scholarly journals Identification of a specific surface epitope of OmpC for Escherichia coli O157:H7 with protein topology facilitated affinity mass spectrometry

2021 ◽  
Vol 105 (18) ◽  
pp. 6819-6833
Author(s):  
Wenbin Wang ◽  
Xinyue Zhou ◽  
Yunong Sang ◽  
Xiaxia Liang ◽  
Jianxin Liu ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Escherichia Coli ◽  
High Specificity ◽  
Target Protein ◽  
Two Dimensional Gel Electrophoresis ◽  
E Coli ◽  
Homologous Protein ◽  
Flight Mass Spectrometry ◽  
Key Points ◽  
Liquid Chromatography Tandem Mass

Abstract The goal of this work was to identify the target protein and epitope of a previously reported Escherichia coli O157:H7 (ECO157)–specific monoclonal antibody (mAb) 2G12. mAb 2G12 has shown high specificity for the recovery and detection of ECO157. To achieve this goal, the target protein was first separated by two-dimensional gel electrophoresis (2-DE) and located by Western blot (WB). The protein spots were identified to be the outer membrane protein (Omp) C by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF–MS). After that, the target protein was purified by immunoaffinity chromatography (IAC) and subjected to in situ enzymatic cleavage of the vulnerable peptides. Eight eluted peptides of OmpC identified by liquid chromatography–tandem mass spectrometry (LC–MS/MS) were further mapped onto the homologous protein structure of E. coli OmpC (2IXX). The topology of OmpC showed that three peptides had extracellular loops. Epitope mapping with overlapping peptide library and sequence homology analysis revealed that the epitope consisted of a specific peptide, “LGVING,” and an adjacent conservative peptide, “TQTYNATRVGSLG.” Both peptides loop around the overall structure of the epitope. To test the availability of the epitope when ECO157 was grown under different osmolarity, pH, and nutrition levels, the binding efficacy of mAb 2G12 with ECO157 grown in these conditions was evaluated. Results further demonstrated the good stability of this epitope under potential stressful environmental conditions. In summary, this study revealed that mAb 2G12 targeted one specific and one conservative extracellular loop (peptide) of the OmpC present on ECO157, and the epitope was stable and accessible on ECO157 cells grown in different environment. Key points • OmpC is the target of a recently identified ECO157-specific mAb 2G12. • Eight peptides were identified from the OmpC by using LC–MS/MS. • The specificity of mAb 2G12 is mainly determined by the “LGVING” peptide.

Comparative Proteomics of Commensal and Pathogenic Strains of Escherichia coli

2020 ◽  
Vol 27 (11) ◽  
pp. 1171-1177
Author(s):  
Neelja Singhal ◽  
Divakar Sharma ◽  
Manish Kumar ◽  
Deepa Bisht ◽  
Jugsharan Singh Virdi
Keyword(s):  
Escherichia Coli ◽  
Proteomic Analysis ◽  
Two Dimensional Gel Electrophoresis ◽  
Maldi Tof Ms ◽  
E Coli ◽  
Comparative Proteomic Analysis ◽  
Maldi Tof ◽  
Northern India ◽  
Flight Mass Spectrometry ◽  
Tof Ms

Background: Most of the proteomic studies in Escherichia coli have focussed on pathogenic strains, while very few studies have studied the commensal strains. It is important to study the commensal strains because under the selective pressure of their habitat, commensal strains might serve as reservoirs of virulent and pathogenic strains. Objective: In this study, we have performed a comparative proteomic analysis of commensal and pathogenic strains of E. coli isolated from a major river flowing through northern India. Methods: Proteins were resolved by two dimensional gel electrophoresis and the differentially expressed proteins were identified using matrix-assisted laser desorption ionization-time of flight mass-spectrometry (MALDI-TOF MS). Results: Many proteins of the commensal strain showed an increased expression compared to the pathogenic strain, of which seventeen proteins were identified by MALDI-TOF MS. Functional classification of these proteins revealed that they belonged to different functional pathways like energy metabolism, nucleotide and nucleoside conversions, translation, biosynthesis of amino acids and motility and energytaxis/chemotaxis. Conclusion: As per the best of our knowledge, this is the first report on comparative proteomic analysis of E. coli commensal and pathogenic strains of aquatic origin. Our results suggest that the increased production of these proteins might play an important role in adaptation of E. coli to a commensal/pathogenic lifestyle. However, further experiments are required to understand the precise role of these proteins in regulating the pathogenicity/commensalism of E. coli.

