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

2021 ◽  
Vol 118 (44) ◽  
pp. e2106022118
Author(s):  
Bartłomiej Salamaga ◽  
Lingyuan Kong ◽  
Laia Pasquina-Lemonche ◽  
Lucia Lafage ◽  
Milena von und zur Muhlen ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Turgor Pressure ◽  
Regulatory System ◽  
Bactericidal Effect ◽  
Hydrolase Activity ◽  
Peptidoglycan Hydrolase ◽  
Peptidoglycan Synthesis ◽  
Peptidoglycan Hydrolases ◽  
The Face

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.

scholarly journals EssH Peptidoglycan Hydrolase EnablesStaphylococcus aureusType VII Secretion across the Bacterial Cell Wall Envelope

2018 ◽  
Vol 200 (20) ◽  
Author(s):  
Maksym Bobrovskyy ◽  
Stephanie E. Willing ◽  
Olaf Schneewind ◽  
Dominique Missiakas
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Bacterial Cell ◽  
Bacterial Cell Wall ◽  
Hydrolase Activity ◽  
Amide Bond ◽  
Peptidoglycan Hydrolase ◽  
Bacterial Membrane ◽  
Secretion Systems ◽  
Content Type

ABSTRACTThe ESAT-6-like secretion system (ESS) ofStaphylococcus aureusis assembled in the bacterial membrane from core components that promote the secretion of WXG-like proteins (EsxA, EsxB, EsxC, and EsxD) and the EssD effector. Genes encoding the ESS secretion machinery components, effector, and WXG-like proteins are located in theesslocus. Here, we identifyessH, a heretofore uncharacterized gene of theesslocus, whose product is secreted via an N-terminal signal peptide into the extracellular medium of staphylococcal cultures. EssH exhibits two peptidoglycan hydrolase activities, cleaving the pentaglycine cross bridge and the amide bond ofN-acetylmuramyl-l-alanine, thereby separating glycan chains and wall peptides with cleaved cross bridges. Unlike other peptidoglycan hydrolases, EssH does not promote the lysis of staphylococci. EssH residues Cys199and His254, which are conserved in other CHAP domain enzymes, are required for peptidoglycan hydrolase activity and forS. aureusESS secretion. These data suggest that EssH and its murein hydrolase activity are required for protein secretion by the ESS pathway.IMPORTANCEGene clusters encoding WXG-like proteins and FtsK/SpoIIIE-like P loop ATPases inFirmicutesencode type 7b secretion systems (T7bSS) for the transport of select protein substrates. TheStaphylococcus aureusT7bSS assembles in the bacterial membrane and promotes the secretion of WXG-like proteins and effectors. The mechanisms whereby staphylococci extend the T7SS across the bacterial cell wall envelope are not known. Here, we show that staphylococci secrete EssH to cleave their peptidoglycan, thereby enabling T7bSS transport of proteins across the bacterial cell wall envelope.

scholarly journals Class A PBPs have a distinct and unique role in the construction of the pneumococcal cell wall

2020 ◽  
Vol 117 (11) ◽  
pp. 6129-6138 ◽  
Author(s):  
Daniel Straume ◽  
Katarzyna Wiaroslawa Piechowiak ◽  
Silje Olsen ◽  
Gro Anita Stamsås ◽  
Kari Helene Berg ◽  
...  
Keyword(s):  
Cell Wall ◽  
Peptidoglycan Hydrolase ◽  
Unique Role ◽  
Penicillin Binding Proteins ◽  
The Core ◽  
Class A ◽  
Peptidoglycan Synthesis ◽  
Resistant Form ◽  
Class B ◽  
Open Question

In oval-shapedStreptococcus pneumoniae, septal and longitudinal peptidoglycan syntheses are performed by independent functional complexes: the divisome and the elongasome. Penicillin-binding proteins (PBPs) were long considered the key peptidoglycan-synthesizing enzymes in these complexes. Among these were the bifunctional class A PBPs, which are both glycosyltransferases and transpeptidases, and monofunctional class B PBPs with only transpeptidase activity. Recently, however, it was established that the monofunctional class B PBPs work together with transmembrane glycosyltransferases (FtsW and RodA) from the shape, elongation, division, and sporulation (SEDS) family to make up the core peptidoglycan-synthesizing machineries within the pneumococcal divisome (FtsW/PBP2x) and elongasome (RodA/PBP2b). The function of class A PBPs is therefore now an open question. Here we utilize the peptidoglycan hydrolase CbpD that targets the septum ofS. pneumoniaecells to show that class A PBPs have an autonomous role during pneumococcal cell wall synthesis. Using assays to specifically inhibit the function of PBP2x and FtsW, we demonstrate that CbpD attacks nascent peptidoglycan synthesized by the divisome. Notably, class A PBPs could process this nascent peptidoglycan from a CbpD-sensitive to a CbpD-resistant form. The class A PBP-mediated processing was independent of divisome and elongasome activities. Class A PBPs thus constitute an autonomous functional entity which processes recently formed peptidoglycan synthesized by FtsW/PBP2×. Our results support a model in which mature pneumococcal peptidoglycan is synthesized by three functional entities, the divisome, the elongasome, and bifunctional PBPs. The latter modify existing peptidoglycan but are probably not involved in primary peptidoglycan synthesis.

