Structures of trehalose-6-phosphate synthase, Tps1, from the fungal pathogen Cryptococcus neoformans: A target for antifungals.

Invasive fungal diseases are a major threat to human health, resulting in more than 1.5 million annual deaths worldwide. The arsenal of antifungal therapeutics remains limited and is in dire need of drugs that target additional biosynthetic pathways that are absent from humans. One such pathway involves the biosynthesis of trehalose. Trehalose is a disaccharide that is required for pathogenic fungi to survive in their human hosts. In the first step of trehalose biosynthesis, trehalose-6-phosphate synthase (Tps1) converts UDP-glucose and glucose-6-phosphate to trehalose-6-phosphate. Here, we report the structures of full-length Cryptococcus neoformans Tps1 (CnTps1) in unliganded form and in complex with uridine diphosphate and glucose-6-phosphate. Comparison of these two structures reveals significant movement toward the catalytic pocket by the N terminus upon ligand binding and identifies residues required for substrate binding, as well as residues that stabilize the tetramer. Intriguingly, an intrinsically disordered domain (IDD), which is conserved among Cryptococcal species and closely related basidiomycetes, extends from each subunit of the tetramer into the "solvent" but is not visible in density maps. We determined that the IDD is not required for C. neoformans Tps1-dependent thermotolerance and osmotic stress survival. Studies with UDP-galactose highlight the exquisite substrate specificity of CnTps1. In toto, these studies expand our knowledge of trehalose biosynthesis in Cryptococcus and highlight the potential of developing antifungal therapeutics that disrupt the synthesis of this disaccharide or the formation of a functional tetramer and the use of cryo-EM in the structural characterization of CnTps1-ligand/drug complexes.

A food color-based colorimetric assay for Cryptococcus neoformans laccase activity.

Cryptococcus neoformans is a fungal pathogen that causes cryptococcosis primarily in immunocompromised patients, such as those with HIV/AIDS. One survival mechanism of C. neoformans during infection is melanin production, which catalyzed by laccase and protects fungal cells against immune attack. Hence, the comparative assessment of laccase activity is useful for characterizing cryptococcal strains. We serendipitously observed that culturing C. neoformans with food coloring resulted in degradation of some dyes with phenolic structures. Consequently, we investigated the color changes for the food dyes metabolized by C. neoformans laccase and by using this effect explored the development of a colorimetric assay to measure laccase activity. We developed several versions of a food dye-based colorimetric laccase assay that can be used to compare the relative laccase activities between different C. neoformans strains. We found that phenolic color degradation was glucose-dependent, which may reflect changes in the reduction properties of the media. Our food color-based colorimetric assay has several advantages, including lower cost, irreversibility, and not requiring constant monitoring , over the commonly used 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay for determining laccase activity. This method has potential applications to bioremediation of water pollutants in addition to its use in determining laccase virulence factor expression.IMPORTANCECryptococcus neoformans is present in the environment, and while infection is common, disease occurs mostly in immunocompromised individuals. C. neoformans infection in the lungs results in symptoms like pneumonia, and consequently, cryptococcal meningitis occurs if the fungal infection spreads to the brain. The laccase enzyme catalyzes the melanization reaction that serves as a virulence factor for C. neoformans. Developing a simple and less costly assay to determine the laccase activity in C. neoformans strains can be useful for a variety of procedures ranging from studying the relative virulence of cryptococci to environmental pollution studies.

Unraveling the cryptic functions of mitogen-activated protein kinases Cpk2 and Mpk2 in Cryptococcus neoformans.

