scholarly journals A peptide of a type I toxin−antitoxin system inducesHelicobacter pylorimorphological transformation from spiral shape to coccoids

2020 ◽  
Vol 117 (49) ◽  
pp. 31398-31409
Author(s):  
Lamya El Mortaji ◽  
Alejandro Tejada-Arranz ◽  
Aline Rifflet ◽  
Ivo G. Boneca ◽  
Gérard Pehau-Arnaudet ◽  
...  
Keyword(s):  
Antisense Rna ◽  
Type I ◽  
Morphological Transformation ◽  
Bacterial Chromosomes ◽  
Concentration Result ◽  
Expression Activity ◽  
Spiral Shape ◽  
H Pylori ◽  
Plasmid Stabilization

Toxin−antitoxin systems are found in many bacterial chromosomes and plasmids with roles ranging from plasmid stabilization to biofilm formation and persistence. In these systems, the expression/activity of the toxin is counteracted by an antitoxin, which, in type I systems, is an antisense RNA. While the regulatory mechanisms of these systems are mostly well defined, the toxins’ biological activity and expression conditions are less understood. Here, these questions were investigated for a type I toxin−antitoxin system (AapA1−IsoA1) expressed from the chromosome of the human pathogenHelicobacter pylori. We show that expression of the AapA1 toxin inH. pyloricauses growth arrest associated with rapid morphological transformation from spiral-shaped bacteria to round coccoid cells. Coccoids are observed in patients and during in vitro growth as a response to different stress conditions. The AapA1 toxin, first molecular effector of coccoids to be identified, targetsH. pyloriinner membrane without disrupting it, as visualized by cryoelectron microscopy. The peptidoglycan composition of coccoids is modified with respect to spiral bacteria. No major changes in membrane potential or adenosine 5′-triphosphate (ATP) concentration result from AapA1 expression, suggesting coccoid viability. Single-cell live microscopy tracking the shape conversion suggests a possible association of this process with cell elongation/division interference. Oxidative stress induces coccoid formation and is associated with repression of the antitoxin promoter and enhanced processing of its transcript, leading to an imbalance in favor of AapA1 toxin expression. Our data support the hypothesis of viable coccoids with characteristics of dormant bacteria that might be important inH. pyloriinfections refractory to treatment.

scholarly journals A peptide of a type I toxin-antitoxin system induces Helicobacter pylori morphological transformation from spiral-shape to coccoids

2019 ◽  
Author(s):  
Lamya El Mortaji ◽  
Alejandro Tejada-Arranz ◽  
Aline Rifflet ◽  
Ivo G Boneca ◽  
Gérard Pehau-Arnaudet ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Helicobacter Pylori ◽  
Membrane Integrity ◽  
Type I ◽  
Morphological Transformation ◽  
Bacterial Chromosomes ◽  
Concentration Result ◽  
H Pylori ◽  
Plasmid Stabilization

SummaryToxin-antitoxin systems are found in many bacterial chromosomes and plasmids with roles ranging from plasmid stabilization to biofilm formation and persistence. In these systems, the expression/activity of the toxin is counteracted by an antitoxin, which in type I systems is an antisense-RNA. While the regulatory mechanisms of these systems are mostly well-defined, the toxins’ biological activity and expression conditions are less understood. Here, these questions were investigated for a type I toxin-antitoxin system (AapA1-IsoA1) expressed from the chromosome of the human pathogen Helicobacter pylori. We show that expression of the AapA1 toxin in H. pylori causes growth arrest associated with rapid morphological transformation from spiral-shaped bacteria to round coccoid cells. Coccoids are observed in patients and during in vitro growth as a response to different stress conditions. The AapA1 toxin, first molecular effector of coccoids to be identified, targets H. pylori inner membrane without disrupting it, as visualized by Cryo-EM. The peptidoglycan composition of coccoids is modified with respect to spiral bacteria. No major changes in membrane potential or ATP concentration result from AapA1 expression, suggesting coccoid viability. Single-cell live microscopy tracking the shape conversion suggests a possible association of this process with cell elongation/division interference. Oxidative stress induces coccoid formation and is associated with repression of the antitoxin promoter and enhanced processing of its transcript, leading to an imbalance in favor of AapA1 toxin expression.Our data support the hypothesis of viable coccoids with characteristics of dormant bacteria that might be important in H. pylori infections refractory to treatment.Significance StatementHelicobacter pylori, a gastric pathogen causing 800,000 deaths in the world annually, is encountered both in vitro and in patients as spiral-shaped bacteria and as round cells named coccoids. We discovered that the toxin from a chromosomal type I toxin-antitoxin system is targeting H. pylori membrane and acting as an effector of H. pylori morphological conversion to coccoids. We showed that these round cells maintain their membrane integrity and metabolism, strongly suggesting that they are viable dormant bacteria. Oxidative stress was identified as a signal inducing toxin expression and coccoid formation. Our findings reveal new insights into a form of dormancy of this bacterium that might be associated with H. pylori infections refractory to treatment.

