Yeast Lsm Pro-Apoptotic Mutants Show Defects in Autophagy.

LSM4 is an essential yeast gene encoding a component of different LSM complexes involved in the regulation of mRNA splicing, stability, and translation. In previous papers, we reported that the expression in S. cerevisiae of the K. lactis LSM4 gene lacking the C-terminal Q/N-rich domain in an Lsm4 null strain S. cerevisiae (Sclsm4Δ1) restored cell viability. Nevertheless, in this transformed strain, we observed some phenotypes that are typical markers of regulated cell death, reactive oxygen species (ROS), and oxidated RNA accumulation. In this paper, we report that a similar truncation operated in the S. cerevisiae LSM4 gene confers on cells the same phenotypes observed with the K. lactis lsm4Δ1 gene. Up until now, there was no evidence of the direct involvement of LSM4 in autophagy. Here we found that the Sclsm4Δ1 mutant showed a block in the autophagic process and was very sensitive to nitrogen starvation or treatment with low doses of rapamycin, an inducer of autophagy. Moreover, both during nitrogen starvation and aging, the Sclsm4Δ1 mutant accumulated cytoplasmic autophagy-related structures, suggesting a role of Lsm4 in a later step of the autophagy process.

Inappropriate expression of the translation elongation factor 1A disrupts genome stability and metabolism.

The translation elongation factor eEF1A is one of the most abundant proteins found within cells, and its role within protein synthesis is well documented. Levels of eEF1A are tightly controlled, with inappropriate expression linked to oncogenesis. However, the mechanisms by which increased eEF1A expression alters cell behaviour are unknown. Our analyses in yeast suggest that elevation of eEF1A levels leads to stabilisation of the spindle pole body and changes in nuclear organisation. Elevation of the eEF1A2 isoform also leads to altered nuclear morphology in cultured human cells, suggesting a conserved role in maintaining genome stability. Gene expression and metabolomic analyses reveal that the level of eEF1A is crucial for the maintenance of metabolism and amino acid levels in yeast, most likely because of its role in the control of vacuole function. Increased eEF1A2 levels trigger lysosome biogenesis in cultured human cells, also suggesting a conserved role within metabolic control mechanisms. Taken together, our data suggest that the control of eEF1A levels is important for the maintenance of a number of cell functions beyond translation and that its de-regulation might contribute to its oncogenic properties.

Increased levels of RNA oxidation enhance the reversion frequency in aging pro-apoptotic yeast mutants.

Despite recent advances in understanding the complexity of RNA processes, regulation of the metabolism of oxidized cellular RNAs and the mechanisms through which oxidized ribonucleotides affect mRNA translation, and consequently cell viability, are not well characterized. We show here that the level of oxidized RNAs is markedly increased in a yeast decapping Kllsm4Δ1 mutant, which accumulates mRNAs, ages much faster that the wild type strain and undergoes regulated-cell-death. We also found that in Kllsm4Δ1 cells the mutation rate increases during chronological life span indicating that the capacity to handle oxidized RNAs in yeast declines with aging. Lowering intracellular ROS levels by antioxidants recovers the wild-type phenotype of mutant cells, including reduced amount of oxidized RNAs and lower mutation rate. Since mRNA oxidation was reported to occur in different neurodegenerative diseases, decapping-deficient cells may represent a useful tool for deciphering molecular mechanisms of cell response to such conditions, providing new insights into RNA modification-based pathogenesis.

Annurca apple (M. pumila Miller cv Annurca) extracts act against stress and ageing in S. cerevisiae yeast cells.

BACKGROUND: During the past years, a number of studies have demonstrated the positive effect of apple on ageing and different diseases such as cancer, degenerative and cardiovascular diseases. The unicellular yeast Saccharomyces cerevisiae represents a simple eukaryotic model to study the effects of different compounds on lifespan. We previously demonstrated that apple extracts have anti-ageing effects in this organism because of their antioxidant properties. In particular, the effect is related to the presence in this fruit of polyphenols, which give a large contribution to the antioxidant activity of apples. METHODS: We we used a clonogenic assay to assess the viability and the resistance to oxidative stress of S. cerevisiae cells in the presence of Annurca apple extracts. The production of ROS and the aberrant morphology of nuclei were detected by cell staining with the fluorescent dies Dihydrorhodamine 123 and DAPI, respectively. Mitochondrial morphology was analyzed by following the localization of the mito-GFP protein into the mitochondrial matrix. RESULTS: In this study, we show that apple extracts can increase yeast lifespan, reduce the levels of reactive oxygen species and cell sensitivity to oxidative stress, and prevent nuclei and mitochondria fragmentation protecting cells from regulated cell death. CONCLUSIONS: In this paper, by using the yeast S. cerevisiae as a model, we have demonstrated that Annurca extracts possess antioxidant properties thanks to which the extracts can reduce the intracellular ROS levels and have anti-apoptotic functions thus prolonging cell lifespan. These results contribute to knowledge on the effects of natural compounds on ageing and support the use of yeast as a model organism for the development of simple tests to assess the effectiveness of bioactive substances from natural sources.

