Genome-wide characterization of Phytophthora infestans metabolism: a systems biology approach

AbstractGenome-scale metabolic models (GEMs) provide a functional view of the complex network of biochemical reactions in the living cell. Initially mainly applied to reconstruct the metabolism of model organisms, the availability of increasingly sophisticated reconstruction methods and more extensive biochemical databases now make it possible to reconstruct GEMs for less characterized organisms as well, and have the potential to unravel the metabolism in pathogen-host systems. Here we present a GEM for the oomycete plant pathogen Phytophthora infestans as a first step towards an integrative model with its host. We predict the biochemical reactions in different cellular compartments and investigate the gene-protein-reaction associations in this model to get an impression of the biochemical capabilities of P. infestans. Furthermore, we generate life stage-specific models to place the transcriptomic changes of genes encoding metabolic enzymes into a functional context. In sporangia and zoospores there is an overall downregulation, most strikingly reflected in the fatty acid biosynthesis pathway. To investigate the robustness of the GEM, we simulate gene deletions to predict which enzymes are essential for in vitro growth. While there is room for improvement, this first model is an essential step towards an understanding of P. infestans and its interactions with plants as a system, which will help to formulate new hypotheses on infection mechanisms and disease prevention.

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Research of microcystins as inhibitors of Phytophthora infestans development

Naukovì dopovìdì Nacìonalʹnogo unìversitetu bìoresursiv ì prirodokoristuvannâ Ukraïni ◽

10.31548/dopovidi2021.05.001 ◽

2021 ◽

Author(s):

V. V. Nykyforov ◽

◽

О.V. Novokhatko ◽

O.V. Maznitska ◽

О.А. Sakun ◽

...

Keyword(s):

Late Blight ◽

Phytophthora Infestans ◽

Green Algae ◽

Field Experiments ◽

Model Organisms ◽

Time Interval ◽

Complete Disappearance ◽

Blue Green Algae

Information from foreign literary sources regarding microcystins of blue-green algae is presented. The current state of the problem of reservoirs «blooming» and the significance of this phenomenon for humans are reflected. The research was carried out in two stages: laboratory and full-scale. A pure culture of Phytophthora infestans was isolated, on which further studies were carried out. Isolation of isolates was carried out on agar nutrient medium. Potassium permanganate and ethanol were selected from the available antiseptics. Field experiments were carried out on experimental lines of Solanum lycopersicum by diagnosing signs of late blight disease. The determination of the potential negative effect of tomato plants treated with a suspension of cyanobacteria was carried out by the method of biotesting using Achatina fulica as a test object. The effect of blue-green algae toxins – microcystins – on colonies Ph. infestans in vitro is described. Photometric observation of the decrease in the number of colonies with a time interval of three days is presented. The phytophtorostatic effect of microcystins has been established. Degradation of Ph. infestans the next day were fixed after treatment of the colony with a suspension of cyanobacteria. An inhibitory effect was revealed, almost to the complete disappearance of symptoms in plants partially affected by late blight and grown in vivo. It has been determined that plants treated with microcystin can be considered safe for further consumption; the death of model organisms has not been registered.

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Role of Satb1 and Satb2 Transcription Factors in the Glutamate Receptors Expression and Ca2+ Signaling in the Cortical Neurons In Vitro

International Journal of Molecular Sciences ◽

10.3390/ijms22115968 ◽

2021 ◽

Vol 22 (11) ◽

pp. 5968

Author(s):

Egor A. Turovsky ◽

Maria V. Turovskaya ◽

Evgeniya I. Fedotova ◽

Alexey A. Babaev ◽

Viktor S. Tarabykin ◽

...

Keyword(s):