scholarly journals SEDS-bPBP pairs direct Lateral and Septal Peptidoglycan Synthesis in Staphylococcus aureus

2019 ◽  
Author(s):  
Nathalie T. Reichmann ◽  
Andreia C. Tavares ◽  
Bruno M. Saraiva ◽  
Ambre Jousselin ◽  
Patricia Reed ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Cell Division ◽  
Late Stage ◽  
Cell Elongation ◽  
Cell Shape ◽  
Protein A ◽  
Interacting Proteins ◽  
Peptidoglycan Synthesis ◽  
Multiple Rings

Peptidoglycan (PGN) is the major component of the bacterial cell wall, a structure essential for the physical integrity and shape of the cell. Bacteria maintain cell shape by directing PGN incorporation to distinct regions of the cell, namely through the localisation of the late stage PGN synthesis proteins. These include two key protein families, SEDS transglycosylases and the bPBP transpeptidases, proposed to function in cognate pairs. Rod-shaped bacteria have two SEDS-bPBP pairs, involved in cell elongation and cell division. Here, we elucidate why coccoid bacteria, such as Staphylococcus aureus, also possess two SEDS-bPBP pairs. We determined that S. aureus RodA-PBP3 and FtsW-PBP1 likely constitute cognate pairs of interacting proteins. Lack of RodA-PBP3 decreased cell eccentricity due to deficient pre-septal PGN synthesis, whereas the depletion of FtsW-PBP1 arrested normal septal PGN incorporation. Although PBP1 is an essential protein, a mutant lacking PBP1 transpeptidase activity is viable, showing that this protein has a second function. We propose that the FtsW-PBP1 pair has a role in stabilising the divisome at midcell. In the absence of these proteins, the divisome appears as multiple rings/arcs that drive lateral PGN incorporation, leading to cell elongation. We conclude that RodA-PBP3 and FtsW-PBP1 mediate lateral and septal PGN incorporation, respectively, and that the activity of these pairs must be balanced in order to maintain coccoid morphology.

scholarly journals YycH and YycI Regulate Expression of Staphylococcus aureus Autolysins by Activation of WalRK Phosphorylation

2020 ◽  
Vol 8 (6) ◽  
pp. 870
Author(s):  
Mike Gajdiss ◽  
Ian R. Monk ◽  
Ute Bertsche ◽  
Janina Kienemund ◽  
Tanja Funk ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Teichoic Acid ◽  
Regulatory System ◽  
Regulatory Function ◽  
Wall Teichoic Acid ◽  
Dependent Cell ◽  
Severe Infections

Staphylococcus aureus is a facultative pathogen that can encode numerous antibiotic resistance and immune evasion genes and can cause severe infections. Reduced susceptibility to last resort antibiotics such as vancomycin and daptomycin is often associated with mutations in walRK, an essential two-component regulatory system (TCS). This study focuses on the WalK accessory membrane proteins YycH and YycI and their influence on WalRK phosphorylation. Depletion of YycH and YycI by antisense RNA caused an impaired autolysis, indicating a positive regulatory function on WalK as has been previously described. Phosphorylation assays with full-length recombinant proteins in phospholipid liposomes showed that YycH and YycI stimulate WalK activity and that both regulatory proteins are needed for full activation of the WalK kinase. This was validated in vivo through examining the phosphorylation status of WalR using Phos-tag SDS-PAGE with a yycHI deletion mutant exhibiting reduced levels of phosphorylated WalR. In the yycHI knockdown strain, muropeptide composition of the cell wall was not affected, however, the wall teichoic acid content was increased. In conclusion, a direct modulation of WalRK phosphorylation activity by the accessory proteins YycH and YycI is reported both in vitro and in vivo. Taken together, our results show that YycH and YycI are important in the direct regulation of WalRK-dependent cell wall metabolism.

scholarly journals FtsEX is required for CwlO peptidoglycan hydrolase activity during cell wall elongation inBacillus subtilis

