scholarly journals The Molecular Basis for Escherichia coli O157:H7 Phage FAHEc1 Endolysin Function and Protein Engineering to Increase Thermal Stability

Viruses ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 1101
Author(s):  
Michael J. Love ◽  
David Coombes ◽  
Sarah H. Manners ◽  
Gayan S. Abeysekera ◽  
Craig Billington ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Thermal Stability ◽  
Antibacterial Agents ◽  
Catalytic Mechanism ◽  
Bioinformatic Analysis ◽  
Catalytic Triad ◽  
Escherichia Coli O157 ◽  
Hydrophobic Core ◽  
Rational Engineering ◽  
Enzymatic Function

Bacteriophage-encoded endolysins have been identified as antibacterial candidates. However, the development of endolysins as mainstream antibacterial agents first requires a comprehensive biochemical understanding. This study defines the atomic structure and enzymatic function of Escherichia coli O157:H7 phage FAHEc1 endolysin, LysF1. Bioinformatic analysis suggests this endolysin belongs to the T4 Lysozyme (T4L)-like family of proteins and contains a highly conserved catalytic triad. We then solved the structure of LysF1 with x-ray crystallography to 1.71 Å. LysF1 was confirmed to exist as a monomer in solution by sedimentation velocity experiments. The protein architecture of LysF1 is conserved between T4L and related endolysins. Comparative analysis with related endolysins shows that the spatial orientation of the catalytic triad is conserved, suggesting the catalytic mechanism of peptidoglycan degradation is the same as that of T4L. Differences in the sequence illustrate the role coevolution may have in the evolution of this fold. We also demonstrate that by mutating a single residue within the hydrophobic core, the thermal stability of LysF1 can be increased by 9.4 °C without compromising enzymatic activity. Overall, the characterization of LysF1 provides further insight into the T4L-like class of endolysins. Our study will help advance the development of related endolysins as antibacterial agents, as rational engineering will rely on understanding mutable positions within this protein fold.

scholarly journals The Structure and Function of Modular Escherichia coli O157:H7 Bacteriophage FTBEc1 endolysin, LysT84: Defining a New Endolysin Catalytic Subfamily

2021 ◽  
Author(s):  
Michael Love ◽  
David Coombes ◽  
Salim Ismail ◽  
Craig Billington ◽  
Renwick CJ Dobson
Keyword(s):  
Crystal Structure ◽  
Escherichia Coli ◽  
Antibacterial Agents ◽  
Analytical Ultracentrifugation ◽  
Structural Data ◽  
Scattering Data ◽  
Structural Basis ◽  
Escherichia Coli O157 ◽  
Dynamic Simulations ◽  
Enzymatic Function

Bacteriophage endolysins degrade peptidoglycan and have been identified as antibacterial candidates to combat antimicrobial resistance. Considering the catalytic and structural diversity of endolysins, there is a paucity of structural data to inform how these enzymes work at the molecular level—key data that is needed to realize the potential of endolysin-based antibacterial agents. Here, we determine the atomic structure and define the enzymatic function of Escherichia coli O157:H7 phage FTEBc1 endolysin, LysT84. Bioinformatic analysis reveals that LysT84 is a modular endolysin, which is unusual for Gram-negative endolysins, comprising a peptidoglycan binding domain and an enzymatic domain. The crystal structure of LysT84 (2.99 Å) revealed a mostly α-helical protein with two domains connected by a linker region but packed together. LysT84 was determined to be a monomer in solution using analytical ultracentrifugation. Small-angle X-ray scattering data revealed that LysT84 is a flexible protein but does not have the expected bimodal P(r) function of a multidomain protein, suggesting that the domains of LysT84 pack closely creating a globular protein as seen in the crystal structure. Structural analysis reveals two key glutamate residues positioned on either side of the active site cavity; mutagenesis demonstrating these residues are critical for peptidoglycan degradation. Molecular dynamic simulations suggest that the enzymatically active domain is dynamic, allowing the appropriate positioning of these catalytic residues for hydrolysis of the β(1–4) bond. Overall, our study defines the structural basis for peptidoglycan degradation by LysT84 which supports rational engineering of related endolysins into effective antibacterial agents.

