Search Results (14)

The antimicrobial function of citral, one of the main compounds of the essential oils (EO) of the Citrus genus, and widely used by the food industry toward spoilage yeast, was previously proven. In this study, the possible mode of action of citral against yeast cells was evaluated by using a global deletome approach. Firstly, the suitability of Saccharomyces cerevisiae Σ1278b to serve as model yeast was assessed by determining its sensitivity to citral (MIC = 0.5 μL/mL). Subsequently, the complete library of Σ1278b haploid mutants deleted in 4019 non-essential genes was screened to identify potential molecular targets of citral. Finally, the deleted genes in the 590 mutants showing increased citral resistance was analyzed with an in-silico approach (Gene Ontology). The significantly enriched GO Terms were “cytoplasm”, “vacuole”, and “mitochondrion” (cellular components); “catalytic activity” (molecular function); “pseudohyphal growth” (biological process). For molecular function, resistant mutants were grouped into thiosulfate sulfur transferase activity, transferase activity, and oxidoreductase activity; for cellular components, resistant mutants were grouped as: cytoplasm, intracellular organelle, membrane-bounded organelle, mitochondrion, organelle membrane, and vacuole; and finally, with regard to biological process, deleted genes were grouped as: pseudohyphal growth, mitochondrion organization, lipid metabolic process, DNA recombination and repair, and proteolysis. Interestingly, many identified genes were associated with the cellular response to oxidative stress and ROS scavenging. These findings have important implications for the development of citral-based antimicrobials and the elucidation of its mechanism of action. Full article

Annual surveys of Irish soil samples identified three isolates, CBS 16921 (UCD88), CBS 18246 (UCD443), and CBS 18247 (UCD483), of an apiculate yeast species within the Hanseniaspora genus. The internal transcribed spacer (ITS) and D1/D2 region of the large subunit (LSU) rRNA sequences showed that these are isolates of the recently described species Hanseniaspora menglaensis, first isolated from Southwest China. No genome sequence for H. menglaensis is currently available. The genome sequences of the three Irish isolates were determined using short-read (Illumina) sequencing, and the sequence of one isolate (CBS 16921) was assembled to chromosome level using long-read sequencing (Oxford Nanopore Technologies). Phylogenomic analysis shows that H. menglaensis belongs to the fast-evolving lineage (FEL) of Hanseniaspora. Only one MAT idiomorph (encoding MATα1) was identified in all three sequenced H. menglaensis isolates, consistent with one mating type of a heterothallic species. Genome comparisons showed that there has been a rearrangement near MATα of FEL species compared to isolates from the slowly evolving lineage (SEL). Full article

Autophagy is a process conserved from yeast to humans. Since the discovery of autophagy, its physiological role in cell survival and cell death has been intensively investigated. The inherent ability of the autophagy machinery to sequester, deliver, and degrade cytoplasmic components enables autophagy to participate in cell survival and cell death in multiple ways. The primary role of autophagy is to send cytoplasmic components to the vacuole or lysosomes for degradation. By fine-tuning autophagy, the cell regulates the removal and recycling of cytoplasmic components in response to various stress or signals. Recent research has shown the implications of the autophagy machinery in other pathways independent of lysosomal degradation, expanding the pro-survival role of autophagy. Autophagy also facilitates certain forms of regulated cell death. In addition, there is complex crosstalk between autophagy and regulated cell death pathways, with a number of genes shared between them, further suggesting a deeper connection between autophagy and cell death. Finally, the mitochondrion presents an example where the cell utilizes autophagy to strike a balance between cell survival and cell death. In this review, we consider the current knowledge on the physiological role of autophagy as well as its regulation and discuss the multiple functions of autophagy in cell survival and cell death. Full article

Autolysis is a common physiological process in eukaryotic cells that is often prevented or applied, especially in yeast expression systems. In this study, an antimicrobial peptide from chicken (AMP) was recombinantly expressed in the Pichia pastoris expression system, which induced a series of cellular autolysis phenotypes after methanol treatment, such as the aggregated, lysed, irregular, and enlarged cell morphology, while the cells expressing a recombinant aflatoxin-detoxifizyme (ADTZ) were not autolyzed. A comparative transcriptomic analysis showed that the transcriptomic profiles of cells derived from the autolysis and non-autolysis groups were well discriminated, suggesting that the mechanisms of autolysis were at the transcriptional level. A further differential expression gene (DEG) analysis showed that the DEGs from the two groups were involved mainly in autophagy, the MAPK signaling pathway, transcriptional factors, the central carbon metabolism, anti-stress functions, and so on. In the autolysis group, the cell activity was significantly reduced with the MAPK signaling pathway, the central carbon metabolism was down-regulated, and components of the cytoplasm-to-vacuole targeting (CVT) and mitophagy pathways were up-regulated, suggesting that the autophagy involved in the trafficking of intracellular molecules in the vacuole and mitochondrion contributed to autolysis, which was regulated by transcriptional factors and signal pathways at the transcriptional level. This study provides a theoretical basis for genetic modifications to prevent or utilize cell autolysis in the recombinant protein expression system. Full article

