scholarly journals Comparative Proteomic Analysis of Extracellular Proteins of Enterohemorrhagic and Enteropathogenic Escherichia coli Strains and Their ihf and ler Mutants

2004 ◽  
Vol 70 (9) ◽  
pp. 5274-5282 ◽  
Author(s):  
M. Li ◽  
I. Rosenshine ◽  
S. L. Tung ◽  
X. H. Wang ◽  
D. Friedberg ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Polyacrylamide Gel Electrophoresis ◽  
Integration Host Factor ◽  
Extracellular Proteins ◽  
Major Protein ◽  
Human Pathogens ◽  
Protein Database ◽  
Flight Mass Spectrometry ◽  
Proteomic Approach ◽  
In The Wild

ABSTRACT Enterohemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC, respectively) strains are closely related human pathogens that are responsible for food-borne epidemics in many countries. Integration host factor (IHF) and the locus of enterocyte effacement-encoded regulator (Ler) are needed for the expression of virulence genes in EHEC and EPEC, including the elicitation of actin rearrangements for attaching and effacing lesions. We applied a proteomic approach, using two-dimensional polyacrylamide gel electrophoresis in combination with matrix-assisted laser desorption ionization-time of flight mass spectrometry and a protein database search, to analyze the extracellular protein profiles of EHEC EDL933, EPEC E2348/69, and their ihf and ler mutants. Fifty-nine major protein spots from the extracellular proteomes were identified, including six proteins of unknown function. Twenty-six of them were conserved between EHEC EDL933 and EPEC E2348/69, while some of them were strain-specific proteins. Four common extracellular proteins (EspA, EspB, EspD, and Tir) were regulated by both IHF and Ler in EHEC EDL933 and EPEC E2348/69. TagA in EHEC EDL933 and EspC and EspF in EPEC E2348/69 were present in the wild-type strains but absent from their respective ler and ihf mutants, while FliC was overexpressed in the ihf mutant of EPEC E2348/69. Two dominant forms of EspB were found in EHEC EDL933 and EPEC E2348/69, but the significance of this is unknown. These results show that proteomics is a powerful platform technology for accelerating the understanding of EPEC and EHEC pathogenesis and identifying markers for laboratory diagnoses of these pathogens.

scholarly journals The Intake of a Cafeteria Diet in Nursing Rats Alters the Breast Milk Concentration of Proteins Important for the Development of Offspring

Nutrients ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 2470 ◽  
Author(s):  
Catalina Amadora Pomar ◽  
Juana Sánchez ◽  
Andreu Palou
Keyword(s):  
Breast Milk ◽  
Energy Homeostasis ◽  
Maternal Diet ◽  
Relative Concentration ◽  
Polyacrylamide Gel Electrophoresis ◽  
Cafeteria Diet ◽  
Phenotypic Traits ◽  
Flight Mass Spectrometry ◽  
Proteomic Approach ◽  
Altered Proteins

We aimed to analyse the effects of maternal intake of an unbalanced diet during lactation in the composition and the levels of proteins present in milk. Milk samples from control nursing dams (C-dams) or from nursing dams fed a cafeteria diet during lactation (CAF-dams) were obtained. We conducted a proteomic approach to identify significantly altered proteins in breast milk of C- and CAF-dams, and evaluated the levels of leptin, adiponectin and irisin for their implication in energy homeostasis. One-dimensional SDS-polyacrylamide gel electrophoresis (SDS-PAGE), followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), revealed that the bands that presented a lower intensity in CAF-dams than control contain some caseins (α-S1-casein, α-S2-casein like B, and β-casein), α-lactalbumin and haptoglobin. Leptin and adiponectin levels were greater in the breast milk of CAF-dams than in controls, while levels of irisin were lower. In summary, the relative concentration of bioactive peptides was influenced by maternal diet consumption during lactation; these changes at early stages of life could influence the phenotypic traits of the offspring.

