scholarly journals A genome-scale study of metabolic complementation in endosymbiotic consortia: the case of the cedar aphid

2017 ◽  
Author(s):  
Miguel Ponce-de-Leon ◽  
Daniel Tamarit ◽  
Jorge Calle-Espinosa ◽  
Matteo Mori ◽  
Amparo Latorre ◽  
...  
Keyword(s):  
Essential Amino Acids ◽  
Bacterial Consortium ◽  
Buchnera Aphidicola ◽  
Knock Out ◽  
A Genome ◽  
Complete Networks ◽  
Small Set ◽  
Bacterial Endosymbionts ◽  
Serratia Symbiotica ◽  
Genome Scale

AbstractBacterial endosymbionts and their insect hosts establish an intimate metabolic relationship. Bacteria offer a variety of essential nutrients to their hosts, whereas insect cells provide the necessary sources of matter and energy to their tiny metabolic allies. These nutritional complementations sustain themselves on a diversity of metabolite exchanges between the cell host and the reduced yet highly specialized bacterial metabolism –which, for instance, overproduces a small set of essential amino acids and vitamins. A well-known case of metabolic complementation is provided by the cedar aphid Cinara cedri that harbors two co-primary endosymbionts, Buchnera aphidicola BCc and Ca. Serratia symbiotica SCc, and in which some metabolic pathways are partitioned between different partners. Here we present a genome scale metabolic network (GEM) for the bacterial consortium from the cedar aphid iBSCc. The analysis of this GEM allows us the confirmation of cases of metabolic complementation previously described by genome analysis (i.e. tryptophan and biotin biosynthesis) and the proposal of a hitherto unnoticed event of metabolic pathway sharing between the two endosymbionts, namely the biosynthesis of tetrahydrofolate. In silico knock-out experiments with iBSCc showed that the consortium metabolism is a highly integrated yet fragile network. We also have explored the evolutionary pathways leading to the emergence of metabolic complementation between reduced metabolisms starting from individual, complete networks. Our results suggest that, during the establishment of metabolic complementation in endosymbionts, adaptive evolution is more significant than previously thought.

scholarly journals Further evidence that mechanisms of host/symbiont integration are dissimilar in the maternal versus embryonic Acyrthosiphon pisum bacteriome

EvoDevo ◽  
2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Celeste R. Banfill ◽  
Alex C. C. Wilson ◽  
Hsiao-ling Lu
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Acyrthosiphon Pisum ◽  
Essential Amino Acids ◽  
Developmental Time ◽  
Follicular Epithelium ◽  
Future Research ◽  
Buchnera Aphidicola ◽  
Bacterial Endosymbionts ◽  
Asexual Embryogenesis

Abstract Background Host/symbiont integration is a signature of evolutionarily ancient, obligate endosymbioses. However, little is known about the cellular and developmental mechanisms of host/symbiont integration at the molecular level. Many insects possess obligate bacterial endosymbionts that provide essential nutrients. To advance understanding of the developmental and metabolic integration of hosts and endosymbionts, we track the localization of a non-essential amino acid transporter, ApNEAAT1, across asexual embryogenesis in the aphid, Acyrthosiphon pisum. Previous work in adult bacteriomes revealed that ApNEAAT1 functions to exchange non-essential amino acids at the A. pisum/Buchnera aphidicola symbiotic interface. Driven by amino acid concentration gradients, ApNEAAT1 moves proline, serine, and alanine from A. pisum to Buchnera and cysteine from Buchnera to A. pisum. Here, we test the hypothesis that ApNEAAT1 is localized to the symbiotic interface during asexual embryogenesis. Results During A. pisum asexual embryogenesis, ApNEAAT1 does not localize to the symbiotic interface. We observed ApNEAAT1 localization to the maternal follicular epithelium, the germline, and, in late-stage embryos, to anterior neural structures and insect immune cells (hemocytes). We predict that ApNEAAT1 provisions non-essential amino acids to developing oocytes and embryos, as well as to the brain and related neural structures. Additionally, ApNEAAT1 may perform roles related to host immunity. Conclusions Our work provides further evidence that the embryonic and adult bacteriomes of asexual A. pisum are not equivalent. Future research is needed to elucidate the developmental time point at which the bacteriome reaches maturity.

scholarly journals Identification of Imatinib-Sensitizing Genes in Chronic Myeloid Leukemia with a Genome-Scale Crispr Knock-out Screen