scholarly journals Development of a Mucus Gland Bioreactor in Loach Paramisgurnus dabryanus

2021 ◽  
Vol 22 (2) ◽  
pp. 687
Author(s):  
Tong Zhou ◽  
Bolan Zhou ◽  
Yasong Zhao ◽  
Qing Li ◽  
Guili Song ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Grass Carp ◽  
Single Copy ◽  
Type I ◽  
Western Blots ◽  
Paramisgurnus Dabryanus ◽  
Expression Activity ◽  
Transgenic Construct ◽  
Mucus Gland

Most currently available bioreactors have some defects in the expression, activity, or purification of target protein and peptide molecules, whereas the mucus gland of fish can overcome these defects to become a novel bioreactor for the biopharmaceutical industry. In this study, we have evaluated the practicability of developing a mucus gland bioreactor in loach (Paramisgurnus dabryanus). A transgenic construct pT2-krt8-IFN1 was obtained by subcloning the promoter of zebrafish keratin 8 gene and the type I interferon (IFN1) cDNA of grass carp into the SB transposon. The IFN1 expressed in CIK cells exhibited an antiviral activity against the replication of GCRV873 and activated two genes downstream of JAK-STAT signaling pathway. A transgenic loach line was then generated by microinjection of the pT2-krt8-IFN1 plasmids and in vitro synthesized capped SB11 mRNA. Southern blots indicated that a single copy of IFN1 gene was stably integrated into the genome of transgenic loach. The expression of grass carp IFN1 in transgenic loaches was detected with RT-PCR and Western blots. About 0.0825 µg of grass carp IFN1 was detected in 20 µL mucus from transgenic loaches. At a viral titer of 1 × 103 PFU/mL, plaque numbers on plates containing mucus from transgenic loaches reduced by 18% in comparison with those of the control, indicating that mucus of IFN1-transgenic loaches exhibited an antiviral activity. Thus, we have successfully created a mucus gland bioreactor that has great potential for the production of various proteins and peptides.

scholarly journals Antisense RNA Modulation of Alkyl Hydroperoxide Reductase Levels in Helicobacter pylori Correlates with Organic Peroxide Toxicity but Not Infectivity

2007 ◽  
Vol 189 (9) ◽  
pp. 3359-3368 ◽  
Author(s):  
Matthew A. Croxen ◽  
Peter B. Ernst ◽  
Paul S. Hoffman
Keyword(s):  
Helicobacter Pylori ◽  
Rna Interference ◽  
Gene Function ◽  
Antisense Rna ◽  
In Vitro Growth ◽  
Protein Levels ◽  
Alkyl Hydroperoxide ◽  
Gastric Colonization ◽  
H Pylori

ABSTRACT Much of the gene content of the human gastric pathogen Helicobacter pylori (∼1.7-Mb genome) is considered essential. This view is based on the completeness of metabolic pathways, infrequency of nutritional auxotrophies, and paucity of pathway redundancies typically found in bacteria with larger genomes. Thus, genetic analysis of gene function is often hampered by lethality. In the absence of controllable promoters, often used to titrate gene function, we investigated the feasibility of an antisense RNA interference strategy. To test the antisense approach, we targeted alkyl hydroperoxide reductase (AhpC), one of the most abundant proteins expressed by H. pylori and one whose function is essential for both in vitro growth and gastric colonization. Here, we show that antisense ahpC (as-ahpC) RNA expression from shuttle vector pDH37::as-ahpC achieved an ∼72% knockdown of AhpC protein levels, which correlated with increased susceptibilities to hydrogen peroxide, cumene, and tert-butyl hydroperoxides but not with growth efficiency. Compensatory increases in catalase levels were not observed in the knockdowns. Expression of single-copy antisense constructs (expressed under the urease promoter and containing an fd phage terminator) from the rdxA locus of mouse-colonizing strain X47 achieved a 32% knockdown of AhpC protein levels (relative to wild-type X47 levels), which correlated with increased susceptibility to organic peroxides but not with mouse colonization efficiency. Our studies indicate that high levels of AhpC are not required for in vitro growth or for primary gastric colonization. Perhaps AhpC, like catalase, assumes a greater role in combating exogenous peroxides arising from lifelong chronic inflammation. These studies also demonstrate the utility of antisense RNA interference in the evaluation of gene function in H. pylori.