NEM1 acts as a suppressor of apoptotic phenotypes in LSM4 yeast mutants.

Saccharomyces cerevisiae mutants in the essential gene LSM4, involved in messenger RNA decapping, and expressing a truncated form of the LSM4 gene of the yeast Kluyveromyces lactis (Kllsm4Δ1), show premature aging accompanied by the presence of typical markers of apoptosis and high sensitivity to oxidative stressing agents. We isolated multicopy extragenic suppressors of these defects, transforming the Kllsm4Δ1 mutant with a yeast DNA library and selecting clones showing resistance to acetic acid. Here we present one of these clones, carrying a DNA fragment containing the NEM1 gene (Nuclear Envelope Morphology protein 1), which encodes the catalytic subunit of the Nem1p-Spo7p phosphatase holoenzyme. Nem1p regulates nuclear growth by controlling phospholipid biosynthesis and it is required for normal nuclear envelope morphology and sporulation. The data presented here correlate the mRNA metabolism with the biosynthesis of phospholipids and with the functionality of the endoplasmic reticulum.

The Multifunctional Role of Poloxamer P338 as a Biofilm Disrupter and Antibiotic Enhancer: A Small Step forward against the Big Trouble of Catheter-Associated Escherichia coli Urinary Tract Infections.

Poloxamer 338 (P338), a nonionic surfactant amphiphilic copolymer, is herein proposed as an anti-biofilm compound for the management of catheter-associated urinary tract infections (CAUTIs). P338's ability to disrupt Escherichia coli biofilms on silicone urinary catheters and to serve as antibiotic enhancer was evaluated for biofilm-producing E. coli Ec5FSL and Ec9FSL clinical strains, isolated from urinary catheters. In static conditions, quantitative biofilm formation assay allowed us to determine the active P338 concentration. In dynamic conditions, the BioFlux system, combined with confocal laser scanning microscopy, allowed us to investigate the P338 solution's ability to detach biofilm, alone or in combination with sub-MIC concentrations of cefoxitin (FOX). The 0.5% P338 solution was able to destroy the structure of E. coli biofilms, to reduce the volume and area fraction covered by adherent cells (41.42 ± 4.79% and 56.20 ± 9.22% reduction for the Ec5FSL and Ec9FSL biofilms, respectively), and to potentiate the activity of 1\2 MIC FOX in disaggregating biofilms (19.41 ± 7.41% and 34.66 ± 3.75% reduction in the area fraction covered by biofilm for Ec5FSL and Ec9FSL, respectively) and killing cells (36.85 ± 7.13% and 32.33 ± 4.65% increase in the biofilm area covered by dead Ec5FSL and Ec9FSL cells, respectively).

The C-terminal region of yeast ubiquitin-protein ligase Not4 mediates its cellular localization and stress response.

Transient modification of the environment involves the expression of specific genes and degradation of mRNAs and proteins. How these events are linked is poorly understood. CCR4-NOT is an evolutionary conserved complex involved in transcription initiation and mRNA degradation. In this paper, we report that the yeast Not4 localizes in cytoplasmic foci after cellular stress. We focused our attention on the functional characterization of the C-terminus of the Not4 protein. Molecular dissection of this region indicates that the removal of the last 120 amino acids, does not affect protein localization and function, in that the protein is still able to suppress the thermosensitivity observed in the not4Δ mutant. In addition, such shortened form of Not4, as well its absence, increases the transcription of stress-responsive genes conferring to the cell high resistance to the oxidative stress. On the contrary, the last C-terminal 211 amino acids are required for proper Not4 localization at cytoplasmic foci after stress. This truncated version of Not4 fails to increase the transcription of the stress genes, is more stable and seems to be toxic to cells undergoing oxidative stress.

Poloxamer 338 Affects Cell Adhesion and Biofilm Formation in Escherichia coli: Potential Applications in the Management of Catheter-Associated Urinary Tract Infections.