scholarly journals Assembly of the Type III Secretion Apparatus of Enteropathogenic Escherichia coli

2006 ◽  
Vol 188 (8) ◽  
pp. 2801-2811 ◽  
Author(s):  
Tomoaki Ogino ◽  
Ryuta Ohno ◽  
Kachiko Sekiya ◽  
Asaomi Kuwae ◽  
Takeshi Matsuzawa ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Basal Body ◽  
Type Iii Secretion ◽  
Outer Ring ◽  
Major Protein ◽  
Type Iii ◽  
Flight Mass Spectrometry ◽  
Needle Protein ◽  
Inner Membrane Protein ◽  
Protein Components

ABSTRACT Enteropathogenic Escherichia coli (EPEC) secretes many Esps (E. coli-secreted proteins) and effectors via the type III secretion (TTS) system. We previously identified a novel needle complex (NC) composed of a basal body and a needle structure containing an expandable EspA sheath-like structure as a central part of the EPEC TTS apparatus. To further investigate the structure and protein components of the EPEC NC, we purified it in successive centrifugal steps. Finally, NCs with long EspA sheath-like structures could be separated from those with short needle structures on the basis of their densities. Although the highly purified NC appeared to lack an inner ring in the basal body, its core structure, composed of an outer ring and a central rod, was observed by transmission electron microscopy. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry, Western blot, and immunoelectron microscopic analyses revealed that EscC was a major protein component of the outer ring in the core basal body. To investigate the mechanisms of assembly of the basal body, interactions between the presumed components of the EPEC TTS apparatus were analyzed by a glutathione S-transferase pulldown assay. The EscC outer ring protein was associated with both the EscF needle protein and EscD, a presumed inner membrane protein. EscF was also associated with EscJ, a presumed inner ring protein. Furthermore, escC, escD, and escJ mutant strains were unable to produce the TTS apparatus, and thereby the secretion of the Esp proteins and Tir effector was abolished. These results indicate that EscC, EscD, and EscJ are required for the formation of the TTS apparatus.

scholarly journals Escherichia coli Ribosome-Associated Protein SRA, Whose Copy Number Increases during Stationary Phase

2001 ◽  
Vol 183 (9) ◽  
pp. 2765-2773 ◽  
Author(s):  
Kaori Izutsu ◽  
Chieko Wada ◽  
Yuriko Komine ◽  
Tomoyuki Sako ◽  
Chiharu Ueguchi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Stationary Phase ◽  
Copy Number ◽  
Polyacrylamide Gel Electrophoresis ◽  
Ribosomal Subunit ◽  
Integration Host Factor ◽  
Transcriptional Level ◽  
Global Regulators ◽  
Its Gene ◽  
Ribosomal Particle

ABSTRACT Protein D has previously been demonstrated to be associated withEscherichia coli ribosomes by the radical-free and highly reducing method of two-dimensional polyacrylamide gel electrophoresis. In this study, we show that protein D is exclusively present in the 30S ribosomal subunit and that its gene is located at 33.6 min on theE. coli genetic map, between ompC andsfcA. The gene consists of 45 codons, coding for a protein of 5,096 Da. The copy number of protein D per ribosomal particle varied during growth and increased from 0.1 in the exponential phase to 0.4 in the stationary phase. For these reasons, protein D was named SRA (stationary-phase-induced ribosome-associated) protein and its gene was named sra. The amount of SRA protein within the cell was found to be controlled mainly at the transcriptional level: its transcription increased rapidly upon entry into the stationary phase and was partly dependent on an alternative sigma factor (sigma S). In addition, global regulators, such as factor inversion stimulation (FIS), integration host factor (IHF), cyclic AMP, and ppGpp, were found to play a role either directly or indirectly in the transcription ofsra in the stationary phase.

scholarly journals Purification of IHF-like protein from gram-negative bacteria in one chromatographic step.

