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The spores of fungi come in a wide variety of forms and sizes, highly adapted to the route of dispersal and to survival under specific environmental conditions. The ascomycete Ashbya gossypii produces needle shaped spores with a length of 30 μm and a diameter of 1 μm. Formation of these spores relies on actin and actin regulatory proteins and is, therefore, distinct from the minor role that actin plays for spore formation in Saccharomyces cerevisiae. Using in vivo FRET-measurements of proteins labeled with fluorescent proteins, we investigate how the formin AgBnr2, a protein that promotes actin polymerization, integrates into the structure of the spindle pole body during sporulation. We also investigate the role of the A. gossypii homologs to the S. cerevisiae meiotic outer plaque proteins Spo74, Mpc54 and Ady4 for sporulation in A. gossypii. We found highest FRET of AgBnr2 with AgSpo74. Further experiments indicated that AgSpo74 is a main factor for targeting AgBnr2 to the spindle pole body. In agreement with these results, the Agspo74 deletion mutant produces no detectable spores, whereas deletion of Agmpc54 only has an effect on spore length and deletion of Agady4 has no detectable sporulation phenotype. Based on this study and in relation to previous results we suggest a model where AgBnr2 resides within an analogous structure to the meiotic outer plaque of S. cerevisiae. There it promotes formation of actin cables important for shaping the needle shaped spore structure. [ABSTRACT FROM AUTHOR]

eceptor for ctivated inase-1 (RACK1) is a multifunctional eukaryotic scaffolding protein with a seven WD repeat structure. Among their many cellular roles, RACK1 homologs have been shown to serve as alternative Gβ subunits during heterotrimeric G protein signaling in many systems. We investigated genetic interactions between the RACK1 homolog cpc-2, the previously characterized Gβ subunit gnb-1 and other G protein signaling components in the multicellular filamentous fungus Neurospora crassa. Results from cell fractionation studies and from fluorescent microscopy of a strain expressing a CPC-2-GFP fusion protein revealed that CPC-2 is a cytoplasmic protein. Genetic epistasis experiments between cpc-2, the three Gα genes (gna-1, gna-2 and gna-3) and gnb-1 demonstrated that cpc-2 is epistatic to gna-2 with regards to basal hyphae growth rate and aerial hyphae height, while deletion of cpc-2 mitigates the increased macroconidiation on solid medium observed in Δgnb-1 mutants. Δcpc-2 mutants inappropriately produce conidiophores during growth in submerged culture and mutational activation of gna-3 alleviates this defect. Δcpc-2 mutants are female-sterile and fertility could not be restored by mutational activation of any of the three Gα genes. With the exception of macroconidiation on solid medium, double mutants lacking cpc-2 and gnb-1 exhibited more severe defects for all phenotypic traits, supporting a largely synergistic relationship between GNB-1 and CPC-2 in N. crassa. [ABSTRACT FROM AUTHOR]

Chromosome replication in Saccharomyces cerevisiae is initiated from ~300 origins that are regulated by DNA sequence and by the limited abundance of six trans-acting initiation proteins (Sld2, Sld3, Dpb11, Dbf4, Sld7 and Cdc45). We set out to determine how the levels of individual factors contribute to time of origin activation and/or origin efficiency using induced depletion of single factors and overexpression of sets of multiple factors. Depletion of Sld2 or Sld3 slows growth and S phase progression, decreases origin efficiency across the genome and impairs viability as a result of incomplete replication of the rDNA. We find that the most efficient early origins are relatively unaffected by depletion of either Sld2 or Sld3. However, Sld3 levels, and to a lesser extent Sld2 levels, are critical for firing of the less efficient early origins. Overexpression of Sld3 simultaneously with Sld2, Dpb11 and Dbf4 preserves the relative efficiency of origins. Only when Cdc45 and Sld7 are also overexpressed is origin efficiency equalized between early- and late-firing origins. Our data support a model in which Sld3 together with Cdc45 (and/or Sld7) is responsible for the differential efficiencies of origins across the yeast genome. [ABSTRACT FROM AUTHOR]

Replication stress poses a serious threat to genome stability. Recombination-Dependent-Replication (RDR) promotes DNA synthesis resumption from arrested forks. Despite the identification of chromatin restoration pathways after DNA repair, crosstalk coupling RDR and chromatin assembly is largely unexplored. The fission yeast Chromatin Assembly Factor-1, CAF-1, is known to promote RDR. Here, we addressed the contribution of histone deposition to RDR. We expressed a mutated histone, H3-H113D, to genetically alter replication-dependent chromatin assembly by destabilizing (H3-H4)2 tetramer. We established that DNA synthesis-dependent histone deposition, by CAF-1 and Asf1, promotes RDR by preventing Rqh1-mediated disassembly of joint-molecules. The recombination factor Rad52 promotes CAF-1 binding to sites of recombination-dependent DNA synthesis, indicating that histone deposition occurs downstream Rad52. Histone deposition and Rqh1 activity act synergistically to promote cell resistance to camptothecin, a topoisomerase I inhibitor that induces replication stress. Moreover, histone deposition favors non conservative recombination events occurring spontaneously in the absence of Rqh1, indicating that the stabilization of joint-molecules by histone deposition also occurs independently of Rqh1 activity. These results indicate that histone deposition plays an active role in promoting RDR, a benefit counterbalanced by stabilizing at-risk joint-molecules for genome stability. [ABSTRACT FROM AUTHOR]

Accurate segregation of chromosomes to daughter cells is a critical aspect of cell division. It requires the kinetochores on duplicated chromosomes to biorient, attaching to microtubules from opposite poles of the cell. Bioriented attachments come under tension, while incorrect attachments lack tension and must be released to allow proper attachments to form. A well-studied error correction pathway is mediated by the Aurora B kinase, which destabilizes low tension-bearing attachments. We recently discovered that in vitro, kinetochores display an additional intrinsic tension-sensing pathway that utilizes Stu2. The contribution of kinetochore-associated Stu2 to error correction in cells, however, was unknown. Here, we identify a Stu2 mutant that abolishes its kinetochore function and show that it causes biorientation defects in vivo. We also show that this Stu2-mediated pathway functions together with the Aurora B-mediated pathway. Altogether, our work indicates that cells employ multiple pathways to ensure biorientation and the accuracy of chromosome segregation. [ABSTRACT FROM AUTHOR]

Compounds belonging to the group of 5-substituted 4-(1,3,4-thiadiazol-2-yl) benzene-1,3-diols exhibit a broad spectrum of biological activity, including antibacterial, antifungal, and anticancer properties. The mechanism of the antifungal activity of compounds from this group has not been described to date. Among the large group of 5-substituted 4-(1,3,4-thiadiazol-2-yl) benzene-1,3-diol derivatives, the compound 4-(5-methyl-1,3,4-thiadiazole-2-yl) benzene-1,3-diol, abbreviated as C1, was revealed to be one of the most active agents against pathogenic fungi, simultaneously with the lowest toxicity to human cells. The C1 compound is a potent antifungal agent against different Candida species, including isolates resistant to azoles, and molds, with MIC100 values ranging from 8 to 96 μg/ml. The antifungal activity of the C1 compound involves disruption of the cell wall biogenesis, as evidenced by the inability of cells treated with C1 to maintain their characteristic cell shape, increase in size, form giant cells and flocculate. C1-treated cells were also unable to withstand internal turgor pressure causing protoplast material to leak out, exhibited reduced osmotic resistance and formed buds that were not covered with chitin. Disturbances in the chitin septum in the neck region of budding cells was observed, as well as an uneven distribution of chitin and β(1→3) glucan, and increased sensitivity to substances interacting with wall polymerization. The ATR-FTIR spectral shifts in cell walls extracted from C. albicans cells treated with the C1 compound suggested weakened interactions between the molecules of β(1→3) glucans and β(1→6) glucans, which may be the cause of impaired cell wall integrity. Significant spectral changes in the C1-treated cells were also observed in bands characteristic for chitin. The C1 compound did not affect the ergosterol content in Candida cells. Given the low cytotoxicity of the C1 compound to normal human dermal fibroblasts (NHDF), it is possible to use this compound as a therapeutic agent in the treatment of surface and gastrointestinal tract mycoses. [ABSTRACT FROM AUTHOR]

