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

ABSTRACT:

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Most 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 of Staphylococcus 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.

Importance

Understanding 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. Using Staphylococcus 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.

SUBMITTER: Wheeler R

PROVIDER: S-EPMC4551982 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Peptidoglycan (PG) hydrolases, due to their crucial role in the metabolism of the bacterial cell wall (CW), are increasingly being considered suitable targets for therapies, and a potent alternative to conventional antibiotics. In the light of contradictory data reported, detailed mechanism of regulation of enzymes activity based on electrostatic interactions between hydrolase molecule and bacterial CW surface remains unknown. Here, we report a comprehensive study on this phenomenon using as a model two novel PG hydrolases, SpM23_A, and SpM23_B, which although share the same bacterial host, similarities in sequence conservation, domain architecture, and structure, display surprisingly distinct net charges (in 2D electrophoresis, pI 6.8, and pI 9.7, respectively). We demonstrate a strong correlation between hydrolases surface net charge and the enzymes activity by modulating the charge of both, enzyme molecule and bacterial cell surface. Teichoic acids, anionic polymers present in the bacterial CW, are shown to be involved in the mechanism of enzymes activity regulation by the electrostatics-based interplay between charged bacterial envelope and PG hydrolases. These data serve as a hint for the future development of chimeric PG hydrolases of desired antimicrobial specificity. IMPORTANCE This study shows direct relationship between the surface charge of two recently described enzymes, SpM23_A and SpM23_B, and bacterial cell walls. We demonstrate that by (i) surface charge probing of bacterial strains collection, (ii) reduction of the net charge of the positively charged enzyme, and (iii) altering the net charge of the bacterial surface by modifying the content and composition of teichoic acids. In all cases, we observed that lytic activity and binding strength of SpM23 enzymes, are regulated by electrostatic interactions with the bacterial cell envelope and that this interaction contributes to the determination of the spectrum of susceptible bacterial species. Moreover, we revealed the regulatory role of charged cell wall components, namely, teichoic and lipoteichoic acids, over the SpM23 enzymes. We believe that our findings make an important contribution to understand the means of hydrolases activity regulation in the complex environment of the bacterial cell wall.

Project description:UnlabelledBacterial SH3 (SH3b) domains are commonly fused with papain-like Nlp/P60 cell wall hydrolase domains. To understand how the modular architecture of SH3b and NlpC/P60 affects the activity of the catalytic domain, three putative NlpC/P60 cell wall hydrolases were biochemically and structurally characterized. These enzymes all have γ-d-Glu-A2pm (A2pm is diaminopimelic acid) cysteine amidase (or dl-endopeptidase) activities but with different substrate specificities. One enzyme is a cell wall lysin that cleaves peptidoglycan (PG), while the other two are cell wall recycling enzymes that only cleave stem peptides with an N-terminal l-Ala. Their crystal structures revealed a highly conserved structure consisting of two SH3b domains and a C-terminal NlpC/P60 catalytic domain, despite very low sequence identity. Interestingly, loops from the first SH3b domain dock into the ends of the active site groove of the catalytic domain, remodel the substrate binding site, and modulate substrate specificity. Two amino acid differences at the domain interface alter the substrate binding specificity in favor of stem peptides in recycling enzymes, whereas the SH3b domain may extend the peptidoglycan binding surface in the cell wall lysins. Remarkably, the cell wall lysin can be converted into a recycling enzyme with a single mutation.ImportancePeptidoglycan is a meshlike polymer that envelops the bacterial plasma membrane and bestows structural integrity. Cell wall lysins and recycling enzymes are part of a set of lytic enzymes that target covalent bonds connecting the amino acid and amino sugar building blocks of the PG network. These hydrolases are involved in processes such as cell growth and division, autolysis, invasion, and PG turnover and recycling. To avoid cleavage of unintended substrates, these enzymes have very selective substrate specificities. Our biochemical and structural analysis of three modular NlpC/P60 hydrolases, one lysin, and two recycling enzymes, show that they may have evolved from a common molecular architecture, where the substrate preference is modulated by local changes. These results also suggest that new pathways for recycling PG turnover products, such as tracheal cytotoxin, may have evolved in bacteria in the human gut microbiome that involve NlpC/P60 cell wall hydrolases.

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

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