scholarly journals In situ reprogramming of gut bacteria by oral delivery

2020 ◽  
Author(s):  
Bryan B. Hsu ◽  
Isaac N. Plant ◽  
Lorena Lyon ◽  
Frances M. Anastassacos ◽  
Jeffrey C. Way ◽  
...  
Keyword(s):  
Oral Delivery ◽  
Multidisciplinary Approach ◽  
Gut Bacteria ◽  
Release Mechanism ◽  
Bacterial Gene ◽  
E Coli ◽  
Bacterial Gene Expression ◽  
Bacterial Function

AbstractAbundant links between the gut microbiota and human health indicate that the modification of bacterial function could be a powerful therapeutic strategy. The inaccessibility of the gut and inter-connections between gut bacteria and the host make it difficult to precisely target bacterial functions without disrupting the microbiota and/or host physiology. Herein we describe a multidisciplinary approach to modulate the expression of a specific bacterial gene within the gut by oral administration. We first demonstrate that an engineered temperate phage λ expressing a programmable dCas9 represses a targeted E. coli gene in the mammalian gut. To facilitate phage administration while minimizing disruption to host processes, we develop an aqueous-based encapsulation formulation with a microbiota-based release mechanism and show that it facilitates the oral delivery of phage in vivo. Finally we combine these technologies and show that bacterial gene expression in the mammalian gut can be precisely modified in situ with a single oral dose.

scholarly journals In situ reprogramming of gut bacteria by oral delivery

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Bryan B. Hsu ◽  
Isaac N. Plant ◽  
Lorena Lyon ◽  
Frances M. Anastassacos ◽  
Jeffrey C. Way ◽  
...  
Keyword(s):  
Oral Delivery ◽  
Multidisciplinary Approach ◽  
Gut Bacteria ◽  
Release Mechanism ◽  
Bacterial Gene ◽  
E Coli ◽  
Bacterial Gene Expression ◽  
Bacterial Function

Abstract Abundant links between the gut microbiota and human health indicate that modification of bacterial function could be a powerful therapeutic strategy. The inaccessibility of the gut and inter-connections between gut bacteria and the host make it difficult to precisely target bacterial functions without disrupting the microbiota and/or host physiology. Herein we describe a multidisciplinary approach to modulate the expression of a specific bacterial gene within the gut by oral administration. We demonstrate that an engineered temperate phage λ expressing a programmable dCas9 represses a targeted E. coli gene in the mammalian gut. To facilitate phage administration while minimizing disruption to host processes, we develop an aqueous-based encapsulation formulation with a microbiota-based release mechanism and show that it facilitates oral delivery of phage in vivo. Finally we combine these technologies and show that bacterial gene expression in the mammalian gut can be precisely modified in situ with a single oral dose.

Concurrence between the gene expression pattern of Actinobacillus actinomycetemcomitans in localized aggressive periodontitis and in human epithelial cells

2005 ◽  
Vol 54 (5) ◽  
pp. 497-504 ◽  
Author(s):  
Joseph Richardson ◽  
Justin Corey Craighead ◽  
Sam Linsen Cao ◽  
Martin Handfield
Keyword(s):  
Gene Expression ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Expression Pattern ◽  
Gene Expression Pattern ◽  
Aggressive Periodontitis ◽  
Actinobacillus Actinomycetemcomitans ◽  
Bacterial Gene ◽  
Bacterial Gene Expression

Actinobacillus actinomycetemcomitans is a facultatively intracellular pathogen and the aetiological agent of localized aggressive periodontitis. Screening of the genome of A. actinomycetemcomitans for in vivo-induced antigen determinants previously demonstrated that the proteome of this organism differs in laboratory culture compared with conditions found during active infection. The aim of the present study was to determine whether the bacterial gene expression pattern inferred with in vivo-induced antigen technology (IVIAT) in human infections was consistent with the gene expression pattern occurring upon epithelial cell association. To this end, a real-time PCR method was developed and used to quantify absolute and relative bacterial gene expression of A. actinomycetemcomitans grown extra- and intracellularly in two human epithelial cell lines (HeLa and IHGK). The amount of template used in the assay was normalized using the total count of viable bacteria (c.f.u.) as a reference point and performed in duplicate in at least two independent experiments. Controls for this experiment included 16S rRNA and gapdh. Transcription of all eight ORFs tested increased significantly (P < 0.05) in HeLa and IHGK cells compared with bacteria grown extracellularly. The concurrence of gene expression patterns found in the two models suggests that these epithelial cells are valid in vitro models of infection for the genes tested. IVIAT is an experimental platform that can be used as a validation tool to assess the reliability of animal and other models of infection and is applicable to most pathogens.

