Sugar-induced cell death (SICD) is a phenomenon observed in Saccharomyces cerevisiae whereby cells rapidly lose viability in glucose-only solutions. One theory suggests that SICD occurs due to an imbalance in nitrogen and carbon, however, limited studies are available to support this. When stationary phase cells are transferred to glucose-only solutions, cell death resembles that of apoptosis, while exponential phase cells show hallmarks of primary necrosis. Apoptosis in stationary phase cells is independent of the yeast metacaspase, YCA1, however, it remains unknown if SICD occurs through a caspase-independent pathway. Using stationary phase S. cerevisiae BY4741, we showed that SICD can be induced to the same degree with 10 mM or 110 mM glucose. Interestingly, SICD induced by 10 mM of glucose can be protected by supplementation with low concentrations of highly preferred organic nitrogen sources, namely glutamate, glutamine, and arginine, as well as high concentrations of non-preferred organic nitrogen sources. Additionally, cell death can be rescued by deletion of YCA1 and genes involved in caspase-independent apoptosis-STE20, NMA111, AIF1, or NUC1. On the other hand, when S. cerevisiae BY4741 is challenged with 110 mM glucose, SICD can only be rescued by supplementation with the same preferred organic nitrogen sources or deletion of AIF1 or NMA111. In all cases, protection is associated with a decrease in intracellular ROS and preservation of membrane integrity. Taken together, 110 mM glucose results in a catastrophic cell death phenotype that is more difficult to rescue, and nuclear localization of Aif1p and Nma111p is important for cell death in response to glucose.
Non-invasive spectroscopic methods are increasingly valued in life sciences, where preserving the native state of biomolecules is essential for accurate interpretation. Traditional analyses of microbial compounds typically involve solvent-based extraction and chromatographic separation processes, which are time consuming, damaging to samples, and can alter biomolecular structures of complexes. To overcome these limitations, we developed a novel spectroscopic workflow for direct metabolite monitoring in microbial cells. In this study, we established a combined spectroscopic methodology that allows direct pigment and polyhydroxyalkanoates (PHAs) analysis in complex biological samples without requiring chemical extraction procedures. The UV-Vis spectroscopy technique using an integrating sphere enables direct monitoring of pigments even in turbid whole cell suspensions, providing detailed fingerprints of bacteriochlorophyll a and carotenoids in their natural environment. Together, these techniques provide consistent information about cellular composition. Using the photosynthetic bacterium Rhodospirillum rubrum as a model organism, we demonstrate that our combined spectroscopic approach can resolve pigment states, reveal intracellular PHA content and crystallinity, and measure carotenoids and bacteriochlorophylls directly in native whole cell suspensions. Furthermore, advanced data processing provided an improved interpretation of pigment and PHA states in different cellular forms. This innovative combination of spectroscopic techniques reduces sample manipulation, preserves cellular integrity and provides rapid, precise, and environmentally friendly analysis of microbial metabolites in their natural physiological conditions. The demonstrated workflow is broadly applicable to biological samples where maintaining biomolecular integrity is crucial, and it has strong potential for applications in process analytical technology and industrial biotechnology.
Cell-to-cell communication in microbial systems is known for its vital role in cellular signalling and gene expression. A specific form termed Quorum Sensing (QS) has received considerable attention since its discovery in the marine symbiont Aliivibrio fischeri. QS-controlled microbial functions are associated with bacterial virulence, pathogenicity, host-microbe interactions, and biofilm development. Interference in these signalling systems can modulate microbial virulence and pathogenicity, and microbial infection caused by drug-resistant pathogens. Plant-derived phytochemicals are considered a promising candidate, with coumarins emerging as significant plant-derived signalling molecules shaping microbiome dynamics and pathogen behaviours from a broad spectrum of ecosystems. Here we explored the role of natural and synthetic coumarin compounds in the control of signalling and virulence traits in Pseudomonas aeruginosa and other priority bacterial pathogens, including the fungal opportunist Aspergillus fumigatus. We uncovered an important 'hydroxylation-bias' favouring coumarin, umbelliferone (7-OH), and 6-hydroxy-coumarin (6-OH) in the specific competitive inhibition of the Pseudomonas Quinolone Signal (PQS), associated with reduced activity of a PqsR translational fusion and suppression of pyocyanin production. Conversely, while esculetin (6,7-OH) was most effective at Acyl Homoserine Lactone (AHL) QS biosensor inhibition, it did not affect PQS production. Anti-biofilm activity of coumarins against P. aeruginosa was independent of initial attachment but linked to changes in exopolysaccharide production. As the very real threat posed by antimicrobial resistance persists, these data support a role for phytochemicals such as coumarins in delivering an ecological solution to dysbiosis in the host-microbe interaction.