Phenotypic switching between 2 opposing cellular states is a fundamental aspect of biology, and fungi provide facile systems to analyze the interactions between regulons that control this type of switch. A long-standing mystery in fungal pathogens of humans is how thermally dimorphic fungi switch their developmental form in response to temperature. These fungi, including the subject of this study, Histoplasma capsulatum, are temperature-responsive organisms that utilize unknown regulatory pathways to couple their cell shape and associated attributes to the temperature of their environment. H. capsulatum grows as a multicellular hypha in the soil that switches to a pathogenic yeast form in response to the temperature of a mammalian host. These states can be triggered in the laboratory simply by growing the fungus either at room temperature (RT; which promotes hyphal growth) or at 37 °C (which promotes yeast-phase growth). Prior worked revealed that 15% to 20% of transcripts are differentially expressed in response to temperature, but it is unclear which transcripts are linked to specific phenotypic changes, such as cell morphology or virulence. To elucidate temperature-responsive regulons, we previously identified 4 transcription factors (required for yeast-phase growth [Ryp]1–4) that are required for yeast-phase growth at 37 °C; in each ryp mutant, the fungus grows constitutively as hyphae regardless of temperature, and the cells fail to express genes that are normally induced in response to growth at 37 °C. Here, we perform the first genetic screen to identify genes required for hyphal growth of H. capsulatum at RT and find that disruption of the signaling mucin MSB2 results in a yeast-locked phenotype. RNA sequencing (RNAseq) experiments reveal that MSB2 is not required for the majority of gene expression changes that occur when cells are shifted to RT. However, a small subset of temperature-responsive genes is dependent on MSB2 for its expression, thereby implicating these genes in the process of filamentation. Disruption or knockdown of an multicopy suppression of a budding defect 2 (Msb2)-dependent mitogen-activated protein (MAP) kinase (HOG2) and an ASM-1/Phd1/StuA/EFG1/SOK2 (APSES) transcription factor (STU1) prevents hyphal growth at RT, validating that the Msb2 regulon contains genes that control filamentation. Notably, the Msb2 regulon shows conserved hyphal-specific expression in other dimorphic fungi, suggesting that this work defines a small set of genes that are likely to be conserved regulators and effectors of filamentation in multiple fungi. In contrast, a few yeast-specific transcripts, including virulence factors that are normally expressed only at 37 °C, are inappropriately expressed at RT in the msb2 mutant, suggesting that expression of these genes is coupled to growth in the yeast form rather than to temperature. Finally, we find that the yeast-promoting transcription factor Ryp3 associates with the MSB2 promoter and inhibits MSB2 transcript expression at 37 °C, whereas Msb2 inhibits accumulation of Ryp transcripts and proteins at RT. These findings indicate that the Ryp and Msb2 circuits antagonize each other in a temperature-dependent manner, thereby allowing temperature to govern cell shape and gene expression in this ubiquitous fungal pathogen of humans. [ABSTRACT FROM AUTHOR]

A single cancer genome can harbor thousands of clustered mutations. Mutation signature analyses have revealed that the origin of clusters are lesions in long tracts of single-stranded (ss) DNA damaged by apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) cytidine deaminases, raising questions about molecular mechanisms that generate long ssDNA vulnerable to hypermutation. Here, we show that ssDNA intermediates formed during the repair of gamma-induced bursts of double-strand breaks (DSBs) in the presence of APOBEC3A in yeast lead to multiple APOBEC-induced clusters similar to cancer. We identified three independent pathways enabling cluster formation associated with repairing bursts of DSBs: 5′ to 3′ bidirectional resection, unidirectional resection, and break-induced replication (BIR). Analysis of millions of mutations in APOBEC-hypermutated cancer genomes revealed that cancer tolerance to formation of hypermutable ssDNA is similar to yeast and that the predominant pattern of clustered mutagenesis is the same as in resection-defective yeast, suggesting that cluster formation in cancers is driven by a BIR-like mechanism. The phenomenon of genome-wide burst of clustered mutagenesis revealed by our study can play an important role in generating somatic hypermutation in cancers as well as in noncancerous cells. [ABSTRACT FROM AUTHOR]

Aim: To investigate the correlation between the dorsal tongue porphyrin autofluorescence, revealed using VELscope, and Candida saprophytism. Material and methods: Consecutive patients underwent an autofluorescence examination by the VELscope device to establish the presence or absence of porphyrin fluorescence. A tongue swab was collected for the Candida cultural test. Sensitivity, specificity, accuracy, negative predictive value and positive predictive value were calculated considering the oral swab as the gold standard. The degree of agreement between the two tests was calculated using Cohen's K coefficient. Results: One hundred twenty-six patients were enrolled. Porphyrin fluorescence method showed a sensitivity of 78%, specificity of 76% and an accuracy of 78%. Negative predictive value and positive predictive value were respectively 90% and 59%. The strength of agreement between the two methods resulted to be moderate (k = 0.551). Conclusions: Off-label use of tongue autofluorescence examination to detect the presence of Candida species is characterized by a loss of porphyrin fluorescence. The high negative predictive value of porphyrin fluorescence loss suggests its use in preliminary selection of Candida carriers, in order to plan preventive and therapeutic strategies. [ABSTRACT FROM AUTHOR]

Iron homeostasis is important for growth, reproduction and other metabolic processes in all eukaryotes. However, the functions of ATP-binding cassette (ABC) transporters in iron homeostasis are largely unknown. Here, we found that one ABC transporter (named FgAtm1) is involved in regulating iron homeostasis, by screening sensitivity to iron stress for 60 ABC transporter mutants of Fusarium graminearum, a devastating fungal pathogen of small grain cereal crops worldwide. The lack of FgAtm1 reduces the activity of cytosolic Fe-S proteins nitrite reductase and xanthine dehydrogenase, which causes high expression of FgHapX via activating transcription factor FgAreA. FgHapX represses transcription of genes for iron-consuming proteins directly but activates genes for iron acquisition proteins by suppressing another iron regulator FgSreA. In addition, the transcriptional activity of FgHapX is regulated by the monothiol glutaredoxin FgGrx4. Furthermore, the phosphorylation of FgHapX, mediated by the Ser/Thr kinase FgYak1, is required for its functions in iron homeostasis. Taken together, this study uncovers a novel regulatory mechanism of iron homeostasis mediated by an ABC transporter in an important pathogenic fungus. [ABSTRACT FROM AUTHOR]

Aggregation of the prion protein has strong implications in the human prion disease. Sup35p is a yeast prion, and has been used as a model protein to study the disease mechanism. We have studied the pattern of Sup35p aggregation inside live yeast cells under stress, by using confocal microscopy, fluorescence activated cell sorting and western blotting. Heat shock proteins are a family of proteins that are produced by yeast cells in response to exposure to stressful conditions. Many of the proteins behave as chaperones to combat stress-induced protein misfolding and aggregation. In spite of this, yeast also produce small molecules called osmolytes during stress. In our work, we tried to find the reason as to why yeast produce osmolytes and showed that the osmolytes are paramount to ameliorate the long-term effects of lethal stress in Saccharomyces cerevisiae, either in the presence or absence of Hsp104p. [ABSTRACT FROM AUTHOR]

The canonical model of eukaryotic translation posits that efficient translation initiation increases protein expression and mRNA stability. Contrary to this model, we find that increasing initiation rate can decrease both protein expression and stability of certain mRNAs in the budding yeast Saccharomyces cerevisiae. These mRNAs encode a stretch of polybasic residues that cause ribosome stalling. Our computational modeling predicts that the observed decrease in gene expression at high initiation rates occurs when ribosome collisions at stalls stimulate abortive termination of the leading ribosome or cause endonucleolytic mRNA cleavage. Consistent with this prediction, the collision-associated quality-control factors Asc1 and Hel2 (orthologs of human RACK1 and ZNF598, respectively) decrease gene expression from stall-containing mRNAs only at high initiation rates. Remarkably, hundreds of S. cerevisiae mRNAs that contain ribosome stall sequences also exhibit lower translation efficiency. We propose that inefficient translation initiation allows these stall-containing endogenous mRNAs to escape collision-stimulated reduction in gene expression. [ABSTRACT FROM AUTHOR]

Courtship is pivotal for successful mating. However, courtship is challenging for the Cryptococcus neoformans species complex, comprised of opportunistic fungal pathogens, as the majority of isolates are α mating type. In the absence of mating partners of the opposite mating type, C. deneoformans can undergo unisexual reproduction, during which a yeast-to-hyphal morphological transition occurs. Hyphal growth during unisexual reproduction is a quantitative trait, which reflects a strain’s ability to undergo unisexual reproduction. In this study, we determined whether unisexual reproduction confers an ecological benefit by promoting foraging for mating partners. Through competitive mating assays using strains with different abilities to produce hyphae, we showed that unisexual reproduction potential did not enhance competition for mating partners of the same mating type, but when cells of the opposite mating type were present, cells with enhanced hyphal growth were more competitive for mating partners of either the same or opposite mating type. Enhanced mating competition was also observed in a strain with increased hyphal production that lacks the mating repressor gene GPA3, which contributes to the pheromone response. Hyphal growth in unisexual strains also enables contact between adjacent colonies and enhances mating efficiency during mating confrontation assays. The pheromone response pathway activation positively correlated with unisexual reproduction hyphal growth during bisexual mating and exogenous pheromone promoted bisexual cell fusion. Despite the benefit in competing for mating partners, unisexual reproduction conferred a fitness cost. Taken together, these findings suggest C. deneoformans employs hyphal growth to facilitate contact between colonies at long distances and utilizes pheromone sensing to enhance mating competition. [ABSTRACT FROM AUTHOR]