Mitogen-activated protein kinase (MAPK) pathways are fundamental to the regulation of biological processes in eukaryotic organisms. The basidiomycete Cryptococcus neoformans, known for causing fungal meningitis worldwide, possesses five MAPKs. Among these, Cpk1, Hog1, and Mpk1 have established roles in sexual reproduction, stress responses, and cell wall integrity. However, the roles of Cpk2 and Mpk2 are less understood. Our study elucidates the functional interplay between the Cpk1/Cpk2 and Mpk1/Mpk2 MAPK pathways in C. neoformans. We discovered that CPK2 overexpression compensates for cpk1Δ mating deficiencies via the Mat2 transcription factor, revealing functional redundancy between Cpk1 and Cpk2. We also found that Mpk2 is phosphorylated in response to cell wall stress, a process regulated by the MAPK kinase (MAP2K) Mkk2 and MAP2K kinases (MAP3Ks) Ssk2 and Ste11. Overexpression of MPK2 partially restores cell wall integrity in mpk1Δ by influencing key cell wall components, such as chitin and the polysaccharide capsule. Contrarily, MPK2 overexpression cannot restore thermotolerance and cell membrane integrity in mpk1Δ. These results suggest that Mpk1 and Mpk2 have redundant and opposing roles in the cellular response to cell wall and membrane stresses. Most notably, the dual deletion of MPK1 and MPK2 restores wild-type mating efficiency in cpk1Δ mutants via upregulation of the mating-regulating transcription factors MAT2 and ZNF2, suggesting that the Mpk1 and Mpk2 cooperate to negatively regulate the pheromone-responsive Cpk1 MAPK pathway. Our research collectively underscores a sophisticated regulatory network of cryptococcal MAPK signaling pathways that intricately govern sexual reproduction and cell wall integrity, thereby controlling fungal development and pathogenicity.IMPORTANCEIn the realm of fungal biology, our study on Cryptococcus neoformans offers pivotal insights into the roles of specific proteins called mitogen-activated protein kinases (MAPKs). Here, we discovered the cryptic functions of Cpk2 and Mpk2, two MAPKs previously overshadowed by their dominant counterparts Cpk1 and Mpk1, respectively. Our findings reveal that these "underdog" proteins are not just backup players; they play crucial roles in vital processes like mating and cell wall maintenance in C. neoformans. Their ability to step in and compensate when their dominant counterparts are absent showcases the adaptability of C. neoformans. This newfound understanding not only enriches our knowledge of fungal MAPK mechanisms but also underscores the intricate balance and interplay of proteins in ensuring the organism's survival and adaptability.

Broad Protection against Invasive Fungal Disease from a Nanobody Targeting the Active Site of Fungal ß-1,3-Glucanosyltransferases.

Invasive fungal disease accounts for about 3.8 million deaths annually, an unacceptable rate that urgently prompts the discovery of new knowledge-driven treatments. We report the use of camelid single-domain nanobodies (Nbs) against fungal ß-1,3-glucanosyltransferases (Gel) involved in ß-1,3-glucan transglycosylation. Crystal structures of two Nbs with Gel4 from Aspergillus fumigatus revealed binding to a dissimilar CBM43 domain and a highly conserved catalytic domain across fungal species, respectively. Anti-Gel4 active site Nb3 showed significant antifungal efficacy in vitro and in vivo prophylactically and therapeutically against different A. fumigatus and Cryptococcus neoformans isolates, reducing the fungal burden and disease severity, thus significantly improving immunocompromised animal survival. Notably, C. deneoformans (serotype D) strains were more susceptible to Nb3 and genetic Gel deletion than C. neoformans (serotype A) strains, indicating a key role for ß-1,3-glucan remodelling in C. deneoformans survival. These findings add new insight about the role of ß-1,3-glucan in fungal biology and demonstrate the potential of nanobodies in targeting fungal enzymes to combat invasive fungal diseases.

Cryptococcus neoformans trehalose-6-phosphate synthase (tps1) promotes organ-specific virulence and fungal protection against multiple lines of host defenses.

