Genetic Selection of Peptide Aptamers That Interact and Inhibit Both Small Protein B and Alternative Ribosome-Rescue Factor A of Aeromonas veronii C4

Abstract

Aeromonas veronii is a pathogenic gram-negative bacterium, which infects a variety of animals and results in mass mortality. The stalled-ribosome rescues are reported to ensure viability and virulence under stress conditions, of which primarily include trans-translation and alternative ribosome-rescue factor A (ArfA) in A. veronii. For identification of specific peptides that interact and inhibit the stalled-ribosome rescues, peptide aptamer library (pTRG-SN-peptides) was constructed using pTRG as vector and Staphylococcus aureus nuclease (SN) as scaffold protein, in which 16 random amino acids were introduced to form an exposed surface loop. In the meantime both Small Protein B (SmpB) which acts as one of the key components in trans-translation, and ArfA were inserted to pBT to constitute pBT-SmpB and pBT-ArfA, respectively. The peptide aptamer PA-2 was selected from pTRG-SN-peptides by bacterial two-hybrid system (B2H) employing pBT-SmpB or pBT-ArfA as baits. The conserved sites G133K134 and D138K139R140 of C-terminal SmpB were identified by interacting with N-terminal SN, and concurrently the residue K62 of ArfA was recognized by interacting with the surface loop of the specific peptide aptamer PA-2. The expression plasmids pN-SN or pN-PA-2, which combined the duplication origin of pRE112 with the neokanamycin promoter expressing SN or PA-2, were created and transformed into A. veronii C4, separately. The engineered A. veronii C4 which endowing SN or PA-2 expression impaired growth capabilities under stress conditions including temperatures, sucrose, glucose, potassium chloride (KCl) and antibiotics, and the stress-related genes rpoS and nhaP were down-regulated significantly by Quantitative Real-time PCR (qRT-PCR) when treating in 2.0% KCl. Thus, the engineered A. veronii C4 conferring PA-2 expression might be potentially attenuated vaccine, and also the peptide aptamer PA-2 could develop as anti-microbial drugs targeted to the ribosome rescued factors in A. veronii.

First-in-class matrix anti-assembly peptide prevents staphylococcal biofilmin vitroandin vivo

“…Briefly, we monitored the real-time molecular self-assembly reaction by measuring the optical density at 600 nm (OD 600 ) as a function of time with or without capsicumicine. As a negative control, we used PA-1, a similarly sized peptide ( 40, 41 ). OD increases when capsicumicine is present, which shows that the molecular self-assembly process is quicker overall (Fig.…”

Targeting Inhibition of SmpB by Peptide Aptamer Attenuates the Virulence to Protect Zebrafish against Aeromonas veronii Infection

“…All plasmids and primers used in this study were listed in Table 1 and Supplementary Table S1 , respectively. The truncations and mutants of pBT-SmpB and pN-SN were from our previous work ( Liu et al, 2016 ). The peptide aptamer library (pTRG-SN-peptides) was constructed and comprised of approximate 2 × 10 7 clones which expressed the scaffold protein and the random exposed loop ( Liu et al, 2016 ).…”

“…The truncations and mutants of pBT-SmpB and pN-SN were from our previous work ( Liu et al, 2016 ). The peptide aptamer library (pTRG-SN-peptides) was constructed and comprised of approximate 2 × 10 7 clones which expressed the scaffold protein and the random exposed loop ( Liu et al, 2016 ). In brief, the DNA fragment encoding SN was inserted into pTRG, and expressed as a fusion protein with α-subunit of RNA polymerase as scaffold protein, in which the constrained loop composed of the residues S 63 L 64 R 65 K 66 A 67 was replaced by 16 random amino acids.…”

“…Trans-translation mediated by transfer-messenger RNA (tmRNA) and Small protein B (SmpB) is the primary stalled-ribosome rescue system in bacteria in which SmpB functions as an essential component, to protect tmRNA from degradation, enhance tmRNA alanylation, and help tmRNA to bind with stalled ribosomes in vivo ( Felden and Gillet, 2011 ). In addition, SmpB regulates both the RNA polymerase RpoS as a RNA chaperone ( Liu et al, 2016 ) and the virulence sensor protein BvgS as a transcription factor ( Liu et al, 2015 ), successively affecting protein synthesis, growth and adaptation to cellular stress, and pathogenic virulence. Recent reports show that smpB mutants serve as a live attenuated vaccine to provide effective immune protection.…”

“…Frequently the affinity with peptide aptamer disturbs the functions of the target protein and causes distinct phenotypes at intracellular level ( Cobbert et al, 2015 ). Previously we constructed fabricated peptide aptamer libraries (pTRG-SN-peptides), which included both a scaffold protein Staphylococcus aureus nuclease (SN) and an loop consisted of random 16 amino acids ( Liu et al, 2016 ). In this study, the conserved SmpB of A. veronii was considered as a potential antibacterial target.…”