Glyoxalase I – structure, function and a critical role in the enzymatic defence against glycation

2003 ◽  
Vol 31 (6) ◽  
pp. 1343-1348 ◽  
Author(s):  
P.J. Thornalley
Keyword(s):  
Metal Ion ◽  
Catalytic Mechanism ◽  
Critical Role ◽  
Glyoxalase I ◽  
Water Molecules ◽  
Glyoxalase System ◽  
E Coli ◽  
Antimalarial Agents

Glyoxalase I is part of the glyoxalase system present in the cytosol of cells. The glyoxalase system catalyses the conversion of reactive, acyclic α-oxoaldehydes into the corresponding α-hydroxyacids. Glyoxalase I catalyses the isomerization of the hemithioacetal, formed spontaneously from α-oxoaldehyde and GSH, to S-2-hydroxyacylglutathione derivatives [RCOCH(OH)-SG→RCH(OH)CO-SG], and in so doing decreases the steady-state concentrations of physiological α-oxoaldehydes and associated glycation reactions. Physiological substrates of glyoxalase I are methylglyoxal, glyoxal and other acyclic α-oxoaldehydes. Human glyoxalase I is a dimeric Zn2+ metalloenzyme of molecular mass 42 kDa. Glyoxalase I from Escherichia coli is a Ni2+ metalloenzyme. The crystal structures of human and E. coli glyoxalase I have been determined to 1.7 and 1.5 Å resolution. The Zn2+ site comprises two structurally equivalent residues from each domain – Gln-33A, Glu-99A, His-126B, Glu-172B and two water molecules. The Ni2+ binding site comprises His-5A, Glu-56A, His-74B, Glu-122B and two water molecules. The catalytic reaction involves base-catalysed shielded-proton transfer from C-1 to C-2 of the hemithioacetal to form an ene-diol intermediate and rapid ketonization to the thioester product. R- and S-enantiomers of the hemithioacetal are bound in the active site, displacing the water molecules in the metal ion primary co-ordination shell. It has been proposed that Glu-172 is the catalytic base for the S-substrate enantiomer and Glu-99 the catalytic base for the R-substrate enantiomer; Glu-172 then reprotonates the ene-diol stereospecifically to form the R-2-hydroxyacylglutathione product. By analogy with the human enzyme, Glu-56 and Glu-122 may be the bases involved in the catalytic mechanism of E. coli glyoxalase I. The suppression of α-oxoaldehyde-mediated glycation by glyoxalase I is particularly important in diabetes and uraemia, where α-oxoaldehyde concentrations are increased. Decreased glyoxalase I activity in situ due to the aging process and oxidative stress results in increased glycation and tissue damage. Inhibition of glyoxalase I pharmacologically with specific inhibitors leads to the accumulation of α-oxoaldehydes to cytotoxic levels; cell-permeable glyoxalase I inhibitors are antitumour and antimalarial agents. Glyoxalase I has a critical role in the prevention of glycation reactions mediated by methylglyoxal, glyoxal and other α-oxoaldehydes in vivo.

Oxytocin stimulates LH production by the anterior pituitary gland of the rat

1992 ◽  
Vol 132 (2) ◽  
pp. 277-283 ◽  
Author(s):  
G. Robinson ◽  
J. J. Evans ◽  
K. J. Catt
Keyword(s):  
Female Rats ◽  
Release Mechanism ◽  
Protein Synthesis Inhibitor ◽  
Pituitary Cells ◽  
Synthesis Inhibitor ◽  
Mechanical Dispersion ◽  
Lh Release