Transcription Factors ◽

Nmda Receptor ◽

Cortical Neurons ◽

Target Genes ◽

Cortical Cell ◽

Inhibitory System ◽

Selective Agonist ◽

Genes Encoding ◽

Deficient Mice

Transcription factors Satb1 and Satb2 are involved in the processes of cortex development and maturation of neurons. Alterations in the expression of their target genes can lead to neurodegenerative processes. Molecular and cellular mechanisms of regulation of neurotransmission by these transcription factors remain poorly understood. In this study, we have shown that transcription factors Satb1 and Satb2 participate in the regulation of genes encoding the NMDA-, AMPA-, and KA- receptor subunits and the inhibitory GABA(A) receptor. Deletion of gene for either Satb1 or Satb2 homologous factors induces the expression of genes encoding the NMDA receptor subunits, thereby leading to higher amplitudes of Ca2+-signals in neurons derived from the Satb1-deficient (Satb1fl/+ * NexCre/+) and Satb1-null mice (Satb1fl/fl * NexCre/+) in response to the selective agonist reducing the EC50 for the NMDA receptor. Simultaneously, there is an increase in the expression of the Gria2 gene, encoding the AMPA receptor subunit, thus decreasing the Ca2+-signals of neurons in response to the treatment with a selective agonist (5-Fluorowillardiine (FW)). The Satb1 deletion increases the sensitivity of the KA receptor to the agonist (domoic acid), in the cortical neurons of the Satb1-deficient mice but decreases it in the Satb1-null mice. At the same time, the Satb2 deletion decreases Ca2+-signals and the sensitivity of the KA receptor to the agonist in neurons from the Satb1-null and the Satb1-deficient mice. The Satb1 deletion affects the development of the inhibitory system of neurotransmission resulting in the suppression of the neuron maturation process and switching the GABAergic responses from excitatory to inhibitory, while the Satb2 deletion has a similar effect only in the Satb1-null mice. We show that the Satb1 and Satb2 transcription factors are involved in the regulation of the transmission of excitatory signals and inhibition of the neuronal network in the cortical cell culture.

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An In Vitro Cell Culture Model for Pyoverdine-Mediated Virulence

Pathogens ◽

10.3390/pathogens10010009 ◽

2020 ◽

Vol 10 (1) ◽

pp. 9

Author(s):

Donghoon Kang ◽

Natalia V. Kirienko

Keyword(s):

Cell Culture ◽

Virulence Factors ◽

Model Organisms ◽

Cell Culture Model ◽

Chronic Infections ◽

In Vitro Cell Culture ◽

Mitochondrial Homeostasis ◽

Culture Model ◽

Wide Range

Pseudomonas aeruginosa is a multidrug-resistant, opportunistic pathogen that utilizes a wide-range of virulence factors to cause acute, life-threatening infections in immunocompromised patients, especially those in intensive care units. It also causes debilitating chronic infections that shorten lives and worsen the quality of life for cystic fibrosis patients. One of the key virulence factors in P. aeruginosa is the siderophore pyoverdine, which provides the pathogen with iron during infection, regulates the production of secreted toxins, and disrupts host iron and mitochondrial homeostasis. These roles have been characterized in model organisms such as Caenorhabditis elegans and mice. However, an intermediary system, using cell culture to investigate the activity of this siderophore has been absent. In this report, we describe such a system, using murine macrophages treated with pyoverdine. We demonstrate that pyoverdine-rich filtrates from P. aeruginosa exhibit substantial cytotoxicity, and that the inhibition of pyoverdine production (genetic or chemical) is sufficient to mitigate virulence. Furthermore, consistent with previous observations made in C. elegans, pyoverdine translocates into cells and disrupts host mitochondrial homeostasis. Most importantly, we observe a strong correlation between pyoverdine production and virulence in P. aeruginosa clinical isolates, confirming pyoverdine’s value as a promising target for therapeutic intervention. This in vitro cell culture model will allow rapid validation of pyoverdine antivirulents in a simple but physiologically relevant manner.

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In vivo transcriptome analysis provides insights into host-dependent expression of virulence factors by Yersinia entomophaga MH96, during infection of Galleria mellonella

G3 Genes|Genome|Genetics ◽

10.1093/g3journal/jkaa024 ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Amber R Paulson ◽

Maureen O’Callaghan ◽

Xue-Xian Zhang ◽

Paul B Rainey ◽

Mark R H Hurst

Keyword(s):

