Construction of multicellular yeast networks using the communication toolkit with variable specificity and attenuation

The key next step in synthetic biology is to extend cellular network engineering to the multicellular level by utilizing cell-cell communication for information processing. To facilitate the implementation of multicellular networks in the most commonly used eukaryotic chassis, Saccharomyces cerevisiae, we developed the yeast communication toolkit (YCTK). This toolkit is based on the fungal mating pathway and contains five pheromone-inducible promoters (response parts), eleven pheromones (α-factors; sender parts), eleven pheromone receptors (Ste2; receiver parts), as well as five Bar1 proteases (suppressor parts). All YCTK parts were thoroughly characterized and are compatible with the commonly used yeast Golden Gate cloning standard. We demonstrated the application of the YCTK by implementing several different logic gate-like population networks. Furthermore, we used this toolkit to investigate the pheromone-receptor promiscuity patterns among different yeast species. This toolkit extends currently available resources for construction of complex multicellular eukaryotic networks with varying degrees of promiscuity and attenuation.

Insights into Nuclear G-Protein-Coupled Receptors as Therapeutic Targets in Non-Communicable Diseases

G-protein-coupled receptors (GPCRs) comprise a large protein superfamily divided into six classes, rhodopsin-like (A), secretin receptor family (B), metabotropic glutamate (C), fungal mating pheromone receptors (D), cyclic AMP receptors (E) and frizzled (F). Until recently, GPCRs signaling was thought to emanate exclusively from the plasma membrane as a response to extracellular stimuli but several studies have challenged this view demonstrating that GPCRs can be present in intracellular localizations, including in the nuclei. A renewed interest in GPCR receptors’ superfamily emerged and intensive research occurred over recent decades, particularly regarding class A GPCRs, but some class B and C have also been explored. Nuclear GPCRs proved to be functional and capable of triggering identical and/or distinct signaling pathways associated with their counterparts on the cell surface bringing new insights into the relevance of nuclear GPCRs and highlighting the nucleus as an autonomous signaling organelle (triggered by GPCRs). Nuclear GPCRs are involved in physiological (namely cell proliferation, transcription, angiogenesis and survival) and disease processes (cancer, cardiovascular diseases, etc.). In this review we summarize emerging evidence on nuclear GPCRs expression/function (with some nuclear GPCRs evidencing atypical/disruptive signaling pathways) in non-communicable disease, thus, bringing nuclear GPCRs as targets to the forefront of debate.

The master regulator MAT1-1-1 of fungal mating binds to its targets via a conserved motif in the human pathogen Aspergillus fumigatus

Mating-type transcription factors are master regulators of sexually related signal transduction pathways in fungi; however, their recognition of specific DNA sequences from target genes is widely undetermined. Here, we identified and characterized the DNA-binding sequence of the MAT1-1-1 alpha-box domain transcription factor from the human pathogen Aspergillus fumigatus. In order to explore MAT1-1-1 DNA-binding targets, we used the previously reported MAT1-1-1 binding motif from Penicillium chrysogenum, in a bioinformatics approach. We identified 18 A. fumigatus genes carrying the MAT1.1 sequence in their upstream region, among them genes for the α-pheromone precursor (PpgA), G-protein-coupled pheromone receptor (PreA), and for TomA, an unidentified protein. To validate our prediction further, quantification of transcript levels showed a decrease in expression of ppgA, tomA, and others in a MAT1-1 deletion strain. For a functional analysis of the binding sites, truncated variants of the A. fumigatus MAT1-1-1 gene were introduced into Escherichia coli for heterologous expression. The yield of recombinant protein was further optimized for the AfMAT1-1-178–235 variant that harbors an extended alpha-box domain. AfMAT1-1-178–235 bound to a subset of the most strongly upregulated genes: ppgA, preA, and tomA. The DNA-binding specificity was confirmed by testing mutated binding sequences, as well as performing competition experiments with specific and non-specific sequences. Finally, equilibrium dissociation constants of 1.83 ± 0.1 and 1.45 ± 0.26 µM were determined for AfMAT1-1-178–235 and fusion protein GST-AfMAT1-1-178–235. Collectively, these findings provide further insights into AfMAT1-1-1-mediated gene expression and imply that alpha-box domain regulators from other members of Eurotiales control fungal development in a conserved manner.