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4233-4233 ◽  
Author(s):  
Matthieu Lewis ◽  
Valerie Prouzet-Mauleon ◽  
Elodie Richard ◽  
Beatrice Turcq ◽  
Richard Iggo ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Kinase Inhibitors ◽  
Knock Out ◽  
Individual Gene ◽  
Oncogenic Pathways ◽  
A Genome ◽  
Tki Resistance ◽  
Genome Scale

Abstract Background: Resistance to tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML) can either originate from mutations in the BCR-ABL1 gene, which are mostly well characterized, or emerge from unknown alternative mutations elsewhere in the genome. Small hairpin (sh)RNA screens have been used to discover such genes but are becoming limited due to sup-optimal protein depletion and non-reliable off-target effects. More efficient screening techniques in human cells are now available as a result of the increasing understanding of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) /Cas9 system. Aims: Our goal is to uncover imatinib (IM)-sensitizing genes that cause IM resistance when knocked-out. Characterizing these genes may help understand the mechanisms of IM uptake, metabolism, degradation and/or activity in CML cells. Additionally, we also expect to unveil alternative, BCR-ABL1 independent, oncogenic pathways in CML cells. Methods: In order to find other genes involved in IM resistance, we performed a genome scale CRISPR knock-out (GeCKO) screen, which contains 121,413 sgRNAs that target 20,914 protein coding genes and miRNAs. We transduced one sgRNA per cell and challenged the K562-GeCKO cell pool to IM selection. We compared the abundance of sgRNAs between pre/post-IM treatment by next generation sequencing (NGS). Results: After IM selection, the sgRNAs from surviving cells were identified by NGS and unveiled potential IM-sensitizing genes. The most enriched sgRNAs (FDR < 0.01) targeted genes involved in transcriptional (KLF1, MED24) and translational (EIF2AK1, UBE2M) regulation, apoptosis (BAX, BCL2L11) and cell cycle regulation (BAP1, SPRED2). Subsequent screens on LAMA84 cells are currently underway in order to validate our findings. Additionally, the establishment of individual gene knock-out cell lines are in progress in order to fully understand the role of each gene in IM resistance. Summary/Conclusion: Using a CRISPR knock-out screen, we produced a list of 19 genes (FDR < 0.05) that may play a role in IM resistance. Encouragingly, a subset of these genes (BAX, BAP1, BCL2L11 and SPRED2) have already been correlated to CML progression and/or TKI resistance in the past. We aim to bolster our findings by establishing individual gene KO cell lines and study resistance in LAMA84 cells. The utilization of CRISPR libraries may not only help understand TKI resistance in CML, but also help identify numerous novel genes involved in drug resistances for a myriad of different diseases. Disclosures Mahon: ARIAD: Honoraria; PFIZER: Honoraria; BMS: Consultancy, Honoraria; NOVARTIS PHARMA: Consultancy, Honoraria, Research Funding.

scholarly journals A Freeloader?: The Highly Eroded Yet large Genome of the Serratia symbiotica symbiont of Cinara strobi

2018 ◽  
Author(s):  
Alejandro Manzano-Marín ◽  
Armelle Coeur d’acier ◽  
Anne-Laure Clamens ◽  
Céline Orvain ◽  
Corinne Cruaud ◽  
...  
Keyword(s):  
Amplicon Sequencing ◽  
Genome Reduction ◽  
The Novel ◽  
Bacterial Symbionts ◽  
Buchnera Aphidicola ◽  
Essential Nutrients ◽  
Apparent Lack ◽  
Obligate Symbiont ◽  
Bacterial Endosymbionts ◽  
Serratia Symbiotica

ABSTRACTGenome reduction is pervasive among maternally-inherited bacterial endosymbionts. This genome reduction can eventually lead to serious deterioration of essential metabolic pathways, thus rendering an obligate endosymbiont unable to provide essential nutrients to its host. This loss of essential pathways can lead to either symbiont complementation (sharing of the nutrient production with a novel co-obligate symbiont) or symbiont replacement (complete takeover of nutrient production by the novel symbiont). However, the process by which these two evolutionary events happen remains somewhat enigmatic by the lack of examples of intermediate stages of this process. Cinara aphids (Hemiptera: Aphididae) typically harbour two obligate bacterial symbionts: Buchnera and Serratia symbiotica. However, the latter has been replaced by different bacterial taxa in specific lineages, and thus species within this aphid lineage could provide important clues into the process of symbiont replacement. In the present study, using 16S rRNA high-throughput amplicon sequencing, we determined that the aphid Cinara strobi harbours not two, but three fixed bacterial symbionts: Buchnera aphidicola, a Sodalis sp., and S. symbiotica. Through genome assembly and genome-based metabolic inference, we have found that only the first two symbionts (Buchnera and Sodalis) actually contribute to the hosts’ supply of essential nutrients while S. symbiotica has become unable to contribute towards this task. We found that S. symbiotica has a rather large and highly eroded genome which codes only for a few proteins and displays extensive pseudogenisation. Thus, we propose an ongoing symbiont replacement within C. strobi, in which a once ‘‘competent” S. symbiotica does no longer contribute towards the beneficial association. These results suggest that in dual symbiotic systems, when a substitute co-symbiont is available, genome deterioration can precede genome reduction and a symbiont can be maintained despite the apparent lack of benefit to its host.