1996 ◽  
Vol 43 (2) ◽  
pp. 379-382 ◽  
Author(s):  
B Krawczyk ◽  
J Kur
Keyword(s):  
Escherichia Coli ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Integration Host Factor ◽  
Gram Negative Bacteria ◽  
Proteus Vulgaris ◽  
Gram Negative ◽  
Acinetobacter Junii ◽  
E Coli ◽  
Alpha Beta

We describe a fast and very efficient method of purification which yields highly purified integration host factor-like proteins in one chromatographic step. IHF-like proteins from Acinetobacter junii or Proteus vulgaris are each an alpha beta heterodimer (subunits of 10 and 11 kDa) similar to the IHF of Escherichia coli when analyzed by polyacrylamide gel electrophoresis. The purified IHF are able to bind to the same ihf sites as IHF of E. coli. The results presented confirm that IHF is conserved during evolution in gram-negative bacteria.

Mass Spectrometry-Based Identification of Urinary Antimicrobial Peptides in Dairy Cows

2020 ◽  
Vol 27 (3) ◽  
pp. 225-235
Author(s):  
Ambika Sharma ◽  
Rajesh Nigam ◽  
Ashish Kumar ◽  
Simmi Singh
Keyword(s):  
Mass Spectrometry ◽  
Antimicrobial Peptides ◽  
Polyacrylamide Gel Electrophoresis ◽  
Radial Diffusion ◽  
Protein Database ◽  
Antimicrobial Effects ◽  
Cow Urine ◽  
Radial Diffusion Assay ◽  
Promising Source ◽  
Diffusion Assay

Background:: Urine is considered one of the biological fluids in which antimicrobial peptides are secreted or expressed. Cow urine has not been investigated for the presence of these peptides using MALDI-TOF-MS. Objective:: The aim of this study is to isolate, identify and assess the antimicrobial activity of urinary antimicrobial peptides from healthy normal cycling cows. Method:: We analyzed the urine sample using diafiltration, ion exchange chromatography, Reverse Phase High-Performance Liquid Chromatography (RP-HPLC), acid urea polyacrylamide gel electrophoresis (AU-PAGE) coupled with identification through Peptide Mass Fingerprinting (PMF) by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDITOF- MS). The in vitro antimicrobial effects of purified fractions were assessed using Radial Diffusion Assay (RDA) and microtitre broth dilution assay against Gram-positive and Gramnegative bacteria. Results: : Proteins corresponding to the peaks were identified using SWISSPROT protein database. This study revealed constitutive expression of β-Defensin-1 (DEFB1), β-Defensin-4A (DFB4A), Neutrophil Defensin-1 (DEF1), Neutrophil Defensin-3 (DEF3) in cow urine. The identified peptides are cationic antimicrobial peptides of the defensin family. The purified fractions exhibited antimicrobial effects in radial diffusion assay and MIC values in the range of 2.93-29.3 &*#181;M/L. Conclusion:: This study concludes that cow urine, previously unexplored with regard to antimicrobial peptides, would be a promising source of highly potent AMPs and an effective alternative to the resistant antibiotics.

scholarly journals Escherichia coli MutM Suppresses Illegitimate Recombination Induced by Oxidative Stress

Genetics ◽  
1999 ◽  
Vol 151 (2) ◽  
pp. 439-446 ◽  
Author(s):  
Masaaki Onda ◽  
Katsuhiro Hanada ◽  
Hirokazu Kawachi ◽  
Hideo Ikeda
Keyword(s):  
Oxidative Stress ◽  
Escherichia Coli ◽  
Hydrogen Peroxide ◽  
Dna Damage ◽  
Double Strand Breaks ◽  
Illegitimate Recombination ◽  
Recombination Analysis ◽  
Wild Type ◽  
Strand Breaks ◽  
In The Wild

