Phenotypic landscape of an invasive fungal pathogen reveals its unique biology.

Cryptococcus neoformans is the most common cause of fungal meningitis and the top-ranking WHO fungal priority pathogen. Only distantly related to model fungi, C. neoformans is also a powerful experimental system for exploring conserved eukaryotic mechanisms lost from specialist model yeast lineages.

Near 100% efficient homology-dependent genome engineering in the human fungal pathogen Cryptococcus neoformans.

We recently described CRISPR/Cas9-based short homology-dependent genome engineering in the human fungal pathogen Cryptococcus neoformans, a haploid budding yeast that is the most common cause of fungal meningitis and an emerging model organism. This was achieved by electroporation of strains stably expressing a codon-optimized Cas9 with 2 separate DNA molecules, one encoding a selectable marker flanked by short homology arms and a second encoding a sgRNA under the control of the U6 snRNA promoter. However, the efficiency of desired homology-dependent repair relative to undesired non-homologous end-joining (NHEJ) events can be low and variable.

On-Resin Assembly of Macrocyclic Inhibitors of May1: A Pathway to Potent Antifungal Agents.

Macrocyclic inhibitors have emerged as a privileged scaffold in medicinal chemistry, offering enhanced selectivity, stability, and pharmacokinetic profiles compared to their linear counterparts. Here, we describe a novel, on-resin macrocyclization strategy for the synthesis of potent inhibitors targeting the secreted protease Major Aspartyl Peptidase 1 in , a pathogen responsible for life-threatening fungal infections. By employing diverse aliphatic linkers and statine-based transition-state mimics, we constructed a focused library of 624 macrocyclic compounds.

An auxin-inducible degron system for conditional mutation in the fungal meningitis pathogen Cryptococcus neoformans.

Cryptococcus neoformans is the top-ranked W.H.O.

Mechanisms of Inheritance of Chromatin States: From Yeast to Human.

In this article I review mechanisms that underpin epigenetic inheritance of CpG methylation and histone H3 lysine 9 methylation (H3K9me) in chromatin in fungi and mammals. CpG methylation can be faithfully inherited epigenetically at some sites for a lifetime in vertebrates and, remarkably, can be propagated for millions of years in some fungal lineages. Transmission of methylation patterns requires maintenance-type DNA methyltransferases (DNMTs) that recognize hemimethylated CpG DNA produced by replication.

Phenotypic landscape of a fungal meningitis pathogen reveals its unique biology.

is the most common cause of fungal meningitis and the top-ranked W.H.O.

Deep learning meets histones at the replication fork.

Epigenetic inheritance of heterochromatin requires transfer of parental H3-H4 tetramers to both daughter duplexes during replication. Three recent papers exploit yeast genetics coupled to inheritance assays and AlphaFold2-multimer predictions coupled to biochemistry to reveal that a replisome component (Mrc1/CLASPIN) is an H3-H4 tetramer chaperone important for parental histone transfer to daughters.

Understanding the dynamic design of the spliceosome.

The spliceosome catalyzes the splicing of pre-mRNAs. Although the spliceosome evolved from a prokaryotic self-splicing intron and an associated protein, it is a vastly more complex and dynamic ribonucleoprotein (RNP) whose function requires at least eight ATPases and multiple RNA rearrangements. These features afford stepwise opportunities for multiple inspections of the intron substrate, coupled with spliceosome disassembly for substrates that fail inspection.

Convergent evolution of innate immune-modulating effectors in invasive fungal pathogens.

Invasive fungal infections pose a major threat to human health. Bacterial and protozoan pathogens secrete protein effectors that overcome innate immune barriers to promote microbial colonization, yet few such molecules have been identified in human fungal pathogens. Recent studies have begun to reveal these long-sought effectors and have illuminated how they subvert key cellular pathways, including apoptosis, myeloid cell polarization, Toll-like receptor signaling, and phagosome action.

Targeted high-throughput mutagenesis of the human spliceosome reveals its in vivo operating principles.

The spliceosome is a staggeringly complex machine, comprising, in humans, 5 snRNAs and >150 proteins. We scaled haploid CRISPR-Cas9 base editing to target the entire human spliceosome and investigated the mutants using the U2 snRNP/SF3b inhibitor, pladienolide B. Hypersensitive substitutions define functional sites in the U1/U2-containing A complex but also in components that act as late as the second chemical step after SF3b is dissociated.

