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Human epithelial organoids-3D spheroids derived from adult tissue stem cells-enable investigation of epithelial physiology and disease and host interactions with microorganisms, viruses and bioactive molecules. One challenge in using organoids is the difficulty in accessing the apical, or luminal, surface of the epithelium, which is enclosed within the organoid interior. This protocol describes a method we previously developed to control human and mouse organoid polarity in suspension culture such that the apical surface faces outward to the medium (apical-out organoids). Our protocol establishes apical-out polarity rapidly (24-48 h), preserves epithelial integrity, maintains secretory and absorptive functions and allows regulation of differentiation. Here, we provide a detailed description of the organoid polarity reversal method, compatible characterization assays and an example of an application of the technology-specifically the impact of host-microbe interactions on epithelial function. Control of organoid polarity expands the possibilities of organoid use in gastrointestinal and respiratory health and disease research., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

The distal lung contains terminal bronchioles and alveoli that facilitate gas exchange. Three-dimensional in vitro human distal lung culture systems would strongly facilitate the investigation of pathologies such as interstitial lung disease, cancer and coronavirus disease 2019 (COVID-19) pneumonia caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here we describe the development of a long-term feeder-free, chemically defined culture system for distal lung progenitors as organoids derived from single adult human alveolar epithelial type II (AT2) or KRT5 + basal cells. AT2 organoids were able to differentiate into AT1 cells, and basal cell organoids developed lumens lined with differentiated club and ciliated cells. Single-cell analysis of KRT5 + cells in basal organoids revealed a distinct population of ITGA6 + ITGB4 + mitotic cells, whose offspring further segregated into a TNFRSF12A hi subfraction that comprised about ten per cent of KRT5 + basal cells. This subpopulation formed clusters within terminal bronchioles and exhibited enriched clonogenic organoid growth activity. We created distal lung organoids with apical-out polarity to present ACE2 on the exposed external surface, facilitating infection of AT2 and basal cultures with SARS-CoV-2 and identifying club cells as a target population. This long-term, feeder-free culture of human distal lung organoids, coupled with single-cell analysis, identifies functional heterogeneity among basal cells and establishes a facile in vitro organoid model of human distal lung infections, including COVID-19-associated pneumonia.

Enteroaggregative Escherichia coli (EAEC) is a significant cause of acute and chronic diarrhea, foodborne outbreaks, infections of the immunocompromised, and growth stunting in children in developing nations. There is no vaccine and resistance to antibiotics is rising. Unlike related E. coli pathotypes that are often associated with acute bouts of infection, EAEC is associated with persistent diarrhea and subclinical long-term colonization. Several secreted virulence factors have been associated with EAEC pathogenesis and linked to disease in humans, less certain are the molecular drivers of adherence to the intestinal mucosa. We previously established human intestinal enteroids (HIEs) as a model system to study host-EAEC interactions and aggregative adherence fimbriae A (AafA) as a major driver of EAEC adherence to HIEs. Here, we report a large-scale assessment of the host response to EAEC adherence from all four segments of the intestine across at least three donor lines for five E. coli pathotypes. The data demonstrate that the host response in the duodenum is driven largely by the infecting pathotype, whereas the response in the colon diverges in a patient-specific manner. Major pathways altered in gene expression in each of the four enteroid segments differed dramatically, with responses observed for inflammation, apoptosis and an overwhelming response to different mucin genes. In particular, EAEC both associated with large mucus droplets and specific mucins at the epithelial surface, binding that was ameliorated when mucins were removed, a process dependent on AafA. Pan-screening for glycans for binding to purified AafA identified the human ligand as heparan sulfate proteoglycans (HSPGs). Removal of HSPG abrogated EAEC association with HIEs. These results may mean that the human intestine responds remarkably different to distinct pathobionts that is dependent on the both the individual and intestinal segment in question, and uncover a major role for surface heparan sulfate proteoglycans as tropism-driving factor in adherence and/or colonization., Competing Interests: The authors have declared that no competing interests exist.