Poloxamers are nontoxic, amphiphilic copolymers used in different formulations. Due to its surfactant properties, Poloxamer 338 (P388) is herein proposed as a strategy to avoid biofilm formation often causing recalcitrant catheter-associated urinary tract infections (CAUTI). The aim is to evaluate the ability of P388 coatings to affect the adhesion of Ec5FSL and Ec9FSL Escherichia coli strains on silicone urinary catheters. Attenuated total reflection infrared spectroscopy, atomic force microscopy, and static water contact angle measurement were employed to characterize the P388-coated silicone catheter in terms of amount of P388 layered, coating thickness, homogeneity, and hydrophilicity. In static conditions, the antifouling power of P388 was defined by comparing the E. coli cells adherent on a hydrophilic P388-adsorbed catheter segment with those on an uncoated one. A P388-coated catheter, having a homogeneous coverage of 35 nm in thickness, reduced of 0.83 log10 and 0.51 log10 the biofilm of Ec5FSL and Ec9FSL, respectively. In dynamic conditions, the percentage of cell adhesion on P388-adsorbed silicone channels was investigated by a microfluidic system, simulating the in vivo conditions of catheterized patients. As a result, both E. coli isolates were undetected. The strong and stable antifouling property against E. coli biofilm lead us to consider P388 as a promising anti-biofilm agent for CAUTIs control.

Acid Stress Triggers Resistance to Acetic Acid-Induced Regulated Cell Death through Hog1 Activation Which Requires RTG2 in Yeast.

Acid stress causes resistance to acetic acid-induced regulated cell death (AA-RCD) in budding yeast, resulting in catalase activation. In order to explore the molecular determinants of evasion of AA-RCD triggered by acid stress adaptation, we studied the involvement and the possible interplay of the master regulator of transcription high-osmolarity glycerol 1 (HOG1) and RTG2, a positive regulator of the RTG-dependent mitochondrial retrograde signaling. Viability, DNA fragmentation, and ROS accumulation have been analyzed in wild-type and mutant cells lacking HOG1 and/or RTG2. Catalase activity and transcription of CTT1 and CTA1, coding the cytosolic and peroxisomal/mitochondrial catalase, respectively, as well as Hog1 phosphorylation, were also analyzed. Our results show that HOG1 is essential for resistance to AA-RCD and its activation results in the upregulation of CTT1, but not CTA1, transcription during acid stress adaptation. RTG2 is required for Hog1-dependent CTT1 upregulation upon acid stress, despite failure of RTG pathway activation. We give evidence that Rtg2 has a cytoprotective role and can act as a general cell stress sensor independent of Rtg1/3-dependent transcription.

A role for microbial selection in frescoes' deterioration in Tomba degli Scudi in Tarquinia, Italy.

Mural paintings in the hypogeal environment of the Tomba degli Scudi in Tarquinia, Italy, show a quite dramatic condition: the plaster mortar lost his cohesion and a white layer coating is spread over almost all the wall surfaces. The aim of this research is to verify if the activity of microorganisms could be one of the main causes of deterioration and if the adopted countermeasures (conventional biocide treatments) are sufficient to stop it. A biocide treatment of the whole environment has been carried out before the conservative intervention and the tomb has been closed for one month. When the tomb was opened again, we sampled the microorganisms present on the frescoes and we identified four Bacillus species and one mould survived to the biocide treatment. These organisms are able to produce spores, a highly resistant biological form, which has permitted the survival despite the biocide treatment. We show that these Bacillus strains are able to produce calcium carbonate and could be responsible for the white deposition that was damaging and covering the entire surface of the frescoes. Our results confirm that the sanitation intervention is non always resolutive and could even be deleterious in selecting harmful microbial communities.

Functional screening of antibiotic resistance genes from a representative metagenomic library of food fermenting microbiota.

Lactic acid bacteria (LAB) represent the predominant microbiota in fermented foods. Foodborne LAB have received increasing attention as potential reservoir of antibiotic resistance (AR) determinants, which may be horizontally transferred to opportunistic pathogens. We have previously reported isolation of AR LAB from the raw ingredients of a fermented cheese, while AR genes could be detected in the final, marketed product only by PCR amplification, thus pointing at the need for more sensitive microbial isolation techniques. We turned therefore to construction of a metagenomic library containing microbial DNA extracted directly from the food matrix. To maximize yield and purity and to ensure that genomic complexity of the library was representative of the original bacterial population, we defined a suitable protocol for total DNA extraction from cheese which can also be applied to other lipid-rich foods. Functional library screening on different antibiotics allowed recovery of ampicillin and kanamycin resistant clones originating from Streptococcus salivarius subsp. thermophilus and Lactobacillus helveticus genomes. We report molecular characterization of the cloned inserts, which were fully sequenced and shown to confer AR phenotype to recipient bacteria. We also show that metagenomics can be applied to food microbiota to identify underrepresented species carrying specific genes of interest.