scholarly journals Random and combinatorial mutagenesis for improved total production of secretory target protein in Escherichia coli

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
David Gonzalez-Perez ◽  
James Ratcliffe ◽  
Shu Khan Tan ◽  
Mary Chen May Wong ◽  
Yi Pei Yee ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein Secretion ◽  
Protein Production ◽  
Secretory Protein ◽  
Target Protein ◽  
Carrier Protein ◽  
Signal Peptides ◽  
Carrier Proteins ◽  
E Coli ◽  
Combinatorial Mutagenesis

AbstractSignal peptides and secretory carrier proteins are commonly used to secrete heterologous recombinant protein in Gram-negative bacteria. The Escherichia coli osmotically-inducible protein Y (OsmY) is a carrier protein that secretes a target protein extracellularly, and we have previously applied it in the Bacterial Extracellular Protein Secretion System (BENNY) to accelerate directed evolution. In this study, we reported the first application of random and combinatorial mutagenesis on a carrier protein to enhance total secretory target protein production. After one round of random mutagenesis followed by combining the mutations found, OsmY(M3) (L6P, V43A, S154R, V191E) was identified as the best carrier protein. OsmY(M3) produced 3.1 ± 0.3 fold and 2.9 ± 0.8 fold more secretory Tfu0937 β-glucosidase than its wildtype counterpart in E. coli strains BL21(DE3) and C41(DE3), respectively. OsmY(M3) also produced more secretory Tfu0937 at different cultivation temperatures (37 °C, 30 °C and 25 °C) compared to the wildtype. Subcellular fractionation of the expressed protein confirmed the essential role of OsmY in protein secretion. Up to 80.8 ± 12.2% of total soluble protein was secreted after 15 h of cultivation. When fused to a red fluorescent protein or a lipase from Bacillus subtillis, OsmY(M3) also produced more secretory protein compared to the wildtype. In this study, OsmY(M3) variant improved the extracellular production of three proteins originating from diverse organisms and with diverse properties, clearly demonstrating its wide-ranging applications. The use of random and combinatorial mutagenesis on the carrier protein demonstrated in this work can also be further extended to evolve other signal peptides or carrier proteins for secretory protein production in E. coli.

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 Metabolic Profiling of Pitaya (Hylocereus polyrhizus) during Fruit Development and Maturation

Molecules ◽  
2019 ◽  
Vol 24 (6) ◽  
pp. 1114 ◽  
Author(s):  
Yawei Wu ◽  
Juan Xu ◽  
Yizhong He ◽  
Meiyan Shi ◽  
Xiumei Han ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Metabolic Profiling ◽  
Developmental Stages ◽  
Principal Component ◽  
Nutrient Content ◽  
Flight Mass Spectrometry ◽  
Potential Biomarker ◽  
Fruit Samples ◽  
Liquid Chromatography Tandem Mass ◽  
Hylocereus Polyrhizus