Interspecific hybridization can introduce genetic variation that aids in adaptation to new or changing environments. Here, we investigate how hybrid adaptation to temperature and nutrient limitation may alter parental genome representation over time. We evolved Saccharomyces cerevisiae x Saccharomyces uvarum hybrids in nutrient-limited continuous culture at 15°C for 200 generations. In comparison to previous evolution experiments at 30°C, we identified a number of responses only observed in the colder temperature regime, including the loss of the S. cerevisiae allele in favor of the cryotolerant S. uvarum allele for several portions of the hybrid genome. In particular, we discovered a genotype by environment interaction in the form of a loss of heterozygosity event on chromosome XIII; which species’ haplotype is lost or maintained is dependent on the parental species’ temperature preference and the temperature at which the hybrid was evolved. We show that a large contribution to this directionality is due to a temperature dependent fitness benefit at a single locus, the high affinity phosphate transporter gene PHO84. This work helps shape our understanding of what forces impact genome evolution after hybridization, and how environmental conditions may promote or disfavor the persistence of hybrids over time. [ABSTRACT FROM AUTHOR]

Efficient adaptation to iron starvation is an essential virulence determinant of the most common human mold pathogen, Aspergillus fumigatus. Here, we demonstrate that the cytosolic monothiol glutaredoxin GrxD plays an essential role in iron sensing in this fungus. Our studies revealed that (i) GrxD is essential for growth; (ii) expression of the encoding gene, grxD, is repressed by the transcription factor SreA in iron replete conditions and upregulated during iron starvation; (iii) during iron starvation but not iron sufficiency, GrxD displays predominant nuclear localization; (iv) downregulation of grxD expression results in de-repression of genes involved in iron-dependent pathways and repression of genes involved in iron acquisition during iron starvation, but did not significantly affect these genes during iron sufficiency; (v) GrxD displays protein-protein interaction with components of the cytosolic iron-sulfur cluster biosynthetic machinery, indicating a role in this process, and with the transcription factors SreA and HapX, which mediate iron regulation of iron acquisition and iron-dependent pathways; (vi) UV-Vis spectra of recombinant HapX or the complex of HapX and GrxD indicate coordination of iron-sulfur clusters; (vii) the cysteine required for iron-sulfur cluster coordination in GrxD is in vitro dispensable for interaction with HapX; and (viii) there is a GrxD-independent mechanism for sensing iron sufficiency by HapX; (ix) inactivation of SreA suppresses the lethal effect caused by GrxD inactivation. Taken together, this study demonstrates that GrxD is crucial for iron homeostasis in A. fumigatus. [ABSTRACT FROM AUTHOR]

In all kingdoms of life, is used to encode hereditary information. Propagation of the genetic material between generations requires timely and accurate duplication of DNA by prior to cell division to ensure each daughter cell receives the full complement of . DNA synthesis of daughter strands starts at discrete sites, termed replication origins, and proceeds in a bidirectional manner until all genomic DNA is replicated. Despite the fundamental nature of these events, organisms have evolved surprisingly divergent strategies that control replication onset. Here, we discuss commonalities and differences in replication origin organization and recognition in the three domains of life. [ABSTRACT FROM AUTHOR]

Background: Ocular involvement of candidemia can result in serious complications, including vision loss. This study investigated the risk factors for ocular involvement in patients with candidemia and the outcomes of treatment. Methods: Episodes of candidemia in hospitalized adults who underwent ophthalmic examinations within 2 weeks of candidemia onset between January 2014 and May 2017 were retrospectively reviewed. Their demographic characteristics, antifungal treatments, and visual outcomes were evaluated. Results: During the study period, 438 adults were diagnosed with candidemia, with 275 (62.8%) undergoing ophthalmic examinations within 2 weeks. Of these 275 patients, 59 (21.5%) had fundoscopic abnormalities suggestive of ocular involvement, including 51 with chorioretinitis and eight with Candida endophthalmitis. Eleven patients were symptomatic. Persistent candidemia (adjusted odd ratio [aOR], 2.55; 95% confidence interval [CI], 1.29–5.08; P = 0.01), neutropenia during the preceding 2 weeks (aOR, 2.92; 95% CI, 1.14–7.53; P = 0.03), and C. albicans infection (aOR, 2.15; 95% CI, 1.09–4.24; P = 0.03) were independently associated with ocular involvement. Among the 24 patients with neutropenia, 41.7% had ocular involvements at the initial examination. Ophthalmologic examination even before the neutrophil recovery was positive in one-third of neutropenic patients. Out of the 37 patients in whom ocular outcomes after 6 weeks were available, 35 patients showed favorable or stable fundoscopic findings. Two patients had decreased visual acuity despite the stable fundoscopic finding. Conclusion: Neutropenia within two weeks of candidemia was a risk factor for ocular involvement. More than 80 percent of patients with ocular involvements were asymptomatic, emphasizing the importance of routine ophthalmic examinations. The median 6 weeks of systemic antifungal treatment resulted in favorable outcomes in 89.2% of patients. [ABSTRACT FROM AUTHOR]

Cellular microscopy images contain rich insights about biology. To extract this information, researchers use features, or measurements of the patterns of interest in the images. Here, we introduce a convolutional neural network (CNN) to automatically design features for fluorescence microscopy. We use a self-supervised method to learn feature representations of single cells in microscopy images without labelled training data. We train CNNs on a simple task that leverages the inherent structure of microscopy images and controls for variation in cell morphology and imaging: given one cell from an image, the CNN is asked to predict the fluorescence pattern in a second different cell from the same image. We show that our method learns high-quality features that describe protein expression patterns in single cells both yeast and human microscopy datasets. Moreover, we demonstrate that our features are useful for exploratory biological analysis, by capturing high-resolution cellular components in a proteome-wide cluster analysis of human proteins, and by quantifying multi-localized proteins and single-cell variability. We believe paired cell inpainting is a generalizable method to obtain feature representations of single cells in multichannel microscopy images. [ABSTRACT FROM AUTHOR]

L-aspartate aminotransferase is a pyridoxal 5ʹ-phosphate-dependent transaminase that catalyzes reversible transfer of an α-amino group from aspartate to α-ketoglutarate or from glutamate to oxaloacetate. L-aspartate aminotransferase not only mediates amino acid and carbohydrate metabolism but also regulates the cellular level of amino acids by catalyzing amino acid degradation and biosynthesis. To expand our structural information, we determined the crystal structure of L-aspartate aminotransferase from Schizosaccharomyces pombe at 2.1 Å resolution. A structural comparison between two yeast L-aspartate aminotransferases revealed conserved enzymatic mechanism mediated by the open–closed conformational change. Compared with higher eukaryotic species, L-aspartate aminotransferases showed distinguishable inter-subunit interaction between the N-terminal arm and a large domain of the opposite subunit. Interestingly, structural homology search showed varied conformation of the N-terminal arm among 71 structures of the family. Therefore, we classified pyridoxal 5ʹ-phosphate-dependent enzymes into eight subclasses based on the structural feature of N-terminal arms. In addition, structure and sequence comparisons showed strong relationships among the eight subclasses. Our results may provide insights into structure-based evolutionary aspects of pyridoxal 5ʹ-phosphate-dependent enzymes. [ABSTRACT FROM AUTHOR]

Background: Genomic data have become major resources to understand complex mechanisms at fine-scale temporal and spatial resolution in functional and evolutionary genetic studies, including human diseases, such as cancers. Recently, a large number of whole genomes of evolving populations of yeast (Saccharomyces cerevisiae W303 strain) were sequenced in a time-dependent manner to identify temporal evolutionary patterns. For this type of study, a chromosome-level sequence assembly of the strain or population at time zero is required to compare with the genomes derived later. However, there is no fully automated computational approach in experimental evolution studies to establish the chromosome-level genome assembly using unique features of sequencing data. Methods and results: In this study, we developed a new software pipeline, the integrative meta-assembly pipeline (IMAP), to build chromosome-level genome sequence assemblies by generating and combining multiple initial assemblies using three de novo assemblers from short-read sequencing data. We significantly improved the continuity and accuracy of the genome assembly using a large collection of sequencing data and hybrid assembly approaches. We validated our pipeline by generating chromosome-level assemblies of yeast strains W303 and SK1, and compared our results with assemblies built using long-read sequencing and various assembly evaluation metrics. We also constructed chromosome-level sequence assemblies of S. cerevisiae strain Sigma1278b, and three commonly used fungal strains: Aspergillus nidulans A713, Neurospora crassa 73, and Thielavia terrestris CBS 492.74, for which long-read sequencing data are not yet available. Finally, we examined the effect of IMAP parameters, such as reference and resolution, on the quality of the final assembly of the yeast strains W303 and SK1. Conclusions: We developed a cost-effective pipeline to generate chromosome-level sequence assemblies using only short-read sequencing data. Our pipeline combines the strengths of reference-guided and meta-assembly approaches. Our pipeline is available online at including a Docker image, as well as a Perl script, to help users install the IMAP package, including several prerequisite programs. Users can use IMAP to easily build the chromosome-level assembly for the genome of their interest. [ABSTRACT FROM AUTHOR]

