Translation‐dependent mRNA cleavage by YhaV in Escherichia coli

Yoon

Journal of Microbiology and Biotechnology

2018

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Bacterial programmed cell death is regulated by the toxin-antitoxin (TA) system. YhaV (toxin) and Pr1F (antitoxin) have been recently identified as a type II TA system in Escherichia coli. YhaV homologs have conserved active residues within the C-terminus, and to characterize the function of this region, we purified native YhaV protein (without denaturing) and constructed YhaV proteins of varying lengths. Here, we report a new low-temperature method of purifying native YhaV, which is notable given the existing challenges of purifying this highly toxic protein. The secondary structures and thermostability of the purified native protein were characterized and no significant structural destruction was observed, suggesting that the observed inhibition of cell growth in vivo was not the result of structural protein damage. However, it has been reported that excessive levels of protein expression may result in protein misfolding and changes in cell growth and mRNA stability. To exclude this possibility, we used an [ 3 5S]-methionine prokaryotic cell-free protein synthesis system in vitro in the presence of purified YhaV, and two C-terminal truncated forms of this protein (YhaV-L and YhaV-S). Our results suggest that the YhaV C-terminal region is essential for mRNA interferase activity, and the W143 or H154 residues may play an analogous role to Y87 of RelE. S S 988 Choi et al. J. Microbiol. Biotechnol.

Section: Bacterial Strains Plasmids and Primersmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Functional Characterization of the C-Terminus of YhaV in the Escherichia coli PrlF-YhaV Toxin-Antitoxin System

Yoon

Journal of Microbiology and Biotechnology

2018

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“…Aliquots of fresh medium were inoculated with E. coli 3D8scFv or with the control strain, and the OD 600 and the number of CFUs in the inoculated cultures were measured. Aliquots of the cell cultures were collected at the same time intervals (0 h, 1 h, 3 h, 6 h, 9 h, 12 h, 1 d, 3 d, 6 d, 15 d, 18 d, and 21 d) used for the collection of stools in mice, and the OD 600 and CFU of the cell culture aliquots were measured (Choi et al, 2017). The OD 600 values of the E. coli 3D8scFv culture samples were measured and normalized to the blank value ( Fig.…”

Section: Effect Of 3d8 Scfv On E Coli 3d8scfv Cell Growthmentioning

confidence: 99%

Survival of Escherichia coli harboring nucleic acid-hydrolyzing 3D8 scFv during RNA virus infection

Lee

Regulatory Toxicology and Pharmacology

et al. 2018

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Previously, Escherichia coli harboring the codon-optimized 3D8scFv gene (E. coli 3D8scFv) was developed as a feed additive for use in preventing norovirus infection. Here, we evaluated whether the 3D8scFv gene affects the colonization of E coli when E. coli 3D8scFv passes through the mouse gastrointestinal tract. To determine the colonization ability of E. coli 3D8scFv, E. coli cells with or without the 3D8scFv gene were fed to mice. Total DNA was extracted from the animals' stools, stomach, small intestine and colon. All samples were amplified using 3D8scFv gene-specific primer sets. E. coli 3D8scFv begins to be excreted 1 h after feeding and that all E. coli 3D8scFv cells were excreted between 12 and 24 h after the last feeding of the cells. The previously measured gastrointestinal transit time of the mice was between 8 h and 22 h. The results of this study therefore show that E. coli 3D8scFv cannot colonize the gastrointestinal tracts of mice. In addition, if the purified 3D8 scFv protein is used as a feed additive, any associated E. coli 3D8scFv bacteria will not colonize the gastrointestinal tracts of the livestock. Thus, this feed additive meets the safety assessment criteria for the commercial use of bacteria.

“…Most bacteria have the toxin-antitoxin (Ta) system which is a mechanism to control the cell growth. Toxin induces cell growth arrest and even cause cell death without the co-expression their cognate antitoxins 13,14 . Thus, virus proteases have been considered ideal drug targets.…”

Section: Introductionmentioning

confidence: 99%

Specific elimination of coxsackievirus B3 infected cells with a protein engineered toxin-antitoxin system

et al. 2019

Mol. Cell. Toxicol.

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Backgrounds:Coxsackievirus B3 (CVB3) is a member of the family Picornaviridae, and along with polioviruses, belongs to the Enterovirus genus. The CVB3 genome is composed single-stranded rna encoding polyproteins, which are cleaved to individual functional proteins by 2a and 3C proteases proteins which have been targeted for drug development. Here, we showed that protease activity required to activate a toxic protein may be used to prevent viral infection. Methods: We modified the MazE-MazF antitoxin-toxin system of Escherichia coli to fuse a C-terminal fragment of MazE to the N-terminal end of toxin MazF with a linker having a specific protease cleavage site for CVB3. This fusion protein formed a stable dimer and was capable of inactivating the mrna interferase activity of MazF which cleaves the ACA sequence in mRNA substrates. Results: The incubation of 2a proteases with the fusion proteins induced cleavage between the MazE and MazF fragments from the fusion proteins; the subsequent release of MazF significantly inhibited virus replication. additionally, we note that, CVB3 infected HeLa cells quickly died through a MazF toxin mediated effect before virus protein expression. Conclusion: These findings suggest that the MazEF fusion protein has a strong potential to be developed as an anti-virus therapy following CVB3 infection.