Identification and Characterization of an Intergenic “Safe Haven” Region in Human Fungal Pathogen Cryptococcus gattii

Li, Yeqi; Pham, Tuyetnhu; Xie, Xianan; Lin, Xiaorong

doi:10.3390/jof8020178

Cited by 3 publications

(2 citation statements)

References 46 publications

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“…To integrate the overexpression constructs at loci other than the established SH2 site, additional safe haven sites need to be identified. We used the same procedures to identify the additional safe heaven sites as we described previously ( 73 – 75 ). Briefly, the genomic sequence and annotation files of C. neoformans H99 were downloaded from NCBI (GenBank assembly accession: GCA_000149245.3 ).…”

Section: Methodsmentioning

confidence: 99%

“…Briefly, the genomic sequence and annotation files of C. neoformans H99 were downloaded from NCBI (GenBank assembly accession: GCA_000149245.3 ). We classified the intergenic regions and calculated the sizes of these intergenic regions based on the orientation and positions of two neighboring genes ( 73 – 75 ). BAM (binary alignment map) files and FPKM values were generated using Trim_Galore (0.6.5), STAR (2.7.1a), and Gufflink (2.2.1).…”

Section: Methodsmentioning

confidence: 99%

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

Li,

Chadwick,

Pham

et al. 2024

mBio

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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 CO 2 , we serendipitously discovered that the aspartyl peptidase May1 contributes to cryptococcal sensitivity to high levels of CO 2 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 MAY1 oe 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.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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

Li,

Chadwick,

Pham

et al. 2024

mBio

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Genetic Transformation of Cryptococcus Species with Agrobacterium Transfer DNA

Chong,

Idnurm,

Nugent

2024

Methods in Molecular Biology

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Illuminating the Cryptococcus neoformans species complex: unveiling intracellular structures with fluorescent-protein-based markers

Shi,

Lin

2024

Genetics

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Cryptococcus neoformans is a fungal pathogen of the top critical priority recognized by the World Health Organization. This clinically important fungus also serves as a eukaryotic model organism. A variety of resources have been generated to facilitate investigation of the C. neoformans species complex, including congenic pairs, well-annotated genomes, genetic editing tools, and gene deletion sets. Here, we generated a set of strains with all major organelles fluorescently marked. We tested short organelle-specific targeting sequences and successfully labeled the following organelles by fusing the targeting sequences with a fluorescence protein: the plasma membrane, the nucleus, the peroxisome, and the mitochondrion. We used native cryptococcal Golgi and late endosomal proteins fused with a fluorescent protein to label these two organelles. These fluorescence markers were verified via colocalization using organelle-specific dyes. All the constructs for the fluorescent protein tags were integrated in an intergenic safe haven region. These organelle-marked strains were examined for growth and various phenotypes. We demonstrated that these tagged strains could be employed to track cryptococcal interaction with the host in phagocytosis assays. These strains also allowed us to discover remarkable differences in the dynamics of proteins targeted to different organelles during sexual reproduction. Additionally, we revealed that “dormant” spores transcribed and synthesized their own proteins and trafficked the proteins to the appropriate subcellular compartments, demonstrating that spores are metabolically active. We anticipate that these newly generated fluorescent markers will greatly facilitate further investigation of cryptococcal biology and pathogenesis.

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Identification and Characterization of an Intergenic “Safe Haven” Region in Human Fungal Pathogen Cryptococcus gattii

Cited by 3 publications

References 46 publications

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

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

Genetic Transformation of Cryptococcus Species with Agrobacterium Transfer DNA

Illuminating the Cryptococcus neoformans species complex: unveiling intracellular structures with fluorescent-protein-based markers

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