scholarly journals In Vitro Activity of Sertraline, an Antidepressant, Against Antibiotic-Susceptible and Antibiotic-Resistant Helicobacter pylori Strains

Pathogens ◽  
2019 ◽  
Vol 8 (4) ◽  
pp. 228 ◽  
Author(s):  
Paweł Krzyżek ◽  
Roman Franiczek ◽  
Barbara Krzyżanowska ◽  
Łukasz Łaczmański ◽  
Paweł Migdał ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Morphological Transformation ◽  
Gastric Diseases ◽  
Additive Interaction ◽  
Antibiotic Resistant ◽  
Minimal Inhibitory Concentrations ◽  
Killing Assay ◽  
H Pylori ◽  
Light Fluorescence

Antibiotic resistance of Helicobacter pylori, a spiral bacterium associated with gastric diseases, is a topic that has been intensively discussed in last decades. Recent discoveries indicate promising antimicrobial and antibiotic-potentiating properties of sertraline (SER), an antidepressant substance. The aim of the study, therefore, was to determine the antibacterial activity of SER in relation to antibiotic-sensitive and antibiotic-resistant H. pylori strains. The antimicrobial tests were performed using a diffusion-disk method, microdilution method, and time-killing assay. The interaction between SER and antibiotics (amoxicillin, clarithromycin, tetracycline, and metronidazole) was determined by using a checkerboard method. In addition, the study was expanded to include observations by light, fluorescence, and scanning electron microscopy. The growth inhibition zones were in the range of 19–37 mm for discs impregnated with 2 mg of SER. The minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) counted for 2–8 µg/mL and 4–8 µg/mL, respectively. The time-killing assay showed the time-dependent and concentration-dependent bactericidal activity of SER. Bacteria exposed to MBCs (but not sub-MICs and MICs ≠ MBCs) underwent morphological transformation into coccoid forms. This mechanism, however, was not protective because these cells after a 24-h incubation had a several-fold reduced green/red fluorescence ratio compared to the control. Using the checkerboard assay, a synergistic/additive interaction of SER with all four antibiotics tested was demonstrated. These results indicate that SER may be a promising anti-H. pylori compound.

scholarly journals Helicobacter pylori Infection: Diagnosis and Therapy

2021 ◽  
Vol 12 (4) ◽  
pp. 145-148
Author(s):  
Darius Hartanto
Keyword(s):  
Helicobacter Pylori ◽  
Acquired Resistance ◽  
Helicobacter Pylori Infection ◽  
Living Standard ◽  
Growth Media ◽  
Negative Bacillus ◽  
Spiral Shape ◽  
H Pylori ◽  
Slow Growing

Helicobacter pylori (H. pylori) is a spiral shape gram-negative bacillus and have flagella for motility in mucus environment. H. pylori is microaerophilic organism, slow growing and requires complex growth media in vitro. H. pylori infecting more than 50% populations in worldwide. Prevalence of H. pylori infection is higher in developing countries compared to developed one, and indicates that socioeconomic and living standard may play a major role in the distribution. This study aims to provide an overview of how to diagnose and manage Helicobacter pylori infection. This study reviewed various sources then reviewed as a literature review. The most successful regimens are triple and quadruple combinations, which consist of a PPI and two or three antibiotics for 7 – 14 days. Patient’s compliance and the use of drug to which strain of H. pylori has not acquired resistance are the most important factors in successful H. pylori treatment.

scholarly journals Identification of an LGP2-associated MDA5 agonist in picornavirus-infected cells

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Safia Deddouche ◽  
Delphine Goubau ◽  
Jan Rehwinkel ◽  
Probir Chakravarty ◽  
Sharmin Begum ◽  
...  
Keyword(s):  
Antisense Rna ◽  
Rna Synthesis ◽  
In Vitro Transcription ◽  
Encephalomyocarditis Virus ◽  
Type I Interferons ◽  
Type I ◽  
Specific Sequence ◽  
Infected Cells ◽  
Rna Complexes

