Role of peptidoglycan deacetylase in Staphylococcus aureus virulence and survival

Staphylococcus aureus is an opportunist human pathogen that colonises the anterior nares and is shed onto skin. The bacterium is most frequently a cause of skin and soft-tissue infections, yet is also a major cause of respiratory, urinary tract, bone, eye and brain infections. Evolution of the bacterium has selected strains that are more antibiotic resistant, particularly in the hospital environment and more recently in the community. The pathogen has the capacity for secreting a wide range of proteins that contribute to survival from host immunity and competing microflora. The bacterial cell wall is a vital structural polymer serving mechanical roles to protect bacteria from osmotic challenges and it serves as a scaffold for the attachment of many anchored proteins and anionic polymers for interaction with extracellular components. Peptidoglycan of S. aureus is modified by enzyme catalysed decoration of N-acetyl muramic acid with teichoic acid and O-acetylation which promotes lysozyme resistance. Further modification of teichoic acid engenders properties linked to colonisation and survival during the life-cycle. This research study sought to assign roles of S. aureus secreted proteins of unknown function using bioinformatics. From this approach the study identified a peptidoglycan deacetylase (Pgd) and this enzyme catalyses a reduction in acetylation of N-acetyl glucosamine in biosynthesis of the cell wall. An allelic replacement mutant revealed altered muropeptide composition that was consistent with its proposed N-deacetylase activity, and transmission electron microscopy (TEM) revealed a reduction in cell wall thickness. S. aureus pgd exhibited increased surface charge to influence a range of phenotypes, including autolysis, resistance and biofilm formation. Resistance was increased to lysozyme, and most notably there was rescue of the lysozyme sensitivity phenotype of an oatA mutant (lacking peptidoglycan O-acetyltransferase [oatA] activity). Hydrodynamic flow biofilm assay revealed that both S. aureus pgd and oatA mutants exhibited negligible biofilm formation on a glass surface; treatment of the surface with plasma fluid increased biofilm capability. Biofilms of S. aureus pgd cultured in static conditions revealed an opposite phenotype with a thicker, but loosely adherent biofilm. Confocal laser scanning microscopy (CSLM) combined with viability staining it was revealed that in the biofilm, there were increased dead cells compared with wild-type. An increased lag phase of growth of S. aureus pgd was identified, and quantitative label free proteomics was used to determine that multiple metabolism and cell wall proteins were differentially expressed, compared with the wild-type strain. The proteome analysis revealed that the pgd mutant expressed increased levels of the lytic transglycosylase, IsaA and this might explain the biofilm phenotype. Further contributions to the biofilm phenotype could be due to decreased expression of protein A and reduced pyruvate metabolism enzymes. S. aureus pgd was killed more rapidly in an opsonophagocytosis assay compared with its isogenic parent but showed equivalent levels of complement deposition and serum survival. Infection models of pneumonia and bacteraemia were tested and revealed reduced bacterial loads for lung and kidney, respectively. The S. aureus pgd mutant phenotype requires further study and it should provide insights into cell wall structure and function. Pgd could represent a future candidate vaccine antigen due to its likely cell surface exposure and contribution to key cellular processes. Abbreviations APS Ammonium persulphate BHI Brain heart infusion BLAST Basic local alignment search tool CWS Cell wall sorting DNA Deoxyribonucleic acid EDTA Ethylenediaminetetraacetic acid et al. Et alia (and others) Fig. Figure Glu Glucose GlcNAC N-acetylglucosamine IPTG Isopropylthiogalactopyranoside LTA Lipoteichoic acid LC-MS/MS Liquid chromatography tandem mass spectrometry LSM Laser scanning microscopy NMR Nuclear magnetic resonance spectroscopy MurNAC N acetylmuramic acid PAGE Polyacrylamide gel electrophoresis Pbp Penicillin binding protein PBS Phosphate buffered saline PCR Polymerase chain reaction PGT Peptidoglycan glycosyl transferase PMN Polymorphonuclear neutrophils RT Room temperature SDTE SDS, DTT, Tris-HCl, EDTA buffer SDS Sodium dodecylsulphate TAE Tris-HCl, acetate EDTA buffer TEM Transmission electron microscopy TSB Tryptone soy broth ATP Adenosine triphosphate UDP Uridine diphosphate UV Ultraviolet WTA Wall-teichoic acid 3D Three dimensional Measurement and Units bp Base pair oC Degree Celsius cfu Colony forming units Da Dalton G Gram H Hour kDa Kilo dalton kV Kilovolt L Litre M Molar mg Milligram min Minute ml Millilitre mM Millimolar Mw Molecular weight μg Microgram μl Micro litre μM Micro molar μF Micro faraday ng Nanogram nm Nanometre rcf Relative centrifugal force rpm Revolutions per minute sec Second V Volt v/v Volume per volume Table of