Abstract DNA damage by oxidative stress is one of the causes of mutagenesis. However, whether or not DNA damage induces illegitimate recombination has not been determined. To study the effect of oxidative stress on illegitimate recombination, we examined the frequency of λbio transducing phage in the presence of hydrogen peroxide and found that this reagent enhances illegitimate recombination. To clarify the types of illegitimate recombination, we examined the effect of mutations in mutM and related genes on the process. The frequency of λbio transducing phage was 5- to 12-fold higher in the mutM mutant than in the wild type, while the frequency in the mutY and mutT mutants was comparable to that of the wild type. Because 7,8-dihydro-8-oxoguanine (8-oxoG) and formamido pyrimidine (Fapy) lesions can be removed from DNA by MutM protein, these lesions are thought to induce illegitimate recombination. Analysis of recombination junctions showed that the recombination at Hotspot I accounts for 22 or 4% of total λbio transducing phages in the wild type or in the mutM mutant, respectively. The preferential increase of recombination at nonhotspot sites with hydrogen peroxide in the mutM mutant was discussed on the basis of a new model, in which 8-oxoG and/or Fapy residues may introduce double-strand breaks into DNA.

scholarly journals Antimicrobial Resistance Genes and Diversity of Clones among ESBL- and Acquired AmpC-Producing Escherichia coli Isolated from Fecal Samples of Healthy and Sick Cats in Portugal

Antibiotics ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 262
Author(s):  
Isabel Carvalho ◽  
Nadia Safia Chenouf ◽  
Rita Cunha ◽  
Carla Martins ◽  
Paulo Pimenta ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Public Health Problem ◽  
Phylogenetic Groups ◽  
Disk Diffusion Test ◽  
Extended Spectrum Beta Lactamase ◽  
E Coli ◽  
Specific Pcr ◽  
Flight Mass Spectrometry ◽  
Antimicrobial Resistance Genes ◽  
Esbl Producers

The aim of the study was to analyze the mechanisms of resistance in extended-spectrum beta-lactamase (ESBL)- and acquired AmpC (qAmpC)-producing Escherichia coli isolates from healthy and sick cats in Portugal. A total of 141 rectal swabs recovered from 98 sick and 43 healthy cats were processed for cefotaxime-resistant (CTXR) E. coli recovery (in MacConkey agar supplemented with 2 µg/mL cefotaxime). The matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) method was used for E. coli identification and antimicrobial susceptibility was performed by a disk diffusion test. The presence of resistance/virulence genes was tested by PCR sequencing. The phylogenetic typing and multilocus sequence typing (MLST) were determined by specific PCR sequencing. CTXRE. coli isolates were detected in seven sick and six healthy cats (7.1% and 13.9%, respectively). Based on the synergy tests, 11 of 13 CTXRE. coli isolates (one/sample) were ESBL-producers (ESBL total rate: 7.8%) carrying the following ESBL genes: blaCTX-M-1 (n = 3), blaCTX-M-15 (n = 3), blaCTX-M-55 (n = 2), blaCTX-M-27 (n = 2) and blaCTX-M-9 (n = 1). Six different sequence types were identified among ESBL-producers (sequence type/associated ESBLs): ST847/CTX-M-9, CTX-M-27, CTX-M-1; ST10/CTX-M-15, CTX-M-27; ST6448/CTX-M-15, CTX-M-55; ST429/CTX-M-15; ST101/CTX-M-1 and ST40/CTX-M-1. Three of the CTXR isolates were CMY-2-producers (qAmpC rate: 2.1%); two of them were ESBL-positive and one ESBL-negative. These isolates were typed as ST429 and ST6448 and were obtained in healthy or sick cats. The phylogenetic groups A/B1/D/clade 1 were detected among ESBL- and qAmpC-producing isolates. Cats are carriers of qAmpC (CMY-2)- and ESBL-producing E. coli isolates (mostly of variants of CTX-M group 1) of diverse clonal lineages, which might represent a public health problem due to the proximity of cats with humans regarding a One Health perspective.

scholarly journals Inhibition of Escherichia coli O157:H7 and Salmonella enterica Isolates on Spinach Leaf Surfaces Using Eugenol-Loaded Surfactant Micelles