Genomic rearrangements (gross chromosomal rearrangements, GCRs) threatens genome integrity and cause cell death or tumor formation. At the terminus of linear chromosomes, a telomere-binding protein complex, called shelterin, ensures chromosome stability by preventing chromosome end-to-end fusions and regulating telomere length homeostasis. As such, shelterin-mediated telomere functions play a pivotal role in suppressing GCR formation. However, it remains unclear whether the shelterin proteins play any direct role in inhibiting GCR at non-telomeric regions. Here, we have established a GCR assay for the first time in fission yeast and measured GCR rates in various mutants. We found that fission yeast cells lacking shelterin components Taz1 or Rap1 (mammalian TRF1/2 or RAP1 homologues, respectively) showed higher GCR rates compared to wild-type, accumulating large chromosome deletions. Genetic dissection of Rap1 revealed that Rap1 contributes to inhibiting GCRs via two independent pathways. The N-terminal BRCT-domain promotes faithful DSB repair, as determined by I-SceI-mediated DSB-induction experiments; moreover, association with Poz1 mediated by the central Poz1-binding domain regulates telomerase accessibility to DSBs, leading to suppression of de novo telomere additions. Our data highlight unappreciated functions of the shelterin components Taz1 and Rap1 in maintaining genome stability, specifically by preventing non-telomeric GCRs. [ABSTRACT FROM AUTHOR]

The NAD+-dependent histone deacetylase Sir2 was originally identified in Saccharomyces cerevisiae as a silencing factor for HML and HMR, the heterochromatic cassettes utilized as donor templates during mating-type switching. MATa cells preferentially switch to MATα using HML as the donor, which is driven by an adjacent cis-acting element called the recombination enhancer (RE). In this study we demonstrate that Sir2 and the condensin complex are recruited to the RE exclusively in MATa cells, specifically to the promoter of a small gene within the right half of the RE known as RDT1. We also provide evidence that the RDT1 promoter functions as a locus control region (LCR) that regulates both transcription and long-range chromatin interactions. Sir2 represses RDT1 transcription until it is removed from the promoter in response to a dsDNA break at the MAT locus induced by HO endonuclease during mating-type switching. Condensin is also recruited to the RDT1 promoter and is displaced upon HO induction, but does not significantly repress RDT1 transcription. Instead condensin appears to promote mating-type donor preference by maintaining proper chromosome III architecture, which is defined by the interaction of HML with the right arm of chromosome III, including MATa and HMR. Remarkably, eliminating Sir2 and condensin recruitment to the RDT1 promoter disrupts this structure and reveals an aberrant interaction between MATa and HMR, consistent with the partially defective donor preference for this mutant. Global condensin subunit depletion also impairs mating-type switching efficiency and donor preference, suggesting that modulation of chromosome architecture plays a significant role in controlling mating-type switching, thus providing a novel model for dissecting condensin function in vivo. [ABSTRACT FROM AUTHOR]

Candidemia has been considered a persistent public health problem with great impact on hospital costs and high mortality. We aimed to evaluate the epidemiology and prognostic factors of candidemia in a tertiary hospital in Northeast Brazil from January 2011 to December 2016. Demographic and clinical data of patients were retrospectively obtained from medical records and antifungal susceptibility profiling was performed using the broth microdilution method. A total of 68 episodes of candidemia were evaluated. We found an average incidence of 2.23 episodes /1000 admissions and a 30-day mortality rate of 55.9%. The most prevalent species were Candida albicans (35.3%), Candida tropicalis (27.4%), Candida parapsilosis (21.6%) and Candida glabrata (11.8%). Higher mortality rates were observed in cases of candidemia due to C. albicans (61.1%) and C. glabrata (100%), especially when compared to C. parapsilosis (27.3%). Univariate analysis revealed some variables which significantly increased the probability of death: older age (P = 0.022; odds ratio [OR] = 1.041), severe sepsis (P < 0.001; OR = 8.571), septic shock (P = 0.035; OR = 3.792), hypotension (P = 0.003; OR = 9.120), neutrophilia (P = 0.046; OR = 3.080), thrombocytopenia (P = 0.002; OR = 6.800), mechanical ventilation (P = 0.009; OR = 8.167) and greater number of surgeries (P = 0.037; OR = 1.920). Multivariate analysis showed that older age (P = 0.040; OR = 1.055), severe sepsis (P = 0.009; OR = 9.872) and hypotension (P = 0.031; OR = 21.042) were independently associated with worse prognosis. There was no resistance to amphotericin B, micafungin or itraconazole and a low rate of resistance to fluconazole (5.1%). However, 20.5% of the Candida isolates were susceptible dose-dependent (SDD) to fluconazole and 7.7% to itraconazole. In conclusion, our results could assist in the adoption of strategies to stratify patients at higher risk for developing candidemia and worse prognosis, in addition to improve antifungal management. [ABSTRACT FROM AUTHOR]

Introns are a prevalent feature of eukaryotic genomes, yet their origins and contributions to genome function and evolution remain mysterious. In budding yeast, repression of the highly transcribed intron-containing ribosomal protein genes (RPGs) globally increases splicing of non-RPG transcripts through reduced competition for the spliceosome. We show that under these “hungry spliceosome” conditions, splicing occurs at more than 150 previously unannotated locations we call protointrons that do not overlap known introns. Protointrons use a less constrained set of splice sites and branchpoints than standard introns, including in one case AT-AC in place of GT-AG. Protointrons are not conserved in all closely related species, suggesting that most are not under positive selection and are fated to disappear. Some are found in non-coding RNAs (e. g. CUTs and SUTs), where they may contribute to the creation of new genes. Others are found across boundaries between noncoding and coding sequences, or within coding sequences, where they offer pathways to the creation of new protein variants, or new regulatory controls for existing genes. We define protointrons as (1) nonconserved intron-like sequences that are (2) infrequently spliced, and importantly (3) are not currently understood to contribute to gene expression or regulation in the way that standard introns function. A very few protointrons in S. cerevisiae challenge this classification by their increased splicing frequency and potential function, consistent with the proposed evolutionary process of “intronization”, whereby new standard introns are created. This snapshot of intron evolution highlights the important role of the spliceosome in the expansion of transcribed genomic sequence space, providing a pathway for the rare events that may lead to the birth of new eukaryotic genes and the refinement of existing gene function. [ABSTRACT FROM AUTHOR]

Drug resistance is a rapidly emerging concern, thus prompting the development of novel therapeutics or combinatorial therapy. Currently, combinatorial therapy targets are based on knowledge of drug mode of action and/or resistance mechanisms, constraining the number of target proteins. Unbiased genome-wide screens could reveal novel genetic components within interaction networks as potential targets in combination therapies. Testing this, in the context of antimicrobial resistance, we implemented an unbiased genome-wide screen, performed in Saccharomyces cerevisiae expressing a Candida glabrata PDR1+ gain-of-function allele. Gain-of-function mutations in this gene are the principal mediators of fluconazole resistance in this human fungal pathogen. Eighteen synthetically lethal S. cerevisiae genetic mutants were identified in cells expressing C. glabrata PDR1+. One mutant, lacking the histone acetyltransferase Gcn5, was investigated further. Deletion or drug-mediated inhibition of Gcn5 caused a lethal phenotype in C. glabrata cells expressing PDR1+ alleles. Moreover, deletion or drug-mediated inactivation of Gcn5, inhibited the emergence of fluconazole-resistant C. glabrata isolates in evolution experiments. Thus, taken together, the data generated in this study provides proof of concept that synthetically lethal genetic screens can identify novel candidate proteins that when therapeutically targeted could allow effective treatment of drug-resistant infections. [ABSTRACT FROM AUTHOR]

DNA methyltransferases are ubiquitous enzymes conserved in bacteria, plants and opisthokonta. These enzymes, which methylate cytosines, are involved in numerous biological processes, notably development. In mammals and higher plants, methylation patterns established and maintained by the cytosine DNA methyltransferases (DMTs) are essential to zygotic development. In fungi, some members of an extensively conserved fungal-specific DNA methyltransferase class are both mediators of the Repeat Induced Point mutation (RIP) genome defense system and key players of sexual reproduction. Yet, no DNA methyltransferase activity of these purified RID (RIP deficient) proteins could be detected in vitro. These observations led us to explore how RID-like DNA methyltransferase encoding genes would play a role during sexual development of fungi showing very little genomic DNA methylation, if any. To do so, we used the model ascomycete fungus Podospora anserina. We identified the PaRid gene, encoding a RID-like DNA methyltransferase and constructed knocked-out ΔPaRid defective mutants. Crosses involving P. anserina ΔPaRid mutants are sterile. Our results show that, although gametes are readily formed and fertilization occurs in a ΔPaRid background, sexual development is blocked just before the individualization of the dikaryotic cells leading to meiocytes. Complementation of ΔPaRid mutants with ectopic alleles of PaRid, including GFP-tagged, point-mutated and chimeric alleles, demonstrated that the catalytic motif of the putative PaRid methyltransferase is essential to ensure proper sexual development and that the expression of PaRid is spatially and temporally restricted. A transcriptomic analysis performed on mutant crosses revealed an overlap of the PaRid-controlled genetic network with the well-known mating-types gene developmental pathway common to an important group of fungi, the Pezizomycotina. [ABSTRACT FROM AUTHOR]

