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

Peng Liu,Yanqiong Tang,Zhu Liu,Yueling Zhang,Haichao Song,Qun Sun,Yong Chen,Dan Wang,Hongqian Tang

doi:10.3389/fmicb.2016.01228

Peng Liu, Yanqiong Tang + Show 7 more

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https://doi.org/10.3389/fmicb.2016.01228

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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.

Highlights

Aeromonas veronii is a type of important pathogenic microbes to human and aquatic animals (Graf, 1999), which is classified as gram-negative bacterium and distributed in freshwater and sewage (Sugita et al, 1995)
A catalytically inactive version of Staphylococcus aureus nuclease (SN) was chosen as scaffold protein, wherein the residues S63L64R65K66A67 of SN were replaced by 16 random amino acids exhibiting surface loop (Figure 1A)
After pTRG-SN-peptides were transformed into competent E. coli XL1-Blue MRF’, the library titer was determined as 1.5–2.0×107 colony forming units (CFU)/ml, which was sufficient for subsequent library screen

Summary

Introduction

Aeromonas veronii is a type of important pathogenic microbes to human and aquatic animals (Graf, 1999), which is classified as gram-negative bacterium and distributed in freshwater and sewage (Sugita et al, 1995). Widespread consumptions of antibiotics in fish farming result in more and more multidrug resistant strains of A. veronii (Varela et al, 2016), which resist to tetracycline, ampicillin, and streptomycin (Castro-Escarpulli et al, 2003; Nawaz et al, 2006). The emergence of multidrug resistant strains of A. veronii in fish farming should not be ignored, as they seriously impact on the economic benefit of aquaculture industry, and jeopardize the health and life of human beings (Castro-Escarpulli et al, 2003; Yücel and Balci, 2010). The development of new antibiotics and protein drugs is becoming an exigent task

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