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.

Central European Institute of Technology Masaryk University Brno 60177 Czech Republic

Department of Infection Immunity and Cardiovascular Diseases University of Sheffield Sheffield S102RX United Kingdom

Department of Physics and Astronomy University of Sheffield Sheffield S37RH United Kingdom

M G DeGroote Institute for Infectious Disease Research David Braley Centre for Antibiotic Discovery Department of Biochemistry and Biomedical Sciences McMaster University Hamilton ON L8S4L8 Canada

School of Biosciences University of Sheffield Sheffield S102TN United Kingdom

School of Biosciences University of Sheffield Sheffield S102TN United Kingdom;

State Key Laboratory of Cellular Stress Biology School of Life Sciences Xiamen University Xiamen 361102 China

State Key Laboratory of Cellular Stress Biology School of Life Sciences Xiamen University Xiamen 361102 China;

The Bateson Centre University of Sheffield Sheffield S102TN United Kingdom

The Florey Institute for Host Pathogen Interactions University of Sheffield Sheffield S102TN United Kingdom

The Florey Institute for Host Pathogen Interactions University of Sheffield Sheffield S102TN United Kingdom;

See more in PubMed

Koch A. L., Growth and form of the bacterial cell wall. Am. Sci. 78, 327–341 (1990).

Yadav A. K., Espaillat A., Cava F., Bacterial strategies to preserve cell wall integrity against environmental threats. Front. Microbiol. 9, 2064 (2018). PubMed PMC

Dörr T., Moynihan P. J., Mayer C., Editorial: Bacterial cell wall structure and dynamics. Front. Microbiol. 10, 2051 (2019). PubMed PMC

Turner R. D., Vollmer W., Foster S. J., Different walls for rods and balls: The diversity of peptidoglycan. Mol. Microbiol. 91, 862–874 (2014). PubMed PMC

Vollmer W., Blanot D., de Pedro M. A., Peptidoglycan structure and architecture. FEMS Microbiol. Rev. 32, 149–167 (2008). PubMed

Schneider T., Sahl H. G., An oldie but a goodie—Cell wall biosynthesis as antibiotic target pathway. Int. J. Med. Microbiol. 300, 161–169 (2010). PubMed

Cho H., Uehara T., Bernhardt T. G., Beta-lactam antibiotics induce a lethal malfunctioning of the bacterial cell wall synthesis machinery. Cell 159, 1300–1311 (2014). PubMed PMC

Giesbrecht P., Kersten T., Maidhof H., Wecke J., Staphylococcal cell wall: Morphogenesis and fatal variations in the presence of penicillin. Microbiol. Mol. Biol. Rev. 62, 1371–1414 (1998). PubMed PMC

Kawai Y., et al. ., Crucial role for central carbon metabolism in the bacterial L-form switch and killing by β-lactam antibiotics. Nat. Microbiol. 4, 1716–1726 (2019). PubMed PMC

Kohanski M. A., Dwyer D. J., Hayete B., Lawrence C. A., Collins J. J., A common mechanism of cellular death induced by bactericidal antibiotics. Cell 130, 797–810 (2007). PubMed

Tomasz A., The mechanism of the irreversible antimicrobial effects of penicillins: How the beta-lactam antibiotics kill and lyse bacteria. Annu. Rev. Microbiol. 33, 113–137 (1979). PubMed

Tomasz A., Albino A., Zanati E., Multiple antibiotic resistance in a bacterium with suppressed autolytic system. Nature 227, 138–140 (1970). PubMed

Lee T. K., Huang K. C., The role of hydrolases in bacterial cell-wall growth. Curr. Opin. Microbiol. 16, 760–766 (2013). PubMed PMC

Vollmer W., Joris B., Charlier P., Foster S., Bacterial peptidoglycan (murein) hydrolases. FEMS Microbiol. Rev. 32, 259–286 (2008). PubMed

Goffin C., Ghuysen J. M., Multimodular penicillin-binding proteins: An enigmatic family of orthologs and paralogs. Microbiol. Mol. Biol. Rev. 62, 1079–1093 (1998). PubMed PMC

Wada A., Watanabe H., Penicillin-binding protein 1 of Staphylococcus aureus is essential for growth. J. Bacteriol. 180, 2759–2765 (1998). PubMed PMC