ABSTRACT Gonadotrophin-releasing activity of oxytocin has previously been demonstrated in vitro and in vivo. This study investigated whether oxytocin is also able to induce LH accumulation in pituitary cells. Following trypsin digestion and mechanical dispersion, pituitary cells from female rats were incubated with oxytocin (100 nmol/l) for 24 h. LH release stimulated by oxytocin increased (P < 0·001) progressively during the incubation indicating a different secretory pattern from the more rapid but less sustained secretion stimulated by gonadotrophin-releasing hormone. Oxytocin also enhanced (P < 0·01) total LH accumulation in the incubation system (released plus cell contents) which was apparent after 7–11 h of stimulation. The release of LH stimulated by oxytocin was reduced by the protein synthesis inhibitor cycloheximide (10 μmol/l). However, cycloheximide did not completely block oxytocin-stimulated LH release; there remained some LH release above that seen in non-stimulated controls (P < 0·01) revealing the presence of a cycloheximide-resistant component in the release mechanism. Furthermore, accumulation of total LH in 24 h incubations was suppressed (P < 0·01) by cycloheximide. The advancement in LH release which oxytocin has been shown to induce in vivo in pro-oestrous rats was accompanied by an early reduction of pituitary LH stores. However, the fall normally observed in LH content during the surge was markedly attenuated by the oxytocin treatment. Thus, loss of pituitary LH stores was less in oxytocin-treated rats than in saline-treated controls, even though net LH release into plasma was increased. Therefore, oxytocin stimulated the replenishment of LH stores. Although the mechanism(s) remains to be defined and the relationships between in-vitro and in-vivo results are as yet uncharacterized, the present study demonstrates that oxytocin treatment stimulates LH production in both dispersed cells and intact pituitaries in situ. Journal of Endocrinology (1992) 132, 277–283

scholarly journals Evaluation of Live Bacterial Prophylactics to Decrease IncF Plasmid Transfer and Association With Intestinal Small RNAs

2021 ◽  
Vol 11 ◽  
Author(s):  
Graham A. J. Redweik ◽  
Mary Kate Horak ◽  
Ryley Hoven ◽  
Logan Ott ◽  
Melha Mellata
Keyword(s):  
Virulence Genes ◽  
Positive Association ◽  
Multidrug Resistant ◽  
Plasmid Transfer ◽  
Siderophore Production ◽  
Causal Mechanism ◽  
Bacterial Gene ◽  
E Coli ◽  
Bacterial Gene Expression

Chicken intestinal Escherichia coli are a reservoir for virulence and antimicrobial resistance (AMR) genes that are often carried on incompatibility group F (IncF) plasmids. The rapid transfer of these plasmids between bacteria in the gut contributes to the emergence of new multidrug-resistant and virulent bacteria that threaten animal agriculture and human health. Thus, the aim of the present study was to determine whether live bacterial prophylactics could affect the distribution of large virulence plasmids and AMR in the intestinal tract and the potential role of smRNA in this process. In this study, we tested ∼100 randomly selected E. coli from pullet feces (n = 3 per group) given no treatment (CON), probiotics (PRO), a live Salmonella vaccine (VAX), or both (P + V). E. coli isolates were evaluated via plasmid profiles and several phenotypic (siderophore production and AMR), and genotypic (PCR for virulence genes and plasmid typing) screens. P + V isolates exhibited markedly attenuated siderophore production, lack of AMR and virulence genes, which are all related to the loss of IncF and ColV plasmids (P &lt; 0.0001). To identify a causal mechanism, we evaluated smRNA levels in the ceca mucus and found a positive association between smRNA concentrations and plasmid content, with both being significantly reduced in P + V birds compared to other groups (P &lt; 0.01). To test this positive association between IncF plasmid transfer and host smRNA concentration, we evenly pooled smRNA per group and treated E. coli mating pairs with serial concentrations of smRNA in vitro. Higher smRNA concentrations resulted in greater rates of IncF plasmid transfer between E. coli donors (APEC O2 or VAX isolate IA-EC-001) and recipient (HS-4) (all groups; P &lt; 0.05). Finally, RNAHybrid predictive analyses detected several chicken miRNAs that hybridize with pilus assembly and plasmid transfer genes on the IncF plasmid pAPEC-O2-R. Overall, we demonstrated P + V treatment reduced smRNA levels in the chicken ceca, which was associated with a reduction in potentially virulent E. coli. Furthermore, we propose a novel mechanism in which intestinal smRNAs signal plasmid exchange between E. coli. Investigations to understand the changes in bacterial gene expression as well as smRNAs responsible for this phenomenon are currently underway.

scholarly journals Role of Hfq in Genome Evolution: Instability of G-Quadruplex Sequences in E. coli

2019 ◽  
Vol 8 (1) ◽  
pp. 28 ◽  
Author(s):  
Virali J. Parekh ◽  
Brittany A. Niccum ◽  
Rachna Shah ◽  
Marisa A. Rivera ◽  
Mark J. Novak ◽  
...  
Keyword(s):  
Genomic Instability ◽  
Dna Repeats ◽  
Loop Formation ◽  
Bacterial Gene ◽  
Quadruplex Dna ◽  
E Coli ◽  
Alternative Structures ◽  
Bacterial Gene Expression ◽  
G Quadruplex