Human enteroids-epithelial spheroids derived from primary gastrointestinal tissue-are a promising model to study pathogen-epithelial interactions. However, accessing the apical enteroid surface is challenging because it is enclosed within the spheroid. We developed a technique to reverse enteroid polarity such that the apical surface everts to face the media. Apical-out enteroids maintain proper polarity and barrier function, differentiate into the major intestinal epithelial cell (IEC) types, and exhibit polarized absorption of nutrients. We used this model to study host-pathogen interactions and identified distinct polarity-specific patterns of infection by invasive enteropathogens. Salmonella enterica serovar Typhimurium targets IEC apical surfaces for invasion via cytoskeletal rearrangements, and Listeria monocytogenes, which binds to basolateral receptors, invade apical surfaces at sites of cell extrusion. Despite different modes of entry, both pathogens exit the epithelium within apically extruding enteroid cells. This model will enable further examination of IECs in health and disease., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

The lactic acid bacterium Oenococcus oeni is the most important species involved in malolactic fermentation due to its capability to survive in presence of ethanol and in the acidic environment of wine. In order to identify novel genes involved in adaptation to wine, a new approach using genome-wide analysis based on stress-related genes was performed in strain O. oeni PSU-1, and 106 annotated stress genes were identified. The in silico analysis revealed the high similarity of all those genes through 57 O. oeni genomes; however, seven variable regions of genomic plasticity could be determined for their different presence observed among these strains. Regions 3 and 5 had the typical hallmarks of horizontal transfer, suggesting that the strategy of acquiring genes from other bacteria enhanced the fitness of O. oeni strains. Certain genes related to stress resistance were described in these regions, and similarities of putative acquired regions with other lactic acid bacteria species were found. Some genomic fragments present in all the strains were described and another new genomic island harbouring a threonine dehydrogenase was found. The association of selected sequences with adaptation to wine was assessed by screening 31 O. oeni strains using PCR of single genes, but no sequences were found to be exclusive to highly performing malolactic fermentation strains. This study provides new information about the genomic variability of O. oeni strains contributing to a further understanding of this species and the relationship of its genomic traits with the ability to adapt to stress conditions., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Although Oenococcus oeni is the main species that is responsible for malolactic fermentation (MLF), harsh wine conditions can limit its performance. Although several mechanisms underlying the response to stress have been studied in this species, little is known regarding the cellular systems that protect against oxidative stress in other bacteria, such as glutathione (GSH). O. oeni cannot synthesize GSH but contains several genes related to its utilization. In this study, the relative expression (RE) of the seven genes involved in the GSH redox system found in O. oeni PSU-1 (gshR, gpo, three glutaredoxin-like genes and two subunits of an hypothetical transporter) has been measured. The study was performed using three strains, with each exhibiting a different GSH uptake capacity. The strains were grown in a stress-adaptation medium supplemented with 5mM GSH and under different adaptation stress conditions (pH4 and 6% ethanol). The RE showed that only some of these genes, including one for a possible glutaredoxin (OEOE_RS04215) and cydC for a subunit of a putative GSH transporter (OEOE_RS1995), responded to the addition of GSH. The presence of ethanol had a relevant effect on gene expression. Among the studied genes, the one for a NrdH-redoxin (OEOE_RS00645) showed a common response to ethanol in the strains, being over-expressed when grown with GSH. In most cases, the transcriptional changes were more evident for the strain with a higher capacity of GSH uptake. Malolactic performance of the three strains after pre-adaptation was evaluated in wine-like media (12% ethanol and pH3.4). It was observed that the addition of GSH during pre-adaptation growth had a protective role in the cells exposed to low pH and ethanol, resulting in a quicker MLF., (Copyright © 2016. Published by Elsevier B.V.)

