Characterization of serine acetyltransferase (CysE) from methicillin-resistant Staphylococcus aureus and inhibitory effect of two natural products on CysE

Highlights

• A colorimetric assay was developed to detect the serine acetyltransferase activity of MRSA CysE.

• Molecular docking analysis showed that Residue Asp94 and His95 were essential for serine acetyltransferase activity of CysE.

• The colorimetric assay was used to screen natural products and six compounds which inhibited CysE activity were found.

• Two of these compounds had inhibitory effect on the growth of MRSA and mature biofilm.

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA) is a major hospital-acquired infective pathogen that has developed resistance to many antibiotics. It is imperious to develop novel anti-MRSA drugs to control the emergence of drug resistance. The biosynthesis of cysteine in bacteria is catalyzed by CysE and CysK. CysE was predicted to be important for bacterial viability, it could be a potential drug target. The serine acetyltransferase activity of CysE was detected and its catalytic properties were also determined. CysE homology model was built to investigate interaction sites between CysE and substrate L-Ser or inhibitors by molecular docking. Docking data showed that residues Asp94 and His95 were essential for serine acetyltransferase activity of CysE, which were confirmed by site-directed mutagenesis. Colorimetric assay was used to screen natural products and six compounds which inhibited CysE activity (IC₅₀ ranging from 29.83 μM to 203.13 μM) were found. Inhibition types of two compounds 4 (11-oxo-ebracteolatanolide B) and 30 ((4R,4aR)-dihydroxy-3-hydroxymethyl-7,7,10a-trimethyl-2,4,4a,5,6,6a,7,8,9,10,10a,l0b-dodecahydrophenanthro[3,2-b]furan-2-one) on CysE were determined. Compounds 4 and 30 showed inhibitory effect on MRSA growth (MIC at 12.5 μg/ml and 25 μg/ml) and mature biofilm. The established colorimetric assay will facilitate further high-throughput screening of CysE inhibitors from different compound libraries. The compounds 4 and 30 may offer structural basis for developing new anti-MRSA drugs.

Introduction

Staphylococcus aureus is an opportunistic pathogen, which can cause skin and soft-tissue infections, sepsis, and necrotizing pneumonia in the world [1,2]. Methicillin-resistant Staphylococcus aureus (MRSA) emerged in the 1960s, spreading rapidly due to the emergence of drug resistance to β-lactam antibiotics [[3], [4], [5]]. Moreover, MRSA clinical strains showed decreased susceptibility to vancomycin and became more resistant to daptomycin and linezolid [6,7]. As a consequence, it is a matter of urgency to find more effective candidates of novel anti-MRSA drugs.

Sulfur element is essential for life and plays a core role in numerous microbial metabolic processes. It is used in the biosynthesis of cysteine and methionine in its reduced form. Cysteine can be converted to important coenzymes and mycothiol involved in the redox defense [[8], [9], [10], [11]]. The serine acetyltransferase (CysE) and O-acetylserine sulfhydrylase (CysK) involved in the biosynthetic pathway of cysteine had been characterized in Escherichia coli, Salmonella typhimurium, Mycobacterium tuberculosis, Corynebacterium glutamicum and Entamoeba histolytica [[12], [13], [14], [15], [16], [17], [18]]. However, the CysE and CysK of Staphylococcus aureus have not been identified. The amino acid sequences of CysE from seven bacterial species, Staphylococcus aureus, MRSA, Escherichia coli, Salmonella typhimurium, Mycobacterium tuberculosis, Corynebacterium glutamicum and Entamoeba histolytica were compared in this study (Fig. 1). The alignment results showed that the amino acids sequences of CysE highly conserved between MRSA and Staphylococcus aureus, but the low homology of CysE existed between MRSA and other bacterial species. Moreover, since microbial and plants sulfur metabolic pathways are largely absent in humans and CysE involved in the cysteine synthesis is essential [19], it is assumed that CysE could serve as a potential drug target for anti-Staphylococcus aureus. It is necessary to understand the catalytic mechanism of MRSA CysE and find inhibitors on MRSA CysE. S. aureus and MRSA are etiological intermediary to lead to countless human acute infections and their biofilm is the cause of chronic and even recalcitrant disease [20,21]. Therefore, it is also needed to find inhibitors which are capable to effectively inhibit biofilm formation and/or destroy the biofilm.

In this study, MRSA cysE gene was cloned and MRSA CysE protein was expressed in E. coli, and purified CysE protein was obtained through Ni²⁺ affinity chromatography. The enzymatic activity of CysE protein was detected and the catalytic properties of CysE were determined. The colorimetric assay of CysE was developed to screen natural products and six CysE inhibitors were found as well as their action mechanism was explored in this study.