Bisphenol A (BPA) is a major component of the most commonly used plastic products, such as disposable plastics, Tetra Paks, cans, sport protective equipment, or medical devices. Due to the accumulation of excessive amounts of plastic waste and the subsequent release of BPA into the environment, BPA is classified as a pollutant that is undesirable in the environment. To date, the most interesting finding is the ability of BPA to act as an endocrine disrupting compound due to its binding to estrogen receptors (ERs), and adverse physiological effects on living organisms may result from this action. Since evidence of the potential pro-oxidizing effects of BPA has accumulated over the last years, herein, we focus on the detection of oxidative stress and its origin following BPA exposure using pulsed-field gel electrophoresis, flow cytometry, fluorescent microscopy, and Western blot analysis. Saccharomyces cerevisiae cells served as a model system, as these cells lack ERs allowing us to dissect the ER-dependent and -independent effects of BPA. Our data show that high concentrations of BPA affect cell survival and cause increased intracellular oxidation in yeast, which is primarily generated in the mitochondrion. However, an acute BPA exposure does not lead to significant oxidative damage to DNA or proteins. Full article

Mitochondria and peroxisomes are ubiquitous subcellular organelles that are highly dynamic and possess a high degree of plasticity. These organelles proliferate through division of pre-existing organelles. Studies on yeast, mammalian cells, and unicellular algae have led to a surprising finding that mitochondria and peroxisomes share the components of their division machineries. At the heart of the mitochondrial and peroxisomal division machineries is a GTPase dynamin-like protein, Dnm1/Drp1, which forms a contractile ring around the neck of the dividing organelles. During division, Dnm1/Drp1 functions as a motor protein and constricts the membrane. This mechanochemical work is achieved by utilizing energy from GTP hydrolysis. Over the last two decades, studies have focused on the structure and assembly of Dnm1/Drp1 molecules around the neck. However, the regulation of GTP during the division of mitochondrion and peroxisome is not well understood. Here, we review the current understanding of Dnm1/Drp1-mediated divisions of mitochondria and peroxisomes, exploring the mechanisms of GTP regulation during the Dnm1/Drp1 function, and provide new perspectives on their potential contribution to mitochondrial and peroxisomal biogenesis. Full article

The order Trypanosomatida has been well studied due to its pathogenicity and the unique biology of the mitochondrion. In Trypanosoma brucei, four DNA polymerases, namely PolIA, PolIB, PolIC, and PolID, related to bacterial DNA polymerase I (PolI), were shown to be localized in mitochondria experimentally. These mitochondrion-localized DNA polymerases are phylogenetically distinct from other family A DNA polymerases, such as bacterial PolI, DNA polymerase gamma (Polγ) in human and yeasts, “plant and protist organellar DNA polymerase (POP)” in diverse eukaryotes. However, the diversity of mitochondrion-localized DNA polymerases in Euglenozoa other than Trypanosomatida is poorly understood. In this study, we discovered putative mitochondrion-localized DNA polymerases in broad members of three major classes of Euglenozoa—Kinetoplastea, Diplonemea, and Euglenida—to explore the origin and evolution of trypanosomatid PolIA-D. We unveiled distinct inventories of mitochondrion-localized DNA polymerases in the three classes: (1) PolIA is ubiquitous across the three euglenozoan classes, (2) PolIB, C, and D are restricted in kinetoplastids, (3) new types of mitochondrion-localized DNA polymerases were identified in a prokinetoplastid and diplonemids, and (4) evolutionarily distinct types of POP were found in euglenids. We finally propose scenarios to explain the inventories of mitochondrion-localized DNA polymerases in Kinetoplastea, Diplonemea, and Euglenida. Full article