Emerging evidence suggests that the upper female reproductive tract is not sterile and that microbial signals within follicular fluid (FF) may influence oocyte competence. However, previous studies have largely relied on pooled FF samples or dominant follicles, limiting insight into follicle-specific associations with fertilization outcomes. In this exploratory paired study, follicular fluid samples were collected from 24 women undergoing IVF/ICSI treatment. For each patient, two FF samples were analyzed individually: one associated with a fertilized oocyte, and one associated with an oocyte that failed fertilization. Bacterial DNA and total bacterial load (TBL) were assessed using quantitative real-time PCR targeting predefined microbial taxa. Bacterial DNA above the predefined detection threshold was identified in 39.6% of all FF samples. Notably, within this exploratory cohort, FF samples associated with fertilization failure were more frequently TBL-positive compared with FF samples linked to successful fertilization (70.8% vs. 8.3%). Follicles from the same patient often differed in bacterial DNA presence, indicating substantial intra-individual variability. Several bacterial taxa, including Fannyhessea vaginae, Ureaplasma spp., and Lactobacillus spp., were more frequently detected in FF samples associated with failed fertilization; however, no individual taxon showed a consistent association with outcome across all samples. In this paired follicle-level analysis, the absence of detectable bacterial DNA in follicular fluid was associated with fertilization outcome. These findings highlight follicle-level heterogeneity in microbial DNA detection and underscore the importance of follicle-specific analyses in reproductive microbiome research. Larger prospective studies are required to validate these observations and to clarify the biological mechanisms underlying follicular microbial signals.
Skin barrier dysfunction is central to inflammation and susceptibility to infection in atopic dermatitis (AD). Cutaneous T-cell lymphoma (CTCL) shares clinical similarities with AD and is also associated with a high prevalence of Staphylococcus aureus (S. aureus) colonisation. However, the mechanisms driving skin barrier damage in CTCL and the contribution of bacteria remain poorly understood. We investigate how the interplay between S. aureus (and staphylococcal enterotoxins (SEs)) and primary malignant- and non-malignant T cells affects keratinocyte expression of skin barrier proteins; in vitro, in an EL4 murine lymphoma model of bacteria-driven tumour progression, and in CTCL patient lesions colonised with SE-producing S. aureus before and after bacterial eradication by antibiotic treatment. S. aureus and SEs activate malignant and non-malignant T cells to release barrier-repressing cytokines, including IL-4, IL-13, IL-22, and OSM, and JAK-dependent downregulation of filaggrin and loricrin in keratinocytes. In the EL4 model, bacteria-colonised tumour-bearing mice show significant filaggrin loss in tumour-adjacent epidermis, whereas antibiotic-treated mice maintain near-normal expression. Clinically, antibiotic eradication of SE-producing S. aureus partially restores filaggrin and loricrin expression in three of four patients, paralleling reduced inflammatory signalling. SE-producing S. aureus promotes skin barrier impairment in CTCL through cytokine-driven, JAK-dependent repression of structural proteins in keratinocytes. These findings identify microbial-immune crosstalk as a contributor to CTCL skin pathology and provide mechanistic rationale for strategies targeting S. aureus colonisation as adjunctive therapy in CTCL.
