Staphylococcus aureus cell wall structure and dynamics during host-pathogen interaction

Abstract

Peptidoglycan is the major structural component of the Staphylococcus aureus cell wall, in which it maintains cellular integrity, is the interface with the host, and its synthesis is targeted by some of the most crucial antibiotics developed. Despite this importance, and the wealth of data from in vitro studies, we do not understand the structure and dynamics of peptidoglycan during infection. In this study we have developed methods to harvest bacteria from an active infection in order to purify cell walls for biochemical analysis ex vivo. Isolated ex vivo bacterial cells are smaller than those actively growing in vitro, with thickened cell walls and reduced peptidoglycan crosslinking, similar to that of stationary phase cells. These features suggested a role for specific peptidoglycan homeostatic mechanisms in disease. As S. aureus penicillin binding protein 4 (PBP4) has reduced peptidoglycan crosslinking in vitro its role during infection was established. Loss of PBP4 resulted in an increased recovery of S. aureus from the livers of infected mice, which correlated with enhanced fitness within murine and human macrophages. Thicker cell walls correlate with reduced activity of peptidoglycan hydrolases. S. aureus has a family of 4 putative glucosaminidases, that are collectively crucial for growth. Loss of the major enzyme SagB, led to attenuation during murine infection and reduced survival in human macrophages. However, loss of the other three enzymes Atl, SagA and ScaH resulted in clustering dependent attenuation, in a zebrafish embryo, but not a murine, model of infection. A combination of pbp4 and sagB deficiencies resulted in a restoration of parental virulence. Our results, demonstrate the importance of appropriate cell wall structure and dynamics during pathogenesis, providing new insight to the mechanisms of disease.

Human serum triggers antibiotic tolerance in Staphylococcus aureus

“…Importantly, this increase in peptidoglycan is in line with the results of other studies that have investigated the differences between S. aureus grown in culture medium and in model host environments. For example, Hines et al demonstrated that the cell walls of S. aureus grown in 20 % human serum were twice as thick as those grown in TSB 33 , while Sutton et al observed that the cell walls of S. aureus isolated from an experimental murine kidney abscess model were significantly thicker than those of cells grown in vitro 35 . Therefore, we are confident that the results from our ex vivo model system are applicable to the host environment during infection.…”

“…Laboratory media lack host-derived nutrients and macromolecules, resulting in significant differences between the metabolism and physiology of S. aureus in vitro and in vivo. Additionally, phenotypic studies have identified that growth in model host environments affects properties of the staphylococcal cell envelope, including affecting the structure of the cell wall and the composition and properties of the cell membrane [33][34][35] . For example, incorporation of serum unsaturated fatty acids affected membrane fluidity, a factor known to influence the susceptibility of S. aureus to membrane-targeting antimicrobials 34,36 .…”

Rotating Magnetic Field Increases β-Lactam Antibiotic Susceptibility of Methicillin-Resistant Staphylococcus aureus Strains

“…These changes included mostly the shape and size of cells; also collapse of cell walls and cellular leakage was observed (Figure 8a,b, Supplementary Figure S8). Noteworthy is that with the exception of the last type of alteration, of a rather irreversible character, one may assume that changes in such altered cells as those pictured in Figure 8a (and marked with numbers 2 and 3) could be reversed [69] if the cells were seeded in a fresh microbiological medium and cultivated without RMF presence. Such a phenomenon largely explains the lack of changes in the number between cultivable bacteria exposed and unexposed to RMF (Figure 5) and a drop in live cells (dyed with SYTO9) in the RMF-exposed vs unexposed setting (Figure 6).…”

PBP1 ofStaphylococcus aureushas multiple essential functions in cell division

“…S. aureus strains were prepared for electron microscopy as previously described (Sutton et al, 2021).…”

“…Peptidoglycan from S. aureus cells was extracted and if required HF treated to remove cell wall polymers as previously described (Sutton et al, 2021).…”