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Electrostatic Interaction with the Bacterial Cell Envelope Tunes the Lytic Activity of Two Novel Peptidoglycan Hydrolases.

ABSTRACT: 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.

SUBMITTER: Wysocka A

PROVIDER: S-EPMC9241647 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:UnlabelledMost 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.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. 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.

Project description:Bdellovibrio are predatory bacteria that have evolved to invade virtually all gram-negative bacteria, including many prominent pathogens. Upon invasion, prey bacteria become rounded up into an osmotically stable niche for the Bdellovibrio, preventing further superinfection and allowing Bdellovibrio to replicate inside without competition, killing the prey bacterium and degrading its contents. Historically, prey rounding was hypothesized to be associated with peptidoglycan (PG) metabolism; we found two Bdellovibrio genes, bd0816 and bd3459, expressed at prey entry and encoding proteins with limited homologies to conventional dacB/PBP4 DD-endo/carboxypeptidases (responsible for peptidoglycan maintenance during growth and division). We tested possible links between Bd0816/3459 activity and predation. Bd3459, but not an active site serine mutant protein, bound ?-lactam, exhibited DD-endo/carboxypeptidase activity against purified peptidoglycan and, importantly, rounded up E. coli cells upon periplasmic expression. A ?Bd0816 ?Bd3459 double mutant invaded prey more slowly than the wild type (with negligible prey cell rounding) and double invasions of single prey by more than one Bdellovibrio became more frequent. We solved the crystal structure of Bd3459 to 1.45 Å and this revealed predation-associated domain differences to conventional PBP4 housekeeping enzymes (loss of the regulatory domain III, alteration of domain II and a more exposed active site). The Bd3459 active site (and by similarity the Bd0816 active site) can thus accommodate and remodel the various bacterial PGs that Bdellovibrio may encounter across its diverse prey range, compared to the more closed active site that "regular" PBP4s have for self cell wall maintenance. Therefore, during evolution, Bdellovibrio peptidoglycan endopeptidases have adapted into secreted predation-specific proteins, preventing wasteful double invasion, and allowing activity upon the diverse prey peptidoglycan structures to sculpt the prey cell into a stable intracellular niche for replication.

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Electrostatic Interaction with the Bacterial Cell Envelope Tunes the Lytic Activity of Two Novel Peptidoglycan Hydrolases.

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