The RIG-I-like receptors RIG-I, LGP2, and MDA5 initiate an antiviral response that includes production of type I interferons (IFNs). The nature of the RNAs that trigger MDA5 activation in infected cells remains unclear. Here, we purify and characterise LGP2/RNA complexes from cells infected with encephalomyocarditis virus (EMCV), a picornavirus detected by MDA5 and LGP2 but not RIG-I. We show that those complexes contain RNA that is highly enriched for MDA5-stimulatory activity and for a specific sequence corresponding to the L region of the EMCV antisense RNA. Synthesis of this sequence by in vitro transcription is sufficient to generate an MDA5 stimulatory RNA. Conversely, genomic deletion of the L region in EMCV generates viruses that are less potent at stimulating MDA5-dependent IFN production. Thus, the L region antisense RNA of EMCV is a key determinant of innate immunity to the virus and represents an RNA that activates MDA5 in virally-infected cells.

scholarly journals Riboregulation in the Major Gastric Pathogen Helicobacter pylori

2021 ◽  
Vol 12 ◽  
Author(s):  
Alejandro Tejada-Arranz ◽  
Hilde De Reuse
Keyword(s):  
Helicobacter Pylori ◽  
Virulence Factors ◽  
Antisense Rna ◽  
Small Rnas ◽  
Genetic Regulation ◽  
Type I ◽  
Control Of Gene Expression ◽  
Ulcer Disease ◽  
Interesting Study ◽  
H Pylori

Helicobacter pylori is a Gram-negative bacterial pathogen that colonizes the stomach of about half of the human population worldwide. Infection by H. pylori is generally acquired during childhood and this bacterium rapidly establishes a persistent colonization. H. pylori causes chronic gastritis that, in some cases, progresses into peptic ulcer disease or adenocarcinoma that is responsible for about 800,000 deaths in the world every year. H. pylori has evolved efficient adaptive strategies to colonize the stomach, a particularly hostile acidic environment. Few transcriptional regulators are encoded by the small H. pylori genome and post-transcriptional regulation has been proposed as a major level of control of gene expression in this pathogen. The transcriptome and transcription start sites (TSSs) of H. pylori strain 26695 have been defined at the genome level. This revealed the existence of a total of 1,907 TSSs among which more than 900 TSSs for non-coding RNAs (ncRNAs) including 60 validated small RNAs (sRNAs) and abundant anti-sense RNAs, few of which have been experimentally validated. An RNA degradosome was shown to play a central role in the control of mRNA and antisense RNA decay in H. pylori. Riboregulation, genetic regulation by RNA, has also been revealed and depends both on antisense RNAs and small RNAs. Known examples will be presented in this review. Antisense RNA regulation was reported for some virulence factors and for several type I toxin antitoxin systems, one of which controls the morphological transition of H. pylori spiral shape to round coccoids. Interestingly, the few documented cases of small RNA-based regulation suggest that their mechanisms do not follow the same rules that were well established in the model organism Escherichia coli. First, the genome of H. pylori encodes none of the two well-described RNA chaperones, Hfq and ProQ that are important for riboregulation in several organisms. Second, some of the reported small RNAs target, through “rheostat”-like mechanisms, repeat-rich stretches in the 5′-untranslated region of genes encoding important virulence factors. In conclusion, there are still many unanswered questions about the extent and underlying mechanisms of riboregulation in H. pylori but recent publications highlighted original mechanisms making this important pathogen an interesting study model.

Collagen fibrillogenesis in vitro: interaction of types I and V collagen

1986 ◽  
Vol 44 ◽  
pp. 210-211
Author(s):  
Arthur J. Wasserman ◽  
Kathy C. Kloos ◽  
David E. Birk
Keyword(s):  
Type I Collagen ◽  
Corneal Stroma ◽  
Minor Component ◽  
Homogeneous Distribution ◽  
Type I ◽  
Type V Collagen ◽  
Fibril Morphology ◽  
Type V ◽  
In Vitro Interaction

Type I collagen is the predominant collagen in the cornea with type V collagen being a quantitatively minor component. However, the content of type V collagen (10-20%) in the cornea is high when compared to other tissues containing predominantly type I collagen. The corneal stroma has a homogeneous distribution of these two collagens, however, immunochemical localization of type V collagen requires the disruption of type I collagen structure. This indicates that these collagens may be arranged as heterpolymeric fibrils. This arrangement may be responsible for the control of fibril diameter necessary for corneal transparency. The purpose of this work is to study the in vitro assembly of collagen type V and to determine whether the interactions of these collagens influence fibril morphology.

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