Pitaya (Hylocereus polyrhizus) has attracted much interest from consumers as it is a novelty fruit with high nutrient content and a tolerance to drought stress. As a group of attractive pigment- and health-promoting natural compounds, betalains represent a visual feature for pitaya fruit quality. However, little information on the correlation between betalains and relevant metabolites exists so far. Currently, color (Commission International del’Eclairage, CIE) parameters, betalain contents, and untargeted metabolic profiling (gas chromatography-time-of-flight-mass spectrometry, GC–MS and liquid chromatography tandem mass spectrometry, LC–MS) have been examined on ‘Zihonglong’ fruits at nine different developmental stages, and the variation character of the metabolite contents was simultaneously investigated between peel and pulp. Furthermore, principal component analysis (PCA) and partial least-squares discriminant analysis (PLS-DA) were used to explore metabolite profiles from the fruit samples. Our results demonstrated that the decrease of amino acid, accompanied by the increase of sugars and organic acid, might contribute to the formation of betalains. Notably, as one of four potential biomarker metabolites, citramalic acid might be related to betalain formation.

scholarly journals Structural insights into Escherichia coli phosphopantothenoylcysteine synthetase by native ion mobility–mass spectrometry

2019 ◽  
Vol 476 (21) ◽  
pp. 3125-3139 ◽  
Author(s):  
Daniel Shiu-Hin Chan ◽  
Jeannine Hess ◽  
Elen Shaw ◽  
Christina Spry ◽  
Robert Starley ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Escherichia Coli ◽  
Structural Biology ◽  
Ion Mobility ◽  
Biosynthetic Pathway ◽  
Screening Tool ◽  
Native Mass Spectrometry ◽  
Ion Mobility Mass Spectrometry ◽  
E Coli ◽  
Structural Insights

Abstract CoaBC, part of the vital coenzyme A biosynthetic pathway in bacteria, has recently been validated as a promising antimicrobial target. In this work, we employed native ion mobility–mass spectrometry to gain structural insights into the phosphopantothenoylcysteine synthetase domain of E. coli CoaBC. Moreover, native mass spectrometry was validated as a screening tool to identify novel inhibitors of this enzyme, highlighting the utility and versatility of this technique both for structural biology and for drug discovery.

scholarly journals Qualitative analysis of 7- and 8-hydroxyzolpidem and discovery of novel zolpidem metabolites in postmortem urine using liquid chromatography–tandem mass spectrometry

2022 ◽  
Author(s):  
Koji Yamaguchi ◽  
Hajime Miyaguchi ◽  
Youkichi Ohno ◽  
Yoshimasa Kanawaku
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Pyridine Ring ◽  
Multiple Reaction Monitoring ◽  
Fragmentation Pattern ◽  
The Novel ◽  
Flight Mass Spectrometry ◽  
Urine Extract ◽  
Liquid Chromatography Tandem Mass ◽  
Characteristic Fragmentation

Abstract Purpose Zolpidem (ZOL) is a hypnotic sometimes used in drug-facilitated crimes. Understanding ZOL metabolism is important for proving ZOL intake. In this study, we synthesized standards of hydroxyzolpidems with a hydroxy group attached to the pyridine ring and analyzed them to prove their presence in postmortem urine. We also searched for novel ZOL metabolites in the urine sample using liquid chromatography–triple quadrupole mass spectrometry (LC-QqQMS) and liquid chromatography–quadrupole time-of-flight mass spectrometry (LC-QqTOFMS). Methods 7- and 8-Hydroxyzolpidem (7OHZ and 8OHZ, respectively) were synthesized and analyzed using LC-QqQMS. Retention times were compared between the synthetic standards and extracts of postmortem urine. To search for novel ZOL metabolites, first, the urine extract was analyzed with data-dependent acquisition, and the peaks showing the characteristic fragmentation pattern of ZOL were selected. Second, product ion spectra of these peaks at various collision energies were acquired and fragments that could be used for multiple reaction monitoring (MRM) were chosen. Finally, MRM parameters were optimized using the urine extract. These peaks were also analyzed using LC-QqTOFMS. Results The presence of 7OHZ and 8OHZ in urine was confirmed. The highest peak among hydroxyzolpidems was assigned to 7OHZ. The novel metabolites found were zolpidem dihydrodiol and its glucuronides, cysteine adducts of ZOL and dihydro(hydroxy)zolpidem, and glucuronides of hydroxyzolpidems. Conclusions The presence of novel metabolites revealed new metabolic pathways, which involve formation of an epoxide on the pyridine ring as an intermediate.

