The Species-Specific Acquisition and Diversification of a K1-like Family of Killer Toxins in Budding Yeasts of the Saccharomycotina.

Killer toxins are extracellular antifungal proteins that are produced by a wide variety of fungi, including Saccharomyces yeasts. Although many Saccharomyces killer toxins have been previously identified, their evolutionary origins remain uncertain given that many of these genes have been mobilized by double-stranded RNA (dsRNA) viruses. A survey of yeasts from the Saccharomyces genus has identified a novel killer toxin with a unique spectrum of activity produced by Saccharomyces paradoxus. The expression of this killer toxin is associated with the presence of a dsRNA totivirus and a satellite dsRNA. Genetic sequencing of the satellite dsRNA confirmed that it encodes a killer toxin with homology to the canonical ionophoric K1 toxin from Saccharomyces cerevisiae and has been named K1-like (K1L). Genomic homologs of K1L were identified in six non-Saccharomyces yeast species of the Saccharomycotina subphylum, predominantly in subtelomeric regions of the genome. When ectopically expressed in S. cerevisiae from cloned cDNAs, both K1L and its homologs can inhibit the growth of competing yeast species, confirming the discovery of a family of biologically active K1-like killer toxins. The sporadic distribution of these genes supports their acquisition by horizontal gene transfer followed by diversification. The phylogenetic relationship between K1L and its genomic homologs suggests a common ancestry and gene flow via dsRNAs and DNAs across taxonomic divisions. This appears to enable the acquisition of a diverse arsenal of killer toxins by different yeast species for potential use in niche competition.

In vivo structure of the Ty1 retrotransposon RNA genome.

Long terminal repeat (LTR)-retrotransposons constitute a significant part of eukaryotic genomes and influence their function and evolution. Like other RNA viruses, LTR-retrotransposons efficiently utilize their RNA genome to interact with host cell machinery during replication. Here, we provide the first genome-wide RNA secondary structure model for a LTR-retrotransposon in living cells. Using SHAPE probing, we explore the secondary structure of the yeast Ty1 retrotransposon RNA genome in its native in vivo state and under defined in vitro conditions. Comparative analyses reveal the strong impact of the cellular environment on folding of Ty1 RNA. In vivo, Ty1 genome RNA is significantly less structured and more dynamic but retains specific well-structured regions harboring functional cis-acting sequences. Ribosomes participate in the unfolding and remodeling of Ty1 RNA, and inhibition of translation initiation stabilizes Ty1 RNA structure. Together, our findings support the dual role of Ty1 genomic RNA as a template for protein synthesis and reverse transcription. This study also contributes to understanding how a complex multifunctional RNA genome folds in vivo, and strengthens the need for studying RNA structure in its natural cellular context.

Identification and Molecular Characterization of Novel Mycoviruses in Saccharomyces and Non-Saccharomyces Yeasts of Oenological Interest.

Wine yeasts can be natural hosts for dsRNA, ssRNA viruses and retrotransposon elements. In this study, high-throughput RNA sequencing combined with bioinformatic analyses unveiled the virome associated to 16 Saccharomyces cerevisiae and 8 non-Saccharomyces strains of oenological interest. Results showed the presence of six viruses and two satellite dsRNAs from four different families, two of which-Partitiviridae and Mitoviridae-were not reported before in yeasts, as well as two ORFan contigs of viral origin. According to phylogenetic analysis, four new putative mycoviruses distributed in Totivirus, Cryspovirus, and Mitovirus genera were identified. The majority of commercial S. cerevisiae strains were confirmed to be the host for helper L-A type totiviruses and satellite M dsRNAs associated with the killer phenotype, both in single and mixed infections with L-BC totiviruses, and two viral sequences belonging to a new cryspovirus putative species discovered here for the first time. Moreover, single infection by a narnavirus 20S-related sequence was also found in one S. cerevisiae strain. Considering the non-Saccharomyces yeasts, Starmerella bacillaris hosted four RNAs of viral origin-two clustering in Totivirus and Mitovirus genera, and two ORFans with putative satellite behavior. This study confirmed the infection of wine yeasts by viruses associated with useful technological characteristics and demonstrated the presence of complex mixed infections with unpredictable biological effects.

A rAAV2-producing yeast screening model to identify host proteins enhancing rAAV DNA replication and vector yield.