Galleria Mellonella ◽

Functional Enrichment ◽

Natural Environments ◽

Insecticidal Toxin ◽

Heat Stable ◽

Expression Of Genes ◽

Genes Encoding ◽

Cell Densities

Abstract The function of microbes can be inferred from knowledge of genes specifically expressed in natural environments. Here, we report the in vivo transcriptome of the entomopathogenic bacterium Yersinia entomophaga MH96, captured during initial, septicemic, and pre-cadaveric stages of intrahemocoelic infection in Galleria mellonella. A total of 1285 genes were significantly upregulated by MH96 during infection; 829 genes responded to in vivo conditions during at least one stage of infection, 289 responded during two stages of infection, and 167 transcripts responded throughout all three stages of infection compared to in vitro conditions at equivalent cell densities. Genes upregulated during the earliest infection stage included components of the insecticidal toxin complex Yen-TC (chi1, chi2, and yenC1), genes for rearrangement hotspot element containing protein yenC3, cytolethal distending toxin cdtAB, and vegetative insecticidal toxin vip2. Genes more highly expressed throughout the infection cycle included the putative heat-stable enterotoxin yenT and three adhesins (usher-chaperone fimbria, filamentous hemagglutinin, and an AidA-like secreted adhesin). Clustering and functional enrichment of gene expression data also revealed expression of genes encoding type III and VI secretion system-associated effectors. Together these data provide insight into the pathobiology of MH96 and serve as an important resource supporting efforts to identify novel insecticidal agents.

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PRO: Carbapenems should be used for ALL infections caused by ceftriaxone-resistant Enterobacterales

JAC-Antimicrobial Resistance ◽

10.1093/jacamr/dlab013 ◽

2021 ◽

Vol 3 (1) ◽

Author(s):

David L Paterson ◽

Burcu Isler ◽

Patrick N A Harris

Keyword(s):

Antibiotic Resistance ◽

Observational Studies ◽

Randomized Trials ◽

Antibiotic Resistance Genes ◽

Definitive Treatment ◽

In Vitro Activity ◽

Genes Encoding ◽

Amoxicillin Clavulanate ◽

Trial Outcomes

Abstract Ceftriaxone resistance in the Enterobacterales is typically the result of production of ESBLs or AmpC β-lactamases. The genes encoding these enzymes are often co-located with other antibiotic resistance genes leading to resistance to aminoglycosides, quinolones and trimethoprim/sulfamethoxazole. Carbapenems are stable to ESBLs and AmpC giving them reliable in vitro activity against producers of these β-lactamases. In contrast, piperacillin/tazobactam and amoxicillin/clavulanate are compromised by co-production of OXA-1, which is not inhibited by tazobactam or clavulanate. These in vitro findings provide an explanation for the MERINO trial outcomes, where 3.7% (7/191) randomized to meropenem died compared with 12.3% (23/187) randomized to piperacillin/tazobactam as definitive treatment of bloodstream infection due to ceftriaxone-resistant organisms. No randomized trials have yet put cefepime and carbapenems head to head, but some observational studies have shown worse outcomes with cefepime. We argue that carbapenems are the antibiotics of choice for ceftriaxone-resistant Enterobacterales.

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HGG-26. H3G34V MUTATION AFFECTS GENOMIC H3K36 METHYLATION IN PEDIATRIC GLIOMA

Neuro-Oncology ◽

10.1093/neuonc/noaa222.310 ◽

2020 ◽

Vol 22 (Supplement_3) ◽

pp. iii348-iii348

Author(s):

Tina Huang ◽

Andrea Piunti ◽

Elizabeth Bartom ◽

Jin Qi ◽

Rintaro Hashizume ◽

...

Keyword(s):

Western Blot ◽

Allelic Frequency ◽

Glioma Cell Line ◽

Fluorescent Imaging ◽

Wild Type ◽

Gene Expressions ◽

Pediatric Glioma ◽

Genes Encoding ◽

Normal Human

Abstract BACKGROUND Histone H3.3 mutation (H3F3A) occurs in 50% of cortical pediatric high-grade gliomas. This mutation replaces glycine 34 with arginine or valine (G34R/V), impairing SETD2 activity (H3K36-specific trimethyltransferase), resulting in reduced H3K36me on H3G34V nucleosomes relative to wild-type. This contributes to genomic instability and drives distinct gene expressions associated with tumorigenesis. However, it is not known if this differential H3K36me3 enrichment is due to H3G34V mutant protein alone. Therefore, we set to elucidate the effect of H3G34V on genomic H3K36me3 enrichment in vitro. METHODS Doxycycline-inducible short hairpin RNA (shRNA) against H3F3A was delivered via lentivirus to established H3G34V mutant pediatric glioma cell line KNS42, and H3G34V introduced into H3.3 wild type normal human astrocytes (NHA). Transfections were confirmed by western blot, fluorescent imaging, and flow cytometry, with resulting H3.3WT and H3K36me3 expression determined by western blot. H3.3WT, H3K36me3, and H3G34V ChIP-Seq was performed to evaluate genomic enrichment. RESULTS Complete knockdown of H3G34V was achieved with DOX-induced shRNA, with no change in total H3.3, suggesting disproportionate allelic frequency of genes encoding H3.3 (H3F3A and H3F3B). Modest increase in H3K36me3 occurred after H3F3A-knockdown from KNS42, suggesting H3G34V alone impacts observed H3K36me3 levels. Distinct H3K36me3 genomic enrichment was observed with H3G34V knock-in. CONCLUSIONS We demonstrate that DOX-inducible knockdown of H3F3A in an H3G34V mutant pediatric glioma cells and H3G34V mutation transduction in wild-type astrocytes affects H3K36me3 expression. Further evaluation by ChIP-Seq analysis for restoration of wild-type genomic H3K36me3 enrichment patterns with H3G34V knockdown, and mutant H3K36me3 patterns with H3G34V transduction, is currently underway.