Platelets and mast cells promote pathogenic eosinophil recruitment during invasive fungal infection via the 5-HIAA-GPR35 ligand-receptor system.

Cryptococcus neoformans is the leading cause of fungal meningitis and is characterized by pathogenic eosinophil accumulation in the context of type-2 inflammation. The chemoattractant receptor GPR35 is expressed by granulocytes and promotes their migration to the inflammatory mediator 5-hydroxyindoleacetic acid (5-HIAA), a serotonin metabolite. Given the inflammatory nature of cryptococcal infection, we examined the role of GPR35 in the circuitry underlying cell recruitment to the lung.

Deep tissue infection by an invasive human fungal pathogen requires lipid-based suppression of the IL-17 response.

Candida albicans is the most common cause of fungal infection in humans. IL-17 is critical for defense against superficial fungal infections, but the role of this response in invasive disease is less understood. We show that C.

Secreted fungal virulence effector triggers allergic inflammation via TLR4.

Invasive fungal pathogens are major causes of human mortality and morbidity. Although numerous secreted effector proteins that reprogram innate immunity to promote virulence have been identified in pathogenic bacteria, so far, there are no examples of analogous secreted effector proteins produced by human fungal pathogens. Cryptococcus neoformans, the most common cause of fungal meningitis and a major pathogen in AIDS, induces a pathogenic type 2 response characterized by pulmonary eosinophilia and alternatively activated macrophages.

Structural insights into DNMT5-mediated ATP-dependent high-fidelity epigenome maintenance.

Epigenetic evolution occurs over million-year timescales in Cryptococcus neoformans and is mediated by DNMT5, the first maintenance type cytosine methyltransferase identified in the fungal or protist kingdoms, the first dependent on adenosine triphosphate (ATP), and the most hemimethyl-DNA-specific enzyme known. To understand these novel properties, we solved cryo-EM structures of CnDNMT5 in three states. These studies reveal an elaborate allosteric cascade in which hemimethylated DNA binding first activates the SNF2 ATPase domain by a large rigid body rotation while the target cytosine partially flips out of the DNA duplex.

Short homology-directed repair using optimized Cas9 in the pathogen Cryptococcus neoformans enables rapid gene deletion and tagging.

Cryptococcus neoformans, the most common cause of fungal meningitis, is a basidiomycete haploid budding yeast with a complete sexual cycle. Genome modification by homologous recombination is feasible using biolistic transformation and long homology arms, but the method is arduous and unreliable. Recently, multiple groups have reported the use of CRISPR-Cas9 as an alternative to biolistics, but long homology arms are still necessary, limiting the utility of this method.

Coupling of spliceosome complexity to intron diversity.

We determined that over 40 spliceosomal proteins are conserved between many fungal species and humans but were lost during the evolution of S. cerevisiae, an intron-poor yeast with unusually rigid splicing signals. We analyzed null mutations in a subset of these factors, most of which had not been investigated previously, in the intron-rich yeast Cryptococcus neoformans.

Re-emerging Aspartic Protease Targets: Examining 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.

Unbelievable but True: Epigenetics and Chromatin in Fungi.

Evolutionary innovations in chromatin biology have been recently discovered through the study of fungi. In Saccharomyces cerevisiae, a prion form of a deacetylase complex assembles over subtelomeric domains that produces a heritable gene expression state that enables resistance to stress. In Candida albicans, stress triggers adaptive chromosome destabilization via erasure a centromeric histone H3, CENP-A; a process that cooperates with a newly evolved H2A variant lacking a mitotic phosphorylation site.

Coordinate genomic association of transcription factors controlled by an imported quorum sensing peptide in Cryptococcus neoformans.

Qsp1 is a secreted quorum sensing peptide required for virulence of the fungal meningitis pathogen Cryptococcus neoformans. Qsp1 functions to control cell wall integrity in vegetatively growing cells and also functions in mating. Rather than acting on a cell surface receptor, Qsp1 is imported to act intracellularly via the predicted oligopeptide transporter Opt1.

ATP Hydrolysis by the SNF2 Domain of Dnmt5 Is Coupled to Both Specific Recognition and Modification of Hemimethylated DNA.

C.neoformans Dnmt5 is an unusually specific maintenance-type CpG methyltransferase (DNMT) that mediates long-term epigenome evolution. It harbors a DNMT domain and SNF2 ATPase domain.