Recombinant adeno-associated viral vectors (rAAV) are promising therapies for genetic diseases. Although current platforms for recombinant vector production can generate drug material for pre-clinical and clinical studies, rAAV biomanufacturing will eventually face commercial supply challenges if per cell vector productivity and process scalability are not improved. Because considerable efforts have traditionally focused on optimizing rAAV plasmid design, herein we investigate the impact of host cell proteins on vector production to identify proteins that may enhance rAAV yield. Using a rAAV2-GFP-producing Saccharomyces cerevisiae model in combination with the yeast Tet Hughes Collection screening library, we identified 22 gene candidates that improved rAAV DNA replication (rAAV-GFP/18s rDNA ratio) and vector yield (benzonase-resistant rAAV DNA vector genome titer) as high as 6-fold and 15-fold relative to control, respectively. The candidate proteins participate in biological processes such as DNA replication, ribosome biogenesis, and RNA and protein processing. The best five candidates (PRE4, HEM4, TOP2, GPN3, and SDO1) were further screened by generating overexpression mutants in the YPH500 yeast strain. Subsequent clone evaluation was performed to confirm the rAAV-promoting activity of selected candidates under plate-based and bioreactor-controlled fermentation conditions. Digital droplet PCR analysis of cell lysate and AVB resin-purified material confirmed HEM4 and TOP2 overexpression mutants displayed the highest per cell total rAAV DNA productivity (1.6 and 1.7-fold increase over control, respectively) and per cell vector productivity (3 and 4-fold over control, respectively). This evaluation confirmed that overexpression of HEM4 and TOP2 proteins enhanced total and benzonase-resistant rAAV DNA yield. Further studies are needed to understand their mechanism of action and to assess their potential application in molecular strategies for rAAV production. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2725, 2019.

Saccharomyces paradoxus K66 Killer System Evidences Expanded Assortment of Helper and Satellite Viruses.

The Saccharomycetaceae yeast family recently became recognized for expanding of the repertoire of different dsRNA-based viruses, highlighting the need for understanding of their cross-dependence. We isolated the Saccharomyces paradoxus AML-15-66 killer strain from spontaneous fermentation of serviceberries and identified helper and satellite viruses of the family Totiviridae, which are responsible for the killing phenotype. The corresponding full dsRNA genomes of viruses have been cloned and sequenced. Sequence analysis of SpV-LA-66 identified it to be most similar to S. paradoxus LA-28 type viruses, while SpV-M66 was mostly similar to the SpV-M21 virus. Sequence and functional analysis revealed significant differences between the K66 and the K28 toxins. The structural organization of the K66 protein resembled those of the K1/K2 type toxins. The AML-15-66 strain possesses the most expressed killing property towards the K28 toxin-producing strain. A genetic screen performed on S. cerevisiae YKO library strains revealed 125 gene products important for the functioning of the S. paradoxus K66 toxin, with 85% of the discovered modulators shared with S. cerevisiae K2 or K1 toxins. Investigation of the K66 protein binding to cells and different polysaccharides implies the ß-1,6 glucans to be the primary receptors of S. paradoxus K66 toxin. For the first time, we demonstrated the coherent habitation of different types of helper and satellite viruses in a wild-type S. paradoxus strain.

Variation and Distribution of L-A Helper Totiviruses in Saccharomyces sensu stricto Yeasts Producing Different Killer Toxins.

Yeasts within the Saccharomyces sensu stricto cluster can produce different killer toxins. Each toxin is encoded by a medium size (1.5-2.4 Kb) M dsRNA virus, maintained by a larger helper virus generally called L-A (4.6 Kb). Different types of L-A are found associated to specific Ms: L-A in K1 strains and L-A-2 in K2 strains. Here, we extend the analysis of L-A helper viruses to yeasts other than S. cerevisiae, namely S. paradoxus, S. uvarum and S. kudriavzevii. Our sequencing data from nine new L-A variants confirm the specific association of each toxin-producing M and its helper virus, suggesting co-evolution. Their nucleotide sequences vary from 10% to 30% and the variation seems to depend on the geographical location of the hosts, suggesting cross-species transmission between species in the same habitat. Finally, we transferred by genetic methods different killer viruses from S. paradoxus into S. cerevisiae or viruses from S. cerevisiae into S. uvarum or S. kudriavzevii. In the foster hosts, we observed no impairment for their stable transmission and maintenance, indicating that the requirements for virus amplification in these species are essentially the same. We also characterized new killer toxins from S. paradoxus and constructed "superkiller" strains expressing them.

A population study of killer viruses reveals different evolutionary histories of two closely related Saccharomyces sensu stricto yeasts.

