Search Results (6)

Cryptococcus neoformans is a facultative intracellular fungal pathogen. Ten-generation-old (10GEN) C. neoformans cells are more resistant to phagocytosis and killing by macrophages than younger daughter cells. However, mechanisms that mediate this resistance and intracellular parasitism are poorly understood. Here, we identified important factors for the intracellular survival of 10GEN C. neoformans, such as urease activity, capsule synthesis, and DNA content using flow cytometry and fluorescent microscopy techniques. The real-time visualization of time-lapse imaging was applied to determine the phagosomal acidity, membrane permeability, and vomocytosis (non-lytic exocytosis) rate in J774 macrophages that phagocytosed C. neoformans of different generational ages. Our results showed that old C. neoformans exhibited higher urease activity and enhanced Golgi activity. In addition, old C. neoformans were more likely to be arrested in the G2 phase, resulting in the occasional formation of aberrant trimera-like cells. To finish, the advanced generational age of the yeast cells slightly reduced vomocytosis events within host cells, which might be associated with increased phagolysosome pH and membrane permeability. Altogether, our results suggest that old C. neoformans prevail within acidic phagolysosomes and can manipulate the phagosome pH. These strategies may be used by old C. neoformans to resist phagosomal killing and drive cryptococcosis pathogenesis. The comprehension of these essential host–pathogen interactions could further shed light on mechanisms that bring new insights for novel antifungal therapeutic design. Full article

Cryptococcus neoformans causes meningoencephalitis in immunocompromised individuals, which is treated with fluconazole (FLC) monotherapy when resources are limited. This can lead to azole resistance, which can be mediated by overexpression of ABC transporters, a class of efflux pumps. ABC pump-mediated efflux of FLC is also augmented in 10-generation old C. neoformans cells. Here, we describe a new ABC transporter Afr3 (CNAG_06909), which is overexpressed in C. neoformans cells of advanced generational age that accumulate during chronic infection. The Δafr3 mutant strain showed higher FLC susceptibility by FLC E-Test strip testing and also by a killing test that measured survival after 3 h FLC exposure. Furthermore, Δafr3 cells exhibited lower Rhodamine 6G efflux compared to the H99 wild-type cells. Afr3 was expressed in the Saccharomyces cerevisiae ADΔ strain, which lacks several drug transporters, thus reducing background transport. The ADΔ + Afr3 strain demonstrated a higher efflux with both Rhodamine 6G and Nile red, and a higher FLC resistance. Afr3-GFP localized in the plasma membrane of the ADΔ + Afr3 strain, further highlighting its importance as an efflux pump. Characterization of the Δafr3 mutant revealed unattenuated growth but a prolongation (29%) of the replicative life span. In addition, Δafr3 exhibited decreased resistance to macrophage killing and attenuated virulence in the Galleria mellonella infection model. In summary, our data indicate that a novel ABC pump Afr3, which is upregulated in C. neoformans cells of advanced age, may contribute to their enhanced FLC tolerance, by promoting drug efflux. Lastly, its role in macrophage resistance may also contribute to the selection of older C. neoformans cells during chronic infection. Full article

Chronic meningoencephalitis is caused by Cryptococcus neoformans and is treated in many parts of the world with fluconazole (FLC) monotherapy, which is associated with treatment failure and poor outcome. In the host, C. neoformans propagates predominantly under low glucose growth conditions. We investigated whether low glucose, mimicked by growing in synthetic media (SM) with 0.05% glucose (SM^lowglu), affects FLC-resistance. A > 4-fold increase in FLC tolerance was observed in seven C. neoformans strains when minimum inhibitory concentration (MIC) was determined in SM^lowglu compared to MIC in SM with normal (2%) glucose (SM^nlglu). In SM^lowglu, C. neoformans cells exhibited upregulation of efflux pump genes AFR1 (8.7-fold) and AFR2 (2.5-fold), as well as decreased accumulation (2.6-fold) of Nile Red, an efflux pump substrate. Elevated intracellular ATP levels (3.2-fold and 3.4-fold), as well as decreased mitochondrial reactive oxygen species levels (12.8-fold and 17-fold), were found in the presence and absence of FLC, indicating that low glucose altered mitochondrial function. Fluorescence microscopy revealed that mitochondria of C. neoformans grown in SM^lowglu were fragmented, whereas normal glucose promoted a reticular network of mitochondria. Although mitochondrial membrane potential (MMP) was not markedly affected in SM^lowglu, it significantly decreased in the presence of FLC (12.5-fold) in SM^nlglu, but remained stable in SM^lowglu-growing C. neoformans cells. Our data demonstrate that increased FLC tolerance in low glucose-growing C. neoformans is the result of increased efflux pump activities and altered mitochondrial function, which is more preserved in SM^lowglu. This mechanism of resistance is different from FLC heteroresistance, which is associated with aneuploidy of chromosome 1 (Chr1). Full article

