Widespread bacterial diversity within the bacteriome of fungi.

Knowledge of associations between fungal hosts and their bacterial associates has steadily grown in recent years as the number and diversity of examinations have increased, but current knowledge is predominantly limited to a small number of fungal taxa and bacterial partners. Here, we screened for potential bacterial associates in over 700 phylogenetically diverse fungal isolates, representing 366 genera, or a tenfold increase compared with previously examined fungal genera, including isolates from several previously unexplored phyla. Both a 16 S rDNA-based exploration of fungal isolates from four distinct culture collections spanning North America, South America and Europe, and a bioinformatic screen for bacterial-specific sequences within fungal genome sequencing projects, revealed that a surprisingly diverse array of bacterial associates are frequently found in otherwise axenic fungal cultures. We demonstrate that bacterial associations with diverse fungal hosts appear to be the rule, rather than the exception, and deserve increased consideration in microbiome studies and in examinations of microbial interactions.

Democratization of fungal highway columns as a tool to investigate bacteria associated with soil fungi.

Bacteria-fungi interactions (BFIs) are essential in ecosystem functioning. These interactions are modulated not only by local nutritional conditions but also by the physicochemical constraints and 3D structure of the environmental niche. In soils, the unsaturated and complex nature of the substrate restricts the dispersal and activity of bacteria. Under unsaturated conditions, some bacteria engage with filamentous fungi in an interaction (fungal highways) in which they use fungal hyphae to disperse. Based on a previous experimental device to enrich pairs of organisms engaging in this interaction in soils, we present here the design and validation of a modified version of this sampling system constructed using additive printing. The 3D printed devices were tested using a novel application in which a target fungus, the common coprophilous fungus Coprinopsis cinerea, was used as bait to recruit and identify bacterial partners using its mycelium for dispersal. Bacteria of the genera Pseudomonas, Sphingobacterium and Stenotrophomonas were highly enriched in association with C. cinerea. Developing and producing these new easy-to-use tools to investigate how bacteria overcome dispersal limitations in cooperation with fungi is important to unravel the mechanisms by which BFIs affect processes at an ecosystem scale in soils and other unsaturated environments.

Effect of Organic Carbon and Nitrogen on the Interactions of Morchella spp. and Bacteria Dispersing on Their Mycelium.

In this study we investigated how the source of organic carbon (Corg) and nitrogen (Norg) affects the interactions between fungi of the genus Morchella and bacteria dispersing along their hyphae (fungal highways; FH). We demonstrated that bacteria using FH increase the hydrolysis of an organic nitrogen source that only the fungus can degrade. Using purified fungal exudates, we found that this increased hydrolysis was due to bacteria enhancing the activity of proteolytic enzymes produced by the fungus. The same effect was shown for various fungal and bacterial strains. The effect of this enhanced proteolytic activity on bacterial and fungal biomass production varied accordingly to the source of Corg and Norg provided. An increase in biomass for both partners 5 days post-inoculation was only attained with a Norg source that the bacterium could not degrade and when additional Corg was present in the medium. In contrast, all other combinations yielded a decrease on biomass production in the co-cultures compared to individual growth. The coupled cycling of Corg and Norg is rarely considered when investigating the role of microbial activity on soil functioning. Our results show that cycling of these two elements can be related through cross-chemical reactions in independent, albeit interacting microbes. In this way, the composition of organic material could greatly alter nutrient turnover due to its effect on the outcome of interactions between fungi and bacteria that disperse on their mycelia.

Oxalic acid, a molecule at the crossroads of bacterial-fungal interactions.

Oxalic acid is the most ubiquitous and common low molecular weight organic acid produced by living organisms. Oxalic acid is produced by fungi, bacteria, plants, and animals. The aim of this review is to give an overview of current knowledge about the microbial cycling of oxalic acid through ecosystems. Here we review the production and degradation of oxalic acid, as well as its implications in the metabolism for fungi, bacteria, plants, and animals. Indeed, fungi are well known producers of oxalic acid, while bacteria are considered oxalic acid consumers. However, this framework may need to be modified, because the ability of fungi to degrade oxalic acid and the ability of bacteria to produce it, have been poorly investigated. Finally, we will highlight the role of fungi and bacteria in oxalic acid cycling in soil, plant and animal ecosystems.

