scholarly journals Investigation of the regulation of expression of E. Coli Common Pilus subunit, ECPA, of enterohaemorrhagic E. Coli 0157:H7 under acid stress

2021 ◽  
Author(s):  
Hannah Tollman
Keyword(s):  
Translational Regulation ◽  
Bacterial Species ◽  
Acid Stress ◽  
Regulation Of Expression ◽  
Western Blots ◽  
E Coli ◽  
Severe Diarrhea ◽  
Regulatory Systems ◽  
Uremic Syndrome ◽  
Isogenic Mutants

Escherichia coli (EHEC) 0157:H7 is a pathogenic bacterial species that is most commonly linked to severe diarrhea, but is also the leading cause of the potentially fatal hemolytic-uremic syndrome (HUS). In order to establish infection in the colon, EHEC must endure different stresses encountered in the gastrointestinal (GI) tract, such as acid stress in the stomach, bile salt stress in the small intestine, and short-chain fatty acid (SCFA) stress in the colon. These bacteria are likely able to use GI stresses as indicators of their location, impacting gene expression of adhesion, motility, and virulence factors. The E. coli Common Pilus (ECP) has been shown to be an important factor for EHEC adhesion to epithelial cells, which is increased after either acid or SCFA stress. It has also been demonstrated via microarray that genes of this operon are upregulated after acid stress. The aim of this study is to determine how expression of the main subunit of this structure, EcpA, is regulated upon exposure of EHEC 0157:H7 to acid or SCFA-stress. Both transcriptional and translational regulation are hypothesized to be involved. Isogenic mutants have been constructed that lacked key regulators suspected to be important for each system. Two approaches are used to determine if the predicted regulatory systems are playing a role in response to stress: observing EcpA protein expression analysis through Western blotting with anti-EcpA antibodies, and examining differences in ecp operon promoter activity in regulatory mutants. In this study Western blots reconfirmed H-NS does not modulate ecpA expression in direct response to acute acid stress. This suggests an alternate regulatory response in EHEC 0157:H7 to acute acid stress resulting in the upregulation of ecpA expression previously observed with microarray analysis.

scholarly journals Investigation of the regulation of expression of E. Coli Common Pilus subunit, ECPA, of enterohaemorrhagic E. Coli 0157:H7 under acid stress

2021 ◽  
Author(s):  
Hannah Tollman
Keyword(s):  
Translational Regulation ◽  
Bacterial Species ◽  
Acid Stress ◽  
Regulation Of Expression ◽  
Western Blots ◽  
E Coli ◽  
Severe Diarrhea ◽  
Regulatory Systems ◽  
Uremic Syndrome ◽  
Isogenic Mutants

Escherichia coli (EHEC) 0157:H7 is a pathogenic bacterial species that is most commonly linked to severe diarrhea, but is also the leading cause of the potentially fatal hemolytic-uremic syndrome (HUS). In order to establish infection in the colon, EHEC must endure different stresses encountered in the gastrointestinal (GI) tract, such as acid stress in the stomach, bile salt stress in the small intestine, and short-chain fatty acid (SCFA) stress in the colon. These bacteria are likely able to use GI stresses as indicators of their location, impacting gene expression of adhesion, motility, and virulence factors. The E. coli Common Pilus (ECP) has been shown to be an important factor for EHEC adhesion to epithelial cells, which is increased after either acid or SCFA stress. It has also been demonstrated via microarray that genes of this operon are upregulated after acid stress. The aim of this study is to determine how expression of the main subunit of this structure, EcpA, is regulated upon exposure of EHEC 0157:H7 to acid or SCFA-stress. Both transcriptional and translational regulation are hypothesized to be involved. Isogenic mutants have been constructed that lacked key regulators suspected to be important for each system. Two approaches are used to determine if the predicted regulatory systems are playing a role in response to stress: observing EcpA protein expression analysis through Western blotting with anti-EcpA antibodies, and examining differences in ecp operon promoter activity in regulatory mutants. In this study Western blots reconfirmed H-NS does not modulate ecpA expression in direct response to acute acid stress. This suggests an alternate regulatory response in EHEC 0157:H7 to acute acid stress resulting in the upregulation of ecpA expression previously observed with microarray analysis.