Trehalose-6-phosphate synthase (TPS1) was identified as a virulence factor for Cryptococcus neoformans and a promising therapeutic target. This study reveals previously unknown roles of TPS1 in evasion of host defenses during pulmonary and disseminated phases of infection. In the pulmonary infection model, TPS1-deleted (tps1Δ) Cryptococci are rapidly cleared by mouse lungs whereas TPS1-sufficent WT (H99) and revertant (tps1Δ:TPS1) strains expand in the lungs and disseminate, causing 100% mortality. Rapid pulmonary clearance of tps1Δ mutant is T-cell independent and relies on its susceptibility to lung resident factors and innate immune factors, exemplified by tps1Δ but not H99 inhibition in a coculture with dispersed lung cells and its rapid clearance coinciding with innate leukocyte infiltration. In the disseminated model of infection, which bypasses initial lung-fungus interactions, tps1Δ strain remains highly attenuated. Specifically, tps1Δ mutant is unable to colonize the lungs from the bloodstream or expand in spleens but is capable of crossing into the brain, where it remains controlled even in the absence of T cells. In contrast, strains H99 and tps1Δ:TPS1 rapidly expand in all studied organs, leading to rapid death of the infected mice. Since the rapid pulmonary clearance of tps1Δ mutant resembles a response to acapsular strains, the effect of tps1 deletion on capsule formation in vitro and in vivo was examined. Tps1Δ cryptococci form capsules but with a substantially reduced size. In conclusion, TPS1 is an important virulence factor, allowing C. neoformans evasion of resident pulmonary and innate defense mechanisms, most likely via its role in cryptococcal capsule formation.

Characterization of a methyltransferase for iterative N-methylation at the leucinostatin termini in Purpureocillium lilacinum.

N-methyltransferase (NMT)-catalyzed methylation at the termini of nonribosomal peptides (NRPs) has rarely been reported. Here, we discover a fungal NMT LcsG for the iterative terminal N-methylation of a family of NRPs, leucinostatins. Gene deletion results suggest that LcsG is essential for leucinostatins methylation. Results from in vitro assays and HRESI-MS-MS analysis reveal the methylation sites as NH2, NHCH3 and N(CH3)2 in the C-terminus of various leucinostatins. LcsG catalysis yields new lipopeptides, some of which demonstrate effective antibiotic properties against the human pathogen Cryptococcus neoformans and the plant pathogen Phytophthora infestans. Multiple sequence alignments and site-directed mutagenesis of LcsG indicate the presence of a highly conserved SAM-binding pocket, along with two possible active site residues (D368 and D395). Molecular dynamics simulations show that the targeted N can dock between these two residues. Thus, this study suggests a method for increasing the variety of natural bioactivity of NPRs and a possible catalytic mechanism underlying the N-methylation of NRPs.

Aspartyl peptidase May1 induces host inflammatory response by altering cell wall composition in the fungal pathogen Cryptococcus neoformans.

Cryptococcus neoformans causes cryptococcal meningoencephalitis, a disease that kills more than 180,000 people annually. Contributing to its success as a fungal pathogen is its cell wall surrounded by a capsule. When the cryptococcal cell wall is compromised, exposed pathogen-associated molecular pattern molecules (PAMPs) could trigger host recognition and initiate attack against this fungus. Thus, cell wall composition and structure are tightly regulated. The cryptococcal cell wall is unusual in that chitosan, the acetylated form of chitin, is predominant over chitin and is essential for virulence. Recently, it was shown that acidic pH weakens the cell wall and increases exposure of PAMPs partly due to decreased chitosan levels. However, the molecular mechanism responsible for the cell wall remodeling in acidic pH is unknown. In this study, by screening for genes involved in cryptococcal tolerance to high levels of CO2, we serendipitously discovered that the aspartyl peptidase May1 contributes to cryptococcal sensitivity to high levels of CO2 due to acidification of unbuffered media. Overexpression of MAY1 increases the cryptococcal cell size and elevates PAMP exposure, causing a hyper-inflammatory response in the host while MAY1 deletion does the opposite. We discovered that May1 weakens the cell wall and reduces the chitosan level, partly due to its involvement in the degradation of Chs3, the sole chitin synthase that supplies chitin to be converted to chitosan. Consistently, overexpression of CHS3 largely rescues the phenotype of MAY1oe in acidic media. Collectively, we demonstrate that May1 remodels the cryptococcal cell wall in acidic pH by reducing chitosan levels through its influence on Chs3. IMPORTANCE: The fungal cell wall is a dynamic structure, monitoring and responding to internal and external stimuli. It provides a formidable armor to the fungus. However, in a weakened state, the cell wall also triggers host immune attack when PAMPs, including glucan, chitin, and mannoproteins, are exposed. In this work, we found that the aspartyl peptidase May1 impairs the cell wall of Cryptococcus neoformans and increases the exposure of PAMPs in the acidic environment by reducing the chitosan level. Under acidic conditions, May1 is involved in the degradation of the chitin synthase Chs3, which supplies chitin to be deacetylated to chitosan. Consistently, the severe deficiency of chitosan in acidic pH can be rescued by overexpressing CHS3. These findings improve our understanding of cell wall remodeling and reveal a potential target to compromise the cell wall integrity in this important fungal pathogen.