Cryptococcus gattii is a capsular fungal pathogen, which causes life-threatening cryptococcosis in immunocompetent individuals. This emerging pathogen is less likely to be recognized by innate immunity compared to traditional Cryptococcus neoformans strains. Previous studies indicate that C-type lectin receptors (CLRs), including dectin-1 and dectin-2, play a role in recognizing cryptococcal cells; however, it remains to be elucidated whether the receptors physically associate with C. gattii yeast cell surfaces. Based on the previous findings, we hypothesized that culture conditions influence the expression or exposure of CLR ligands on C. gattii. Therefore, in the present study, we first investigated the culture conditions that induce exposure of CLR ligands on C. gattii yeast cells via the binding assay using recombinant fusion proteins of mouse CLR and IgG Fc, Fc dectin-1 and Fc dectin-2. Common fungal culture media, such as yeast extract–peptone–dextrose (YPD) broth, Sabouraud broth, and potato dextrose agar, did not induce the exposure of dectin-1 ligands, including β-1,3-glucan, on both capsular and acapsular C. gattii strains, in contrast to Fc dectin-1 and Fc dectin-2 bound to C. gattii cells growing in the conventional synthetic dextrose (SD) medium [may also be referred to as a yeast nitrogen base with glucose medium]. The medium also induced the exposure of dectin-1 ligands on C. neoformans, whereas all tested media induced dectin-1 and dectin-2 ligands in a control fungus Candida albicans. Notably, C. gattii did not expose dectin-1 ligands in SD medium supplemented with yeast extract or neutral buffer. In addition, compared to YPD medium-induced C. gattii, SD medium-induced C. gattii more efficiently induced the phosphorylation of Syk, Akt, and Erk1/2 in murine dendritic cells (DCs). Afterwards, the cells were considerably engulfed by DCs and remarkably induced DCs to secrete the inflammatory cytokines. Overall, the findings suggest that C. gattii alters its immunostimulatory potential in response to the environment. [ABSTRACT FROM AUTHOR]

Histone H3 and its variants regulate gene expression but the latter are absent in most ascomycetous fungi. Here, we report the identification of a variant histone H3, which we have designated H3VCTG because of its exclusive presence in the CTG clade of ascomycetes, including Candida albicans, a human pathogen. C. albicans grows both as single yeast cells and hyphal filaments in the planktonic mode of growth. It also forms a three-dimensional biofilm structure in the host as well as on human catheter materials under suitable conditions. H3VCTG null (hht1/hht1) cells of C. albicans are viable but produce more robust biofilms than wild-type cells in both in vitro and in vivo conditions. Indeed, a comparative transcriptome analysis of planktonic and biofilm cells reveals that the biofilm circuitry is significantly altered in H3VCTG null cells. H3VCTG binds more efficiently to the promoters of many biofilm-related genes in the planktonic cells than during biofilm growth, whereas the binding of the core canonical histone H3 on the corresponding promoters largely remains unchanged. Furthermore, biofilm defects associated with master regulators, namely, biofilm and cell wall regulator 1 (Bcr1), transposon enhancement control 1 (Tec1), and non-dityrosine 80 (Ndt80), are significantly rescued in cells lacking H3VCTG. The occupancy of the transcription factor Bcr1 at its cognate promoter binding sites was found to be enhanced in the absence of H3VCTG in the planktonic form of growth resulting in enhanced transcription of biofilm-specific genes. Further, we demonstrate that co-occurrence of valine and serine at the 31st and 32nd positions in H3VCTG, respectively, is essential for its function. Taken together, we show that even in a unicellular organism, differential gene expression patterns are modulated by the relative occupancy of the specific histone H3 type at the chromatin level. [ABSTRACT FROM AUTHOR]

Phagocytosis is a receptor-mediated process critical to innate immune clearance of pathogens. It proceeds in a regulated sequence of stages: (a) migration of phagocytes towards pathogens, (b) recognition of PAMPs and binding through PRRs, (c) engulfment and internalisation into phagosomes, (d) phagosome maturation, and (e) killing of pathogen or host cells. However, little is known about the role that individual receptors play in these discrete stages in the recognition of fungal cells. In a previous study, we found that dectin-2 deficiency impacted some but not all stages of macrophage-mediated phagocytosis of Candida glabrata. Because the C-type lectin receptor dectin-2 critically requires coupling to the FcRγ chain for signalling, we hypothesised that this coupling may be important for regulating phagocytosis of fungal cargo. We therefore examined how deficiency in FcRγ itself or two receptors to which it couples (dectin-2 and mincle) impacts phagocytosis of six fungal organisms representing three different fungal taxa. Our data show that deficiency in these proteins impairs murine bone marrow-derived macrophage migration, engulfment, and phagosome maturation, but not macrophage survival. Therefore, FcRγ engagement with selective C-type lectin receptors (CLRs) critically affects the spatio-temporal dynamics of fungal phagocytosis. [ABSTRACT FROM AUTHOR]

Candida auris is an emerging, multi-drug resistant, health care-associated fungal pathogen. Its predominant prevalence in hospitals and nursing homes indicates its ability to adhere to and colonize the skin, or persist in an environment outside the host—a trait unique from other Candida species. Besides being associated globally with life-threatening disseminated infections, C. auris also poses significant clinical challenges due to its ability to adhere to polymeric surfaces and form highly drug-resistant biofilms. Here, we performed bioinformatic studies to identify the presence of adhesin proteins in C. auris, with sequence as well as 3-D structural homologies to the major adhesin/invasin of C. albicans, Als3. Anti-Als3p antibodies generated by vaccinating mice with NDV-3A (a vaccine based on the N-terminus of Als3 protein formulated with alum) recognized C. auris in vitro, blocked its ability to form biofilms and enhanced macrophage-mediated killing of the fungus. Furthermore, NDV-3A vaccination induced significant levels of C. auris cross-reactive humoral and cellular immune responses, and protected immunosuppressed mice from lethal C. auris disseminated infection, compared to the control alum-vaccinated mice. The mechanism of protection is attributed to anti-Als3p antibodies and CD4+ T helper cells activating tissue macrophages. Finally, NDV-3A potentiated the protective efficacy of the antifungal drug micafungin, against C. auris candidemia. Identification of Als3-like adhesins in C. auris makes it a target for immunotherapeutic strategies using NDV-3A, a vaccine with known efficacy against other Candida species and safety as well as efficacy in clinical trials. Considering that C. auris can be resistant to almost all classes of antifungal drugs, such an approach has profound clinical relevance. [ABSTRACT FROM AUTHOR]

The S-phase checkpoint plays an essential role in regulation of the ribonucleotide reductase (RNR) activity to maintain the dNTP pools. How eukaryotic cells respond appropriately to different levels of replication threats remains elusive. Here, we have identified that a conserved GSK-3 kinase Mck1 cooperates with Dun1 in regulating this process. Deleting MCK1 sensitizes dun1Δ to hydroxyurea (HU) reminiscent of mec1Δ or rad53Δ. While Mck1 is downstream of Rad53, it does not participate in the post-translational regulation of RNR as Dun1 does. Mck1 phosphorylates and releases the Crt1 repressor from the promoters of DNA damage-inducible genes as RNR2-4 and HUG1. Hug1, an Rnr2 inhibitor normally silenced, is induced as a counterweight to excessive RNR. When cells suffer a more severe threat, Mck1 inhibits HUG1 transcription. Consistently, only a combined deletion of HUG1 and CRT1, confers a dramatic boost of dNTP levels and the survival of mck1Δdun1Δ or mec1Δ cells assaulted by a lethal dose of HU. These findings reveal the division-of-labor between Mck1 and Dun1 at the S-phase checkpoint pathway to fine-tune dNTP homeostasis. [ABSTRACT FROM AUTHOR]

Variation in synonymous codon usage is abundant across multiple levels of organization: between codons of an amino acid, between genes in a genome, and between genomes of different species. It is now well understood that variation in synonymous codon usage is influenced by mutational bias coupled with both natural selection for translational efficiency and genetic drift, but how these processes shape patterns of codon usage bias across entire lineages remains unexplored. To address this question, we used a rich genomic data set of 327 species that covers nearly one third of the known biodiversity of the budding yeast subphylum Saccharomycotina. We found that, while genome-wide relative synonymous codon usage (RSCU) for all codons was highly correlated with the GC content of the third codon position (GC3), the usage of codons for the amino acids proline, arginine, and glycine was inconsistent with the neutral expectation where mutational bias coupled with genetic drift drive codon usage. Examination between genes’ effective numbers of codons and their GC3 contents in individual genomes revealed that nearly a quarter of genes (381,174/1,683,203; 23%), as well as most genomes (308/327; 94%), significantly deviate from the neutral expectation. Finally, by evaluating the imprint of translational selection on codon usage, measured as the degree to which genes’ adaptiveness to the tRNA pool were correlated with selective pressure, we show that translational selection is widespread in budding yeast genomes (264/327; 81%). These results suggest that the contribution of translational selection and drift to patterns of synonymous codon usage across budding yeasts varies across codons, genes, and genomes; whereas drift is the primary driver of global codon usage across the subphylum, the codon bias of large numbers of genes in the majority of genomes is influenced by translational selection. [ABSTRACT FROM AUTHOR]

