Browsing by Subject "Mycoses"

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    Aerobic Uptake of Cholesterol by Ergosterol Auxotrophic Strains in Candida glabrata & Random and Site-Directed Mutagenesis of ERG25 in Saccharomyces cerevisiae
    (2012-09-27) Whybrew, Jennafer Marie; Bard, Martin; Lees, N. Douglas; Blacklock, Brenda
    Candida albicans and Candida glabrata are opportunistic human pathogens that are the leading cause of fungal infections, which are increasingly becoming the leading cause of sepsis in immunosuppressed individuals. C. glabrata in particular has become a significant concern due to the increase in clinical isolates that demonstrate resistance to triazole antifungal drugs, the most prevalent treatment for such infections. Triazole drugs target the ERG11 gene product and prevent C-14 demethylation of the first sterol intermediate, lanosterol, preventing the production of the pathways end product ergosterol. Ergosterol is required by yeast for cell membrane fluidity and cell signaling. Furthermore, C. glabrata, and not C. albicans, has been reported to utilize cholesterol as a supplement for growth. Although drug resistance is known to be caused by an increase in expression of drug efflux pumps, we hypothesize a second mechanism: that the overuse of triazole drugs has lead to the increase of resistance by C. glabrata through a 2-step process: 1) the accumulation of ergosterol auxotrophic mutations and 2) mutants able to take up exogenous cholesterol anaerobically in the body acquire a second mutation allowing uptake of cholesterol aerobically. Two groups of sterol auxotrophic C. glabrata clinical isolates have been reported to take up sterol aerobically but do not produce a sterol precursor. Sterol auxotrophs have been created in C. glabrata by disrupting different essential genes (ERG1, ERG7, ERG11, ERG25, and ERG27) in the ergosterol pathway to assess which ergosterol mutants will take up sterols aerobically. Random and site-directed mutagenesis was also completed in ERG25 of Saccharmoyces cerevisiae. The ERG25 gene encodes a sterol C-4 methyloxidase essential for sterol biosynthesis in plants, animals, and yeast. This gene functions in turn with ERG26, a sterol C-3 dehydrogenase, and ERG27, a sterol C-3 keto reductase, to remove two methyl groups at the C-4 position on the sterol A ring. In S. cerevisiae, ERG25 has four putative histidine clusters, which bind non-heme iron and a C-terminal KKXX motif, which is a Golgi to ER retrieval motif. We have conducted site-directed and random mutagenesis in the S. cerevisiae wild-type strain SCY876. Site-Directed mutagenesis focused on the four histidine clusters, the KKXX C-terminal motif and other conserved amino acids among various plant, animal, and fungal species. Random mutagenesis was completed with a procedure known as gap repair and was used in an effort to find novel changes in enzyme function outside of the parameters utilized for site-directed mutagenesis. The four putative histidine clusters are expected to be essential for gene function by acting as non-heme iron binding ligands bringing in the oxygen required for the oxidation-reduction in the C-4 demethylation reaction.
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    Kazachstania slooffiae Fungemia: A Case Report and Literature Review on an Emerging Opportunistic Pathogen in Humans
    (Oxford University Press, 2025-04-04) Fortna, Sarah N.; Aronoff, David M.; Dysangco, Andrew T.; Medicine, School of Medicine
    Background: We report a case of Kazachstania slooffiae fungemia in a 77-year-old immunocompromised male with gastrointestinal abnormalities including achalasia, gastroparesis, and prior esophagectomy. He presented with sepsis, gastric ischemia, and pleural effusion. To date, only 2 prior cases of Kazachstania slooffiae infection have been reported, both involving nonbloodstream infections. Methods: We reviewed the existing literature and compared clinical, diagnostic, and treatment characteristics across all identified cases. Results: Kazachstania slooffiae has been identified in critically ill, immunocompromised patients with gastrointestinal anatomic abnormalities, underlying malignancy, and disease. Diagnosis relies on advanced methods such as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and DNA sequencing. Antifungal treatments (caspofungin, fluconazole, micafungin) have resulted in favorable outcomes. Conclusions: This review highlights Kazachstania slooffiae as a rare opportunistic pathogen and provides the first evidence for Kazachstania slooffiae as a bloodstream pathogen in humans. Prompt identification and antifungal therapy are critical for managing infections.