Autophagy contributes to cellular homeostasis by degrading and recycling intracellular components, especially under nutrient-limited conditions. While autophagy is well characterized under acute starvation in synthetic media in Saccharomyces cerevisiae, its regulation during the stationary phase of prolonged growth in nutrient-rich complex media, when cells experience gradual metabolic shifts and sustained stress, remains poorly understood. In this study, we identified Sir2, an NAD + -dependent histone deacetylase, as a key suppressor of autophagy during the stationary phase in YPD complex medium. Using GFP-Atg8 processing as a readout of autophagic flux, we demonstrated that SIR2 deletion led to sustained autophagy activation. Notably, Sir2 selectively inhibited mitophagy, pexophagy, and the Cvt pathway, while non-selective autophagy remained largely unaffected. Transcriptomic analysis revealed that Sir2 facilitates a coordinated entry into quiescence, in part by regulating ribosome biogenesis and nutrient-responsive pathways during the stationary phase. Mechanistically, Sir2 stabilized Ume6, a repressor of ATG8 transcription, thereby limiting autophagic activity. Deletion of SIR2 drastically increased the phosphorylation and stabilization of the mitochondrial receptor Atg32 during the stationary phase, leading to enhanced mitophagy. Additionally, we found that ROS generated by mitophagy enhanced autophagy through a positive feedback loop. Collectively, our findings establish Sir2 as a previously unrecognized regulator of selective autophagy during the stationary phase in complex medium and highlight how cells dynamically control organelle degradation to maintain viability under extended metabolic stress.
Liquid biopsies are transforming oncology, enabling earlier diagnosis, dynamic treatment guidance, and personalized precision medicine, yet current approaches focusing mainly on circulating host cell-free DNA (cfDNA) neglect crucial information within co-existing microbial cell-free DNA (mcfDNA). This review argues for the combined potential of simultaneously analyzing host and microbial signals from samples like blood, specifically focusing on circulating tumor DNA (ctDNA) as the key host component. While ctDNA analysis is already used to guide treatment decisions, the detection of mcfDNA-although present in smaller amounts compared to total cfDNA-offers a distinct and complementary opportunity to identify disease-causing microbes and investigate the host-associated microbiome in the context of cancer. Leveraging machine learning strategies is essential to integrate these multi-view data sets and realize their full potential for enhancing liquid biopsy applications, particularly in early cancer detection.
Like other Gram-negative bacteria, Vibrio cholerae, releases bacterial extracellular vesicles (BEVs), which have documented roles along the facultative human-pathogen's lifecycle. Most studies have focused on BEVs released its outermost surface under non-stressed conditions, which are mainly composed of outer membrane and periplasmic components. Herein, we comprehensively characterise stress-induced BEVs released upon exposure to the SOS response-inducing genotoxin mitomycin C or the antimicrobial emulsifier bile, which V. cholerae faces during intestinal colonisation. Compared to control BEVs from non-stressed V. cholerae cultures, MMC and bile trigger the release of a high number of enlarged, nucleic acid-rich BEVs with increased cytoplasmic content, a hallmark of cell lysis-derived BEVs. Despite similarities between stress-induced BEVs, our results indicate stressor-specific BEV compositions and divergent SOS response activation, suggesting different biogenesis routes and subtypes of stress-induced BEVs. Stress-induced BEVs promote horizontal gene transfer (HGT) of a chromosomal antibiotic resistance cassette during laboratory cultivation and intestinal colonisation. We provide novel insights in BEV-mediated HGT, which is independent of the PilA-pilus of the competence machinery, but requires the periplasmic ComEA complex and downstream components. BEV-mediated HGT is facilitated under intestinal conditions, that is, exposure to bile and proteases, which highlights the potential of genetic exchange via BEVs during host colonisation.
Cultivating hydrogen-oxidizing bacteria (HOB), such as Cupriavidus necator, using H 2 , CO 2 , and O 2 offers a promising route for CO 2 valorization into chemicals and materials. To enhance cultivation efficiency in a lab-scale gas fermenter lacking a gas recycling system, an automated gas supply strategy based on real-time CO 2 and O 2 monitoring was developed. Fine-tuning gas delivery is essential to ensure an adequate supply for cellular growth while minimizing excess gas, particularly H 2 , that leaves the bioreactor unused, to improve process economics. In the absence of ATEX-compliant H 2 sensors, a soft sensor was implemented to estimate dissolved H 2 concentrations from O 2 uptake rates and growth phase identification. Total gas flow was controlled according to the O 2 requirements of the cells. This strategy reduced overall gas and H 2 consumption by 67%. In addition, a high-cell-density medium was formulated by integrating published recipes with Inductively Coupled Plasma Optical Emission Spectroscopy and nutrient inhibition testing. The optimized medium increased biomass yield from 15 g/L to 53 g/L, with 75% of the dry weight consisting of the bioplastic poly(3-hydroxybutyrate), without requiring nutrient addition or pH control. Together, these strategies improve the scalability, efficiency, and sustainability of CO 2 -based cultivation of hydrogen-oxidizing bacteria.