Pereira S. F., Henriques A. O., Pinho M. G., de Lencastre H., Tomasz A., Role of PBP1 in cell division of Staphylococcus aureus. J. Bacteriol. 189, 3525–3531 (2007). PubMed PMC

Pereira S. F., Henriques A. O., Pinho M. G., de Lencastre H., Tomasz A., Evidence for a dual role of PBP1 in the cell division and cell separation of Staphylococcus aureus. Mol. Microbiol. 72, 895–904 (2009). PubMed PMC

Pinho M. G., Filipe S. R., de Lencastre H., Tomasz A., Complementation of the essential peptidoglycan transpeptidase function of penicillin-binding protein 2 (PBP2) by the drug resistance protein PBP2A in Staphylococcus aureus. J. Bacteriol. 183, 6525–6531 (2001). PubMed PMC

Wheeler R., et al. ., Bacterial cell enlargement requires control of cell wall stiffness mediated by peptidoglycan hydrolases. MBio 6, e00660 (2015). PubMed PMC

Chan Y. G., Frankel M. B., Missiakas D., Schneewind O., SagB glucosaminidase is a determinant of staphylococcus aureus glycan chain length, antibiotic susceptibility, and protein secretion. J. Bacteriol. 198, 1123–1136 (2016). PubMed PMC

Foster S. J., Molecular characterization and functional analysis of the major autolysin of Staphylococcus aureus 8325/4. J. Bacteriol. 177, 5723–5725 (1995). PubMed PMC

Oshida T., et al. ., A Staphylococcus aureus autolysin that has an N-acetylmuramoyl-L-alanine amidase domain and an endo-beta-N-acetylglucosaminidase domain: Cloning, sequence analysis, and characterization. Proc. Natl. Acad. Sci. U.S.A. 92, 285–289 (1995). PubMed PMC

Kajimura J., et al. ., Identification and molecular characterization of an N-acetylmuramyl-L-alanine amidase Sle1 involved in cell separation of Staphylococcus aureus. Mol. Microbiol. 58, 1087–1101 (2005). PubMed

Delauné A., et al. ., The WalKR system controls major staphylococcal virulence genes and is involved in triggering the host inflammatory response. Infect. Immun. 80, 3438–3453 (2012). PubMed PMC

Delaune A., et al. ., Peptidoglycan crosslinking relaxation plays an important role in Staphylococcus aureus WalKR-dependent cell viability. PLoS One 6, e17054 (2011). PubMed PMC

Dubrac S., Bisicchia P., Devine K. M., Msadek T., A matter of life and death: Cell wall homeostasis and the WalKR (YycGF) essential signal transduction pathway. Mol. Microbiol. 70, 1307–1322 (2008). PubMed

Pasquina-Lemonche L., et al. ., The architecture of the Gram-positive bacterial cell wall. Nature 582, 294–297 (2020). PubMed PMC

Frankel M. B., Hendrickx A. P., Missiakas D. M., Schneewind O., LytN, a murein hydrolase in the cross-wall compartment of Staphylococcus aureus, is involved in proper bacterial growth and envelope assembly. J. Biol. Chem. 286, 32593–32605 (2011). PubMed PMC

Stapleton M. R., et al. ., Characterization of IsaA and SceD, two putative lytic transglycosylases of Staphylococcus aureus. J. Bacteriol. 189, 7316–7325 (2007). PubMed PMC

Zheng L., Yan M., Fan F., Ji Y., The essential WalK histidine kinase and WalR regulator differentially mediate autolysis of Staphylococcus aureus RN4220. J. Nat. Sci. 1, e111 (2015). PubMed PMC

Dubrac S., Msadek T., Identification of genes controlled by the essential YycG/YycF two-component system of Staphylococcus aureus. J. Bacteriol. 186, 1175–1181 (2004). PubMed PMC

Adhikari R. P., Novick R. P., Subinhibitory cerulenin inhibits staphylococcal exoprotein production by blocking transcription rather than by blocking secretion. Microbiology (Reading) 151, 3059–3069 (2005). PubMed

Rebets Y., et al. ., Moenomycin resistance mutations in Staphylococcus aureus reduce peptidoglycan chain length and cause aberrant cell division. ACS Chem. Biol. 9, 459–467 (2014). PubMed PMC

Weidenmaier C., et al. ., Lack of wall teichoic acids in Staphylococcus aureus leads to reduced interactions with endothelial cells and to attenuated virulence in a rabbit model of endocarditis. J. Infect. Dis. 191, 1771–1777 (2005). PubMed

