In vitro Impact of Yeast Expressed Hybrid Peptide CATH-2TP5 as a Prophylactic Measure Toward Sepsis and Inflammation.

Baseer Ahmad,Zhongxuan Li,Junhao Cheng,Quratulain Hanif,Xubiao Wei,Abdul Basit,Muhammad Shahid,Naveed Sabir,Lulu Zhang,Amin Ur Rehman,Dayong Si,Rijun Zhang,Shahzad Akbar Khan

doi:10.3389/fbioe.2020.00454

Abstract

CATH-2TP5 is a linear cationic hybrid peptide, consequent from naturally occurring antimicrobial peptide (AMPs) Cathelicidin-2 (CATH-2) and Immunomodulatory peptide Thymopentin (TP5) having dynamic and potent anti-inflammatory activities without hemolytic effect. The biocompatible mechanism of CATH-2TP5 is favored to explore new methodologies in the direction of biomedical applications. In this retrospectively study, an antiendotoxin and anti-inflammatory hybrid peptide CATH-2TP5 was emulated into pPICZα-A and successfully expressed in Pichia pastoris (P. pastoris). The recombinant CATH-2TP5 was purified through the Ni-affinity column and reversed-phase HPLC. The purified CATH-2TP5 peptide exhibited robust anti-endotoxin activity and significantly (p < 0.05) neutralized the effect of lipopolysaccharide (LPS). Furthermore, the down-regulated effect of CATH-2TP was more pronounced (p < 0.05) on LPS-induced cytotoxic effects, nitric oxide secretion and pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β) in murine RAW264.7 macrophages. As associated to control and parental peptide the number of apoptotic cells was also contracted with the treatment of CATH-2TP5. Thus, we concluded that CATH-2TP5 peptide may be used in various biomedical applications as a therapeutic drug.

Highlights

Antimicrobial peptides (AMPs) act as an imperative component of the innate immune response in vertebrates and invertebrates against microbial infections (Golec, 2007; Mardirossian et al, 2016)
Yeast expression vector pPICZαA-CATH-2TP5 that contains a 96 bp DNA fragment encoding a recombinant C-terminal 6 × His-tagged that facilitate purification and analysis of expressed proteins
The restriction enzyme KpnI and XbaI were attached at its 5 and 3 ends and fragment was cloned in the frame of α-factor signal, downstream promoter alcohol oxidase (AOXI) of the expression vector pPICZαA and the result in recombinant pPICZαA-CATH2TP5 after double digested with a restriction enzyme (Supplementary Figure S1)

Summary

Introduction

Antimicrobial peptides (AMPs) act as an imperative component of the innate immune response in vertebrates and invertebrates against microbial infections (Golec, 2007; Mardirossian et al, 2016). Despite the accessibility of drugs and antibiotics, microbial infections are the quite main cause of death due to the incapability of the particular therapeutics to kill microbes and bind their pathogenic effect like LPS (Lu et al, 2010; Hwang et al, 2013). Gram-negative bacteria contain LPS in the outer leaflet of membrane that usually plays a key role in the pathogenesis of respiratory diseases, chronic illness and septic shock (Golec, 2007; Pompilio et al, 2012; Mardirossian et al, 2016). AMPs have been determined as a potential source in applied medicine as antibacterial, immunomodulatory and anti-inflammatory agents. There is an instantaneous and urgent need for the development of new peptides that have both antibacterial and LPS neutralizing activities

Methods

Results

Discussion

Conclusion