Preparation and Antimicrobial Activity of Antibacterial Silver-Loaded Polyphosphazene Microspheres

2021 ◽  
Vol 21 (10) ◽  
pp. 5120-5130
Author(s):  
Hui Long ◽  
Wei-Cong Kuang ◽  
Shi-Liang Wang ◽  
Jing-Xian Zhang ◽  
Lang-Huan Huang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Thermal Stability ◽  
Staphylococcus Aureus ◽  
Antimicrobial Activity ◽  
Silver Nanoparticles ◽  
Antibacterial Activity ◽  
Antibacterial Agents ◽  
Good Thermal Stability ◽  
Polymerization Method

Poly(cyclotriphosphazene-co-4,4’-diaminodiphenyl ether) (PPO) microspheres were prepared via a precipitation polymerization method, using hexachlorocyclotriphosphazene (HCCP) and 4,4’-diaminodiphenyl ether (ODA) as monomers. Silver-loaded PPO (PPOA) microspheres were generated by the in situ loading of silver nanoparticles onto the surface by Ag+ reduction. Our results showed that PPOA microspheres were successfully prepared with a relatively uniform distribution of silver nanoparticles on microsphere surfaces. PPOA microspheres had good thermal stability and excellent antibacterial activity towards Escherichia coli and Staphylococcus aureus. Furthermore, PPOA microspheres exhibited lower cytotoxicity when compared to citrate-modified silver nanoparticles (c-Ag), and good sustained release properties. Our data indicated that polyphosphazene-based PPOA microspheres are promising antibacterial agents in the biological materials field.

scholarly journals Screening for Antibacterial Inhibitors of the UDP-3-O-(R-3-Hydroxymyristoyl)- N-Acetylglucosamine Deacetylase (LpxC) Using a High-Throughput Mass Spectrometry Assay

2009 ◽  
Vol 15 (1) ◽  
pp. 52-61 ◽  
Author(s):  
Erik F. Langsdorf ◽  
Asra Malikzay ◽  
William A. Lamarr ◽  
Dayna Daubaras ◽  
Cynthia Kravec ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Escherichia Coli ◽  
Thermal Stability ◽  
High Throughput ◽  
High Throughput Screening ◽  
Antibacterial Agents ◽  
Screening Tool ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Mass Spectrometry Assay

A high-throughput mass spectrometry assay to measure the catalytic activity of UDP-3-O-(R-3-hydroxymyristoyl)- Nacetylglucosamine deacetylase, LpxC, is described. This reaction is essential in the biosynthesis of lipopolysaccharide (LPS) of gram-negative bacteria and is an attractive target for the development of new antibacterial agents. The assay uses the RapidFire™ mass spectrometry platform to measure the native LpxC substrate and the reaction product and thereby generates a ratiometric readout with minimal artifacts due to detection interference. The assay was robust in a high-throughput screen of a library of more than 700,000 compounds arrayed as orthogonal mixtures, with a median Z' factor of 0.74. Selected novel inhibitors from the screening campaign were confirmed as binding to LpxC by biophysical measurements using a thermal stability shift assay. Some inhibitors showed whole-cell antimicrobial activity against a sensitive strain of Escherichia coli with reduced LpxC activity (strain D22; minimum inhibitory concentrations ranging from 0.625-20 µg/mL). The results show that mass spectrometry—based screening is a valuable high-throughput screening tool for detecting inhibitors of enzymatic targets involving difficult to detect reactions.

Bedeutung von Escherichia coli O157 beim Schlachtschaf in der Schweiz

2006 ◽  
Vol 148 (6) ◽  
pp. 289-295 ◽  
Author(s):  
C. Zweifel ◽  
M. Kaufmann ◽  
J. Blanco ◽  
R. Stephan
Keyword(s):  
Escherichia Coli ◽  
Escherichia Coli O157

Molecular profiling and antimicrobial susceptibility of Escherichia coli O157:H7 isolated in Bulgaria

2020 ◽  
Vol 23 (3) ◽  
pp. 310-318
Author(s):  
K. Koev ◽  
T. Stoyanchev ◽  
G. Zhelev ◽  
P. Marutsov ◽  
K. Gospodinova ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Escherichia Coli O157 ◽  
Acid Sensitivity ◽  
Lower Extent ◽  
Phenotype Analysis ◽  
Anal Swab ◽  
Amoxicillin Clavulanic Acid ◽  
Primary Identification ◽  
Stx1 Gene ◽  
Cattle Farms

The purpose of this study was to detect the presence of shiga-toxin producing Escherichia coli (STEC) in faeces of healthy dairy cattle and to determine the sensitivity of isolates to several anti­microbial drugs. A total of 1,104 anal swab samples originating from 28 cattle farms were examined. After the primary identification, 30 strains were found to belong to serogroup О157. By means of conventional multiplex PCR, isolates were screened for presence of resistance genes stx1, stx2 and eaeА. Twenty-nine strains possesses amplicons with a size corresponding to genes stx2 and eaeA, one had amplicons also for the stx1 gene and one lacked amplicons of all three genes. Twenty-eight strains demonstrated amplicons equivalent to gene H7. The results from phenotype analysis of resistance showed preserved sensitivity to ceftriaxone, ceftazidime, cefotaxime, cephalothin, streptomycin, gentamicin, tetracycline, enrofloxacin and combinations sulfamethoxazole/trimethoprim and amoxicillin/clavulanic acid. Sensitivity to ampicillin was relatively preserved, although at a lower extent.

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