Arg1 from Cryptococcus neoformans lacks PI3 kinase activity and conveys virulence roles via its IP3-4 kinase activity.

Inositol tris/tetrakis phosphate kinases (IP3-4K) in the human fungal priority pathogens, Cryptococcus neoformans (CnArg1) and Candida albicans (CaIpk2), convey numerous virulence functions, yet it is not known whether the IP3-4K catalytic activity or a scaffolding role is responsible. We therefore generated a C. neoformans strain with a non-functional kinase, referred to as the dead-kinase (dk) CnArg1 strain (dkArg1). We verified that, although dkARG1 cDNA cloned from this strain produced a protein with the expected molecular weight, dkArg1 was catalytically inactive with no IP3-4K activity. Using recombinant CnArg1 and CaIpk2, we confirmed that, unlike the IP3-4K homologs in humans and Saccharomyces cerevisiae, CnArg1 and CaIpk2 do not phosphorylate the lipid-based substrate, phosphatidylinositol 4,5-bisphosphate, and therefore do not function as class I PI3Ks. Inositol polyphosphate profiling using capillary electrophoresis-electrospray ionization-mass spectrometry revealed that IP3 conversion is blocked in the dkArg1 and ARG1 deletion (Cnarg1Δ) strains and that 1-IP7 and a recently discovered isomer (4/6-IP7) are made by wild-type C. neoformans. Importantly, the dkArg1 and Cnarg1Δ strains had similar virulence defects, including suppressed growth at 37°C, melanization, capsule production, and phosphate starvation response, and were avirulent in an insect model, confirming that virulence is dependent on IP3-4K catalytic activity. Our data also implicate the dkArg1 scaffold in transcriptional regulation of arginine metabolism but via a different mechanism to S. cerevisiae since CnArg1 is dispensable for growth on different nitrogen sources. IP3-4K catalytic activity therefore plays a dominant role in fungal virulence, and IPK pathway function has diverged in fungal pathogens.IMPORTANCEThe World Health Organization has emphasized the urgent need for global action in tackling the high morbidity and mortality rates stemming from invasive fungal infections, which are exacerbated by the limited variety and compromised effectiveness of available drug classes. Fungal IP3-4K is a promising target for new therapy, as it is critical for promoting virulence of the human fungal priority pathogens, Cryptococcus neoformans and Candida albicans, and impacts numerous functions, including cell wall integrity. This contrasts to current therapies, which only target a single function. IP3-4K enzymes exert their effect through their inositol polyphosphate products or via the protein scaffold. Here, we confirm that the IP3-4K catalytic activity of CnArg1 promotes all virulence traits in C. neoformans that are attenuated by ARG1 deletion, reinforcing our ongoing efforts to find inositol polyphosphate effector proteins and to create inhibitors targeting the IP3-4K catalytic site, as a new antifungal drug class.

Pleiotropic roles of LAMMER kinase, Lkh1 in stress responses and virulence of Cryptococcus neoformans.

Dual-specificity LAMMER kinases are highly evolutionarily conserved in eukaryotes and play pivotal roles in diverse physiological processes, such as growth, differentiation, and stress responses. Although the functions of LAMMER kinase in fungal pathogens in pathogenicity and stress responses have been characterized, its role in Cryptococcus neoformans, a human fungal pathogen and a model yeast of basidiomycetes, remains elusive. In this study, we identified a LKH1 homologous gene and constructed a strain with a deleted LKH1 and a complemented strain. Similar to other fungi, the lkh1Δ mutant showed intrinsic growth defects. We observed that C. neoformans Lkh1 was involved in diverse stress responses, including oxidative stress and cell wall stress. Particularly, Lkh1 regulates DNA damage responses in Rad53-dependent and -independent manners. Furthermore, the absence of LKH1 reduced basidiospore formation. Our observations indicate that Lkh1 becomes hyperphosphorylated upon treatment with rapamycin, a TOR protein inhibitor. Notably, LKH1 deletion led to defects in melanin synthesis and capsule formation. Furthermore, we found that the deletion of LKH1 led to the avirulence of C. neoformans in a systemic cryptococcosis murine model. Taken together, Lkh1 is required for the stress response, sexual differentiation, and virulence of C. neoformans.

