Discovery of novel S. aureus autolysins and molecular engineering to enhance bacteriolytic activity

Abstract

Staphylococcus aureus is a dangerous bacterial pathogen whose clinical impact has been amplified by the emergence and rapid spread of antibiotic resistance. In the search for more effective therapeutic strategies, great effort has been placed on the study and development of staphylolytic enzymes, which benefit from high potency, activity towards drug-resistant strains, and a low inherent susceptibility to emergence of new resistance phenotypes. To date, the majority of therapeutic candidates have derived from either bacteriophage or environmental competitors of S. aureus. Little to no consideration has been given to cis-acting autolysins that represent key elements in the bacterium's endogenous cell wall maintenance and recycling machinery. In this study, five putative autolysins were cloned from the S. aureus genome, and their activities were evaluated. Four of these novel enzymes, or component domains thereof, demonstrated lytic activity towards live S. aureus cells, but their potencies were 10s to 1000s of times lower than that of the well-characterized therapeutic candidate lysostaphin. We hypothesized that their poor activities were due in part to suboptimal cell wall targeting associated with their native cell wall binding domains, and we sought to enhance their antibacterial potential via chimeragenesis with the peptidoglycan binding domain of lysostaphin. The most potent chimera exhibited a 140-fold increase in lytic rate, bringing it within 8-fold of lysostaphin. While this enzyme was sensitive to certain biologically relevant environmental factors and failed to exhibit a measurable minimal inhibitory concentration, it was able to kill lysostaphin-resistant S. aureus and ultimately proved active in lung surfactant. We conclude that the S. aureus proteome represents a rich and untapped reservoir of novel antibacterial enzymes, and we demonstrate enhanced bacteriolytic activity via improved cell wall targeting of autolysin catalytic domains.

The Staphylococcus aureus ArlRS two‐component system regulates virulence factor expression through MgrA

“…AtlR is a repressor of Atl expression (Houston et al , ); however, this should result in decreased autolytic activity in the arlRS mutant. In addition, we see modest regulation of the LysM/CHAP domain containing enzyme SAUSA300_0739 (Osipovitch et al , ) by ArlRS but not MgrA.…”

Wall Teichoic Acids Are Involved in the Medium-Induced Loss of Function of the Autolysin CD11 against Clostridium difficile

“…Both VALs and endolysins are encoded by bacteriophages and are essential for host infection and progeny release, respectively 1 2 3 4 5 . Autolysins are endogenous cell wall hydrolases and play active roles in cell wall synthesis and remodeling, and cell division 6 7 8 . Bacteriolysins are generated and secreted by certain bacteria to selectively target other competing bacteria 9 10 11 .…”

LytM Fusion with SH3b-Like Domain Expands Its Activity to Physiological Conditions

“… 11 , 39 , 40 Even if similar assays are reported, the reactions are often carried out in different conditions making the comparison of the results very difficult, or even impossible. 48 …”

“…Still, they seem to have the greatest staphylolytic activity among autolysin-based chimeric enzymes. 48 …”