High Level Expression and Purification of Atl, the Major Autolytic Protein of Staphylococcus aureus

Staphylococcus aureus is a major human and animal pathogen. Autolysins regulate the growth, turnover, cell lysis, biofilm formation, and the pathogenicity of S. aureus. Atl is the major autolysin in S. aureus. The biochemical and structural studies of staphylococcal Atl have been limited due to difficulty in cloning, high level overexpression, and purification of this protein. This study describes successful cloning, high level over-expression, and purification of two forms of fully functional Atl proteins. These pure proteins can be used to study the functional and structural properties of this important protein. 1. Introduction Staphylococcus aureus is an aggressive pathogen that is responsible for a wide array of diseases from mild skin infections to life-threatening conditions such as bacteremia, pneumonia, and endocarditis [1–4]. The emergence of multidrug resistance in S. aureus is an enormous public health concern and there is an immediate need for additional and alternative therapeutic targets for infections caused by this bacterium [5]. Autolysins are enzymes that degrade the peptidoglycan cell wall layer and are called peptidoglycan hydrolases. They represent a diverse group of enzymes and appear to have redundant roles and more than one function [6, 7]. These enzymes include N-acetylmuramidase, N-acetyl glucosaminidase, N-acetylmuramyl-L-alanine amidase, and endopeptidase [8–11]. While amidases break the bonds between the glycan strand and the stem peptides, the glycosidases are responsible for the cleavage of the glycan strands [7]. Cellular levels and activities of autolysins are believed to be intricately regulated and are proposed to play key roles in bacterial cell wall metabolism, daughter-cell separation, and antibiotic mediated cell lysis [2, 10]. Bacteria may secrete these autolysins and cause lysis of other bacteria that compete with S. aureus for nutrients [12]. The peptidoglycan hydrolases are important in bacterial pathogenicity [13, 14]. These enzymes modulate muropeptide release which in turn activates host innate immune responses [13]. There is plenty of evidence to suggest that most S. aureus autolysins result from the processing of full-length Atl [9, 15]. The full length Atl is an ~137？kDa protein with two catalytically active domains [15–18]. A 63.3？kDa N-terminal domain possesses amidase activity while a 53.6？kDa N-terminal domain possesses the glucosaminidase activity [15–18]. Many investigators [16, 17, 19] in the past have reported difficulty in cloning the full length S. aureus atl gene. A difficulty in overexpression