scholarly journals A Genome-Scale CRISPR Knock-Out Screen Identifies MicroRNA-5197-5p as a Promising Radiosensitive Biomarker in Colorectal Cancer

2021 ◽  
Vol 11 ◽  
Author(s):  
Shijun Yu ◽  
Li Li ◽  
Kailing Fan ◽  
Yandong Li ◽  
Yong Gao
Keyword(s):  
Colorectal Cancer ◽  
Critical Factor ◽  
Cell Counting ◽  
Knock Out ◽  
Cycle Arrest ◽  
A Genome ◽  
Mechanistic Investigation ◽  
Apoptosis Assay ◽  
Underlying Mechanisms ◽  
Genome Scale

Radioresistance is one of the main reasons causing unsatisfactory curative effects of ionizing radiation (IR) against colorectal cancer (CRC). However, its underlying mechanisms remain unclear yet. In the present study, we applied a genome-scale CRISPR knockout screen in combination of NGS sequencing upon CRC cell lines to explore regulatory factors involved radioresistance of CRC, and 3 candidate genes were identified. Cytotoxicity of IR was determined by Cell Counting Kit-8 (CCK-8) assay, colony formation assay and apoptosis assay, and microRNA-5197-5p (miR-5197) was found to significantly enhance the cytotoxicity of IR to CRC cells. By further mechanistic investigation, we demonstrated that miR-5197 directly targeted CDK6 and inhibited its expression in RKO cells, which induced cell cycle arrest at G1/S phase and inhibited cell division, thereby radiosensitivity was enhanced by miR-5197. Our findings revealed that miR-5197 might be a critical factor regulating CRC cell radiosensitivity and provided novel insights into the development of therapeutic strategies for CRC patients who are resistant to IR.

Genome-Scale CRISPR Screening reveals novel factors regulating Wnt-dependent regeneration of mouse gastric organoids

2020 ◽  
Author(s):  
Kazuhiro Murakami ◽  
Yumi Terakado ◽  
Kikue Saito ◽  
Yoshie Jomen ◽  
Haruna Takeda ◽  
...  
Keyword(s):  
Stem Cell ◽  
Adult Stem Cells ◽  
Ex Vivo ◽  
Critical Role ◽  
Sequencing Analysis ◽  
Self Renewal ◽  
Knock Out ◽  
Epithelial Regeneration ◽  
A Genome ◽  
Genome Scale

ABSTRACTAn ability to safely harness the powerful regenerative potential of adult stem cells for clinical applications is critically dependent on a comprehensive understanding of the underlying mechanisms regulating their activity. Epithelial organoid cultures accurately recapitulate many features of in vivo stem cell-driven epithelial regeneration, providing an excellent ex vivo platform for interrogation of key regulatory mechanisms. Here, we employed a Genome-Scale CRISPR Knock-Out (GeCKO) screening assay using mouse gastric epithelial organoids to identify novel modulators of Wnt-driven stem cell-dependent epithelial regeneration in the gastric mucosa. In addition to known Wnt pathway regulators such as Apc, we found that knock-out (KO) of Alk, Bclaf3 or Prkra supports the Wnt independent self-renewal of gastric epithelial cells ex vivo. In adult mice, expression of these factors is predominantly restricted to non Lgr5-expressing stem cell zones above the gland base, implicating a critical role for these factors in suppressing Wnt-dependent self-renewal of gastric epithelia. Furthermore, using comprehensive RNA-sequencing analysis, we found that these factors influence epithelial regeneration by regulating Wnt signalling, apoptosis and expression of Reg family genes which could contribute to the epithelial regeneration through JAK/STAT3 pathway.

scholarly journals A genome‐scale CRISPR knock‐out screen in chronic myeloid leukemia identifies novel drug resistance mechanisms along with intrinsic apoptosis and MAPK signaling