Microbes have evolved ways of interference competition to gain advantage over their ecological competitors. The use of secreted killer toxins by yeast cells through acquiring double-stranded RNA viruses is one such prominent example. Although the killer behaviour has been well studied in laboratory yeast strains, our knowledge regarding how killer viruses are spread and maintained in nature and how yeast cells co-evolve with viruses remains limited. We investigated these issues using a panel of 81 yeast populations belonging to three Saccharomyces sensu stricto species isolated from diverse ecological niches and geographic locations. We found that killer strains are rare among all three species. In contrast, killer toxin resistance is widespread in Saccharomyces paradoxus populations, but not in Saccharomyces cerevisiae or Saccharomyces eubayanus populations. Genetic analyses revealed that toxin resistance in S. paradoxus is often caused by dominant alleles that have independently evolved in different populations. Molecular typing identified one M28 and two types of M1 killer viruses in those killer strains. We further showed that killer viruses of the same type could lead to distinct killer phenotypes under different host backgrounds, suggesting co-evolution between the viruses and hosts in different populations. Taken together, our data suggest that killer viruses vary in their evolutionary histories even within closely related yeast species.

Incidence of symbiotic dsRNA 'killer' viruses in wild and domesticated yeast.

Viruses are found in almost all organisms and physical habitats. One interesting example is the yeast viral 'killer system'. The virus provides the host with a toxin directed against strains that do not carry it, while the yeast cell enables its propagation. Although yeast viruses are believed to be common, they have been actually described only for a limited number of yeast isolates. We surveyed 136 Saccharomyces cerevisiae and S. paradoxus strains of known origin and phylogenetic relatedness. Of these, 14 (c. 10%) were infected by killer viruses of one of the three types: K1, K2 or K28. As many as 34 strains (c. 25%) were not sensitive to at least one type of the killer toxin. In most cases, resistance did not disappear after attempts to cure the host strains from their viruses, suggesting that it was encoded in the host's genome. In terms of phylogeny, killer strains appear to be more related to each other than to nonkiller ones. No such tendency is observed for the phenotype of toxin resistance. Our results suggest that even if the killer toxins are not always present, they do play significant role in yeast ecology and evolution.

Saccharomyces boulardii: novas perspectivas para a terapêutica em diarreia aguda / Saccharomyces bourlardii: new approaches for treatment of acute diarrhea

Probióticos contendo Saccharomyces boulardii têm sido utilizados no tratamento de diarreias de etiologias diversas, incluindo infecciosa e inflamatória. Os mecanismos de ação e efeitos benéficos desses microrganismos no controle do quadro diarreico consistem na redução da hipersecreção de água e eletrólitos, estimulação da atividade de dissacaridases dos enterócitos, produção de aminopeptidases, secreção de IgA, atividade antitoxina, antiinflamatória, metabólica, e antimicrobiana. Tais propriedades biológicas desses microrganismos não patogênicos permitem a sua utilização no tratamento de doenças gastrintestinais endêmicas especialmente em países em desenvolvimento como gastroenterites por rotavírus, diarreia dos viajantes, além de doenças inflamatórias intestinais como a doença de Crohn e colite ulcerativa. Os objetivos dessa revisão consistem em discutir os aspectos significativos e a aplicabilidade do uso de Saccharomyces boulardii no tratamento das diarreias agudas e o racional para a dose de ataque na fase aguda.

Viral dsRNA in the wine yeast Saccharomyces bayanus var. uvarum.

The presence of viral dsRNA (L and M fractions) in the cryophilic yeast Saccharomyces bayanus var. uvarum is documented here for the first time. Sixty-eight strains of different origins were analyzed. Most of them did not carry dsRNA; the L fraction was found in seven strains, while 11 strains had both L and M fractions. The size of the L fraction was invariable (4.5 kb), as in the cultured yeast Saccharomyces cerevisiae. In contrast to L-dsRNA, the M fraction varied in size from ca. 1.2 to 1.8 kb. In total, seven different M-dsRNA types were recognized (M1-M3 and M8-M11), predominantly among French wine strains of S. bayanus var. uvarum. Phenotypic analysis revealed that the M-dsRNAs found were cryptic and may represent mutant forms of killer plasmids.

[Molecular polymorphism of viral dsRNA of yeast Saccharomyces paradoxus].

An analysis of 53 strains of yeast Saccharomyces paradoxus (YSP) of different geographic origins enabled us, for the first time, to find viral double-stranded RNA (L and M fractions) in YSP and to study natural polymorphism. As in the cultured Scerevisiae, the size of L dsRNA was constant (4.5 kb). The size of minor M dsRNA varied from 1.5 to 2.4 k.b. In YSP, we determined 7 types of M dsRNA (M1-M7), which were not connected with the source of isolation or geographic origin of the host strains.