Measuring Urease and Phospholipase Secretion in Cryptococcus neoformans.

Urease and phospholipase are enzymes that are important virulence factors for Cryptococcus neoformans. These are two of the most studied enzymes involved in how C. neoformans breaches the blood-brain barrier. Additionally, phospholipase secretion also supports dissemination from the lungs. This chapter describes the methods used to measure the secretion of these enzymes, which may be used to characterize strain invasiveness and virulence.

Measuring Laccase Activity and Melanin Production in Cryptococcus neoformans.

Melanin is a complex dark pigment synthetized by the phenoloxidase enzyme laccase in Cryptococcus neoformans. In vitro, this enzyme oxidizes exogenous catecholamines to produce melanin that may be secreted or incorporated into the fungal cell wall. This pigment has multiple roles in C. neoformans virulence during its interaction with different hosts and probably also in protecting fungal cells in the environment against predation and oxidative and radiation stresses, among others. However, it is important to note that laccase also has melanin-independent roles in C. neoformans interactions with host cells. In this chapter, we describe a quantitative laccase assay and a method for evaluating the kinetics of melanin production in C. neoformans colonies.

Ethanolic Extract Propolis-Loaded Niosomes Diminish Phospholipase B1, Biofilm Formation, and Intracellular Replication of Cryptococcus neoformans in Macrophages.

Secretory phospholipase B1 (PLB1) and biofilms act as microbial virulence factors and play an important role in pulmonary cryptococcosis. This study aims to formulate the ethanolic extract of propolis-loaded niosomes (Nio-EEP) and evaluate the biological activities occurring during PLB1 production and biofilm formation of Cryptococcus neoformans. Some physicochemical characterizations of niosomes include a mean diameter of 270 nm in a spherical shape, a zeta-potential of -10.54 ± 1.37 mV, and 88.13 ± 0.01% entrapment efficiency. Nio-EEP can release EEP in a sustained manner and retains consistent physicochemical properties for a month. Nio-EEP has the capability to permeate the cellular membranes of C. neoformans, causing a significant decrease in the mRNA expression level of PLB1. Interestingly, biofilm formation, biofilm thickness, and the expression level of biofilm-related genes (UGD1 and UXS1) were also significantly reduced. Pre-treating with Nio-EEP prior to yeast infection reduced the intracellular replication of C. neoformans in alveolar macrophages by 47%. In conclusion, Nio-EEP mediates as an anti-virulence agent to inhibit PLB1 and biofilm production for preventing fungal colonization on lung epithelial cells and also decreases the intracellular replication of phagocytosed cryptococci. This nano-based EEP delivery might be a potential therapeutic strategy in the prophylaxis and treatment of pulmonary cryptococcosis in the future.

Unraveling the Pathobiological Role of the Fungal KEOPS Complex in Cryptococcus neoformans.