2020 ◽  
Vol 9 (18) ◽  
pp. 6739-6751
Author(s):  
Matthieu Lewis ◽  
Valérie Prouzet‐Mauléon ◽  
Florence Lichou ◽  
Elodie Richard ◽  
Richard Iggo ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Resistance Mechanisms ◽  
Mapk Signaling ◽  
Intrinsic Apoptosis ◽  
Knock Out ◽  
A Genome ◽  
Genome Scale ◽  
Novel Drug

Abstract 1667: Identifying intrinsic regulators of PD-L1 expression in cancer cells: A genome-scale CRISPR knock-out approach

2017 ◽  
Author(s):  
Jacqueline E. Mann ◽  
Megan L. Ludwig ◽  
Rebecca C. Hoesli ◽  
Aditi Kulkarni ◽  
Simmy Patel ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Knock Out ◽  
A Genome ◽  
Genome Scale

scholarly journals Uncovering novel pathways for enhancing hyaluronan synthesis in recombinantLactococcus lactis: Genome-scale metabolic modelling and experimental validation

2018 ◽  
Author(s):  
Abinaya Badri ◽  
Karthik Raman ◽  
Guhan Jayaraman
Keyword(s):  
Metabolic Network ◽  
Fold Increase ◽  
Experimental Results ◽  
Metabolic Modelling ◽  
Knock Out ◽  
Cell Factories ◽  
Naturally Occurring ◽  
A Genome ◽  
Recombinant Cell ◽  
Genome Scale

AbstractHyaluronan (HA) is a naturally occurring high-value polysaccharide with important medical applications. HA is commercially produced from pathogenic microbial sources. HA-producing recombinant cell factories that are being developed with GRAS organisms are comparatively less productive than the best natural producers. The metabolism of these recombinant systems needs to be more strategically engineered to achieve significant improvement. Here, we use a genome-scale metabolic network model to account for the entire metabolic network of the cell to predict strategies for improving HA production. We here analyze the metabolic network ofLactococcus lactisadapted to produce HA, and identify non-conventional overexpression and knock-out strategies to enhance HA flux.To experimentally validate our predictions, we identify an alternate route for enhancement of HA synthesis, originating from the nucleoside inosine, which has the capacity to function in parallel with the traditionally known route from glucose. Adopting this strategy resulted in a 2.8-fold increase in HA yield. The strategies identified and the experimental results show that the cell is capable of involving a larger subset of metabolic pathways in HA production. Apart from being the first report of the use of a nucleoside to improve HA production, our study shows how experimental results enable model refinement. Overall, we point out that well-constructed genome-scale metabolic models could be very potent tools to derive efficient strategies to improve biosynthesis of important high-value products.

scholarly journals Arabidopsis Genes Essential for Seedling Viability: Isolation of Insertional Mutants and Molecular Cloning

Genetics ◽  
2001 ◽  
Vol 159 (4) ◽  
pp. 1765-1778
Author(s):  
Gregory J Budziszewski ◽  
Sharon Potter Lewis ◽  
Lyn Wegrich Glover ◽  
Jennifer Reineke ◽  
Gary Jones ◽  
...  
Keyword(s):  
Large Scale ◽  
Protein Translocation ◽  
Gene Families ◽  
Mutant Phenotype ◽  
Lethal Mutant ◽  
A Genome ◽  
Genes Encoding ◽  
High Level ◽  
Mutant Lines ◽  
Genome Scale

Abstract We have undertaken a large-scale genetic screen to identify genes with a seedling-lethal mutant phenotype. From screening ~38,000 insertional mutant lines, we identified &gt;500 seedling-lethal mutants, completed cosegregation analysis of the insertion and the lethal phenotype for &gt;200 mutants, molecularly characterized 54 mutants, and provided a detailed description for 22 of them. Most of the seedling-lethal mutants seem to affect chloroplast function because they display altered pigmentation and affect genes encoding proteins predicted to have chloroplast localization. Although a high level of functional redundancy in Arabidopsis might be expected because 65% of genes are members of gene families, we found that 41% of the essential genes found in this study are members of Arabidopsis gene families. In addition, we isolated several interesting classes of mutants and genes. We found three mutants in the recently discovered nonmevalonate isoprenoid biosynthetic pathway and mutants disrupting genes similar to Tic40 and tatC, which are likely to be involved in chloroplast protein translocation. Finally, we directly compared T-DNA and Ac/Ds transposon mutagenesis methods in Arabidopsis on a genome scale. In each population, we found only about one-third of the insertion mutations cosegregated with a mutant phenotype.

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