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.

Potential Application of Phage ɸ11 Lytic Proteins in Rapid Detection and Elimination of Staphylococcus aureus

The increasing antibiotic resistance conferred by Staphylococcus aureus to multiple potential antibiotics has become a serious issue of concern and threat to mankind worldwide. In light of this, phage lytic proteins have been reported which show potential antimicrobial activity against pathogenic microorganisms that could be a promising alternative to antibiotics to eradicate the antibiotic resistant problems. This review discusses the various applications of S. aureus phage lytic proteins and the potentiality of aureophage phi 11 endolysin and virion associated peptidoglycan hydrolase (VAPGH) against staphylococcus strains. Phage Phi11 endolysin harbors two enzymatically active domain; cysteine and histidine-dependent amidohydrolase/peptidase (CHAP) and Amidase 2 at the N-terminus and a cell wall binding domain (CBD) SH3 5 at the C-terminus, while virion associated peptidoglycan hydrolase (VAPGH) has two catalytic domains, CHAP and Glucosaminidase (Mannosyl-glycoprotein endo-beta-N-acetylglucosaminidase) at its N-terminal and C-terminal, respectively.

The LbcA lipoprotein and CtpA protease assemble an oligomeric complex to control peptidoglycan hydrolases in Pseudomonas aeruginosa

Pseudomonas aeruginosa CtpA is a carboxyl terminal–processing protease that partners with the outer membrane lipoprotein LbcA to degrade cell wall cross-link hydrolases. This activity plays an important role in supporting P. aeruginosa virulence. However, almost nothing is known about the molecular mechanisms underlying CtpA and LbcA function. Here, we used structural analysis to show that CtpA alone assembles into an inactive hexamer comprising a trimer of dimers, which limits its substrate access and prevents nonspecific degradation. The adaptor protein LbcA is a right-handed open spiral with 11 tetratricopeptide repeats, which might wrap around a substrate to deliver it to CtpA for degradation. We found that up to three LbcA molecules can bind to one CtpA hexamer to assemble a giant, active protease complex that degrades its peptidoglycan hydrolase substrates both in vitro and in vivo. This work reveals an intricate protease activation mechanism that is substrate delivery-dependent and enables targeted removal of the peptidoglycan hydrolase substrates.

Ser/Thr Kinase-Dependent Phosphorylation of the Peptidoglycan Hydrolase CwlA Controls Its Export and Modulates Cell Division in Clostridioides difficile

ABSTRACT Cell growth and division require a balance between synthesis and hydrolysis of the peptidoglycan (PG). Inhibition of PG synthesis or uncontrolled PG hydrolysis can be lethal for the cells, making it imperative to control peptidoglycan hydrolase (PGH) activity. The synthesis or activity of several key enzymes along the PG biosynthetic pathway is controlled by the Hanks-type serine/threonine kinases (STKs). In Gram-positive bacteria, inactivation of genes encoding STKs is associated with a range of phenotypes, including cell division defects and changes in cell wall metabolism, but only a few kinase substrates and associated mechanisms have been identified. We previously demonstrated that STK-PrkC plays an important role in cell division, cell wall metabolism, and resistance to antimicrobial compounds in the human enteropathogen Clostridioides difficile. In this work, we characterized a PG hydrolase, CwlA, which belongs to the NlpC/P60 family of endopeptidases and hydrolyses cross-linked PG between daughter cells to allow cell separation. We identified CwlA as the first PrkC substrate in C. difficile. We demonstrated that PrkC-dependent phosphorylation inhibits CwlA export, thereby controlling hydrolytic activity in the cell wall. High levels of CwlA at the cell surface led to cell elongation, whereas low levels caused cell separation defects. Thus, we provided evidence that the STK signaling pathway regulates PGH homeostasis to precisely control PG hydrolysis during cell division. IMPORTANCE Bacterial cells are encased in a PG exoskeleton that helps to maintain cell shape and confers physical protection. To allow bacterial growth and cell separation, PG needs to be continuously remodeled by hydrolytic enzymes that cleave PG at critical sites. How these enzymes are regulated remains poorly understood. We identify a new PG hydrolase involved in cell division, CwlA, in the enteropathogen C. difficile. Lack or accumulation of CwlA at the bacterial surface is responsible for a division defect, while its accumulation in the absence of PrkC also increases susceptibility to antimicrobial compounds targeting the cell wall. CwlA is a substrate of the kinase PrkC in C. difficile. PrkC-dependent phosphorylation controls the export of CwlA, modulating its levels and, consequently, its activity in the cell wall. This work provides a novel regulatory mechanism by STK in tightly controlling protein export.

Identification and characterization of a peptidoglycan hydrolase from Rhodobacter johrii

The bacterial whole genome sequences are available in the database therefore explored for the varieties of known and unknown proteins. Bacteria harbor various peptidoglycan hydrolases that cleave peptidoglycan and play an important role in the cell division, growth, spore differentiation and development. In the present study, we report a peptidoglycan hydrolase in an endospore producing phototrophic proteobacterium Rhodobacter johrii. The Peptidoglycan Hydrolase of Rba. johrii (PgHR) can actively hydrolyze the intact spore cortex peptidoglycan (sacculi). The protein contains a pre-peptide precursor which has a Hydrolase-2 (PF07486) family conserved domain. PgHR protein has SleB like properties which are spore cortex-lytic enzymes involved in the depolymerization of cortex peptidoglycan present and characterized in Bacillus spp. The expression pattern of PgHR through qRT-PCR suggests its role in stationary phase of Rba. johrii. This is a new type of peptidoglycan hydrolase reported from a proteobacterium.

Structural basis for recognition of bacterial cell wall teichoic acid by pseudo-symmetric SH3b-like repeats of a viral peptidoglycan hydrolase

Combining genetic, biochemical and computational approaches, we elucidated the molecular mechanisms underlying the recognition of Listeria wall teichoic acid by bacteriophage-encoded SH3b repeats.