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Designing P. aeruginosa synthetic phages with reduced genomes

Scientific Reports ◽

10.1038/s41598-021-81580-2 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Diana P. Pires ◽

Rodrigo Monteiro ◽

Dalila Mil-Homens ◽

Arsénio Fialho ◽

Timothy K. Lu ◽

...

Keyword(s):

Galleria Mellonella ◽

Antibacterial Properties ◽

Genetically Engineered ◽

In Vivo Studies ◽

Infection Model ◽

Promising Therapeutic Approach ◽

Genes Encoding ◽

First Time

AbstractIn the era where antibiotic resistance is considered one of the major worldwide concerns, bacteriophages have emerged as a promising therapeutic approach to deal with this problem. Genetically engineered bacteriophages can enable enhanced anti-bacterial functionalities, but require cloning additional genes into the phage genomes, which might be challenging due to the DNA encapsulation capacity of a phage. To tackle this issue, we designed and assembled for the first time synthetic phages with smaller genomes by knocking out up to 48% of the genes encoding hypothetical proteins from the genome of the newly isolated Pseudomonas aeruginosa phage vB_PaeP_PE3. The antibacterial efficacy of the wild-type and the synthetic phages was assessed in vitro as well as in vivo using a Galleria mellonella infection model. Overall, both in vitro and in vivo studies revealed that the knock-outs made in phage genome do not impair the antibacterial properties of the synthetic phages, indicating that this could be a good strategy to clear space from phage genomes in order to enable the introduction of other genes of interest that can potentiate the future treatment of P. aeruginosa infections.

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The Genes Encoding Small Leucine-Rich Proteoglycans Undergo Differential Expression Alterations in Colorectal Cancer, Depending on Tumor Location

Cells ◽

10.3390/cells10082002 ◽

2021 ◽

Vol 10 (8) ◽

pp. 2002

Author(s):

Maria Pilar Solis-Hernandez ◽

Carla Martín ◽

Beatriz García ◽

Natalia Pérez-López ◽

Yolanda García-Mesa ◽

...

Keyword(s):

Protein Expression ◽

Survival Data ◽

Tumor Cell Line ◽

Tumor Location ◽

Anatomical Location ◽

Cell Interior ◽

Expression Of Genes ◽

Genes Encoding ◽

Descending Colon

Small leucine-rich proteoglycans (SLRPs) regulate different processes and undergo significant alterations in various diseases. Colon carcinomas (CCs) are heterogeneous pathologies with important clinical and molecular differences depending on their location, which makes it interesting to analyze the alterations in SLRPs in right- and left-sided tumors (RS- and LSCCs). SLRP transcription levels were studied in 32 CCs using qPCR compared to healthy colon mucosae samples from the same patients, 20 of them from LSCCs and the remaining 12 from RSCCs. Protein expression of genes with significant differences in their transcriptions was analyzed by immunohistochemistry. The alterations observed were related to survival data. The arrangement of transcription of SLRPs was quite similar in ascending and descending colon, but RS- and LSCCs displayed different patterns of alteration, with a greater number of deregulations occurring in the latter. The analysis of protein expression also indicated changes in the location of these molecules, largely moving to the cell interior. While podocan underexpression showed a trend toward better outcomes, no differences were observed in terms of overall survival. In vitro studies using the HT29 tumor cell line suggest that deregulation of SLRPs could affect cell proliferation. SLRPs constitute new differential markers of RS- and LSCCs, showing differences dependent on the anatomical location of the tumor.

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