Corrigan R. M., Abbott J. C., Burhenne H., Kaever V., Gründling A., c-di-AMP is a new second messenger in Staphylococcus aureus with a role in controlling cell size and envelope stress. PLoS Pathog. 7, e1002217 (2011). PubMed PMC

Duthie E. S., Lorenz L. L., Staphylococcal coagulase; mode of action and antigenicity. J. Gen. Microbiol. 6, 95–107 (1952). PubMed

Shafer W. M., Iandolo J. J., Genetics of staphylococcal enterotoxin B in methicillin-resistant isolates of Staphylococcus aureus. Infect. Immun. 25, 902–911 (1979). PubMed PMC

Smith T. J., Blackman S. A., Foster S. J., Autolysins of Bacillus subtilis: Multiple enzymes with multiple functions. Microbiology (Reading) 146, 249–262 (2000). PubMed

Culp E. J., et al. ., Evolution-guided discovery of antibiotics that inhibit peptidoglycan remodelling. Nature 578, 582–587 (2020). PubMed

Biswas R., et al. ., Activity of the major staphylococcal autolysin Atl. FEMS Microbiol. Lett. 259, 260–268 (2006). PubMed

Eirich J., Orth R., Sieber S. A., Unraveling the protein targets of vancomycin in living S. aureus and E. faecalis cells. J. Am. Chem. Soc. 133, 12144–12153 (2011). PubMed

Weidenmaier C., et al. ., Role of teichoic acids in Staphylococcus aureus nasal colonization, a major risk factor in nosocomial infections. Nat. Med. 10, 243–245 (2004). PubMed

Schlag M., et al. ., Role of staphylococcal wall teichoic acid in targeting the major autolysin Atl. Mol. Microbiol. 75, 864–873 (2010). PubMed

Biswas R., et al. ., Proton-binding capacity of Staphylococcus aureus wall teichoic acid and its role in controlling autolysin activity. PLoS One 7, e41415 (2012). PubMed PMC

Gollan B., Grabe G., Michaux C., Helaine S., Bacterial persisters and infection: Past, present, and progressing. Annu. Rev. Microbiol. 73, 359–385 (2019). PubMed

Campbell J., et al. ., Synthetic lethal compound combinations reveal a fundamental connection between wall teichoic acid and peptidoglycan biosyntheses in Staphylococcus aureus. ACS Chem. Biol. 6, 106–116 (2011). PubMed PMC

Farha M. A., et al. ., Inhibition of WTA synthesis blocks the cooperative action of PBPs and sensitizes MRSA to β-lactams. ACS Chem. Biol. 8, 226–233 (2013). PubMed PMC

Lee S. H., et al. ., TarO-specific inhibitors of wall teichoic acid biosynthesis restore β-lactam efficacy against methicillin-resistant staphylococci. Sci. Transl. Med. 8, 329ra32 (2016). PubMed

Sambrook J., Fritsch E. F., Maniatis T., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).

Novick R. P., Morse S. I., In vivo transmission of drug resistance factors between strains of Staphylococcus aureus. J. Exp. Med. 125, 45–59 (1967). PubMed PMC

Schenk S., Laddaga R. A., Improved method for electroporation of Staphylococcus aureus. FEMS Microbiol. Lett. 73, 133–138 (1992). PubMed

Lund V. A., et al. ., Molecular coordination of Staphylococcus aureus cell division. eLife 7, e32057 (2018). PubMed PMC

Turner R. D., Hobbs J. K., Foster S. J., Atomic force microscopy analysis of bacterial cell wall peptidoglycan architecture. Methods Mol. Biol. 1440, 3–9 (2016). PubMed

Maki H., Miura K., Yamano Y., Katanosin B and plusbacin A(3), inhibitors of peptidoglycan synthesis in methicillin-resistant Staphylococcus aureus. Antimicrob. Agents Chemother. 45, 1823–1827 (2001). PubMed PMC

Panchal V. V., et al. ., Evolving MRSA: High-level β-lactam resistance in Staphylococcus aureus is associated with RNA Polymerase alterations and fine tuning of gene expression. PLoS Pathog. 16, e1008672 (2020). PubMed PMC

Boldock E., et al. ., Human skin commensals augment Staphylococcus aureus pathogenesis. Nat. Microbiol. 3, 881–890 (2018). PubMed PMC