2013 ◽  
Vol 89 (6) ◽  
pp. 1069-1083 ◽  
Author(s):  
Jeffrey Meisner ◽  
Paula Montero Llopis ◽  
Lok-To Sham ◽  
Ethan Garner ◽  
Thomas G. Bernhardt ◽  
...  
Keyword(s):  
Cell Wall ◽  
Hydrolase Activity ◽  
Peptidoglycan Hydrolase

scholarly journals Bacterial Cell Enlargement Requires Control of Cell Wall Stiffness Mediated by Peptidoglycan Hydrolases

mBio ◽  
2015 ◽  
Vol 6 (4) ◽  
Author(s):  
Richard Wheeler ◽  
Robert D. Turner ◽  
Richard G. Bailey ◽  
Bartłomiej Salamaga ◽  
Stéphane Mesnage ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Fundamental Problem ◽  
Biochemical Properties ◽  
Major Advance ◽  
Bacterial Cells ◽  
Cell Enlargement ◽  
Content Type ◽  
Wall Stiffness ◽  
Peptidoglycan Hydrolases

ABSTRACTMost bacterial cells are enclosed in a single macromolecule of the cell wall polymer, peptidoglycan, which is required for shape determination and maintenance of viability, while peptidoglycan biosynthesis is an important antibiotic target. It is hypothesized that cellular enlargement requires regional expansion of the cell wall through coordinated insertion and hydrolysis of peptidoglycan. Here, a group of (apparent glucosaminidase) peptidoglycan hydrolases are identified that are together required for cell enlargement and correct cellular morphology ofStaphylococcus aureus, demonstrating the overall importance of this enzyme activity. These are Atl, SagA, ScaH, and SagB. The major advance here is the explanation of the observed morphological defects in terms of the mechanical and biochemical properties of peptidoglycan. It was shown that cells lacking groups of these hydrolases have increased surface stiffness and, in the absence of SagB, substantially increased glycan chain length. This indicates that, beyond their established roles (for example in cell separation), some hydrolases enable cellular enlargement by making peptidoglycan easier to stretch, providing the first direct evidence demonstrating that cellular enlargement occurs via modulation of the mechanical properties of peptidoglycan.IMPORTANCEUnderstanding bacterial growth and division is a fundamental problem, and knowledge in this area underlies the treatment of many infectious diseases. Almost all bacteria are surrounded by a macromolecule of peptidoglycan that encloses the cell and maintains shape, and bacterial cells must increase the size of this molecule in order to enlarge themselves. This requires not only the insertion of new peptidoglycan monomers, a process targeted by antibiotics, including penicillin, but also breakage of existing bonds, a potentially hazardous activity for the cell. UsingStaphylococcus aureus, we have identified a set of enzymes that are critical for cellular enlargement. We show that these enzymes are required for normal growth and define the mechanism through which cellular enlargement is accomplished, i.e., by breaking bonds in the peptidoglycan, which reduces the stiffness of the cell wall, enabling it to stretch and expand, a process that is likely to be fundamental to many bacteria.

scholarly journals Role of VraSR in Antibiotic Resistance and Antibiotic-Induced Stress Response in Staphylococcus aureus

2006 ◽  
Vol 50 (10) ◽  
pp. 3424-3434 ◽  
Author(s):  
S. Gardete ◽  
S. W. Wu ◽  
S. Gill ◽  
A. Tomasz
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Stress Response ◽  
Critical Role ◽  
Regulatory System ◽  
Experimental System ◽  
Cell Wall Synthesis ◽  
Beta Lactam ◽  
Active Inhibitor

ABSTRACT Exposure of Staphylococcus aureus to cell wall inhibitors induces massive overexpression of a number of genes, provided that the VraSR two-component sensory regulatory system is intact. Inactivation of vraS blocks this transcriptional response and also causes a drastic reduction in the levels of resistance to beta-lactam antibiotics and vancomycin. We used an experimental system in which the essential cell wall synthesis gene of S. aureus, pbpB, was put under the control of an isopropyl-β-d-thiogalactopyranoside-inducible promoter in order to induce reversible perturbations in cell wall synthesis without the use of any cell wall-active inhibitor. Changes in the level of transcription of pbpB were rapidly followed by parallel changes in the vraSR signal, and the abundance of the pbpB transcript was precisely mirrored by the abundance of the transcripts of vraSR and some additional genes that belong to the VraSR regulon. Beta-lactam resistance in S. aureus appears to involve a complex stress response in which VraSR performs the critical role of a sentinel system capable of sensing the perturbation of cell wall synthesis and allowing mobilization of genes that are essential for the generation of a highly resistant phenotype. One of the sites in cell wall synthesis “sensed” by the VraSR system appears to be a step catalyzed by PBP 2.

scholarly journals Characterization of IsaA and SceD, Two Putative Lytic Transglycosylases of Staphylococcus aureus