scholarly journals Characterization of the role of yadK in the pathogenesis of enterohaemorrhagic E. coli O157:H7

2021 ◽  
Author(s):  
Yijing Yu
Keyword(s):  
Acid Stress ◽  
Haemolytic Uremic Syndrome ◽  
E Coli ◽  
Enterohaemorrhagic Escherichia Coli ◽  
Immunize Rabbit ◽  
Uremic Syndrome ◽  
Adhesion Level ◽  
Fimbrial Adhesin

Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 can use serious diarrhea and haemolytic uremic syndrome. Various factors including adhesins contribute to pathogenesis of EHEC. Previous studies suggested that yadK gene, which encodes a putative fimbrial adhesin in EHEC, may be involved in response of EHEC to acid stress. To characterize role of yadK protein in the pathogenesis of EHEC, recombinant yadK protein was generated and used to immunize rabbit to obtain anti-yadK antiserum, which was able to specifically recognize over-expressed yadK protein in EHEC. Western blotting with anti-yadK revealed a higher level of yadK expression in EHEC under acid adapted-acid stress compared to EHEC under unstressed conditions, which confirmed earlier yadK mRNA studies and indicated that yadK is upregulated in EHEC under acid stress. Finally, we observed that anti-yadK antiserum was able to specifically reduce adhesion of acid stressed EHEC to human epithelial cells compared to adhesion level of unstressed EHEC.

scholarly journals Characterization of the role of yadK in the pathogenesis of enterohaemorrhagic E. coli O157:H7

2021 ◽  
Author(s):  
Yijing Yu
Keyword(s):  
Acid Stress ◽  
Haemolytic Uremic Syndrome ◽  
E Coli ◽  
Enterohaemorrhagic Escherichia Coli ◽  
Immunize Rabbit ◽  
Uremic Syndrome ◽  
Adhesion Level ◽  
Fimbrial Adhesin

Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 can use serious diarrhea and haemolytic uremic syndrome. Various factors including adhesins contribute to pathogenesis of EHEC. Previous studies suggested that yadK gene, which encodes a putative fimbrial adhesin in EHEC, may be involved in response of EHEC to acid stress. To characterize role of yadK protein in the pathogenesis of EHEC, recombinant yadK protein was generated and used to immunize rabbit to obtain anti-yadK antiserum, which was able to specifically recognize over-expressed yadK protein in EHEC. Western blotting with anti-yadK revealed a higher level of yadK expression in EHEC under acid adapted-acid stress compared to EHEC under unstressed conditions, which confirmed earlier yadK mRNA studies and indicated that yadK is upregulated in EHEC under acid stress. Finally, we observed that anti-yadK antiserum was able to specifically reduce adhesion of acid stressed EHEC to human epithelial cells compared to adhesion level of unstressed EHEC.

scholarly journals Virulent but not temperate bacteriophages display hallmarks of rapid translation initiation

2021 ◽  
Author(s):  
Adam J Hockenberry ◽  
David C Weaver ◽  
Claus O Wilke
Keyword(s):  
Host Cell ◽  
Translational Regulation ◽  
Recombinant Expression ◽  
Bacterial Species ◽  
Start Codon ◽  
Sequence Motif ◽  
E Coli ◽  
Shine Dalgarno Sequence ◽  
Host Genes ◽  
New Strategies