The Yippee-like (YPEL) proteins are a evolutionarily conserved eukaryotic family implicated in proliferation, senescence, and stress adaptation, yet their molecular functions remain poorly defined. Humans possess five paralogs (YPEL1-YPEL5), while the budding yeast S. cerevisiae contains a single ortholog, MOH1, previously linked to stress responses but with an unclear cellular role. Here, we investigated the function of MOH1 in S. cerevisiae. MOH1 deletion resulted in stress-specific phenotypes, including increased sensitivity to sodium azide and sulfuric acid, but enhanced resistance to hydrogen peroxide and acetic acid. Moh1 protein levels were dynamically regulated, decreasing upon hydrogen peroxide treatment and increasing in response to sulfuric acid. Morphological analyses including SEM revealed that moh1 Δ cells are rounder, form aggregates, and exhibit altered surface architecture independently of stress. RNA profiling and FTIR spectroscopy uncovered transcriptional reprogramming and metabolic remodeling, including alterations in lipid, protein, and cell wall polysaccharide levels and composition. Functional analyses showed that increased resistance to hydrogen peroxide is not due to altered mitochondrial ROS production but rather to reduced intracellular ROS accumulation. This effect is attributed to decreased cellular uptake resulting from altered permeability, supported by resistance to Congo red and sensitivity to SDS, consistent with cell envelope remodeling. Collectively, our findings identify Moh1 as a regulatory factor linking gene expression to metabolism and cellular architecture, thereby influencing cell envelope permeability and conferring selective stress resistance in S. cerevisiae.
Fungi were among the first eukaryotes to transition from aquatic to terrestrial life, developing multicellular hyphae, polar growth, and expanded secretomes for nutrient processing, defense, and symbiosis. We present a reliable method for purifying and characterizing extracellular vesicles (EVs) from Aspergillus nidulans and demonstrate that the induction of xylanase C is associated with increased EV release and EV-associated enzymatic activity. Using a mCherry reporter replacing xylanase C, we generalized this effect, showing that reporter induction increases EV production and reporter loading into EVs. This phenomenon primarily depends on the signal peptide (SP), suggesting that the induction of endoplasmic reticulum (ER)- trafficked proteins has a pronounced effect on EV production and cargo loading. We speculate that EV biogenesis may originate at the ER, where ER-translated proteins could be selectively loaded into vesicles and subsequently trafficked directly to the plasma membrane or through multivesicular bodies (MVBs). EV secretion is minimal in the first 24-48 hours but increases later in growth, coinciding with biofilm formation. This timing allows A. nidulans to modify the secretome, adapting it to new nutrient sources.
Soil is a critical ecological contributor to plant and animal health. In this issue of Cell Host & Microbe, Xiong et al. use global metagenomic data to show that human pathogens linked to diseases like tuberculosis, melioidosis, and sepsis are widespread in humid and agricultural soils harboring reduced microbial diversity.