Candida albicans, Candida auris, Candida glabrata, and Cryptococcus neoformans are pathogenic yeasts which can cause systemic infections in immune-compromised as well as immune-competent individuals. These yeasts undergo replicative aging analogous to a process first described in the nonpathogenic yeast Saccharomyces cerevisiae. The hallmark of replicative aging is the asymmetric cell division of mother yeast cells that leads to the production of a phenotypically distinct daughter cell. Several techniques to study aging that have been pioneered in S. cerevisiae have been adapted to study aging in other pathogenic yeasts. The studies indicate that aging is relevant for virulence in pathogenic fungi. As the mother yeast cell progressively ages, every ensuing asymmetric cell division leads to striking phenotypic changes, which results in increased antifungal and antiphagocytic resistance. This review summarizes the various techniques that are used to study replicative aging in pathogenic fungi along with their limitations. Additionally, the review summarizes some key phenotypic variations that have been identified and are associated with changes in virulence or resistance and thus promote persistence of older cells. Full article

Candidiasis can be present as a cutaneous, mucosal or deep-seated organ infection, which is caused by more than 20 types of Candida sp., with C. albicans being the most common. These are pathogenic yeast and are usually present in the normal microbiome. High-risk individuals are patients of human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS), organ transplant, and diabetes. During infection, pathogens can adhere to complement receptors and various extracellular matrix proteins in the oral and vaginal cavity. Oral and vaginal Candidiasis results from the overgrowth of Candida sp. in the hosts, causing penetration of the oral and vaginal tissues. Symptoms include white patches in the mouth, tongue, throat, and itchiness or burning of genitalia. Diagnosis involves visual examination, microscopic analysis, or culturing. These infections are treated with a variety of antifungals that target different biosynthetic pathways of the pathogen. For example, echinochandins target cell wall biosynthesis, while allylamines, azoles, and morpholines target ergosterol biosynthesis, and 5-Flucytosine (5FC) targets nucleic acid biosynthesis. Azoles are commonly used in therapeutics, however, because of its fungistatic nature, Candida sp. evolve azole resistance. Besides azoles, Candida sp. also acquire resistance to polyenes, echinochandins, and 5FC. This review discusses, in detail, the drug resistance mechanisms adapted by Candida sp. Full article

Cryptococcus neoformans successfully replicates in low glucose in infected patients. In the serotype A strain, H99, growth in this condition prolongs lifespan regulated by SIR2, and can be modulated with SIR2-specific drugs. Previous studies show that lifespan modulation of a cryptococcal population affects its sensitivity to antifungals, and survival in an infection model. Sirtuins and their role in longevity are conserved among fungi; however, the effect of glucose starvation is not confirmed even in Saccharomyces cerevisiae. Lifespan analysis of C. neoformans strains in low glucose showed that 37.5% exhibited pro-longevity, and lifespan of a serotype D strain, RC2, was shortened. Transcriptome comparison of H99 and RC2 under calorie restriction demonstrated differences, confirmed by real-time PCR showing that SIR2, TOR1, SCH9, and PKA1 expression correlated with lifespan response to calorie restriction. As expected, RC2-sir2Δ cells exhibited a shortened lifespan, which was reconstituted. However, shortened lifespan from calorie restriction was independent of SIR2. In contrast to H99 but consistent with altered SIR2 regulation, SIR2-specific drugs did not affect outcome of RC2 infection. These data suggest that SIR2 regulation and response to calorie restriction varies in C. neoformans, which should be considered when Sirtuins are investigated as potential therapy targets for fungal infections. Full article