In the alcoholic fermentation process, Saccharomyces cerevisiae strains present differences in their nitrogen consumption profiles, these phenotypic outcomes have complex genetic and molecular architectures. In this sense, variations in nitrogen signaling pathways regulated by TORC1 represent one of the main sources of phenotypic diversity in nitrogen consumption. This emphasizes the possible roles that allelic variants from the TORC1 pathway have in the nitrogen consumption differences observed in yeast during the alcoholic fermentation. Here, we studied the allelic diversity in the TORC1 pathway across four yeast strains and determined how these polymorphisms directly impact nitrogen consumption during alcoholic fermentation. Using a reciprocal hemizygosity approach combined with phenotyping under fermentative conditions, we found that allelic variants of GTR1, TOR2, SIT4, SAP185, EAP1, NPR1 and SCH9 underlie differences in the ammonium and amino acids consumption phenotypes. Among these, GTR1 alleles from the Wine/European and West African genetic backgrounds showed the greatest effects on ammonium and amino acid consumption, respectively. Furthermore, we identified allelic variants of SAP185, TOR2, SCH9 and NPR1 from an oak isolate that increased the amino acid consumption preference over ammonium; representing putative candidates coming from a non-domesticated strain that could be used for genetic improvement programs. In conclusion, our results demonstrated that a large number of allelic variants within the TORC1 pathway significantly impacts on regulatory mechanisms of nitrogen assimilation during alcoholic fermentation. [ABSTRACT FROM AUTHOR]

Disease spread of Pseudocercospora fijiensis, causal agent of the black Sigatoka disease of banana, depends on ascospores produced through the sexual reproductive cycle. We used phylogenetic analysis to identify P. fijiensis homologs (PKS8-4 and Hybrid8-3) to the PKS4 polyketide synthases (PKS) from Neurospora crassa and Sordaria macrospora involved in sexual reproduction. These sequences also formed a clade with lovastatin, compactin, and betaenone-producing PKS sequences. Transcriptome analysis showed that both the P. fijiensis Hybrid8-3 and PKS8-4 genes have higher expression in infected leaf tissue compared to in culture. Domain analysis showed that PKS8-4 is more similar than Hybrid8-3 to PKS4. pPKS8-4:GFP transcriptional fusion transformants showed expression of GFP in flask-shaped structures in mycelial cultures as well as in crosses between compatible and incompatible mating types. Confocal microscopy confirmed expression in spermagonia in leaf substomatal cavities, consistent with a role in sexual reproduction. A disruption mutant of pks8-4 retained normal pathogenicity on banana, and no differences were observed in growth, conidial production, and spermagonia production. GC-MS profiling of the mutant and wild type did not identify differences in polyketide metabolites, but did identify changes in saturated fatty acid methyl esters and alkene and alkane derivatives. To our knowledge, this is the first report of a polyketide synthase pathway associated with spermagonia. [ABSTRACT FROM AUTHOR]

The Brazilian stingless bee Scaptotrigona depilis requires the brood cells-associated fungus Zygosaccharomyces sp. as steroid source for metamorphosis. Besides the presence of Zygosaccharomyces sp., other fungi inhabit S. depilis brood cells, but their biological functions are unknown. Here we show that Candida sp. and Monascus ruber, isolated from cerumen of S. depilis brood provisions, interact with Zygosaccharomyces sp. and modulate its growth. Candida sp. produces volatile organic compounds (VOCs) that stimulate Zygosacchromyces sp. development. Monascus ruber inhibits Zygosacchromyces sp. growth by producing lovastatin, which blocks steroid biosynthesis. We also observed that in co-cultures M. ruber inhibits Candida sp. through the production of monascin. The modulation of Zygosaccharomyces sp. growth by brood cell-associated fungi suggests their involvement in S. depilis larval development. This tripartite fungal community opens new perspectives in the research of microbial interactions with bees. [ABSTRACT FROM AUTHOR]

The enteric bacterial pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium), utilizes two type III secretion systems (T3SSs) to invade host cells, survive and replicate intracellularly. T3SS1 and its dedicated effector proteins are required for bacterial entry into non-phagocytic cells and establishment and trafficking of the nascent Salmonella-containing vacuole (SCV). Here we identify the first T3SS1 effector required to maintain the integrity of the nascent SCV as SopF. SopF associates with host cell membranes, either when translocated by bacteria or ectopically expressed. Recombinant SopF binds to multiple phosphoinositides in protein-lipid overlays, suggesting that it targets eukaryotic cell membranes via phospholipid interactions. In yeast, the subcellular localization of SopF is dependent on the activity of Mss4, a phosphatidylinositol 4-phosphate 5-kinase that generates PI(4,5)P2 from PI(4)P, indicating that membrane recruitment of SopF requires specific phospholipids. Ectopically expressed SopF partially colocalizes with specific phosphoinositide pools present on the plasma membrane in mammalian cells and with cytoskeletal-associated markers at the leading edge of cells. Translocated SopF concentrates on plasma membrane ruffles and around intracellular bacteria, presumably on the SCV. SopF is not required for bacterial invasion of non-phagocytic cells but is required for maintenance of the internalization vacuole membrane as infection with a S. Typhimurium ΔsopF mutant led to increased lysis of the SCV compared to wild type bacteria. Our structure-function analysis shows that the carboxy-terminal seven amino acids of SopF are essential for its membrane association in host cells and to promote SCV membrane stability. We also describe that SopF and another T3SS1 effector, SopB, act antagonistically to modulate nascent SCV membrane dynamics. In summary, our study highlights that a delicate balance of type III effector activities regulates the stability of the Salmonella internalization vacuole. [ABSTRACT FROM AUTHOR]

One mechanism through which S. cerevisiae may improve the performance of pigs is by altering the composition of the gut microbiota, a response that may be enhanced by early postnatal supplementation of probiotics. To test this hypothesis, newborn piglets (16 piglets/group) were treated with either S. cerevisiae yeast (5 x 109 cfu/pig: Low) or (2.5 x 1010 cfu/piglet: High) or equivalent volume of sterile water (Control) by oral gavage every other day starting from day 1 of age until weaning (28±1 days of age). Piglet body weight was recorded on days 1, 3, 7, 10, 17, 24 and 28 and average daily gain (ADG) calculated for the total period. At weaning, piglets were euthanized to collect cecum content for microbial profiling by sequencing of the 16S rRNA gene. ADG was higher in both Low and High yeast groups than in Control group (P<0.05). Alpha diversity analyses indicated a more diverse microbiota in the Control group compared with Low yeast group; the High yeast being intermediate (P < 0.01). Similarly, Beta diversity analyses indicated differences among treatments (P = 0.03), mainly between Low yeast and Control groups (P = 0.02). The sparse Partial Least Squares Discriminant Analysis (sPLS-DA) indicated that Control group was discriminated by a higher abundance of Veillonella, Dorea, Oscillospira and Clostridium; Low yeast treated pigs by higher Blautia, Collinsella and Eubacterium; and High yeast treated pigs by higher Eubacterium, Anaerostipes, Parabacteroides, Mogibacterium and Phascolarctobacterium. Partial Least Squares (PLS) analysis showed that piglet ADG was positively correlated with genus Prevotella in High yeast group. Yeast supplementation significantly affected microbial diversity in cecal contents of suckling piglets associated with an improvement of short chain fatty acid producing bacteria in a dose-dependent manner. In conclusion, yeast treatment improved piglet performance and shaped the piglet cecum microbiota composition in a dose dependent way. [ABSTRACT FROM AUTHOR]

Gene manipulation and epitope tagging are essential tools for understanding the molecular function of specific genes. The opportunistic human pathogen Candida albicans is a diploid fungus that utilizes a non-canonical genetic code. Since selection markers available in this organism are scarce, several tools based on recyclable markers have been developed for gene disruption, such as the Clox system. This system relies on the Cre recombinase, which recycles selection markers flanked by loxP sites with high efficiency, facilitating single marker or multi-marker recycling. However, PCR-based modules for epitope tagging, such the pFA-modules, mainly use limited non-recyclable auxotrophic markers. To solve this problem, we have used a Gibson assembly strategy to construct a set of new plasmids where the auxotrophic markers of the pFA vectors were swapped with five recyclable marker modules of the Clox system, enhancing the versatility of the pFA plasmids. This new toolkit, named pFA-Clox, is composed of 36 new vectors for gene disruption and epitope tagging (GFP, 3xGFP, mCherry, 3xHA, 5xmyc and TAP). These plasmids contain the dominant NAT1 marker, as well as URA3, HIS1 and ARG4 cassettes, thereby permitting functional analysis of laboratory strains as well as clinical isolates of C. albicans. In summary, we have adapted the Clox system to the pFA-backbone vectors. Thus, the set of primers used for the amplification of previously published pFA modules can also be utilized in this new pFA-Clox system. Therefore, this new toolkit harbors the advantages of both systems, allowing accelerated gene modification strategies that could reduce time and costs in strain construction for C. albicans. [ABSTRACT FROM AUTHOR]

