Induction and identification of disulfide-intact and disulfide-reduced β-subunit of Shiga toxin 2 from Escherichia coli O157:H7 using MALDI-TOF-TOF-MS/MS and top-down proteomics

The Analyst ◽  
2011 ◽  
Vol 136 (8) ◽  
pp. 1739 ◽  
Author(s):  
Clifton K. Fagerquist ◽  
Omar Sultan
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Escherichia Coli O157 ◽  
Β Subunit ◽  
Top Down ◽  
Maldi Tof ◽  
Shiga Toxin 2 ◽  
Tof Ms

scholarly journals Top-Down Proteomic Identification of Furin-Cleavedα-Subunit of Shiga Toxin 2 fromEscherichia coliO157:H7 Using MALDI-TOF-TOF-MS/MS

2010 ◽  
Vol 2010 ◽  
pp. 1-11 ◽  
Author(s):  
Clifton K. Fagerquist ◽  
Omar Sultan
Keyword(s):  
Shiga Toxin ◽  
Time Of Flight ◽  
Top Down ◽  
Web Based ◽  
Protein Fragments ◽  
Maldi Tof ◽  
Disulfide Reduction ◽  
Solid Agar ◽  
Shiga Toxin 2 ◽  
Tof Ms

A method has been developed to identify theα-subunit of Shiga toxin 2 (α-Stx2) fromEscherichia coliO157:H7 using matrix-assisted laser desorption/ionization time-of-flight-time-of-flight tandem mass spectrometry (MALDI-TOF-TOF-MS/MS) and top-down proteomics using web-based software developed in-house. Expression of Stx2 was induced by culturingE. coliO157:H7 on solid agar supplemented with an antibiotic that elicits the bacterial SOS-response. Bacterial cell lysates were incubated in the presence of furin, a human enzyme, that cleavesα-Stx2 into A1 (~28 kDa) and A2 (~5 kDa) protein fragments. A subsequent disulfide reduction step unlinked A1 from A2. MALDI-TOF-MS of the furin-digested/disulfide-reduced sample showed a peak at mass-to-charge () 5286 that corresponded to the A2 fragment. No peak was observed that corresponded to the A1 fragment although its presence was confirmed by bottom-up proteomics. The peak at 5286 was definitively identified by MALDI-TOF-TOF-MS/MS and top-down proteomics as the A2 fragment ofα-Stx2.

Prevalence, Characterization, and Genotypic Analysis of Escherichia coli O157:H7/NM fromSelected Beef Exporting Abattoirs of Argentina

2010 ◽  
Vol 73 (4) ◽  
pp. 649-656 ◽  
Author(s):  
M. O. MASANA ◽  
G. A. LEOTTA ◽  
L. L. DEL CASTILLO ◽  
B. A. D'ASTEK ◽  
P. M. PALLADINO ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Phage Type ◽  
Escherichia Coli O157 ◽  
Phage Types ◽  
Clonal Relatedness ◽  
Genotypic Analysis ◽  
Shiga Toxin 2 ◽  
Uremic Syndrome ◽  
Fecal Content

In Argentina, Escherichia coli O157:H7/NM (STEC O157) is the prevalent serotype associated with hemolytic uremic syndrome (HUS), which is endemic in the country with more than 400 cases per year. In order to estimate the prevalence and characteristics of STEC O157 in beef cattle at slaughter, a survey of 1,622 fecal and carcass samples was conducted in nine beef exporting abattoirs from November 2006 to April 2008. A total of 54 samples were found positive for STEC O157, with an average prevalence of 4.1% in fecal content and 2.6% in carcasses. Calves and heifers presented higher percentages of prevalence in feces, 10.5 and 8.5%, respectively. All STEC O157 isolates harbored stx2 (Shiga toxin 2), eae (intimin), ehxA (enterohemolysin), and fliCH7 (H7 flagellin) genes, while stx1 (Shiga toxin 1) was present in 16.7% of the strains. The prevalent (56%) stx genotype identified was stx2 combined with variant stx2c (vh-a), the combination of which is also prevalent (>90%) in STEC O157 post–enteric HUS cases in Argentina. The clonal relatedness of STEC O157 strains was established by phage typing and pulsed-field gel electrophoresis (PFGE). The 54 STEC isolates were categorized into 12 different phage types and in 29 XbaI-PFGE patterns distributed in 27 different lots. STEC O157 strains isolated from 5 of 21 carcasses were identical by PFGE (100% similarity) to strains of the fecal content of the same or a contiguous bovine in the lot. Five phage type–PFGE–stx profiles of 10 strains isolated in this study matched with the profiles of the strains recovered from 18 of 122 HUS cases that occurred in the same period.

scholarly journals Clinically-relevant Shiga toxin 2 subtypes from environmental Shiga toxin-producing Escherichia coli identified by top-down/middle-down proteomics and DNA sequencing

2019 ◽  
Vol 11 ◽  
pp. 27-36 ◽  
Author(s):  
Clifton K. Fagerquist ◽  
William J. Zaragoza ◽  
Bertram G. Lee ◽  
Jaszemyn C. Yambao ◽  
Beatriz Quiñones
Keyword(s):  
Escherichia Coli ◽  
Dna Sequencing ◽  
Shiga Toxin ◽  
Top Down ◽  
Shiga Toxin 2

Distinctiveness of the genomic sequence of Shiga toxin 2-converting phage isolated from Escherichia coli O157:H7 Okayama strain as compared to other Shiga toxin 2-converting phages

Gene ◽  
2003 ◽  
Vol 309 (1) ◽  
pp. 35-48 ◽  
Author(s):  
Toshio Sato ◽  
Takeshi Shimizu ◽  
Masahisa Watarai ◽  
Midori Kobayashi ◽  
Shigeyuki Kano ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Genomic Sequence ◽  
Escherichia Coli O157 ◽  
Shiga Toxin 2

Seasonal Prevalence of Escherichia coli O157:H7 in Beef Cattle Feces†

2006 ◽  
Vol 69 (12) ◽  
pp. 3018-3020 ◽  
Author(s):  
M. J. ALAM ◽  
L. ZUREK
Keyword(s):  
Escherichia Coli ◽  
Beef Cattle ◽  
Shiga Toxin ◽  
Bacterial Colonization ◽  
Feedlot Cattle ◽  
Escherichia Coli O157 ◽  
Human Pathogen ◽  
E Coli ◽  
Cattle Feces ◽  
Shiga Toxin 2

Cattle are an asymptomatic reservoir of Escherichia coli O157:H7, but the bacterial colonization and shedding patterns are poorly understood. The prevalence and shedding of this human pathogen have been reported to be seasonal with rates typically increasing during warm months. The objectives of this study were (i) to assess the prevalence of E. coli O157:H7 in feces of feedlot cattle in Kansas during summer, fall, and winter months, and (ii) to characterize E. coli O157:H7 by screening for virulence factors. Of 891 fecal samples collected, 82 (9.2%) were positive for E. coli O157:H7. No significant differences in prevalence were detected among summer, fall, and winter months. The highest monthly prevalence (18.1%) was detected in February. All tested isolates were positive for stx2 (Shiga toxin 2) and eaeA (intimin) genes; 14 isolates (12.8%) also carried stx1. Our results indicate the prevalence of E. coli O157:H7 in beef cattle feces is not necessarily season dependent.

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