Effects of Hsp90 Inhibitor Ganetespib on Inhibition of Azole-Resistant Candida albicans

Candida albicans is the most common fungal pathogen. Recently, drug resistance of C. albicans is increasingly severe. Hsp90 is a promising antifungal target to overcome this problem. To evaluate the effects of Hsp90 inhibitor ganetespib on the inhibition of azole-resistant C. albicans, the microdilution checkerboard method was used to measure the in vitro synergistic efficacy of ganetespib. The XTT/menadione reduction assay, microscopic observation, and Rh6G efflux assay were established to investigate the effects of ganetespib on azole-resistant C. albicans biofilm formation, filamentation, and efflux pump. Real-time RT-PCR analysis was employed to clarify the mechanism of antagonizing drug resistance. The in vivo antifungal efficacy of ganetespib was determined by the infectious model of azole-resistant C. albicans. Ganetespib showed an excellent synergistic antifungal activity in vitro and significantly inhibited the fungal biofilm formation, whereas it had no inhibitory effect on fungal hypha formation. Expression of azole-targeting enzyme gene ERG11 and efflux pump genes CDR1, CDR2, and MDR1 was significantly down-regulated when ganetespib was used in combination with FLC. In a mouse model infected with FLC-resistant C. albicans, the combination of ganetespib and FLC effectively reversed the FLC resistance and significantly decreased the kidney fungal load of mouse.

Diversity of Endo and Exo-Bacteria Associated With Fungi Isolated From Plant Rhizospheres: A Pilot Study

Background: Over the past several years, the scientific community has described the diversity of microbial communities in a variety of soils associated with plants, but at present, little is known about the specific diversity of the soil fungal microbiome involving bacteria colonizing the surface of fungi (i.e., exo-bacteria) or existing within fungal hypha (i.e., endobacteria). This study aimed to collect, identify, and characterize several fungi and their associated (endo- and exo-) microbiome obtained from the rhizosphere of six different plants. Microcosm devices called fungal highway columns, containing one of four plant-based media as attractants, were placed in the rhizosphere of six different plants. The isolated fungi and their associated endo- and exo- bacteria were identified by sequencing of the ITS (fungi) or 16S (bacteria) rRNA regions, followed by Scanning Electron Microscope (SEM), and fluorescence in situ hybridization (FISH) imaging.Results: Most of the fungi recovered are known plant pathogens, such as Fusarium, Pleosporales, and Cladosporium together with species associated with the soil, e.g. Kalmusia. The exo-bacteria recovered were previously described as plant promoters, such as Bacillus, Rhizobium, Acinetobacter or Ensifer. The interactions between fungi and exo-bacteria recovered from fungal highway columns were further investigated via confrontation assays. From the reconstruction of the potential co-occurring bacterial-fungal associations in the rhizosphere, we discovered that the most promiscuous exo-bacterium group (associated with diverse fungi) was Bacillus. From the study of the endobacterial community, emerged a core of shared endosymbionts with a potential implication in the nitrogen cycle.Conclusions: The present study demonstrated the importance of selecting and studying cultivable fungi and bacteria from the rhizosphere. Our findings demonstrated that at the rhizosphere level, the range of interactions between fungi and bacteria, both internal and external to the fungal hypha, could vary even among closely related species.

Nanoscale STXM imaging of soil fungal exudates and organo-mineral interfaces

<p>Soils act as a major sink for atmospheric carbon (C) and, correctly managed, can help counterbalance the excessive CO<sub>2</sub> emissions. Organic C in soils can be physically stabilized and ‘hidden’ from its decomposers within soil aggregates and it is thought that soil fungi play a decisive role in “gluing together” and redistributing soil mineral particles and existing organic matter to form them (M. W. I. Schmidt et al., Nature 478(7367), 49–56, 2011). A significant contribution to the early aggregation process is adsorption of fungal exudates to the reactive surfaces of mineral particles. To uncover the mechanisms of C stabilization processes and to be able to increase the C sink potential of our soils, we need a deepened understanding of which fungi play key roles in the process, what mineral properties promote it, and what type of fungal exudates are involved.</p><p>For this purpose, we have grown saprotrophic and symbiotic (both arbuscular mycorrhizal (AM) and ectomycorrhizal (EM)) fungi under sterile conditions in contact with different principal soil components: quartz, goethite and muscovite, on top of X-ray transparent silicon nitride membrane windows and analyzed fungal hyphae by high lateral resolution synchrotron based scanning transmission X-ray microscopy (STXM) in combination with near edge X-ray fine structure (NEXAFS) spectroscopy at absorption edges of C(K), K(L), N(K) and Fe(L). We performed our experiments in the SM beamline at Canadian Light Source, Saskatoon, Canada and I08 beamline at Diamond Light Source, Oxfordshire, UK. In the resultant chemical images, we were able to differentiate the mostly proteinaceous hyphal material, the exudate layer constituting of mixtures of polysaccharides and proteins, and the organo-mineral interfaces consisting of a higher protein and carboxyl to sugar ratio than in the exudate layer. We also observed heterogeneous distributions of the exudate materials around the fungal hypha, indicating presence of exudation channels in the cell wall. Finally, we specifically analyzed NEXAFS spectra at Fe(L) absorption edge of goethite containing samples and were able to show changes in iron speciation in the mineral particles that were in contact with the fungal exudates. These results provide us with better insights to both nanoscale processes of fungal exudation and their role in the formation of organo-mineral interfaces subsequently responsible for soil aggregation.</p>