The thioredoxin system protects against oxidative stress through the reversible oxidation of the thioredoxin active center dithiol to a disulphide. The genome of Oenococcus oeni PSU-1 contains three thioredoxin genes (trxA1, trxA2, trxA3), one thioredoxin reductase (trxB) and one ferredoxin reductase (fdr) which, until recently, was annotated as a second thioredoxin reductase. For the first time, the entire thioredoxin system in several O. oeni strains isolated from wine has been analysed. Comparisons at the DNA and protein levels have been undertaken between sequences from O. oeni and other genera and species, and the genera Leuconostoc and Lactobacillus were found to present the highest similarities. The gene most frequently absent from a collection of 34 strains and the sequences annotated in the NCBI database was trxA1. Moreover, phylogenetic analysis suggested that this gene was horizontally transferred from Lactobacillus to O. oeni. Strain-dependent expression profiles were determined in rich and in wine-like media. General over-expression was detected after inoculation into wine-like medium, with trxA3 being the most highly expressed gene. The increased transcriptional levels of the thioredoxin genes are indicative of the crucial role of this system in the O. oeni response to wine harsh conditions., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Oenococcus oeni is the main species responsible for the malolactic fermentation (MLF) of wine due to its ability to survive in this environment. Some wine-related stress factors, such as ethanol and low pH, may alter the cell redox balance of O. oeni. For the first time, the ability to uptake glutathione (GSH), an almost universal tripeptide with antioxidant properties, has been associated to the improvement of stress response in O. oeni. Despite the inability of O. oeni to synthesize GSH, this bacterium can capture it from the media. The ability of 30 O. oeni strains to uptake GSH was assessed in this study. Although all of the strains tested were able to import GSH, substantial variability among them was detected. To assess the physiological function of GSH, three strains with different GSH-import capacities were selected. Significant changes in membrane fatty acids composition were observed due to GSH addition. The most relevant was the increase of cyclopropane fatty acids in cell membrane, in both the exponential and the stationary phases. Cells grown with GSH showed an improved survival against ethanol shock (14% v/v). GSH addition also increased biomass production during the adaptation to wine stress conditions (pH4, pH3.4 and 6% ethanol). The results suggest that GSH enrichment could improve the resistance to stress to O. oeni, which could be useful for the adaptation of MLF starter cultures., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Oenococcus oeni , the main lactic acid bacteria responsible for malolactic fermentation in wine, has to adapt to stressful conditions, such as low pH and high ethanol content. In this study, the changes in the transcriptome and the proteome of O. oeni PSU-1 during the adaptation period before MLF start have been studied. DNA microarrays were used for the transcriptomic analysis and two complementary proteomic techniques, 2-D DIGE and iTRAQ labeling were used to analyze the proteomic response. One of the most influenced functions in PSU-1 due to inoculation into wine-like medium (WLM) was translation, showing the over-expression of certain ribosomal genes and the corresponding proteins. Amino acid metabolism and transport was also altered and several peptidases were up regulated both at gene and protein level. Certain proteins involved in glutamine and glutamate metabolism showed an increased abundance revealing the key role of nitrogen uptake under stressful conditions. A strong transcriptional inhibition of carbohydrate metabolism related genes was observed. On the other hand, the transcriptional up-regulation of malate transport and citrate consumption was indicative of the use of L-malate and citrate associated to stress response and as an alternative energy source to sugar metabolism. Regarding the stress mechanisms, our results support the relevance of the thioredoxin and glutathione systems in the adaptation of O. oeni to wine related stress. Genes and proteins related to cell wall showed also significant changes indicating the relevance of the cell envelop as protective barrier to environmental stress. The differences found between transcriptomic and proteomic data suggested the relevance of post-transcriptional mechanisms and the complexity of the stress response in O. oeni adaptation. Further research should deepen into the metabolisms mostly altered due to wine conditions to elucidate the role of each mechanism in the O. oeni ability to develop MLF.

Recently, different dehydration-based technologies have been evaluated for the purpose of cell and tissue preservation. Although some early results have been promising, they have not satisfied the requirements for large-scale applications. The long experience of using quantitative trait loci (QTLs) with the yeast Saccharomyces cerevisiae has proven to be a good model organism for studying the link between complex phenotypes and DNA variations. Here, we use QTL analysis as a tool for identifying the specific yeast traits involved in dehydration stress tolerance. Three hybrids obtained from stable haploids and sequenced in the Saccharomyces Genome Resequencing Project showed intermediate dehydration tolerance in most cases. The dehydration resistance trait of 96 segregants from each hybrid was quantified. A smooth, continuous distribution of the anhydrobiosis tolerance trait was found, suggesting that this trait is determined by multiple QTLs. Therefore, we carried out a QTL analysis to identify the determinants of this dehydration tolerance trait at the genomic level. Among the genes identified after reciprocal hemizygosity assays, RSM22, ATG18 and DBR1 had not been referenced in previous studies. We report new phenotypes for these genes using a previously validated test. Finally, our data illustrates the power of this approach in the investigation of the complex cell dehydration phenotype.

The yeast Saccharomyces cerevisiae is able to overcome cell dehydration; cell metabolic activity is arrested during this period but restarts after rehydration. The yeast genes encoding hydrophilin proteins were characterised to determine their roles in the dehydration-resistant phenotype, and STF2p was found to be a hydrophilin that is essential for survival after the desiccation-rehydration process. Deletion of STF2 promotes the production of reactive oxygen species and apoptotic cell death during stress conditions, whereas the overexpression of STF2, whose gene product localises to the cytoplasm, results in a reduction in ROS production upon oxidative stress as the result of the antioxidant capacity of the STF2p protein.