The KEOPS (kinase, putative endopeptidase, and other proteins of small size) complex has critical functions in eukaryotes; however, its role in fungal pathogens remains elusive. Herein, we comprehensively analyzed the pathobiological functions of the fungal KEOPS complex in Cryptococcus neoformans (Cn), which causes fatal meningoencephalitis in humans. We identified four CnKEOPS components: Pcc1, Kae1, Bud32, and Cgi121. Deletion of PCC1, KAE1, or BUD32 caused severe defects in vegetative growth, cell cycle control, sexual development, general stress responses, and virulence factor production, whereas deletion of CGI121 led to similar but less severe defects. This suggests that Pcc1, Kae1, and Bud32 are the core KEOPS components, and Cgi121 may play auxiliary roles. Nevertheless, all KEOPS components were essential for C. neoformans pathogenicity. Although the CnKEOPS complex appeared to have a conserved linear arrangement of Pcc1-Kae1-Bud32-Cgi121, as supported by physical interaction between Pcc1-Kae1 and Kae1-Bud32, CnBud32 was found to have a unique extended loop region that was critical for the KEOPS functions. Interestingly, CnBud32 exhibited both kinase activity-dependent and -independent functions. Supporting its pleiotropic roles, the CnKEOPS complex not only played conserved roles in t6A modification of ANN codon-recognizing tRNAs but also acted as a major transcriptional regulator, thus controlling hundreds of genes involved in various cellular processes, particularly ergosterol biosynthesis. In conclusion, the KEOPS complex plays both evolutionarily conserved and divergent roles in controlling the pathobiological features of C. neoformans and could be an anticryptococcal drug target. IMPORTANCE The cellular function and structural configuration of the KEOPS complex have been elucidated in some eukaryotes and archaea but have never been fully characterized in fungal pathogens. Here, we comprehensively analyzed the pathobiological roles of the KEOPS complex in the globally prevalent fungal meningitis-causing pathogen C. neoformans. The CnKEOPS complex, composed of a linear arrangement of Pcc1-Kae1-Bud32-Cgi121, not only played evolutionarily conserved roles in growth, sexual development, stress responses, and tRNA modification but also had unique roles in controlling virulence factor production and pathogenicity. Notably, a unique extended loop structure in CnBud32 is critical for the KEOPS complex in C. neoformans. Supporting its pleiotropic roles, transcriptome analysis revealed that the CnKEOPS complex governs several hundreds of genes involved in carbon and amino acid metabolism, pheromone response, and ergosterol biosynthesis. Therefore, this study provides novel insights into the fungal KEOPS complex that could be exploited as a potential antifungal drug target.

AICAR transformylase/IMP cyclohydrolase (ATIC) is essential for de novo purine biosynthesis and infection by Cryptococcus neoformans.

The fungal pathogen Cryptococcus neoformans is a leading cause of meningoencephalitis in the immunocompromised. As current antifungal treatments are toxic to the host, costly, limited in their efficacy, and associated with drug resistance, there is an urgent need to identify vulnerabilities in fungal physiology to accelerate antifungal discovery efforts. Rational drug design was pioneered in de novo purine biosynthesis as the end products of the pathway, ATP and GTP, are essential for replication, transcription, and energy metabolism, and the same rationale applies when considering the pathway as an antifungal target. Here, we describe the identification and characterization of C. neoformans 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase/5'-inosine monophosphate cyclohydrolase (ATIC), a bifunctional enzyme that catalyzes the final two enzymatic steps in the formation of the first purine base inosine monophosphate. We demonstrate that mutants lacking the ATIC-encoding ADE16 gene are adenine and histidine auxotrophs that are unable to establish an infection in a murine model of virulence. In addition, our assays employing recombinantly expressed and purified C. neoformans ATIC enzyme revealed Km values for its substrates AICAR and 5-formyl-AICAR are 8-fold and 20-fold higher, respectively, than in the human ortholog. Subsequently, we performed crystallographic studies that enabled the determination of the first fungal ATIC protein structure, revealing a key serine-to-tyrosine substitution in the active site, which has the potential to assist the design of fungus-specific inhibitors. Overall, our results validate ATIC as a promising antifungal drug target.

Essential Roles of Ribonucleotide Reductases under DNA Damage and Replication Stresses in Cryptococcus neoformans.