Background: We applied an in vitro model to evaluate the efficacy of a heparinized 40% ethanol-based lock solution in a wide variety of clinical isolates causing C-RBSI. Methods: A total of 100 clinical strains were collected retrospectively from the blood of patients with C-RBSI. The reduction in biomass and metabolic activity of biofilms was measured using the crystal violet (CV) assay and XTT assay, respectively. Regrowth inhibition (RI) was measured within 24 hours and 72 hours of ethanol lock therapy. Percentage reduction of ≥ 85% in RI was considered to be successful. Results: Ethanol lock was more effective in reducing metabolic activity than in reducing biomass (83% vs. 50%, respectively). Percentages of RI diminished as regrowth was prolonged (57% for 24 hours and 17% for 72 hours of regrowth). No statistically significant intraspecies differences were found in biofilm reduction or in RI (p>0.05). Conclusions: The use of heparinized 40% ethanol lock solution for 72 hours significantly reduced biomass and metabolic activity in clinical isolates from patients with C-RBSI. However, as biofilm has an important regrowth rate, 40% ethanol solution was not able to fully eradicate biofilm in vitro. [ABSTRACT FROM AUTHOR]

Hsp90 is a conserved molecular chaperone that assists in the folding and function of diverse cellular regulators, with a profound impact on biology, disease, and evolution. As a central hub of protein interaction networks, Hsp90 engages with hundreds of protein–protein interactions within eukaryotic cells. These interactions include client proteins, which physically interact with Hsp90 and depend on the chaperone for stability or function, as well as co-chaperones and partner proteins that modulate chaperone function. Currently, there are no methods to accurately predict Hsp90 interactors and there has been considerable network rewiring over evolutionary time, necessitating experimental approaches to define the Hsp90 network in the species of interest. This is a pressing challenge for fungal pathogens, for which Hsp90 is a key regulator of stress tolerance, drug resistance, and virulence traits. To address this challenge, we applied a novel biochemical fractionation and quantitative proteomic approach to examine alterations to the proteome upon perturbation of Hsp90 in a leading human fungal pathogen, Candida albicans. In parallel, we performed affinity purification coupled to mass spectrometry to define physical interacting partners for Hsp90 and the Hsp90 co-chaperones and identified 164 Hsp90-interacting proteins, including 111 that are specific to the pathogen. We performed the first analysis of the Hsp90 interactome upon antifungal drug stress and demonstrated that Hsp90 stabilizes processing body (P-body) and stress granule proteins that contribute to drug tolerance. We also describe novel roles for Hsp90 in regulating posttranslational modification of the Rvb1-Rvb2-Tah1-Pih1 (R2TP) complex and the formation of protein aggregates in response to thermal stress. This study provides a global view of the Hsp90 interactome in a fungal pathogen, demonstrates the dynamic role of Hsp90 in response to environmental perturbations, and highlights a novel connection between Hsp90 and the regulation of mRNA-associated protein granules. [ABSTRACT FROM AUTHOR]

The localization of mRNAs encoding secreted/membrane proteins (mSMPs) to the endoplasmic reticulum (ER) likely facilitates the co-translational translocation of secreted proteins. However, studies have shown that mSMP recruitment to the ER in eukaryotes can occur in a manner that is independent of the ribosome, translational control, and the signal recognition particle, although the mechanism remains largely unknown. Here, we identify a cis-acting RNA sequence motif that enhances mSMP localization to the ER and appears to increase mRNA stability, and both the synthesis and secretion of secretome proteins. Termed SECReTE, for ecretion-nhancing is gulatory argeting lement, this motif is enriched in mRNAs encoding secretome proteins translated on the ER in eukaryotes and on the inner membrane of prokaryotes. SECReTE consists of ≥10 nucleotide triplet repeats enriched with pyrimidine (C/U) every third base (i.e. NNY, where N = any nucleotide, Y = pyrimidine) and can be present in the untranslated as well as the coding regions of the mRNA. Synonymous mutations that elevate the SECReTE count in a given mRNA (e.g. SUC2, HSP150, and CCW12) lead to an increase in protein secretion in yeast, while a reduction in count led to less secretion and physiological defects. Moreover, the addition of SECReTE to the 3’UTR of an mRNA for an exogenously expressed protein (e.g. GFP) led to its increased secretion from yeast cells. Thus, SECReTE constitutes a novel RNA motif that facilitates ER-localized mRNA translation and protein secretion. [ABSTRACT FROM AUTHOR]

A new method is introduced allowing seamless assembly of independent, functionally tested, blunt-end double strand nucleic acid (DNA fragments not supplied in vectors such as plasmids) into more complex biological (e.g. protein expression vectors) and higher order multi-device (e.g. biochemical pathways). Individual parts include bacterial selection markers and origins of replication, promoters useful in a variety of species, transcription terminators, shuttle sequences and a variety of “N” and “C” terminal solubility/affinity protein tags. Parts are not subjected to pre-assembly manipulation with nucleic acid modifying enzymes. Instead, they are simply mixed in appropriate pre-defined combinations and concentrations and then seamlessly linked into devices employing a specialized thermostable enzyme blend. Combinatorial assembly of parts is an inherent time-saving feature of the new method, in contrast to hierarchical binary assembly (“one part at a time”) methods. This feature substantially simplifies and speeds optimization of device and system development. The versatility and functionality of the new method was shown by combinatorial assembly of parts into vector devices, one of which optimally expressed protein from a model gene. Also, a four-enzyme biosynthetic pathway system was re-created by combinatorial construction from parts and devices. Concepts discussed in this paper provide synthetic biologists, chemists and bio-engineers with improved and expanded capability to create novel biological molecules and systems. [ABSTRACT FROM AUTHOR]

The present study aimed to evaluate the probiotic characteristics of certain microbial strains for potential use as feed additives. Three bacterial strains and a yeast previously isolated from different environments were investigated. The strains were subjected to molecular identification and established as Lactobacillus paracasei CP133, Lactobacillus plantarum CP134, Bacillus subtilis CP350 and Saccharomyces cerevisiae CP605. Lactobacillus sp. CP133 and CP134 exhibited antibiosis, antibiotic activity, and relative odor reduction ability. Bacillus subtilis CP350 was thermotolerant, reduced hydrogen sulfide gas and showed significant proteolytic activity, whereas Saccharomyces cerevisiae CP605 exhibited high acid and bile salt tolerance. In general, the isolates in this study demonstrated improved functional characteristics, particularly acid and bile tolerance and relative cell adhesion to HT-29 monolayer cell line. Results in this work provides multifunctional probiotic characteristics of the strains for potential development of probiotics and cleaning of the environment. [ABSTRACT FROM AUTHOR]

In eukaryotes, the cell cycle is driven by the actions of several cyclin dependent kinases (CDKs) and an array of regulatory proteins called cyclins, due to the cyclical expression patterns of the latter. In yeast, the accepted pattern of cyclin waves is based on qualitative studies performed by different laboratories using different strain backgrounds, different growing conditions and media, and different kinds of genetic manipulation. Additionally, only the subset of cyclins regulating Cdc28 was included, while the Pho85 cyclins were excluded. We describe a comprehensive, quantitative and accurate blueprint of G1 cyclins in the yeast Saccharomyces cerevisiae that, in addition to validating previous conclusions, yields new findings and establishes an accurate G1 cyclin blueprint. For the purposes of this research, we produced a collection of strains with all G1 cyclins identically tagged using the same and most respectful procedure possible. We report the contribution of each G1 cyclin for a broad array of growing and stress conditions, describe an unknown role for Pcl2 in heat-stress conditions and demonstrate the importance of maintaining the 3’UTR sequence of cyclins untouched during the tagging process. [ABSTRACT FROM AUTHOR]

