Isolation and identification of yeasts from UTI patient's exhibit susceptibility to Agaricus gennadii extract.

Antifungal resistance represents a major clinical challenge to clinicians responsible for treating invasive fungal infections due to the limited arsenal of systemically available antifungal agents. Currently, fungal invasive infections are related to antifungal resistance by pathogenic fungi. The current study assumed to use of natural ingredients such as the fruiting bodies of large fungi, specifically Agaricus gennadii. This study is also a new initial step in the field of producing an antifungal that has a promising and safe future. However, most of the used antifungals such as polyenes, azoles, echinocandins, and flucytosine have side effects and may lead to damage to the human body. To achieve the goal of this study, 120 mid-stream urine samples were collected from urinary tract infection (UTI) patients, who attended Samarra General Hospital, the primary care sector and some medical clinics in the city of Samarra/Salah al-Din during the period from 11-1-2020 to 1-2-2021. Many laboratory examinations were performed including: microbiological, biochemical, and molecular tests of 70 samples of UTI patients, which develop yeast colonies on culture plates and were considered as positive results. Our results showed that the extract of the Agaricus gennadii fruit bodies contained a number of organic compounds including phenolics, flavonoids, saponins, terpenoids and alkaloids. Regarding the susceptibility of the isolated yeast species, many concentrations (100%, 75%, 50% and 25%) of the fungal extract were investigated. Data analysis of the obtained results showed that among all tested yeasts, Trichosporon mucoides and Candida parapsilosis were susceptible to the fungal extract at all concentrations, however, no effect of the fungal extract on the rest of the studied yeasts. Also, our results demonstrated that the susceptibility was increased with the increase of the fungal extract concentration. More studies are needed to separate and test the exact role of these compounds in the inhibition of fungal growth.

Characterisation of Aspergillus fumigatus Endocytic Trafficking within Airway Epithelial Cells Using High-Resolution Automated Quantitative Confocal Microscopy.

The precise characterization of the mechanisms modulating Aspergillus fumigatus survival within airway epithelial cells has been impaired by the lack of live-cell imaging technologies and user-friendly quantification approaches. Here we described the use of an automated image analysis pipeline to estimate the proportion of A. fumigatus spores taken up by airway epithelial cells, those contained within phagolysosomes or acidified phagosomes, along with the fungal factors contributing to these processes. Coupling the use of fluorescent A. fumigatus strains and fluorescent epithelial probes targeting lysosomes, acidified compartments and cell membrane, we found that both the efficacy of lysosome recruitment to phagosomes and phagosome acidification determines the capacity of airway epithelial cells to contain A. fumigatus growth. Overall, the capability of the airway epithelium to prevent A. fumigatus survival was higher in bronchial epithelial than alveolar epithelial cells. Certain A. fumigatus cell wall mutants influenced phagosome maturation in airway epithelial cells. Taken together, this live-cell 4D imaging approach allows observation and measurement of the very early processes of A. fumigatus interaction within live airway epithelial monolayers.

Phagolysosomal Survival Enables Non-lytic Hyphal Escape and Ramification Through Lung Epithelium During Aspergillus fumigatus Infection.

Aspergillus fumigatus is the most important mould pathogen in immunosuppressed patients. Suboptimal clearance of inhaled spores results in the colonisation of the lung airways by invasive hyphae. The first point of contact between A. fumigatus and the host is the lung epithelium. In vitro and ex vivo studies have characterised critical aspects of the interaction of invasive hyphae on the surface of epithelial cells. However, the cellular interplay between internalised A. fumigatus and the lung epithelium remains largely unexplored. Here, we use high-resolution live-cell confocal microscopy, 3D rendered imaging and transmission electron microscopy to define the development of A. fumigatus after lung epithelium internalisation in vitro. Germination, morphology and growth of A. fumigatus were significantly impaired upon internalisation by alveolar (A549) and bronchial (16HBE) lung epithelial cells compared to those growing on the host surface. Internalised spores and germlings were surrounded by the host phagolysosome membrane. Sixty per cent of the phagosomes containing germlings were not acidified at 24 h post infection allowing hyphal development. During escape, the phagolysosomal membrane was not ruptured but likely fused to host plasma membrane allowing hyphal exit from the intact host cell in an non-lytic Manner. Subsequently, escaping hyphae elongated between or through adjacent epithelial lung cells without penetration of the host cytoplasm. Hyphal tips penetrating new epithelial cells were surrounded by the recipient cell plasma membrane. Altogether, our results suggest cells of lung epithelium survive fungal penetration because the phagolysosomal and plasma membranes are never breached and that conversely, fungal spores survive due to phagosome maturation failure. Consequently, fungal hyphae can grow through the epithelial cell layer without directly damaging the host. These processes likely prevent the activation of downstream immune responses alongside limiting the access of professional phagocytes to the invading fungal hypha. Further research is needed to investigate if these events also occur during penetration of fungi in endothelial cells, fibroblasts and other cell types.

Lung colonization by Aspergillus fumigatus is controlled by ZNF77.

Aspergillus fumigatus is a critical pathogen of humans. Exposure to A. fumigatus conidia occurs frequently but is normally cleared from the respiratory airways. In contrast, individuals with respiratory diseases are often highly colonized by fungi. Here, we use genome-edited epithelial cells to show that the genetic variant rs35699176 in ZNF77 causes loss of integrity of the bronchial epithelium and increases levels of extracellular matrix proteins. These changes promote A. fumigatus conidial adhesion, germination and growth. RNA-seq and LC/MS-MS analysis reveal rs35699176 upregulates vesicle trafficking leading to an increment of adhesion proteins. These changes make cells carrying rs35699176 more receptive to A. fumigatus in the early stages of infection. Moreover, patients with fungal asthma carrying rs35699176+/- have higher A. fumigatus loads in their respiratory airway. Our results indicate ZNF77 as a key controller of Aspergillus colonization and suggest its utility as a risk-marker for patient stratification.

Deciphering the mechanism of action of 089, a compound impairing the fungal cell cycle.

Fungal infections represent an increasingly relevant clinical problem, primarily because of the increased survival of severely immune-compromised patients. Despite the availability of active and selective drugs and of well-established prophylaxis, classical antifungals are often ineffective as resistance is frequently observed. The quest for anti-fungal drugs with novel mechanisms of action is thus important. Here we show that a new compound, 089, acts by arresting fungal cells in the G2 phase of the cell cycle through targeting of SWE1, a mechanism of action unexploited by current anti-fungal drugs. The cell cycle impairment also induces a modification of fungal cell morphology which makes fungal cells recognizable by immune cells. This new class of molecules holds promise to be a valuable source of novel antifungals, allowing the clearance of pathogenic fungi by both direct killing of the fungus and enhancing the recognition of the pathogen by the host immune system.