Biology subjects

Huang, M. Y.

Publications and source records attributed to Huang, M. Y..

6 recordsLinked to original sources

bioRxiv2026

Therapeutic vulnerabilities of the drug-resistant fungal meningitis pathogen Cryptococcus neoformans

Treatment options are limited for the priority fungal pathogen Cryptococcus neoformans. We describe CRISPR-insert, a scalable gene inactivation method for global interrogation of drug targets. Genome-wide screens using this methodology combined with machine learning predict 1,297 essential genes under liberal criteria. Unexpectedly absent from this list is FKS1, encoding the target of echinocandin drugs to which C. neoformans is resistant. Contrary to longstanding literature, targeted deletion shows that FKS1 is not essential. Solid-state NMR reveals that fks1{triangleup} mutants lack {beta}-1,3-glucan, arguing against compensation by another synthase. We experimentally test essentiality predictions for genes lacking human orthologs, identifying high-priority drug target candidates. To uncover targets for combination therapies, we establish genome-scale quantitative genetic interaction mapping. We use this method to uncover synthetic lethal dependencies of echinocandin resistance determinants, including the druggable calcineurin and Cdc50-Apt1 pathways. CRISPR-insert illuminates therapeutic vulnerabilities of an invasive fungal pathogen.

microbiology↗

bioRxiv2025

Rational Generation of Monoclonal Antibodies and Intrabodies Selective for Pathogenic TDP-43

TAR DNA-binding protein 43 (TDP-43), encoded by the TARDBP gene, is a ribonucleoprotein associated with the pathogenesis of amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Alzheimers disease (AD). Under physiological conditions, TDP-43 is predominantly localized in the nucleus, where it participates in a variety of cellular functions related to RNA splicing, transport, and stability, as well as miRNA biogenesis. In disease, it is disproportionately mislocalized to the cytoplasm where it forms aggregates, which contribute to neurotoxicity and prion-like cell-to-cell propagation of pathogenic TDP-43. Targeting of misfolded aggregates of TDP-43 represents an attractive therapeutic strategy. However, development of effective immunotherapeutic agents remains a challenge, as they require stringent selectivity for misfolded TDP-43 in order to maintain the essential functions of physiologically native TDP-43. To address this issue, monoclonal antibodies (mAbs) and intrabodies were generated against an epitope in the N-terminal domain of TDP-43 that is only exposed when the protein is misfolded, but not in its properly folded form. We show that mouse and rabbit mAbs against this epitope displayed high binding affinities by surface plasmon resonance analysis and selectively reacted with pathological TDP-43 in post-mortem tissues from ALS, FTD, and AD patients. In a cell line system, human embryonic kidney (HEK) 293T cells, mAbs and corresponding intrabodies specifically reacted with cytoplasmic aggregates of transfected misfolded TDP-43 lacking the nuclear localization signal, TDP-43{Delta}NLS. Functionally, mAbs inhibited cell-to-cell transmission of misfolded TDP-43 and the seeding activity of misfolded TDP-43 from FTLD brain homogenates by a novel RT-QuIC assay. Intrabodies promoted the degradation of intracellular aggregates of TDP-43 in HEK293T cells and in induced pluripotent stem cell-derived motor neurons (iPSC-MNs) from ALS patients. The results provide proof-of-concept evidence that supports selective targeting of misfolded toxic aggregates of TDP-43 as a potentially safe and effective avenue to treat neurodegenerative diseases associated with TDP-43 proteinopathy.

immunology↗

bioRxiv2025

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 two 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. Here, we describe methods and strains enabling extremely efficient ([~]99%) homology-dependent genome editing in C. neoformans. This high-efficiency method requires two manipulations. First, we placed the sgRNA-expressing segment into the marker-containing DNA flanked by targeting homology; thus, only a single DNA molecule is introduced into cells. Second, we used a strain mutant for the non-homologous end-joining factor Ku80 (encoded by YKU80). We also report the engineering of a yku80::amdS mutant strain harboring an insertion mutation that can be removed scarlessly via recombination between direct repeats. This enables the functional restoration of YKU80 after homology- dependent genome editing via selection against the amdS marker using fluoroacetamide. This approach minimizes documented drawbacks of using Ku-defective strains in downstream experiments. Finally, we describe a plasmid series that enables rapid cloning of sgRNA-marker constructs for genomic manipulation of C. neoformans, including gene deletion and C-terminal tagging. These methods, strains, and plasmids accelerate the genomic manipulation of C. neoformans.