Sorbic acid is a lipophilic weak acid with fungistatic activity, and it has been widely used as a food preservative, along with its potassium and calcium salts. Although the fungistatic effect of sorbic acid is thought to be primarily due to acidification within fungal cells, the detailed fungistatic mechanism remains unclear. We investigated the effects of sorbic acid on yeast translation in Saccharomyces cerevisiae. At sublethal concentrations (2-4 mM), sorbic acid quickly repressed translation. Conversely, removal of sorbic acid restored translation activity, indicating that the sorbic acid-induced translational repression is reversible. Pronounced translational repression induced by various stress conditions or nutrient starvation is often accompanied by eIF2 α phosphorylation, eIF2B-body and stress granule (SG) formation, and the sequestration of Ded1 (which plays a role in translation initiation as a DEAD-box RNA helicase) into SGs. We found that sorbic acid stress also induces eIF2 α phosphorylation and the sequestration of Ded1 into SGs. In contrast, sorbic acid stress induced the formation of not eIF2B bodies but eIF2B granules, which colocalized with SGs. These results suggest that the functional arrest of translation-related factors, including eIF2 α , eIF2B, and Ded1, correlates strongly with the translational repression in the presence of sorbic acid. Notably, Gcn2 deficiency delayed translational repression and SG formation, and significantly suppressed eIF2B granule formation, suggesting the involvement of Gcn2 in these stress responses during sorbic acid stress. Our findings provide new insights into the physiological effects of sorbic acid on yeast cells, specifically regarding the regulation of translation-related factors.
Programmed cell death (PCD) in unicellular organisms is not well characterized. This study investigated the transcriptomic response of Acanthamoeba castellanii to G418-induced PCD, focusing on the role of alternative splicing (AS). RNA sequencing revealed extensive transcriptional changes, affecting approximately 70% of annotated genes over six hours of treatment. This analysis also highlighted significant alterations in pathways related to cell cycle, proteolysis, and RNA splicing. Analysis of AS events identified 18,748 differentially spliced events, predominantly intron retention (IR). Interestingly, retained introns displayed a 3' bias in untreated cells, a pattern that shifted towards uniform distribution throughout the gene body during PCD. Additionally, we characterized retained introns during trophozoite stage and during PCD of the amoeba. Correlational analysis revealed a significant negative correlation between IR and transcript levels, suggesting a complex interplay between transcriptional and post-transcriptional regulation. The predominance of IR, coupled with its dynamic positional shift during PCD, points to a novel regulatory mechanism in A. castellanii PCD. These findings provide insights into the molecular mechanisms underlying PCD in this organism, potentially identifying new therapeutic targets and allowing us a better understanding of such process in A. castellanii, a facultative human pathogen.
Our immune system is constantly exposed to fungi, but it mounts a consistent, disease-related, and species-specific inflammatory response only against a few fungal species. Most of the current understanding of fungus-host interactions is based on a limited number of strains, hence neglecting the fungal intra-specific genetic and phenotypic diversity. To expand our knowledge of the spectrum of immune responses to pathogenic and non-pathogenic fungi, we compared the cytokine and transcriptional profiles of human monocyte-derived dendritic cells exposed to Aspergillus fumigatus, Candida albicans, Candida parapsilosis, and Saccharomyces cerevisiae strains. The tested species triggered common and species-specific responses, mostly resulting from the different timing of signaling pathways. Faster phagolysosome acidification was observed for pathogenic species. These results highlight the urgency to redraw the boundaries between pathogenicity and commensalism in fungi, shining a spotlight on the timing of the response, rather than solely on the genes triggered by the stimuli.
Loss of host-microbiota balance promotes gut inflammation, colitis and inflammatory bowel disease. Yet, whether host or microbial factors are the critical driver of the pathology remains unclear. Here, we investigate how cardiolipin maintains metabolic fitness of regulatory T (Treg) cells to preserve gut-immune homeostasis. We discover that deleting the cardiolipin-synthesizing enzyme protein tyrosine phosphatase mitochondrial 1 (PTPMT1) in T cells predisposes mice to colitis due to impaired Treg cell function in the absence of dysbiosis. Subsequent pathobiont infections accelerate the progression and severity of gut inflammation. Mechanistically, the absence of cardiolipin impairs Treg cell metabolic fitness and triggers a maladaptive integrated stress response, which can be reversed pharmacologically or genetically, restoring gut homeostasis and extending lifespan in PTPMT1 ΔT mice. Barth syndrome, a genetic disorder marked by severe cardiolipin deficiency, also exhibits gastrointestinal symptoms and inflammation associated with helper T cell imbalance and an active integrated stress response signature. Overall, these results suggest that a cardiolipin-mediated mitonuclear axis in T cells preserves gut-immune homeostasis and dictates outcome in pathobiont infections.