Certain G-rich DNA repeats can form quadruplex in bacterial chromatin that can present blocks to DNA replication and, if not properly resolved, may lead to mutations. To understand the participation of quadruplex DNA in genomic instability in Escherichia coli (E. coli), mutation rates were measured for quadruplex-forming DNA repeats, including (G3T)4, (G3T)8, and a RET oncogene sequence, cloned as the template or nontemplate strand. We evidence that these alternative structures strongly influence mutagenesis rates. Precisely, our results suggest that G-quadruplexes form in E. coli cells, especially during transcription when the G-rich strand can be displaced by R-loop formation. Structure formation may then facilitate replication misalignment, presumably associated with replication fork blockage, promoting genomic instability. Furthermore, our results also evidence that the nucleoid-associated protein Hfq is involved in the genetic instability associated with these sequences. Hfq binds and stabilizes G-quadruplex structure in vitro and likely in cells. Collectively, our results thus implicate quadruplexes structures and Hfq nucleoid protein in the potential for genetic change that may drive evolution or alterations of bacterial gene expression.

Spatiotemporal control of the bacteria to express interferon-γ by focused ultrasound for tumor immunotherapy

2021 ◽  
Author(s):  
Yuhao Chen ◽  
Meng Du ◽  
Zhen Yuan ◽  
Fei Yan ◽  
Zhiyi Chen
Keyword(s):  
Focused Ultrasound ◽  
Tumor Therapy ◽  
Tumor Immunotherapy ◽  
Interferon Γ ◽  
Genetic Switch ◽  
Bacterial Gene ◽  
Bacterial Gene Expression ◽  
Ifn Γ

Abstract Bacteria-based tumor therapy has recently attracted wide attentions due to its unique capability in targeting tumors and preferentially colonizing the core area of the tumor. Various therapeutic genes were also harbored into these engineering bacteria to enhance their anti-tumor efficacy. However, it is difficult to spatiotemporally control the expression of these inserted genes in the tumor site. Here, we engineered an ultrasound-responsive bacterium (URB) which can induce the expression of exogenous genes in an ultrasound-controllable manner. Owing to the advantage of ultrasound in the tissue penetration, energy focusing into heating, an acoustic remote control of bacterial gene expression can be realized by designing a temperature-actuated genetic switch. Cytokine interferon-γ (IFN-γ), an important immune regulatory molecule that plays a significant role in tumor immunotherapy, was used to test the system. Our results showed a brief hyperthermia by focused ultrasound successfully induced the expression of IFN-γ gene, significantly improving anti-tumor efficacy of URB in vitro and in vivo. Our study provided a novel strategy for bacteria-mediated tumor immunotherapy.

scholarly journals Structure of hibernating ribosomes studied by cryoelectron tomography in vitro and in situ

2010 ◽  
Vol 190 (4) ◽  
pp. 613-621 ◽  
Author(s):  
Julio O. Ortiz ◽  
Florian Brandt ◽  
Valério R.F. Matias ◽  
Lau Sennels ◽  
Juri Rappsilber ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Stress Response ◽  
Spatial Organization ◽  
Three Dimensional ◽  
E Coli ◽  
Escherichia Coli Cells ◽  
Three Dimensional Configuration

Ribosomes arranged in pairs (100S) have been related with nutritional stress response and are believed to represent a “hibernation state.” Several proteins have been identified that are associated with 100S ribosomes but their spatial organization has hitherto not been characterized. We have used cryoelectron tomography to reveal the three-dimensional configuration of 100S ribosomes isolated from starved Escherichia coli cells and we have described their mode of interaction. In situ studies with intact E. coli cells allowed us to demonstrate that 100S ribosomes do exist in vivo and represent an easily reversible state of quiescence; they readily vanish when the growth medium is replenished.

scholarly journals Revealing the in vivo growth and division patterns of mouse gut bacteria

2020 ◽  
Vol 6 (36) ◽  
pp. eabb2531
Author(s):  
Liyuan Lin ◽  
Qiuyue Wu ◽  
Jia Song ◽  
Yahui Du ◽  
Juan Gao ◽  
...  
Keyword(s):  
Amino Acid ◽  
Gut Microbiota ◽  
Growth Dynamics ◽  
Gut Bacteria ◽  
Metabolic Labeling ◽  
Taxonomic Identification ◽  
In Vivo Growth