genetics↗

bioRxiv2024

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

Cryptococcus neoformans is the top-ranked W.H.O. fungal priority pathogen, but tools for generating conditional mutations are limited. Auxin-inducible degron systems permit rapid and effective cellular depletion of a tagged protein of interest upon adding a small molecule. These tools are invaluable, particularly for studying essential genes, which may play important roles in pathogen biology. AID2 is one such system that improves on previous strategies. This system achieves greater sensitivity and specificity using a "bumped" auxin, 5-Ph-IAA, alongside an OsTIR1(F74G) "hole" mutant. We adapted the AID2 system for C. neoformans by codon optimizing OsTIR1(F74G) and tested its use in multiple scenarios. We demonstrate that the C. neoformans optimized AID2 system enables effective degradation of proteins, including essential proteins, and can be used to discriminate essential from non-essential genes. This tool enables the study of unexplored parts of the C. neoformans genome.

genetics↗

bioRxiv2023

Candida albicans stimulates the formation of a multi-receptor complex that mediates epithelial cell invasion during oropharyngeal infection

Fungal invasion of the oral epithelium is central to the pathogenesis of oropharyngeal candidiasis (OPC). Candida albicans invades the oral epithelium by receptor-induced endocytosis but this process is incompletely understood. We found that C. albicans infection of oral epithelial cells induces c-Met to form a multi-protein complex with E-cadherin and the epidermal growth factor receptor (EGFR). E-cadherin is necessary for C. albicans to activate both c-Met and EGFR and to induce the endocytosis of C. albicans. Proteomics analysis revealed that c-Met interacts with C. albicans Hyr1, Als3 and Ssa1. Both Hyr1 and Als3 were required for C. albicans stimulation of c-Met and EGFR in oral epithelial cells in vitro and for full virulence during OPC in mice. Treating mice with small molecule inhibitors of c-Met and EGFR ameliorated OPC, demonstrating the potential therapeutic efficacy of blocking these host receptors for C. albicans. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=161 HEIGHT=200 SRC="FIGDIR/small/529756v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1448372org.highwire.dtl.DTLVardef@111b411org.highwire.dtl.DTLVardef@144f3b8org.highwire.dtl.DTLVardef@1dcf548_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIc-Met is an oral epithelial cell receptor for Candida albicans C_LIO_LIC. albicans infection causes c-Met and the epidermal growth factor receptor (EGFR) to form a complex with E-cadherin, which is required for c-Met and EGFR function C_LIO_LIC. albicans Hyr1 and Als3 interact with c-Met and EGFR, inducing oral epithelial cell endocytosis and virulence during oropharyngeal candidiasis C_LIO_LIDual blockade of c-Met and EGFR ameliorates oropharyngeal candidiasis C_LI

microbiology↗

bioRxiv2021

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. Since the S. pyogenes Cas9 derivatives used in prior studies were not optimized for expression in C. neoformans, we designed, synthesized, and tested a fully C. neoformans-optimized Cas9. We found that a Cas9 harboring only common C. neoformans codons and a consensus C. neoformans intron together with a TEF1 promoter and terminator and a nuclear localization signal (C. neoformans-optimized CAS9 or "CnoCAS9") reliably enabled genome editing in the widely-used KN99 C. neoformans strain. Furthermore, editing was accomplished using donors harboring short (50 bp) homology arms attached to marker DNAs produced with synthetic oligonucleotides and PCR amplification. We also demonstrated that prior stable integration of CnoCAS9 further enhances both transformation and homologous recombination efficiency; importantly, this manipulation does not impact virulence in animals. We also implemented a universal tagging module harboring a codon-optimized fluorescent protein (mNeonGreen) and a tandem Calmodulin Binding Peptide-2X FLAG Tag that allows for both localization and purification studies of proteins for which the corresponding genes are modified by short homology-directed recombination. These tools enable short-homology genome engineering in C. neoformans.

microbiology↗