A balance in the deoxyribonucleotide (dNTPs) intracellular concentration is critical for the DNA replication and repair processes. In the model yeast Saccharomyces cerevisiae, the Mec1-Rad53-Dun1 kinase cascade mainly regulates the ribonucleotide reductase (RNR) gene expression during DNA replication and DNA damage stress. However, the RNR regulatory mechanisms in basidiomycete fungi during DNA replication and damage stress remain elusive. Here, we observed that in C. neoformans, RNR1 (large RNR subunit) and RNR21 (one small RNR subunit) were required for cell viability, but not RNR22 (another small RNR subunit). RNR22 overexpression compensated for the lethality of RNR21 suppression. In contrast to the regulatory mechanisms of RNRs in S. cerevisiae, Rad53 and Chk1 kinases cooperatively or divergently controlled RNR1 and RNR21 expression under DNA damage and DNA replication stress. In particular, this study revealed that Chk1 mainly regulated RNR1 expression during DNA replication stress, whereas Rad53, rather than Chk1, played a significant role in controlling the expression of RNR21 during DNA damage stress. Furthermore, the expression of RNR22, not but RNR1 and RNR21, was suppressed by the Ssn6-Tup1 complex during DNA replication stress. Notably, we observed that RNR1 expression was mainly regulated by Mbs1, whereas RNR21 expression was cooperatively controlled by Mbs1 and Bdr1 as downstream factors of Rad53 and Chk1 during DNA replication and damage stress. Collectively, the regulation of RNRs in C. neoformans has both evolutionarily conserved and divergent features in DNA replication and DNA damage stress, compared with other yeasts. IMPORTANCE Upon DNA replication or damage stresses, it is critical to provide proper levels of deoxynucleotide triphosphates (dNTPs) and activate DNA repair machinery. Ribonucleotide reductases (RNRs), which are composed of large and small subunits, are required for synthesizing dNTP. An imbalance in the intracellular concentration of dNTPs caused by the perturbation of RNR results in a reduction in DNA repair fidelity. Despite the importance of their roles, functions and regulations of RNR have not been elucidated in the basidiomycete fungi. In this study, we found that the roles of RNR1, RNR21, and RNR22 genes encoding RNR subunits in the viability of C. neoformans. Furthermore, their expression levels are divergently regulated by the Rad53-Chk1 pathway and the Ssn6-Tup1 complex in response to DNA replication and damage stresses. Therefore, this study provides insight into the regulatory mechanisms of RNR genes to DNA replication and damage stresses in basidiomycete fungi.

Cryptococcal Protease(s) and the Activation of SARS-CoV-2 Spike (S) Protein.

In this contribution, we report on the possibility that cryptococcal protease(s) could activate the SARS-CoV-2 spike (S) protein. The S protein is documented to have a unique four-amino-acid sequence (underlined, SPRRAR↓S) at the interface between the S1 and S2 sites, that serves as a cleavage site for the human protease, furin. We compared the biochemical efficiency of cryptococcal protease(s) and furin to mediate the proteolytic cleavage of the S1/S2 site in a fluorogenic peptide. We show that cryptococcal protease(s) processes this site in a manner comparable to the efficiency of furin (p > 0.581). We conclude the paper by discussing the impact of these findings in the context of a SARS-CoV-2 disease manifesting while there is an underlying cryptococcal infection.

Structural features of Cryptococcus neoformans bifunctional GAR/AIR synthetase may present novel antifungal drug targets.

Cryptococcus neoformans is a fungus that causes life-threatening systemic mycoses. During infection of the human host, this pathogen experiences a major change in the availability of purines; the fungus can scavenge the abundant purines in its environmental niche of pigeon excrement, but must employ de novo biosynthesis in the purine-poor human CNS. Eleven sequential enzymatic steps are required to form the first purine base, IMP, an intermediate in the formation of ATP and GTP. Over the course of evolution, several gene fusion events led to the formation of multifunctional purine biosynthetic enzymes in most organisms, particularly the higher eukaryotes. In C. neoformans, phosphoribosyl-glycinamide synthetase (GARs) and phosphoribosyl-aminoimidazole synthetase (AIRs) are fused into a bifunctional enzyme, while the human ortholog is a trifunctional enzyme that also includes GAR transformylase. Here we functionally, biochemically, and structurally characterized C. neoformans GARs and AIRs to identify drug targetable features. GARs/AIRs are essential for de novo purine production and virulence in a murine inhalation infection model. Characterization of GARs enzymatic functional parameters showed that C. neoformans GARs/AIRs have lower affinity for substrates glycine and PRA compared with the trifunctional metazoan enzyme. The crystal structure of C. neoformans GARs revealed differences in the glycine- and ATP-binding sites compared with the Homo sapiens enzyme, while the crystal structure of AIRs shows high structural similarity compared with its H. sapiens ortholog as a monomer but differences as a dimer. The alterations in functional and structural characteristics between fungal and human enzymes could potentially be exploited for antifungal development.

Zinc-binding domain mediates pleiotropic functions of Yvh1 in Cryptococcus neoformans.