The Role of Oxidoreductase-Like Protein Olp1 in Sexual Reproduction and Virulence of Cryptococcus neoformans

Cryptococcus neoformans is a basidiomycete human fungal pathogen causing lethal meningoencephalitis, mainly in immunocompromised patients. Oxidoreductases are a class of enzymes that catalyze redox, playing a crucial role in biochemical reactions. In this study, we identified one Cryptococcus oxidoreductase-like protein-encoding gene OLP1 and investigated its role in the sexual reproduction and virulence of C. neoformans. Gene expression patterns analysis showed that the OLP1 gene was expressed in each developmental stage of Cryptococcus, and the Olp1 protein was located in the cytoplasm of Cryptococcus cells. Although it produced normal major virulence factors such as melanin and capsule, the olp1Δ mutants showed growth defects on the yeast extract peptone dextrose (YPD) medium supplemented with lithium chloride (LiCl) and 5-fluorocytosine (5-FC). The fungal mating analysis showed that Olp1 is also essential for fungal sexual reproduction, as olp1Δ mutants show significant defects in hyphae growth and basidiospores production during bisexual reproduction. The fungal nuclei imaging showed that during the bilateral mating of olp1Δ mutants, the nuclei failed to undergo meiosis after fusion in the basidia, indicating that Olp1 is crucial for regulating meiosis during mating. Moreover, Olp1 was also found to be required for fungal virulence in C. neoformans, as the olp1Δ mutants showed significant virulence attenuation in a murine inhalation model. In conclusion, our results showed that the oxidoreductase-like protein Olp1 is required for both fungal sexual reproduction and virulence in C. neoformans.

Higher Gene Flow in Sex-Related Chromosomes than in Autosomes during Fungal Divergence

Nonrecombining sex chromosomes are widely found to be more differentiated than autosomes among closely related species, due to smaller effective population size and/or to a disproportionally large-X effect in reproductive isolation. Although fungal mating-type chromosomes can also display large nonrecombining regions, their levels of differentiation compared with autosomes have been little studied. Anther-smut fungi from the Microbotryum genus are castrating pathogens of Caryophyllaceae plants with largely nonrecombining mating-type chromosomes. Using whole genome sequences of 40 fungal strains, we quantified genetic differentiation among strains isolated from the geographically overlapping North American species and subspecies of Silene virginica and S. caroliniana. We inferred that gene flow likely occurred at the early stages of divergence and then completely stopped. We identified large autosomal genomic regions with chromosomal inversions, with higher genetic divergence than the rest of the genomes and highly enriched in selective sweeps, supporting a role of rearrangements in preventing gene flow in genomic regions involved in ecological divergence. Unexpectedly, the nonrecombining mating-type chromosomes showed lower divergence than autosomes due to higher gene flow, which may be promoted by adaptive introgressions of less degenerated mating-type chromosomes. The fact that both mating-type chromosomes are always heterozygous and nonrecombining may explain such patterns that oppose to those found for XY or ZW sex chromosomes. The specific features of mating-type chromosomes may also apply to the UV sex chromosomes determining sexes at the haploid stage in algae and bryophytes and may help test general hypotheses on the evolutionary specificities of sex-related chromosomes.

The AGC Kinase SsAgc1 Regulates Sporisorium scitamineum Mating/Filamentation and Pathogenicity

ABSTRACT Sporisorium scitamineum is the fungal pathogen causing severe sugarcane smut disease that leads to massive economic losses globally. S. scitamineum invades host cane by dikaryotic hyphae, formed after sexual mating of two haploid sporidia of opposite mating type. Therefore, mating/filamentation is critical for S. scitamineum pathogenicity, while its molecular mechanisms remain largely unknown. The AGC (cyclic AMP [cAMP]-dependent protein kinase 1 [protein kinase A {PKA}], cGMP-dependent protein kinase [PKG], and protein kinase C [PKC]) kinase family is a group of serine/threonine (Ser/Thr) protein kinases conserved among eukaryotic genomes, serving a variety of physiological functions, including cell growth, metabolism, differentiation, and cell death. In this study, we identified an AGC kinase, named SsAgc1 (for S. scitamineum Agc1), and characterized its function by reverse genetics. Our results showed that SsAgc1 is critical for S. scitamineum mating/filamentation and pathogenicity, and oxidative stress tolerance under some circumstances. Transcriptional profiling revealed that the SsAgc1 signaling pathway may control expression of the genes governing fungal mating/filamentation and tryptophan metabolism, especially for tryptophol production. We showed that tryptophan and tryptophol could at least partially restore ssagc1Δ mating/filamentation. Overall, our work revealed a signaling pathway mediated by AGC protein kinases to regulate fungal mating/filamentation, possibly through sensing and responding to tryptophol as signal molecules. IMPORTANCE The AGC signaling pathway represents a conserved distinct signaling pathway in regulation of fungal differentiation and virulence, while it has not been identified or characterized in the sugarcane smut fungus Sporisorium scitamineum. In this study, we identified a PAS domain-containing AGC kinase, SsAgc1, in S. scitamineum. Functional analysis revealed that SsAgc1 plays a regulatory role on the fungal dimorphic switch.