Bacteriophages rely almost exclusively on host-cell machinery to produce their proteins, and their mRNAs must therefore compete with host mRNAs for valuable translational resources. In many bacterial species, highly translated mRNAs are characterized by the presence of a Shine-Dalgarno sequence motif upstream of the start codon and weak secondary structure within the start codon region. However, the general constraints and principles underlying the translation of phage mRNAs are largely unknown. Here, we show that phage mRNAs are highly enriched in strong Shine-Dalgarno sequences and have comparatively weaker secondary structures in the start codon region than host-cell mRNAs. Phage mRNAs appear statistically similar to the most highly expressed host genes in E. coli according to both features, strongly suggesting that they initiate translation at particularly high rates. Interestingly, we find that these observations are driven largely by virulent phages and that temperate phages encode mRNAs with similar start codon features to their host genes. These findings apply broadly across a wide-diversity of host-species and phage genomes. Further study of phage translational regulation — with a particular emphasis on virulent phages — may provide new strategies for engineering phage genomes and recombinant expression systems more generally.

scholarly journals Translocated Intimin Receptors (Tir) of Shiga-Toxigenic Escherichia coli Isolates Belonging to Serogroups O26, O111, and O157 React with Sera from Patients with Hemolytic-Uremic Syndrome and Exhibit Marked Sequence Heterogeneity

1998 ◽  
Vol 66 (11) ◽  
pp. 5580-5586 ◽  
Author(s):  
Adrienne W. Paton ◽  
Paul A. Manning ◽  
Matthew C. Woodrow ◽  
James C. Paton
Keyword(s):  
Escherichia Coli ◽  
Hemolytic Uremic Syndrome ◽  
Amino Acid Sequences ◽  
Receptor Protein ◽  
Receptor Interaction ◽  
Host Cell Membrane ◽  
Western Blots ◽  
Sequence Heterogeneity ◽  
E Coli ◽  
Uremic Syndrome

ABSTRACT The capacity to form attaching and effacing (A/E) lesions on the surfaces of enterocytes is an important virulence trait of several enteric pathogens, including enteropathogenic Escherichia coli (EPEC) and Shiga-toxigenic E. coli (STEC). Formation of such lesions depends upon an interaction between a bacterial outer membrane protein (intimin) and a bacterially encoded receptor protein (Tir) which is exported from the bacterium and translocated into the host cell membrane. Intimin, Tir, and several other proteins necessary for generation of A/E lesions are encoded on a chromosomal pathogenicity island termed the locus for enterocyte effacement (LEE). Reports of sequence heterogeneity and antigenic variation in the region of intimin believed to be responsible for receptor binding raise the possibility that the receptor itself is also heterogeneous. We have examined this by cloning and sequencingtir genes from three different STEC strains belonging to serogroups O26, O111, and O157. The deduced amino acid sequences for the Tir homologues from these strains varied markedly, exhibiting only 65.4, 80.2, and 56.7% identity, respectively, to that recently reported for EPEC Tir. STEC Tir is also highly immunogenic in humans. Western blots of E. coli DH5α expressing the various STECtir genes cloned in pBluescript [but not E. coli DH5α(pBluescript)] reacted strongly with convalescent sera from patients with hemolytic-uremic syndrome (HUS) caused by known LEE-positive STEC. Moreover, no reaction was seen when the various clone lysates were probed with serum from a patient with HUS caused by a LEE-negative STEC or with serum from a healthy individual. Covariation of exposed epitopes on both intimin and Tir may be a means whereby STEC avoid host immune responses without compromising adhesin-receptor interaction.

Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

2019 ◽  
Author(s):  
Priya Prakash ◽  
Travis Lantz ◽  
Krupal P. Jethava ◽  
Gaurav Chopra
Keyword(s):  
Amyloid Beta ◽  
Western Blots ◽  
Force Microscopy ◽  
E Coli ◽  
Atomic Force ◽  
Set Up ◽  
Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

Efficacy of Silver Nanoparticles Synthesized from Hymenocallis species (Spider Lilly) Leaf Extract as Antimicrobial Agents

2019 ◽  
Vol 12 (5) ◽  
pp. 4644-4647
Author(s):  
K.K. Gupta ◽  
Neha Kumari ◽  
Neha Sinha ◽  
Akruti Gupta
Keyword(s):  
Silver Nanoparticles ◽  
Leaf Extract ◽  
Antimicrobial Agents ◽  
Color Change ◽  
Bacterial Species ◽  
Antimicrobial Property ◽  
Biogenic Synthesis ◽  
E Coli ◽  
Light Yellow ◽  
Antibiotic Property

Biogenic synthesis of silver nanoparticles synthesized from Hymenocallis species (Spider Lilly) leaf extract was subjected for investigation of its antimicrobial property against four bacterial species (E. coli, Salmonella sp., Streptococcus sp. & Staphylococcus sp.). The results revealed that synthesized nanoparticles solution very much justify the color change property from initial light yellow to final reddish brown during the synthesis producing a characteristics absorption peak in the range of 434-466 nm. As antimicrobial agents, their efficacy was evaluated by analysis of variance in between the species and among the different concentration of AgNPs solution, which clearly showed that there was significant variation in the antibiotic property between the four different concentrations of AgNPs solution and also among four different species of bacteria taken under studies. However, silver nanoparticles solution of 1: 9 and 1:4 were proved comparatively more efficient as antimicrobial agents against four species of bacteria.

Substituted 6,7-dimethoxy-5-oxo-2,3,5,9b-tetrahydrothiazolo[2,3-a]isoindole- 3-1,1-dioxide Derivatives with Antimicrobial Activity and Docking Assisted Prediction of the Mechanism of their Antibacterial and Antifungal Properties

2020 ◽  
Vol 20 (29) ◽  
pp. 2681-2691
Author(s):  
Athina Geronikaki ◽  
Victor Kartsev ◽  
Phaedra Eleftheriou ◽  
Anthi Petrou ◽  
Jasmina Glamočlija ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
Antifungal Activity ◽  
Drug Targets ◽  
Bacterial Species ◽  
Pharmaceutical Research ◽  
Docking Studies ◽  
Fungal Species ◽  
Docking Analysis ◽  
E Coli ◽  
Antibacterial And Antifungal

Background: Although a great number of the targets of antimicrobial therapy have been achieved, it remains among the first fields of pharmaceutical research, mainly because of the development of resistant strains. Docking analysis may be an important tool in the research for the development of more effective agents against specific drug targets or multi-target agents 1-3. Methods: In the present study, based on docking analysis, ten tetrahydrothiazolo[2,3-a]isoindole derivatives were chosen for the evaluation of the antimicrobial activity. Results: All compounds showed antibacterial activity against eight Gram-positive and Gram-negative bacterial species being, in some cases, more potent than ampicillin and streptomycin against all species. The most sensitive bacteria appeared to be S. aureus and En. Cloacae, while M. flavus, E. coli and P. aeruginosa were the most resistant ones. The compounds were also tested for their antifungal activity against eight fungal species. All compounds exhibited good antifungal activity better than reference drugs bifonazole (1.4 – 41 folds) and ketoconazole (1.1 – 406 folds) against all fungal species. In order to elucidate the mechanism of action, docking studies on different antimicrobial targets were performed. Conclusion: According to docking analysis, the antifungal activity can be explained by the inhibition of the CYP51 enzyme for most compounds with a better correlation of the results obtained for the P.v.c. strain (linear regression between estimated binding Energy and log(1/MIC) with R 2 =0.867 and p=0.000091 or R 2 = 0.924, p= 0.000036, when compound 3 is excluded.

scholarly journals Shiga Toxin 2a Binds to Complement Components C3b and C5 and Upregulates Their Gene Expression in Human Cell Lines