Biofilm Interactions of Candida albicans and Mitis Group Streptococci in a Titanium-Mucosal Interface Model

ABSTRACT Streptococci from the mitis group (represented mainly by Streptococcus mitis, Streptococcus oralis, Streptococcus sanguinis, and Streptococcus gordonii) form robust biofilms with Candida albicans in different experimental models. These microorganisms have been found in polymicrobial biofilms forming on titanium biomaterial surfaces in humans with peri-implant disease. The purpose of this work was to study mutualistic interactions in biofilms forming on titanium and their effect on the adjacent mucosa, using a relevant infection model. Single and mixed biofilms of C. albicans and each Streptococcus species were grown on titanium disks. Bacterial and fungal biovolume and biomass were quantified in these biofilms. Organotypic mucosal constructs were exposed to preformed titanium surface biofilms to test their effect on secretion of proinflammatory cytokines and cell damage. C. albicans promoted bacterial biofilms of all mitis Streptococcus species on titanium surfaces. This relationship was mutualistic since all bacterial species upregulated the efg1 hypha-associated gene in C. albicans. Mixed biofilms caused increased tissue damage but did not increase proinflammatory cytokine responses compared to biofilms comprising Candida alone. Interestingly, spent culture medium from tissues exposed to titanium biofilms suppressed Candida growth on titanium surfaces. IMPORTANCE Our findings provide new insights into the cross-kingdom interaction between C. albicans and Streptococcus species representative of the mitis group. These microorganisms colonize titanium-based dental implant materials, but little is known about their ability to cause inflammation and damage of the adjacent mucosal tissues. Using an in vitro biomaterial-mucosal interface infection model, we showed that mixed biofilms of each species with C. albicans enhance tissue damage. One possible mechanism for this effect is the increased fungal hypha-associated virulence gene expression we observed in mixed biofilms with these species. Interestingly, we also found that the interaction of multispecies biofilms with organotypic mucosal surfaces led to the release of growth-suppressing mediators of Candida, which may represent a homeostatic defense mechanism of the oral mucosa against fungal overgrowth. Thus, our findings provide novel insights into biofilms on biomaterials that may play an important role in the pathogenesis of mucosal infections around titanium implants.

Isolasi, Identifikasi dan Persentase Keberadaan Hifa Jamur Endofit pada Tanaman Gemitir (Tagetes erecta L.) di Beberapa Daerah di Bali

Endophytic fungi are found in roots, stems, leaves, flowers, fruit, until rhizosphere in soil area, where the fungus is widely used as an antifungal, biostimulan, antibacterial, and many more. Gemitir is one plant which widely cultivated in some areas in Bali. In Bali this plants use as a material for upakara, ornamental plants and herbs. This study was conducted to determine the type and different kind of endophytic fungi and to see the percentage of endophytic fungal hyphae in the roots, stems, and leaves on gemitir which taken at different locations. Sampling was taken at the Pangsan-Badung, Angseri-Tabanan Regency and Temesi-Gianyar. The sampling method which used is the diagonal method with five sample points. Isolation and identification process was carried out in the Laboratory of Plant Taxonomy (Mycology), State University of Udayana in January-March 2017. In this study of 15 species successfully identified to the genus level that belongs to the six genera namely Alternaria, Aspergillus, Botrytis, Trichoderma, Penicillium and Syncepalastrum. Total of fungal colonies in most samples were found at research location A (Pangsan-Badung Village) and percentage of highest endophytic fungal hypha existed at location C (Temesi-Gianyar Village).