Yvh1 is a dual-specificity phosphatase (DUSP) that is evolutionarily conserved in eukaryotes, including yeasts and humans. Yvh1 is involved in the vegetative growth, differentiation, and virulence of animal and plant fungal pathogens. All Yvh1 orthologs have a conserved DUSP catalytic domain at the N-terminus and a zinc-binding (ZB) domain with two zinc fingers (ZFs) at the C-terminus. Although the DUSP domain is implicated in the regulation of MAPK signaling in humans, only the ZB domain is essential for most cellular functions of Yvh1 in fungi. This study aimed to analyze the functions of the DUSP and ZB domains of Yvh1 in the human fungal pathogen Cryptococcus neoformans, whose Yvh1 (CnYvh1) contains a DUSP domain at the C-terminus and a ZB domain at the N-terminus. Notably, CnYvh1 has an extended internal domain between the two ZF motifs in the ZB domain. To elucidate the function of each domain, we constructed individual domain deletions and swapping strains by complementing the yvh1Δ mutant with wild-type (WT) or mutated YVH1 alleles and examined their Yvh1-dependent phenotypes, including growth under varying stress conditions, mating, and virulence factor production. Here, we found that the complementation of the yvh1Δ mutant with the mutated YVH1 alleles having two ZFs of the ZB domain, but not the DUSP and extended internal domains, restored the WT phenotypic traits in the yvh1Δ mutant. In conclusion, the ZB domain, but not the N-terminal DUSP domain, plays a pivotal role in the pathobiological functions of cryptococcal Yvh1.

Re-emerging Aspartic Protease Targets: Examining Cryptococcus neoformans Major Aspartyl Peptidase 1 as a Target for Antifungal Drug Discovery.

Cryptococcosis is an invasive infection that accounts for 15% of AIDS-related fatalities. Still, treating cryptococcosis remains a significant challenge due to the poor availability of effective antifungal therapies and emergence of drug resistance. Interestingly, protease inhibitor components of antiretroviral therapy regimens have shown some clinical benefits in these opportunistic infections. We investigated Major aspartyl peptidase 1 (May1), a secreted Cryptococcus neoformans protease, as a possible target for the development of drugs that act against both fungal and retroviral aspartyl proteases. Here, we describe the biochemical characterization of May1, present its high-resolution X-ray structure, and provide its substrate specificity analysis. Through combinatorial screening of 11,520 compounds, we identified a potent inhibitor of May1 and HIV protease. This dual-specificity inhibitor exhibits antifungal activity in yeast culture, low cytotoxicity, and low off-target activity against host proteases and could thus serve as a lead compound for further development of May1 and HIV protease inhibitors.

Transcription factor-driven alternative localization of Cryptococcus neoformans superoxide dismutase.

Cryptococcus neoformans is an opportunistic fungal pathogen whose pathogenic lifestyle is linked to its ability to cope with fluctuating levels of copper (Cu), an essential metal involved in multiple virulence mechanisms, within distinct host niches. During lethal cryptococcal meningitis in the brain, C. neoformans senses a Cu-deficient environment and is highly dependent on its ability to scavenge trace levels of Cu from its host and adapt to Cu scarcity to successfully colonize this niche. In this study, we demonstrate for this critical adaptation, the Cu-sensing transcription factor Cuf1 differentially regulates the expression of the SOD1 and SOD2 superoxide dismutases in novel ways. Genetic and transcriptional analysis reveals Cuf1 specifies 5'-truncations of the SOD1 and SOD2 mRNAs through specific binding to Cu responsive elements within their respective promoter regions. This results in Cuf1-dependent repression of the highly abundant SOD1 and simultaneously induces expression of two isoforms of SOD2, the canonical mitochondrial targeted isoform and a novel alternative cytosolic isoform, from a single alternative transcript produced specifically under Cu limitation. The generation of cytosolic Sod2 during Cu limitation is required to maintain cellular antioxidant defense against superoxide stress both in vitro and in vivo. Further, decoupling Cuf1 regulation of Sod2 localization compromises the ability of C. neoformans to colonize organs in murine models of cryptococcosis. Our results provide a link between transcription factor-mediated alteration of protein localization and cell proliferation under stress, which could impact tissue colonization by a fungal pathogen.