Dataset Information

Iron Metabolism, Pseudohypha Production, and Biofilm Formation through a Multicopper Oxidase in the Human-Pathogenic Fungus Candida parapsilosis.

ABSTRACT: Among all the essential micronutrients, iron plays an important role in mammalian biology. It is also essential for pathogens infecting mammalian hosts, including bacteria, fungi, and protozoans. As the availability of accessible iron is limited within the mammalian host, several human-pathogenic fungal pathogens, such as Candida albicans, Cryptococcus neoformans, Candida glabrata, and Aspergillus fumigatus, have developed various iron uptake mechanisms. Although Candida parapsilosis is the second or third most common non-albicans Candida species associated with systemic and superficial Candida infections in immunocompromised patients, the mechanisms of iron uptake and homoeostasis remain unknown in this fungus. In the current report, we show that a homologue of the multicopper oxidase gene FET3 is present in the genome of C. parapsilosis (CPAR2_603600) and plays a significant role in iron acquisition. We found that homozygous deletion mutants of CPAR2_603600 showed defects under low-iron conditions and were also sensitive to various stressors. Our results also revealed that the levels of pseudohypha formation and biofilm formation were reduced in the null mutants compared to the wild type. This phenotypic defect could be partially rescued by supplementation with excess iron in the growth medium. The expression levels of the orthologues of various iron metabolism-related genes were also altered in the mutants compared to the parental strain. In conclusion, our report describes the role of CPAR2_603600 in iron homoeostasis maintenance as well as morphology and biofilm formation regulation in this pathogenic fungus.IMPORTANCE C. parapsilosis is the second or third most common opportunistic human-pathogenic Candida species, being responsible for severe fungal infections among immunocompromised patients, especially low-birth-weight infants (0 to 2 years of age). Among the major virulence factors that pathogenic fungi possess is the ability to compete with the host for essential micronutrients, including iron. Accessible iron is required for the maintenance of several metabolic processes. In order to obtain accessible iron from the host, pathogenic fungi have developed several iron acquisition and metabolic mechanisms. Although C. parapsilosis is a frequent cause of invasive candidiasis, little is known about what iron metabolic processes this fungus possesses that could contribute to the species' virulent behavior. In this study, we identified the multicopper oxidase FET3 gene that regulates iron homeostasis maintenance and also plays important roles in the morphology of the fungus as well as in biofilm formation, two additional factors in fungal virulence.

SUBMITTER: Chakraborty T

PROVIDER: S-EPMC7227767 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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Iron Metabolism, Pseudohypha Production, and Biofilm Formation through a Multicopper Oxidase in the Human-Pathogenic Fungus Candida parapsilosis.

Chakraborty Tanmoy T Tóth Zsófia Z Tóth Renáta R Vágvölgyi Csaba C Gácser Attila A

mSphere 20200513 3

Among all the essential micronutrients, iron plays an important role in mammalian biology. It is also essential for pathogens infecting mammalian hosts, including bacteria, fungi, and protozoans. As the availability of accessible iron is limited within the mammalian host, several human-pathogenic fungal pathogens, such as Candida albicans, Cryptococcus neoformans, Candida glabrata, and Aspergillus fumigatus, have developed various iron uptake mechanisms. Although C ...[more]

PMID: 32404511

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Project description:Fungi can use a wide variety of nitrogen sources. In the absence of preferred sources such as ammonium, glutamate, and glutamine, secondary sources, including most other amino acids, are used. Expression of the nitrogen utilization pathways is very strongly controlled at the transcriptional level. Here, we investigated the regulation of nitrogen utilization in the pathogenic yeast Candida parapsilosis. We found that the functions of many regulators are conserved with respect to Saccharomyces cerevisiae and other fungi. For example, the core GATA activators GAT1 and GLN3 have a conserved role in nitrogen catabolite repression (NCR). There is one ortholog of GZF3 and DAL80, which represses expression of genes in preferred nitrogen sources. The regulators PUT3 and UGA3 are required for metabolism of proline and ?-aminobutyric acid (GABA), respectively. However, the role of the Dal81 transcription factor is distinctly different. In S. cerevisiae, Dal81 is a positive regulator of acquisition of nitrogen from GABA, allantoin, urea, and leucine, and it is required for maximal induction of expression of the relevant pathway genes. In C. parapsilosis, induction of GABA genes is independent of Dal81, and deleting DAL81 has no effect on acquisition of nitrogen from GABA or allantoin. Instead, Dal81 represses arginine synthesis during growth under preferred nitrogen conditions. IMPORTANCE Utilization of nitrogen by fungi is controlled by nitrogen catabolite repression (NCR). Expression of many genes is switched off during growth on nonpreferred nitrogen sources. Gene expression is regulated through a combination of activation and repression. Nitrogen regulation has been studied best in the model yeast Saccharomyces cerevisiae. We found that although many nitrogen regulators have a conserved function in Saccharomyces species, some do not. The Dal81 transcriptional regulator has distinctly different functions in S. cerevisiae and C. parapsilosis. In the former, it regulates utilization of nitrogen from GABA and allantoin, whereas in the latter, it regulates expression of arginine synthesis genes. Our findings make an important contribution to our understanding of nitrogen regulation in a human-pathogenic fungus.

Project description:Genetic manipulation is often used to study gene function. However, unplanned genome changes (including single nucleotide polymorphisms [SNPs], aneuploidy, and loss of heterozygosity [LOH]) can affect the phenotypic traits of the engineered strains. Here, we compared the effect of classical deletion methods (replacing target alleles with selectable markers by homologous recombination) with CRISPR-Cas9 editing in the diploid human-pathogenic yeast Candida parapsilosis. We sequenced the genomes of 9 isolates that were modified using classic recombination methods and 12 that were edited using CRISPR-Cas9. As a control, the genomes of eight isolates that were transformed with a Cas9-expressing plasmid in the absence of a guide RNA were also sequenced. Following gene manipulation using classic homologous recombination, only one strain exhibited extensive LOH near the targeted gene (8.9 kb), whereas another contained multiple LOH events not associated with the intended modification. In contrast, large regions of LOH (up to >1,100 kb) were observed in most CRISPR-Cas9-edited strains. LOH most commonly occurred adjacent to the Cas9 cut site and extended to the telomere in four isolates. In two isolates, we observed LOH on chromosomes that were not targeted by CRISPR-Cas9. Among the CRISPR-edited isolates, two exhibited cysteine and methionine auxotrophy caused by LOH at a heterozygous site in MET10, approximately 11 and 157 kb downstream from the Cas9 target site, respectively. C. parapsilosis isolates have relatively low levels of heterozygosity. However, our results show that mutation complementation to confirm observed phenotypes is required when using CRISPR-Cas9. IMPORTANCE CRISPR-Cas9 has greatly streamlined gene editing and is now the gold standard and first choice for genetic engineering. However, we show that in diploid species, extra care should be taken in confirming the cause of any phenotypic changes observed. We show that the Cas9-induced double-strand break is often associated with loss of heterozygosity in the asexual diploid human fungal pathogen Candida parapsilosis. This can result in deleterious heterozygous variants (e.g., stop gain in one allele) becoming homozygous, resulting in unplanned phenotypic changes. Our results stress the importance of mutation complementation even when using CRISPR-Cas9.

Dataset Information

Iron Metabolism, Pseudohypha Production, and Biofilm Formation through a Multicopper Oxidase in the Human-Pathogenic Fungus Candida parapsilosis.

Publications

Iron Metabolism, Pseudohypha Production, and Biofilm Formation through a Multicopper Oxidase in the Human-Pathogenic Fungus Candida parapsilosis.

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