Invasive candidiasis, mainly caused by Candida albicans, is a serious healthcare problem with high mortality rates, particularly in immunocompromised patients. Innate immune cells express pathogen recognition receptors (PRRs) including C-type lectin-like receptors (CLRs) that bind C. albicans to initiate an immune response. Multiple CLRs including Dectin-1, Dectin-2 and Mincle have been proposed individually to contribute to the immune response to C. albicans. However how these receptors collaborate to clear a fungal infection is unknown. Herein, we used novel multi-CLR knockout (KO) mice to decipher the individual, collaborative and collective roles of Dectin-1, Dectin-2 and Mincle during systemic C. albicans infection. These studies revealed an unappreciated and profound role for CLR co-operation in anti-fungal immunity. The protective effect of multiple CLRs was markedly greater than any single receptor, and was mediated through inflammatory monocytes via recognition and phagocytosis of C. albicans, and production of C. albicans-induced cytokines and chemokines. These CLRs were dispensable for mediating similar responses from neutrophils, likely due to lower expression of these CLRs on neutrophils compared to inflammatory monocytes. Concurrent deletion of Dectin-1 and Dectin-2, or all three CLRs, resulted in dramatically increased susceptibility to systemic C. albicans infection compared to mice lacking a single CLR. Multi-CLR KO mice were unable to control fungal growth due to an inadequate early inflammatory monocyte-mediated response. In response to excessive fungal growth, the multi-CLR KO mice mounted a hyper-inflammatory response, likely leading to multiple organ failure. Thus, these data reveal a critical role for CLR co-operation in the effective control of C. albicans and maintenance of organ function during infection. [ABSTRACT FROM AUTHOR]

Eukaryotes have evolved elaborate mechanisms to ensure that chromosomes segregate with high fidelity during mitosis and meiosis, and yet specific aneuploidies can be adaptive during environmental stress. Here, we identify a chromatin-based system required for inducible aneuploidy in a human pathogen. Candida albicans utilizes chromosome missegregation to acquire tolerance to antifungal drugs and for nonmeiotic ploidy reduction after mating. We discovered that the ancestor of C. albicans and 2 related pathogens evolved a variant of histone 2A (H2A) that lacks the conserved phosphorylation site for kinetochore-associated Bub1 kinase, a key regulator of chromosome segregation. Using engineered strains, we show that the relative gene dosage of this variant versus canonical H2A controls the fidelity of chromosome segregation and the rate of acquisition of tolerance to antifungal drugs via aneuploidy. Furthermore, whole-genome chromatin precipitation analysis reveals that Centromere Protein A/ Centromeric Histone H3-like Protein (CENP-A/Cse4), a centromeric histone H3 variant that forms the platform of the eukaryotic kinetochore, is depleted from tetraploid-mating products relative to diploid parents and is virtually eliminated from cells exposed to aneuploidy-promoting cues. We conclude that genetically programmed and environmentally induced changes in chromatin can confer the capacity for enhanced evolvability via chromosome missegregation. [ABSTRACT FROM AUTHOR]

Yeast species that colonize the surface of grape berries at harvest time play an important role during the winemaking process. In this study, the use of culturable microbial techniques permitted a quantitative and qualitative inventory of the different yeast species present on the grape berry surfaces of Montepulciano and Verdicchio varieties when treated with conventional and organic fungicides. The results show that the most widespread yeast species at harvest time were Aureobasidium pullulans and Hanseniaspora uvarum, which are considered normal resident species and independent of the grape varieties and treatments applied. Specific differences when comparing the grape varieties were observed in species and were detected at a lower frequency; Pichia spp. were prevalent in Verdicchio, whereas Lachancea thermotolerans and Zygoascus meyerae were found in Montepulciano. In both vineyards, the farming treatments improved the competitiveness of A. pullulans, which was probably due to its reduced susceptibility to treatments that improved the competition toward other fungi. In contrast, the fermenting yeast H. uvarum was negatively affected by fungicide treatments and showed a reduced presence if compared with untreated grapes. Organic treatments directly impacted the occurrence of Issachenkia terricola in Montepulciano grapes and Debaryomyces hansenii and Pichia membranifaciens in Verdicchio. Conversely, a negative effect of organic treatments was found toward Metschnikowia pulcherrima and Starmerella bacillaris. Overall, the data suggest that the yeast community colonizing the grape berry surface was influenced by both grape variety and farming treatments, which characterized the yeast biota of spontaneous must fermentation. [ABSTRACT FROM AUTHOR]

Accurate chromosome segregation during meiosis relies on the prior establishment of at least one crossover recombination event between homologous chromosomes. Most meiotic recombination intermediates that give rise to interhomolog crossovers are embedded within a hallmark chromosomal structure called the synaptonemal complex (SC), but the mechanisms that coordinate the processes of SC assembly (synapsis) and crossover recombination remain poorly understood. Among known structural components of the budding yeast SC, the Zip1 protein is unique for its independent role in promoting crossover recombination; Zip1 is specifically required for the large subset of crossovers that also rely on the meiosis-specific MutSγ complex. Here we report that adjacent regions within Zip1’s N terminus encompass its crossover and synapsis functions. We previously showed that deletion of Zip1 residues 21–163 abolishes tripartite SC assembly and prevents robust SUMOylation of the SC central element component, Ecm11, but allows excess MutSγ crossover recombination. We find the reciprocal phenotype when Zip1 residues 2–9 or 10–14 are deleted; in these mutants SC assembles and Ecm11 is hyperSUMOylated, but MutSγ crossovers are strongly diminished. Interestingly, Zip1 residues 2–9 or 2–14 are required for the normal localization of Zip3, a putative E3 SUMO ligase and pro-MutSγ crossover factor, to Zip1 polycomplex structures and to recombination initiation sites. By contrast, deletion of Zip1 residues 15–20 does not detectably prevent Zip3’s localization at Zip1 polycomplex and supports some MutSγ crossing over but prevents normal SC assembly and Ecm11 SUMOylation. Our results highlight distinct N terminal regions that are differentially critical for Zip1’s roles in crossing over and SC assembly; we speculate that the adjacency of these regions enables Zip1 to serve as a liaison, facilitating crosstalk between the two processes by bringing crossover recombination and synapsis factors within close proximity of one another. [ABSTRACT FROM AUTHOR]

Storage, manipulation and delivery of DNA fragments is crucial for synthetic biology applications, subsequently allowing organisms of interest to be engineered with genes or pathways to produce desirable phenotypes such as disease or drought resistance in plants, or for synthesis of a specific chemical product. However, DNA with high G+C content can be unstable in many host organisms including Saccharomyces cerevisiae. Here, we report the development of Sinorhizobium meliloti, a nitrogen-fixing plant symbioticα-Proteobacterium, as a novel host that can store DNA, and mobilize DNA to E. coli, S. cerevisiae, and the eukaryotic microalgae Phaeodactylum tricornutum. To achieve this, we deleted the hsdR restriction-system in multiple reduced genome strains of S. meliloti that enable DNA transformation with up to 1.4 x 105 and 2.1 x 103 CFU μg-1 of DNA efficiency using electroporation and a newly developed polyethylene glycol transformation method, respectively. Multi-host and multi-functional shuttle vectors (MHS) were constructed and stably propagated in S. meliloti, E. coli, S. cerevisiae, and P. tricornutum. We also developed protocols and demonstrated direct transfer of these MHS vectors via conjugation from S. meliloti to E. coli, S. cerevisiae, and P. tricornutum. The development of S. meliloti as a new host for inter-kingdom DNA transfer will be invaluable for synthetic biology research and applications, including the installation and study of genes and biosynthetic pathways into organisms of interest in industry and agriculture. [ABSTRACT FROM AUTHOR]

H3K9 methylation (H3K9me) is a conserved marker of heterochromatin, a transcriptionally silent chromatin structure. Knowledge of the mechanisms for regulating heterochromatin distribution is limited. The fission yeast JmjC domain-containing protein Epe1 localizes to heterochromatin mainly through its interaction with Swi6, a homologue of heterochromatin protein 1 (HP1), and directs JmjC-mediated H3K9me demethylation in vivo. Here, we found that loss of epe1 (epe1Δ) induced a red-white variegated phenotype in a red-pigment accumulation background that generated uniform red colonies. Analysis of isolated red and white colonies revealed that silencing of genes involved in pigment accumulation by stochastic ectopic heterochromatin formation led to white colony formation. In addition, genome-wide analysis of red- and white-isolated clones revealed that epe1Δ resulted in a heterogeneous heterochromatin distribution among clones. We found that Epe1 had an N-terminal domain distinct from its JmjC domain, which activated transcription in both fission and budding yeasts. The N-terminal transcriptional activation (NTA) domain was involved in suppression of ectopic heterochromatin-mediated red-white variegation. We introduced a single copy of Epe1 into epe1Δ clones harboring ectopic heterochromatin, and found that Epe1 could reduce H3K9me from ectopic heterochromatin but some of the heterochromatin persisted. This persistence was due to a latent H3K9me source embedded in ectopic heterochromatin. Epe1H297A, a canonical JmjC mutant, suppressed red-white variegation, but entirely failed to remove already-established ectopic heterochromatin, suggesting that Epe1 prevented stochastic de novo deposition of ectopic H3K9me in an NTA-dependent but JmjC-independent manner, while its JmjC domain mediated removal of H3K9me from established ectopic heterochromatin. Our results suggest that Epe1 not only limits the distribution of heterochromatin but also controls the balance between suppression and retention of heterochromatin-mediated epigenetic diversification. [ABSTRACT FROM AUTHOR]