2007 ◽  
Vol 189 (20) ◽  
pp. 7316-7325 ◽  
Author(s):  
Melanie R. Stapleton ◽  
Malcolm J. Horsburgh ◽  
Emma J. Hayhurst ◽  
Lynda Wright ◽  
Ing-Marie Jonsson ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Cell Separation ◽  
Dynamic Structure ◽  
Lytic Transglycosylases ◽  
Peptidoglycan Hydrolases ◽  
Cotton Rats ◽  
Autolytic Activity ◽  
Wall Growth

ABSTRACT Bacterial cell wall peptidoglycan is a dynamic structure requiring hydrolysis to allow cell wall growth and division. Staphylococcus aureus has many known and putative peptidoglycan hydrolases, including two likely lytic transglycosylases. These two proteins, IsaA and SceD, were both found to have autolytic activity. Regulatory studies showed that the isaA and sceD genes are partially mutually compensatory and that the production of SceD is upregulated in an isaA mutant. The expression of sceD is also greatly upregulated by the presence of NaCl. Several regulators of isaA and sceD expression were identified. Inactivation of sceD resulted in impaired cell separation, as shown by light microscopy, and “clumping” of bacterial cultures. An isaA sceD mutant is attenuated for virulence, while SceD is essential for nasal colonization in cotton rats, thus demonstrating the importance of cell wall dynamics in host-pathogen interactions.

scholarly journals Enhanced Staphylolytic Activity of the Staphylococcus aureus Bacteriophage vB_SauS-phiIPLA88 HydH5 Virion-Associated Peptidoglycan Hydrolase: Fusions, Deletions, and Synergy with LysH5

2012 ◽  
Vol 78 (7) ◽  
pp. 2241-2248 ◽  
Author(s):  
Lorena Rodríguez-Rubio ◽  
Beatriz Martínez ◽  
Ana Rodríguez ◽  
David M. Donovan ◽  
Pilar García
Keyword(s):  
Staphylococcus Aureus ◽  
Fusion Proteins ◽  
Antimicrobial Agents ◽  
Lytic Activity ◽  
Peptidoglycan Hydrolase ◽  
Content Type ◽  
Gram Positive Bacteria ◽  
Peptidoglycan Hydrolases ◽  
Antimicrobial Synergy

ABSTRACTVirion-associated peptidoglycan hydrolases have potential as antimicrobial agents due to their ability to lyse Gram-positive bacteria on contact. In this work, our aim was to improve the lytic activity of HydH5, a virion-associated peptidoglycan hydrolase from theStaphylococcus aureusbacteriophage vB_SauS-phiIPLA88. Full-length HydH5 and two truncated derivatives containing only the CHAP (cysteine, histidine-dependent amidohydrolase/peptidase) domain exhibited high lytic activity against liveS. aureuscells. In addition, three different fusion proteins were created between lysostaphin and HydH5, each of which showed higher staphylolytic activity than the parental enzyme or its deletion construct. Both parental and fusion proteins lysedS. aureuscells in zymograms and plate lysis and turbidity reduction assays. In plate lysis assays, HydH5 and its derivative fusions lysed bovine and humanS. aureusstrains, the methicillin-resistantS. aureus(MRSA) strain N315, and humanStaphylococcus epidermidisstrains. Several nonstaphylococcal bacteria were not affected. HydH5 and its derivative fusion proteins displayed antimicrobial synergy with the endolysin LysH5in vitro, suggesting that the two enzymes have distinct cut sites and, thus, may be more efficient in combination for the elimination of staphylococcal infections.

Susceptibility of clinical isolates of Staphylococcus aureus to killing by oxacillin

1975 ◽  
Vol 21 (11) ◽  
pp. 1692-1697 ◽  
Author(s):  
Gary K. Best ◽  
Ann V. Koval ◽  
Norma H. Best
Keyword(s):  
Staphylococcus Aureus ◽  
Growth Rate ◽  
Cell Wall ◽  
Minimal Inhibitory Concentration ◽  
Inhibitory Concentration ◽  
Bactericidal Effect ◽  
Clinical Isolates ◽  
Cell Wall Synthesis ◽  
Abnormal Growth ◽  
Killing Action

Sixty clinical isolates of Staphylococcus aureus have been screened for their relative susceptibility to the killing action of oxacillin. Only one of these strains was found to be exceptionally resistant to the bactericidal effect of this and other β-lactam antibiotics. This ability to survive oxacillin inhibition of cell wall synthesis has been called "tolerance." The characteristics of the tolerant organism, which has been designated the Evans strain, in comparison with other isolates of S. aureus indicate that this form of resistance is not apparent from the minimal inhibitory concentration, is not related to an abnormal growth rate, and can be enhanced by treatment with N-methyl-N′-nitro-N-nitrosoguanidine.

Sign in / Sign up

Export Citation Format

Share Document