Quantitative profiling and dynamics of mRNA modifications in Escherichia coli

2021 ◽  
Author(s):  
Dimitar Plamenov Petrov ◽  
Steffen Kaiser ◽  
Stefanie Kaiser ◽  
Kirsten Jung
Keyword(s):  
Mass Spectrometry ◽  
Escherichia Coli ◽  
Stationary Phase ◽  
Exponential Growth ◽  
Gel Electrophoresis ◽  
E Coli ◽  
Physiological Processes ◽  
Mrna Methylation ◽  
Important Regulator ◽  
Quantitative Profiling

mRNA methylation is an important regulator of many physiological processes in eukaryotes but has not been studied in depth in prokaryotes. In contrast to the large number of eukaryotic mRNA modifications that have been described, N6-methyladenosine (m6A) is the only modification of bacterial mRNA identified to date. Here, we used a gel electrophoresis-based RNA separation method and quantitatively analyzed the mRNA-specific modification profile of Escherichia coli using mass spectrometry. In addition to m6A, we provide evidence for the presence of 7-methylguanosine (m7G), and we found first hints for 5-methylcytidine (m5C), N6,N6-dimethyladenosine (m6,6A), 1-methylguanosine (m1G), 5-methyluridine (m5U), and pseudouridine (Ψ) in the mRNA of E. coli, which implies that E. coli has a complex mRNA modification pattern. Furthermore, we observed changes in the abundance of some mRNA modifications during the transition of E. coli from the exponential growth to the stationary phase as well as upon exposure to stress. This study reveals a previously underestimated level of regulation between transcription and translation in bacteria.

scholarly journals Top-Down Proteomic Identification of Shiga Toxin 1 and 2 from Pathogenic Escherichia coli Using MALDI-TOF-TOF Tandem Mass Spectrometry

2019 ◽  
Vol 7 (11) ◽  
pp. 488 ◽  
Author(s):  
Clifton K. Fagerquist ◽  
William J. Zaragoza ◽  
Michelle Q. Carter
Keyword(s):  
Mass Spectrometry ◽  
Escherichia Coli ◽  
Tandem Mass Spectrometry ◽  
Disulfide Bond ◽  
Shiga Toxin ◽  
Tandem Mass ◽  
Top Down ◽  
E Coli ◽  
B Subunit ◽  
Proteomic Identification

Shiga-toxin-producing Escherichia coli (STEC) are a burden on agriculture and a threat to public health. Rapid methods are needed to identify STEC strains and characterize the Shiga toxin (Stx) they produce. We analyzed three STEC strains for Stx expression, using antibiotic induction, matrix-assisted laser desorption/ionization time-of-flight-time-of-flight (MALDI-TOF-TOF) mass spectrometry, and top-down proteomic analysis. E. coli O157:H- strain 493/89 is a clinical isolate linked to an outbreak of hemolytic uremic syndrome (HUS) in Germany in the late 1980s. E. coli O145:H28 strains RM12367-C1 and RM14496-C1 were isolated from an agricultural region in California. The stx operon of the two environmental strains were determined by whole genome sequencing (WGS). STEC strain 493/89 expressed Shiga toxin 2a (Stx2a) as identified by tandem mass spectrometry (MS/MS) of its B-subunit that allowed identification of the type and subtype of the toxin. RM12367-C1 also expressed Stx2a as identified by its B-subunit. RM14496-C1 expressed Shiga toxin 1a (Stx1a) as identified from its B-subunit. The B-subunits of Stx1 and Stx2 both have an intramolecular disulfide bond. MS/MS was obtained on both the disulfide-bond-intact and disulfide-bond-reduced B-subunit, with the latter being used for top-down proteomic identification. Top-down proteomic analysis was consistent with WGS.

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