the eukaryotic ribosome hampers the development of molecules that are selective for the fungal ribosome. Here, we identify stress-dependent ribosome-associated factors in C. neoformans and highlight one identified protein, Sis1, demonstrating its contribution to thermotolerance and virulence. Identification of stress-induced ribosome-associated factors and uncovering their biological role in the cell may identify pathways for selective therapeutic targeting of the fungal ribosome.