Huanglongbing (HLB), also known as citrus greening, is the most destructive disease of citrus worldwide. HLB is associated with the non-culturable bacterium, Candidatus Liberibacter asiaticus (CaLas) in the United States. The virulence mechanism of CaLas is largely unknown, partly because of the lack of a mutant library. In this study, Tobacco mosaic virus (TMV) and Nicotiana benthamiana (N. benthamiana) were used for large-scale screening of the virulence factors of CaLas. Agroinfiltration of 60 putative virulence factors in N. benthamiana led to the identification of four candidates that caused severe symptoms in N. benthamiana, such as growth inhibition and cell death. CLIBASIA_05150 and CLIBASIA_04065C (C-terminal of CLIBASIA_04065) could cause cell death in the infiltrated leaves at five days post infiltration. Two low-molecular-weight candidates, CLIBASIA_00470 and CLIBASIA_04025, could inhibit plant growth. By converting start codon to stop codon or frameshifting, the four genes lost their harmful effects to N. benthamiana. It indicated that the four virulence factors functioned at the protein level rather than at the RNA level. The subcellular localization of the four candidates was determined by confocal laser scanning microscope. CLIBASIA_05150 located in the Golgi apparatus; CLIBASIA_04065 located in the mitochondrion; CLIBASIA_00470 and CLIBASIA_04025 distributed in cells as free GFP. The host proteins interacting with the four virulence factors were identified by yeast two-hybrid. The host proteins interacting with CLIBASIA_00470 and CLIBASIA_04025 were overlapping. Based on the phenotypes, the subcellular localization and the host proteins identified by yeast two-hybrid, CLIBASIA_00470 and CLIBASIA_04025, functioned redundantly. The hypothesis of CaLas virulence was proposed. CaLas affects citrus development and suppresses citrus disease resistance, comprehensively, in a complicated manner. Ubiquitin-mediated protein degradation might play a vital role in CaLas virulence. Deep characterization of the interactions between the identified virulence factors and their prey will shed light on HLB. Eventually, it will help in developing HLB-resistant citrus and save the endangered citrus industry worldwide. Full article

Amongst the various approaches to contain aflatoxin contamination of feed and food commodities, the use of inhibitors of fungal growth and/or toxin biosynthesis is showing great promise for the implementation or the replacement of conventional pesticide-based strategies. Several inhibition mechanisms were found taking place at different levels in the biology of the aflatoxin-producing fungal species such as Aspergillus flavus: compounds that influence aflatoxin production may block the biosynthetic pathway through the direct control of genes belonging to the aflatoxin gene cluster, or interfere with one or more of the several steps involved in the aflatoxin metabolism upstream. Recent findings pointed to mitochondrial functionality as one of the potential targets of some aflatoxin inhibitors. Additionally, we have recently reported that the effect of a compound belonging to the class of thiosemicarbazones might be related to the energy generation/carbon flow and redox homeostasis control by the fungal cell. Here, we report our investigation about a putative molecular target of the 3-isopropylbenzaldehyde thiosemicarbazone (mHtcum), using the yeast Saccharomyces cerevisiae as model system, to demonstrate how the compound can actually interfere with the mitochondrial respiratory chain. Full article

After billions of years of evolution, mitochondrion retains its own genome, which gets expressed in mitochondrial matrix. Mitochondrial translation machinery rather differs from modern bacterial and eukaryotic cytosolic systems. Any disturbance in mitochondrial translation drastically impairs mitochondrial function. In budding yeast Saccharomyces cerevisiae, deletion of the gene coding for mitochondrial translation initiation factor 3—AIM23, leads to an imbalance in mitochondrial protein synthesis and significantly delays growth after shifting from fermentable to non-fermentable carbon sources. Molecular mechanism underlying this adaptation to respiratory growth was unknown. Here, we demonstrate that slow adaptation from glycolysis to respiration in the absence of Aim23p is accompanied by a gradual increase of cytochrome c oxidase activity and by increased levels of Tma19p protein, which protects mitochondria from oxidative stress. Full article

The organelles of endosymbiotic origin, plastids, and mitochondria, evolved through the serial acquisition of endosymbionts by a host cell. These events were accompanied by gene transfer from the symbionts to the host, resulting in most of the organellar proteins being encoded in the cell nuclear genome and trafficked into the organelle via a series of translocation complexes. Much of what is known about organelle protein translocation mechanisms is based on studies performed in common model organisms; e.g., yeast and humans or Arabidopsis. However, studies performed in divergent organisms are gradually accumulating. These studies provide insights into universally conserved traits, while discovering traits that are specific to organisms or clades. Apicomplexan parasites feature two organelles of endosymbiotic origin: a secondary plastid named the apicoplast and a mitochondrion. In the context of the diseases caused by apicomplexan parasites, the essential roles and divergent features of both organelles make them prime targets for drug discovery. This potential and the amenability of the apicomplexan Toxoplasma gondii to genetic manipulation motivated research about the mechanisms controlling both organelles’ biogenesis. Here we provide an overview of what is known about apicomplexan organelle protein import. We focus on work done mainly in T. gondii and provide a comparison to model organisms. Full article

