Design and High Expression of Non-glycosylated Lysostaphins in Pichia pastoris and Their Pharmacodynamic Study.

Wenluan Shen,Da Teng,Na Yang,Ya Hao,Ruoyu Mao,Jianhua Wang,Xuanxuan Ma

doi:10.3389/fmicb.2021.637662

Abstract

Lysostaphin is an effective antimicrobial agent to Staphylococcus, especially for the methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant Staphylococcus aureus (MDRSA). In this study, the seven lysostaphin derived mutants (rLys) were designed to overcome the barrier of glycosylation during expression in Pichia pastoris. Among them, 127A and 127A232Q had highest antimicrobial activity (MIC values 0.07–0.3 μM) to S. aureus than others and the commercial lysostaphins (1–15.8 times). There was no glycosylation during the expression in 5-L fermenter level, with the high yield of 1315 mg/L (127A) and 1141 mg/L (127A232Q), respectively. Meanwhile, 127A and 127A232Q effectively killed 99.9% of S. aureus at low concentration (1 × MIC) within 30 min, without the regrowth of pathogen. They also showed low toxicity, high pH and temperature stability. The results of in vivo therapeutic effect of 127A and 127A232Q against high virulent S. aureus CVCC546 showed that 127A and 127A232Q increased the survival rate of infected mice up to 100% at the dose of 10 mg/kg than the untreated group, reduced the bacterial translocation by 5-7 log CFU (over 99%) in organs compared to the untreated group and alleviated multiple-organ injuries (liver, kidney and spleen). These data indicated that the non-glycosylated lysostaphin 127A and 127A232Q may be a promising therapeutic agent against MDR staphylococcal infections.

Highlights

Due to the chronic overuse of antibiotics, many resistant Staphylococcus aureus (S. aureus) emerged in recent years, such as methicillin-resistant S. aureus (MRSA), vancomycin-resistant S. aureus (VRSA), and multiple-drug-resistant S. aureus (MDRSA), this S. aureus become a formidable pathogen which can cause terrible infections in humans and livestock
The test strains, including S. aureus ATCC 43300, S. aureus ATCC 25923 were purchased from the American Type Culture Collection (ATCC) (Beijing, China), S. aureus CICC 10436, S. aureus CICC 10473, S. aureus CICC 21601, S. epidermidis CICC 23962, S. epidermidis CICC 10294 were purchased from China Center of Industrial Culture Collection (CICC) (Beijing, China), S. aureus CVCC 546 were purchased from China Veterinary Culture Collection Center(CVCC) (Beijing, China), S. epidermidis CGMCC 1.4206 was purchased from China General Microbiological Culture Collection Center (CGMCC) (Beijing, China), S. hyicus NCTC 10350 was purchased from National Collection of Type Cultures (NCTC)
It was confirmed that the residue N125 was thought to be located at the C-terminal end of the catalytic domain, which was essential for the activity of lysostaphin (Zhao et al, 2014)

Summary

Introduction

Due to the chronic overuse of antibiotics, many resistant Staphylococcus aureus (S. aureus) emerged in recent years, such as methicillin-resistant S. aureus (MRSA), vancomycin-resistant S. aureus (VRSA), and multiple-drug-resistant S. aureus (MDRSA), this S. aureus become a formidable pathogen which can cause terrible infections in humans and livestock. Between 2 and 53 million persons are estimated to carry MRSA worldwide (Gleeson et al, 2015)

Methods

Results

Conclusion