Foods ◽  
2019 ◽  
Vol 8 (11) ◽  
pp. 575
Author(s):  
Songsirin Ruengvisesh ◽  
Chris R. Kerth ◽  
T. Matthew Taylor
Keyword(s):  
Escherichia Coli ◽  
Salmonella Enterica ◽  
Limit Of Detection ◽  
Sodium Dodecyl ◽  
Microbial Pathogens ◽  
Escherichia Coli O157 ◽  
Human Pathogens ◽  
Surfactant Micelles ◽  
Leaf Surfaces ◽  
Spinach Leaf

Spinach and other leafy green vegetables have been linked to foodborne disease outbreaks of Escherichia coli O157:H7 and Salmonella enterica around the globe. In this study, the antimicrobial activities of surfactant micelles formed from the anionic surfactant sodium dodecyl sulfate (SDS), SDS micelle-loaded eugenol (1.0% eugenol), 1.0% free eugenol, 200 ppm free chlorine, and sterile water were tested against the human pathogens E. coli O157:H7 and Salmonella Saintpaul, and naturally occurring microorganisms, on spinach leaf surfaces during storage at 5 °C over 10 days. Spinach samples were immersed in antimicrobial treatment solution for 2.0 min at 25 °C, after which treatment solutions were drained off and samples were either subjected to analysis or prepared for refrigerated storage. Whereas empty SDS micelles produced moderate reductions in counts of both pathogens (2.1–3.2 log10 CFU/cm2), free and micelle-entrapped eugenol treatments reduced pathogens by >5.0 log10 CFU/cm2 to below the limit of detection (<0.5 log10 CFU/cm2). Micelle-loaded eugenol produced the greatest numerical reductions in naturally contaminating aerobic bacteria, Enterobacteriaceae, and fungi, though these reductions did not differ statistically from reductions achieved by un-encapsulated eugenol and 200 ppm chlorine. Micelles-loaded eugenol could be used as a novel antimicrobial technology to decontaminate fresh spinach from microbial pathogens.

scholarly journals Influence of Bacterial Competitors on Salmonella enterica and Enterohemorrhagic Escherichia coli Growth in Microbiological Media and Attachment to Vegetable Seeds

Foods ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 285
Author(s):  
Da Liu ◽  
Ronald Walcott ◽  
Kevin Mis Solval ◽  
Jinru Chen
Keyword(s):  
Escherichia Coli ◽  
Foodborne Pathogens ◽  
Salmonella Enterica ◽  
Lactobacillus Rhamnosus ◽  
Probiotic Bacterium ◽  
Enterohemorrhagic Escherichia Coli ◽  
Biocontrol Agents ◽  
Human Pathogens ◽  
Bacterial Strains ◽  
Pathogen Populations

Interests in using biological agents for control of human pathogens on vegetable seeds are rising. This study evaluated whether probiotic bacterium Lactobacillus rhamnosus GG, bacterial strains previously used as biocontrol agents in plant science, as well as a selected plant pathogen could compete with foodborne human pathogens, such as Salmonella enterica and enterohemorrhagic Escherichia coli (EHEC), for growth in microbiological media and attachment to vegetable seeds; and to determine whether the metabolites in cell-free supernatants of competitive bacterial spent cultures could inhibit the growth of the two pathogens. The results suggest that the co-presence of competitive bacteria, especially L. rhamnosus GG, significantly (p < 0.05) inhibited the growth of Salmonella and EHEC. Cell-free supernatants of L. rhamnosus GG cultures significantly reduced the pathogen populations in microbiological media. Although not as effective as L. rhamnosus GG in inhibiting the growth of Salmonella and EHEC, the biocontrol agents were more effective in competing for attachment to vegetable seeds. The study observed the inhibition of human bacterial pathogens by competitive bacteria or their metabolites and the competitive attachment to sprout seeds among all bacteria involved. The results will help strategize interventions to produce vegetable seeds and seed sprouts free of foodborne pathogens.

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