Gram-negative (GN) periprosthetic joint infections (PJIs) are being increasingly reported. However, the role of Klebsiella species in PJIs remains unclear. Therefore, we aimed to analyze the prevalence, clinical presentation, microbial spectrum, antibiogram, treatment strategies and outcomes of Klebsiella-associated PJIs. A total of 1925 culture-positive total joint revision arthroplasties (rTJA) were retrospectively reviewed at a single center. Patient data were extracted from our institutional arthroplasty and PJI database. We identified 20 Klebsiella-positive PJIs (hip/knee, 11/9), representing 1.0% of all culture-positive rTJAs. The cases were predominantly polymicrobial (80%) and chronic (50%). Notably, Klebsiella spp. was rarely detected as an initial infectious event but was predominantly identified in the context of revision or re-revision procedures, frequently in patients with prior or persistent PJIs. Klebsiella pneumoniae was the most frequent species, with 44% showing multi-drug resistance. The antimicrobial susceptibility of Klebsiella isolates showed high resistance to cephalosporines and penicillin, in contrast little to no resistance to meropenem, gentamicin and levofloxacin. The most common initial surgical intervention was a two-stage revision (65%). Infection control (Tier 1) was observed in 11%, while further intervention was needed in 56% (Tier 3). All patients who had already died were classified as Tier 4 (33%). Klebsiella spp. was detected in 10.0% of GN rTJAs and was mainly associated with complex revision settings rather than primary infections. It is often associated with chronic polymicrobial infections and high antimicrobial resistance. The outcomes were generally poor, highlighting the need for pathogen-specific treatment strategies and improved diagnostics.
Chemicals produced through enzymatic reactions play a key role in the transition from a linear petrol-dependent to a circular bioeconomy. One promising approach is the conversion of single carbon (C1) molecules by biocatalysts to value-added products. Although progress has been made, current biological methods remain less cost-competitive than established chemical processes. Here, we review how single and multi-enzyme transformations, natural C1-trophic microorganisms, and organisms with transplanted synthetic C1 assimilation pathways can synergize to strengthen the competitiveness of C1-based biomanufacturing. To explore the current state-of-the-art and assess the potential of C1 biomanufacturing, we highlight the aforementioned bio-based methodologies and evaluate their industrial applicability through an overview of granted patents.
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Plasmids are a common tool in biotechnology to deliver recombinant DNA into microbial cells for the production of enzymes, pharmaceutical proteins, chemicals, or metabolites. Therefore, a stable plasmid system that provides reliable gene expression over generations is essential for the successful utilization of single-cell organisms in research and production applications. Most Komagataella phaffii expression clones are generated by the integration of linear plasmids into the genome, as circular episomal plasmids are not stable under non-selective conditions. The low rate of homology-directed specific integration and the large variation among transformants of random integration limit the organism's application in enzyme engineering approaches or comparative studies where high transformation rates and uniform expression levels are desired. In the yeast Saccharomyces cerevisiae, the problem of circular plasmid stability and partition to the daughter cells during mitosis has been solved by combining centromeric sequences or elements of the 2-micron plasmid with an autonomously replicating sequence (ARS) that serves as an origin of replication. Similar attempts have not yet been successful or widely adapted in K. phaffii; hence, permanent selection pressure is required to maintain episomal plasmids in K. phaffii. There are no reports so far about functional 2-micron plasmids for P. pastoris, and CEN/ARS plasmids for P. pastoris are usually rather large and do not provide the high transformation rates as known for episomal plasmids of S. cerevisiae expression systems. However, the availability of a broad set of resistance, auxotrophic, and carbon source utilization markers facilitates reliable plasmid selection in small-scale screening applications and recently also proved to be successful for bioreactor-scale expression. This allows the combined advantages of high transformation rates and low clonal variability of ARS plasmids to be exploited. This article describes the successful utilization of ARS1-containing plasmids in K. phaffii, including antibiotic-free selection, complementation of knockout strains, or even for the application of CRISPR/Cas by transient gRNA and CAS9 gene expression in K. phaffii.