Toxins ◽  
2020 ◽  
Vol 13 (1) ◽  
pp. 8
Author(s):  
Sára Kellnerová ◽  
Sneha Chatterjee ◽  
Rafael Bayarri-Olmos ◽  
Louise Justesen ◽  
Heribert Talasz ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Cell Lines ◽  
Gene Transcription ◽  
Specific Binding ◽  
Enterohemorrhagic Escherichia Coli ◽  
Complement Components ◽  
Western Blots ◽  
Complement Proteins ◽  
Uremic Syndrome ◽  
Complement Gene

Enterohemorrhagic Escherichia coli (EHEC) infections can cause EHEC-associated hemolytic uremic syndrome (eHUS) via its main virulent factor, Shiga toxins (Stxs). Complement has been reported to be involved in the progression of eHUS. The aim of this study was to investigate the interactions of the most effective subtype of the toxin, Stx2a, with pivotal complement proteins C3b and C5. The study further examined the effect of Stx2a stimulation on the transcription and synthesis of these complement proteins in human target cell lines. Binding of Stx2a to C3b and C5 was evaluated by ELISA. Kidney and gut cell lines (HK-2 and HCT-8) were stimulated with varied concentrations of Stx2a. Subsequent evaluation of complement gene transcription was studied by real-time PCR (qPCR), and ELISAs and Western blots were performed to examine protein synthesis of C3 and C5 in supernatants and lysates of stimulated HK-2 cells. Stx2a showed a specific binding to C3b and C5. Gene transcription of C3 and C5 was upregulated with increasing concentrations of Stx2a in both cell lines, but protein synthesis was not. This study demonstrates the binding of Stx2a to complement proteins C3b and C5, which could potentially be involved in regulating complement during eHUS infection, supporting further investigations into elucidating the role of complement in eHUS pathogenesis.

scholarly journals L-Alanine Prototrophic Suppressors Emerge from L-Alanine Auxotroph through Stress-Induced Mutagenesis in Escherichia coli

2021 ◽  
Vol 9 (3) ◽  
pp. 472
Author(s):  
Harutaka Mishima ◽  
Hirokazu Watanabe ◽  
Kei Uchigasaki ◽  
So Shimoda ◽  
Shota Seki ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Complex ◽  
Spontaneous Mutation ◽  
Point Mutations ◽  
Whole Genome Analysis ◽  
E Coli ◽  
Clone Pair ◽  
Induced Mutagenesis ◽  
Isogenic Mutants

In Escherichia coli, L-alanine is synthesized by three isozymes: YfbQ, YfdZ, and AvtA. When an E. coli L-alanine auxotrophic isogenic mutant lacking the three isozymes was grown on L-alanine-deficient minimal agar medium, L-alanine prototrophic mutants emerged considerably more frequently than by spontaneous mutation; the emergence frequency increased over time, and, in an L-alanine-supplemented minimal medium, correlated inversely with L-alanine concentration, indicating that the mutants were derived through stress-induced mutagenesis. Whole-genome analysis of 40 independent L-alanine prototrophic mutants identified 16 and 18 clones harboring point mutation(s) in pyruvate dehydrogenase complex and phosphotransacetylase-acetate kinase pathway, which respectively produce acetyl coenzyme A and acetate from pyruvate. When two point mutations identified in L-alanine prototrophic mutants, in pta (D656A) and aceE (G147D), were individually introduced into the original L-alanine auxotroph, the isogenic mutants exhibited almost identical growth recovery as the respective cognate mutants. Each original- and isogenic-clone pair carrying the pta or aceE mutation showed extremely low phosphotransacetylase or pyruvate dehydrogenase activity, respectively. Lastly, extracellularly-added pyruvate, which dose-dependently supported L-alanine auxotroph growth, relieved the L-alanine starvation stress, preventing the emergence of L-alanine prototrophic mutants. Thus, L-alanine starvation-provoked stress-induced mutagenesis in the L-alanine auxotroph could lead to intracellular pyruvate increase, which eventually induces L-alanine prototrophy.

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