The pentatricopeptide repeat (PPR) gene family is one of the largest gene families in plants and plays important roles in posttranscriptional regulation. In this study, we combined whole genome sequencing and transcriptomes to systematically investigate PPRs in Salvia miltiorrhiza, which is a well-known material of traditional Chinese medicine and an emerging model system for medicinal plant studies. Among 562 identified SmPPRs, 299 belong to the P subfamily while the others belong to the PLS subfamily. The majority of SmPPRs have only one exon and are localized in the mitochondrion or chloroplast. As many as 546 SmPPRs were expressed in at least one tissue and exhibited differential expression patterns, which indicates they likely play a variety of functions in S. miltiorrhiza. Up to 349 SmPPRs were salicylic acid-responsive and 183 SmPPRs were yeast extract and Ag⁺-responsive, which indicates these genes might be involved in S. miltiorrhiza defense stresses and secondary metabolism. Furthermore, 23 salicylic acid-responsive SmPPRs were co-expressed with phenolic acid biosynthetic enzyme genes only while 16 yeast extract and Ag⁺-responsive SmPPRs were co-expressed with tanshinone biosynthetic enzyme genes only. Two SmPPRs were co-expressed with both phenolic acid and tanshinone biosynthetic enzyme genes. The results provide a useful platform for further investigating the roles of PPRs in S. miltiorrhiza. Full article

Due to the increased number of immunocompromised patients, infections with the pathogen Candida albicans have significantly increased in recent years. C. albicans transition from yeast to germ tubes is one of the essential factors for virulence. In this study we noted that Lee’s medium, commonly used to induce filamentation, contained 500-fold more biotin than needed for growth and 40-fold more biotin than is typically added to growth media. Thus, we investigated the effects of excess biotin on growth rate and filamentation by C. albicans in different media. At 37 °C, excess biotin (4 µM) enhanced germ tube formation (GTF) ca. 10-fold in both Lee’s medium and a defined glucose-proline medium, and ca. 4-fold in 1% serum. Two biotin precursors, desthiobiotin and 7-keto-8-aminopelargonic acid (KAPA), also stimulated GTF. During these studies we also noted an inverse correlation between the number of times the inoculum had been washed and the concentration of serum needed to stimulate GTF. C. albicans cells that had been washed eight times achieved 80% GTF with only 0.1% sheep serum. The mechanism by which 1–4 µM biotin enhances GTF is still unknown except to note that equivalent levels of biotin are needed to create an internal supply of stored biotin and biotinylated histones. Biotin did not restore filamentation for any of the four known filamentation defective mutants tested. C. albicans is auxotrophic for biotin and this biotin auxotrophy was fulfilled by biotin, desthiobiotin, or KAPA. However, biotin auxotrophy is not temperature dependent or influenced by the presence of 5% CO₂. Biotin starvation upregulated the biotin biosynthetic genes BIO2, BIO3, and BIO4 by 11-, 1500-, and 150-fold, respectively, and BIO2p is predicted to be mitochondrion-localized. Based on our findings, we suggest that biotin has two roles in the physiology of C. albicans, one as an enzymatic cofactor and another as a morphological regulator. Finally, we found no evidence supporting prior claims that C. albicans only forms hyphae at very low biotin (0.1 nM) growth conditions. Full article

In this study the mitochondrion is regarded as a target to reveal compounds that may be used to combat various diseases. Consequently, the sexual structures of yeasts (with high mitochondrial activity) were identified as sensors to screen for various anti-mitochondrial drugs that may be toxic to humans and that are directed, amongst others, against fungal diseases and cancer. Strikingly, these sensors indicated that chloroquine is a potent pro-mitochondrial drug which stimulated yeast sexual reproduction. In addition, these sensors also showed that some Non-Steroidal Anti-Inflammatory drugs (NSAIDs), anti-malarial drugs, antifungal and anticancer drugs are anti-mitochondrial. These yeast sensor bio-assays may fast track studies aimed at discovering new drugs as well as their mechanisms and should now be further evaluated for selectivity towards anti-/ pro-mitochondrials, fertility drugs and contraceptives, using in vitro, in vivo, in silico and omics research. Full article