Distinct roles of Candida albicans drug resistance transcription factors TAC1, MRR1, and UPC2 in virulence.

Azoles are widely used in antifungal therapy in medicine. Resistance to azoles can occur in Candida albicans principally by overexpression of multidrug transporter gene CDR1, CDR2, or MDR1 or by overexpression of ERG11, which encodes the azole target. The expression of these genes is controlled by the transcription factors (TFs) TAC1 (involved in the control of CDR1 and CDR2), MRR1 (involved in the control of MDR1), and UPC2 (involved in the control of ERG11). Several gain-of-function (GOF) mutations are present in hyperactive alleles of these TFs, resulting in the overexpression of target genes. While these mutations are beneficial to C. albicans survival in the presence of the antifungal drugs, their effects could potentially alter the fitness and virulence of C. albicans in the absence of the selective drug pressure. In this work, the effect of GOF mutations on C. albicans virulence was addressed in a systemic model of intravenous infection by mouse survival and kidney fungal burden assays. We engineered a set of strains with identical genetic backgrounds in which hyperactive alleles were reintroduced in one or two copies at their genomic loci. The results obtained showed that neither TAC1 nor MRR1 GOF mutations had a significant effect on C. albicans virulence. In contrast, the presence of two hyperactive UPC2 alleles in C. albicans resulted in a significant decrease in virulence, correlating with diminished kidney colonization compared to that by the wild type. In agreement with the effect on virulence, the decreased fitness of an isolate with UPC2 hyperactive alleles was observed in competition experiments with the wild type in vivo but not in vitro. Interestingly, UPC2 hyperactivity delayed filamentation of C. albicans after phagocytosis by murine macrophages, which may at least partially explain the virulence defects. Combining the UPC2 GOF mutation with another hyperactive TF did not compensate for the negative effect of UPC2 on virulence. In conclusion, among the major TFs involved in azole resistance, only UPC2 had a negative impact on virulence and fitness, which may therefore have consequences for the epidemiology of antifungal resistance.

Identification and antifungal susceptibility of a large collection of yeast strains isolated in Tunisian hospitals.

In this study, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used as a rapid method to identify yeasts isolated from patients in Tunisian hospitals. When identification could not be exstablished with this procedure, sequencing of the internal transcribed spacer with 5.8S ribosomal DNA (rDNA) (ITS1-5.8S-ITS2) and D1/D2 domain of large-subunit (LSU rDNA) were employed as a molecular approach for species differentiation. Candida albicans was the dominant species (43.37% of all cases), followed by C. glabrata (16.55%), C. parapsilosis (13.23%), C. tropicalis (11.34%), C. dubliniensis (4.96%), and other species more rarely encountered in human diseases such as C. krusei, C. metapsilosis, C. lusitaniae, C. kefyr, C. palmioleophila, C. guilliermondii, C. intermedia, C. orthopsilosis, and C. utilis. In addition, other yeast species were obtained including Saccharomyces cerevisiae, Debaryomyces hansenii (anamorph known as C. famata), Hanseniaspora opuntiae, Kodamaea ohmeri, Pichia caribbica (anamorph known as C. fermentati), Trichosporon spp. and finally a novel yeast species, C. tunisiensis. The in vitro antifungal activities of fluconazole and voriconazole were determined by the agar disk diffusion test and Etest, while the susceptibility to additional antifungal agents was determined with the Sensititre YeastOne system. Our results showed low incidence of azole resistance in C. albicans (0.54%), C. tropicalis (2.08%) and C. glabrata (4.28%). In addition, caspofungin was active against most isolates of the collection with the exception of two K. ohmeri isolates. This is the first report to describe caspofungin resistant isolates of this yeast.

From selection to caged aptamers: identification of light-dependent ssDNA aptamers targeting cytohesin.

Caged aptamers represent valuable tools for the spatiotemporal control of protein function by light. Here we describe a general route starting with the de novo selection process targeting cytohesin-1 and aiming at the synthesis of caged aptamers without the prior knowledge of detailed structural determinants of aptamer-target binding.