Current techniques for studying gut microbiota are unable to answer some important microbiology questions, like how different bacteria grow and divide in the gut. We propose a method that integrates the use of sequential d-amino acid–based in vivo metabolic labeling with fluorescence in situ hybridization (FISH), for characterizing the growth and division patterns of gut bacteria. After sequentially administering two d-amino acid–based probes containing different fluorophores to mice by gavage, the resulting dual-labeled peptidoglycans provide temporal information on cell wall synthesis of gut bacteria. Following taxonomic identification with FISH probes, the growth and division patterns of the corresponding bacterial taxa, including species that cannot be cultured separately in vitro, are revealed. Our method offers a facile yet powerful tool for investigating the in vivo growth dynamics of the bacterial gut microbiota, which will advance our understanding of bacterial cytology and facilitate elucidation of the basic microbiology of this gut “dark matter.”

scholarly journals A Prophage-Encoded Small RNA Controls Metabolism and Cell Division in Escherichia coli

mSystems ◽  
2016 ◽  
Vol 1 (1) ◽  
Author(s):  
Divya Balasubramanian ◽  
Preethi T. Ragunathan ◽  
Jingyi Fei ◽  
Carin K. Vanderpool
Keyword(s):  
Gene Expression ◽  
Cell Division ◽  
Posttranscriptional Control ◽  
Bacterial Gene ◽  
Content Type ◽  
E Coli ◽  
Bacterial Gene Expression ◽  
Metabolic Gene Expression ◽  
Defective Prophage ◽  
Ectopic Production

ABSTRACT sRNAs are ubiquitous and versatile regulators of bacterial gene expression. A number of well-characterized examples in E. coli are highly conserved and present in the E. coli core genome. In contrast, the sRNA DicF (identified over 20 years ago but remaining poorly characterized) is encoded by a gene carried on a defective prophage element in many E. coli genomes. Here, we characterize DicF in order to better understand how horizontally acquired sRNA regulators impact bacterial gene expression and physiology. Our data confirm the long-hypothesized DicF-mediated regulation of ftsZ, encoding the bacterial tubulin homolog required for cell division. We further uncover DicF-mediated posttranscriptional control of metabolic gene expression. Ectopic production of DicF is highly toxic to E. coli cells, but the toxicity is not attributable to DicF regulation of ftsZ. Further work is needed to reveal the biological roles of and benefits for the host conferred by DicF and other products encoded by defective prophages. Hundreds of small RNAs (sRNAs) have been identified in diverse bacterial species, and while the functions of most remain unknown, some regulate key processes, particularly stress responses. The sRNA DicF was identified over 25 years ago as an inhibitor of cell division but since then has remained uncharacterized. DicF consists of 53 nucleotides and is encoded by a gene carried on a prophage (Qin) in the genomes of many Escherichia coli strains. We demonstrated that DicF inhibits cell division via direct base pairing with ftsZ mRNA to repress translation and prevent new synthesis of the bacterial tubulin homolog FtsZ. Systems analysis using computational and experimental methods identified additional mRNA targets of DicF: xylR and pykA mRNAs, encoding the xylose uptake and catabolism regulator and pyruvate kinase, respectively. Genetic analyses showed that DicF directly base pairs with and represses translation of these targets. Phenotypes of cells expressing DicF variants demonstrated that DicF-associated growth inhibition is not solely due to repression of ftsZ, indicating that the physiological consequences of DicF-mediated regulation extend beyond effects on cell division caused by reduced FtsZ synthesis. IMPORTANCE sRNAs are ubiquitous and versatile regulators of bacterial gene expression. A number of well-characterized examples in E. coli are highly conserved and present in the E. coli core genome. In contrast, the sRNA DicF (identified over 20 years ago but remaining poorly characterized) is encoded by a gene carried on a defective prophage element in many E. coli genomes. Here, we characterize DicF in order to better understand how horizontally acquired sRNA regulators impact bacterial gene expression and physiology. Our data confirm the long-hypothesized DicF-mediated regulation of ftsZ, encoding the bacterial tubulin homolog required for cell division. We further uncover DicF-mediated posttranscriptional control of metabolic gene expression. Ectopic production of DicF is highly toxic to E. coli cells, but the toxicity is not attributable to DicF regulation of ftsZ. Further work is needed to reveal the biological roles of and benefits for the host conferred by DicF and other products encoded by defective prophages.

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