Demonstration of the role of cell wall homeostasis in Staphylococcus aureus growth and the action of bactericidal antibiotics

Bacterial cell wall peptidoglycan is essential, maintaining both cellular integrity and morphology, in the face of internal turgor pressure. Peptidoglycan synthesis is important, as it is targeted by cell wall antibiotics, including methicillin and vancomycin. Here, we have used the major human pathogen Staphylococcus aureus to elucidate both the cell wall dynamic processes essential for growth (life) and the bactericidal effects of cell wall antibiotics (death) based on the principle of coordinated peptidoglycan synthesis and hydrolysis. The death of S. aureus due to depletion of the essential, two-component and positive regulatory system for peptidoglycan hydrolase activity (WalKR) is prevented by addition of otherwise bactericidal cell wall antibiotics, resulting in stasis. In contrast, cell wall antibiotics kill via the activity of peptidoglycan hydrolases in the absence of concomitant synthesis. Both methicillin and vancomycin treatment lead to the appearance of perforating holes throughout the cell wall due to peptidoglycan hydrolases. Methicillin alone also results in plasmolysis and misshapen septa with the involvement of the major peptidoglycan hydrolase Atl, a process that is inhibited by vancomycin. The bactericidal effect of vancomycin involves the peptidoglycan hydrolase SagB. In the presence of cell wall antibiotics, the inhibition of peptidoglycan hydrolase activity using the inhibitor complestatin results in reduced killing, while, conversely, the deregulation of hydrolase activity via loss of wall teichoic acids increases the death rate. For S. aureus, the independent regulation of cell wall synthesis and hydrolysis can lead to cell growth, death, or stasis, with implications for the development of new control regimes for this important pathogen.

How bacteria grow and divide is a fundamental question in microbiology, where many of the essential processes involved are the targets of clinically important antibiotics. The cell wall is crucial for bacterial survival, forming the interface between the external and internal environments and maintaining internal turgor pressure (1, 2). The major cell wall structural component is peptidoglycan (PG), a polymer of glycan strands and peptide cross-links (3–5), the synthesis of which is the target of antibiotics including β-lactams and glycopeptides (6). These cell wall antibiotics inhibit the final stages of PG synthesis where building blocks are incorporated into the existing structure via the action of penicillin-binding proteins (PBPs) (6). Several mechanisms linking the action of antibiotics to the inhibition of essential processes in cell wall growth and division have been suggested, including lytic and nonlytic death, oxidative stress, and futile PG synthesis (7–12).As a single macromolecule that surrounds the cell, PG can increase in surface area to permit growth and division while maintaining cellular integrity. It has been proposed that areal PG growth occurs as a consequence of both synthesis and hydrolysis (4, 13, 14), with new material being covalently bound to the existing macrostructure and hydrolysis of existing bonds allowing expansion. This leads to a simple set of hypotheses for growth but also makes predictions as to the effects of inhibition of PG homeostasis activities, including cell wall antibiotics (Fig. 1A). The lack of either PG synthesis or hydrolysis will result in cell death because of the continued activity of the other, but the loss of both will lead to stasis.Open in a separate windowFig. 1.The role of regulation of PG hydrolases (PGHs) by WalKR in life and death. (A) Predictive model for how cell wall homeostasis governs bacterial life and death. Both cell wall synthesis and hydrolysis are required for growth, loss of either results in death, or both, cell stasis. (B–H) Effect of 10 × minimum inhibitory concentration (MIC) vancomycin for 3 h on conditional lethal strain S. aureus P_spac-walKR (without inducer; WalKR OFF) compared to the control (with inducer; WalKR ON). (B) CFU relative to T = 0; after t test with Welch''s correction: P (WalKR OFF − WalKR OFF + vancomycin, **) = 6.9 × 10⁻³. (C and D) PG synthesis and transpeptidase activity measured by ¹⁴C-GlcNAc and Atto 488 dipeptide (53) incorporation, normalized against WalKR ON. (E) Transmission electron microscopy (TEM) (scale bars, 300 nm). (F) Quantification of bacterial phenotypes (SI Appendix, Fig. S2; dark green: no septum, mid-green: incomplete septum, light green: complete septum, and yellow: growth defects). For samples shown, the number of individual cells quantified was n > 300. (G) AFM topographic images of sacculi (scale bars, 150, 300, and 300 nm; data scales [DS], 85, 200, and 85 nm, respectively, from Left to Right). (i) Insets show sacculus external architecture from Left to Right, (WalKR ON) from dashed box in panel G, (WalKR OFF) from SI Appendix, Fig. S2E, (WalKR OFF+Van) from SI Appendix, Fig. S2D, respectively (scale bars, 50 nm; DS, 30, 52, and 32 nm, respectively, from Left to Right; images were analyzed with NanoscopeAnalysis from Bruker using the default color scale). (H) Thickness distribution values for sacculi with SD (n = 5). For sample size and data reproducibility, see Materials and Methods.Staphylococcus aureus is a major human antimicrobial-resistant pathogen. As a spheroid cell with a simple growth and division cycle, it forms an excellent subject to demonstrate the basic principles underlying growth, division, and the action of antibiotics. Many organisms have multiple PBPs, but S. aureus has only four, of which PBP1 and PBP2 are essential for growth and division (15–19). S. aureus also has many PG hydrolases (PGHs), including SagB, which is involved in cell growth (20, 21). The bifunctional Atl is involved in generalized cell lysis and cell separation after septation and contains both amidase and glucosaminidase domains (22, 23). PGHs often show functional redundancy with several enzymes involved in the same process (20, 24). In S. aureus, no individual PGH alone has been shown to be required for either growth or division, but multiple PGHs are positively regulated by an essential two-component system, WalKR (25–27), further suggesting that their collective activity is required.Recently, using atomic force microscopy (AFM), we have revealed that the molecular architecture of the PG is that of an expanded hydrogel whose mature external surface is a porous open network but with an interior surface characterized by a much smoother and denser mesh of PG material (28). This provides an architectural framework from which to begin to elucidate the roles of PG synthesis and hydrolysis. Here, we have taken an integrated approach to determine the role of PG homeostasis in S. aureus growth, division, and the bactericidal action of cell wall antibiotics.