The Staphylococcus aureus cell division protein, DivIC, interacts with the cell wall and controls its biosynthesis

Bacterial cell division is a complex, dynamic process that requires multiple protein components to orchestrate its progression. Many division proteins are highly conserved across bacterial species alluding to a common, basic mechanism. Central to division is a transmembrane trimeric complex involving DivIB, DivIC and FtsL in Gram-positives. Here, we show a distinct, essential role for DivIC in division and survival of Staphylococcus aureus. DivIC spatially regulates peptidoglycan synthesis, and consequently cell wall architecture, by influencing the recruitment to the division septum of the major peptidoglycan synthetases PBP2 and FtsW. Both the function of DivIC and its recruitment to the division site depend on its extracellular domain, which interacts with the cell wall via binding to wall teichoic acids. DivIC facilitates the spatial and temporal coordination of peptidoglycan synthesis with the developing architecture of the septum during cell division. A better understanding of the cell division mechanisms in S. aureus and other pathogenic microorganisms can provide possibilities for the development of new, more effective treatments for bacterial infections

The architecture of the Gram-positive bacterial cell wall

The primary structural component of the bacterial cell wall is peptidoglycan, which is essential for viability and the synthesis of which is the target for crucial antibiotics1,2. Peptidoglycan is a single macromolecule made of glycan chains crosslinked by peptide side branches that surrounds the cell, acting as a constraint to internal turgor1,3. In Gram-positive bacteria, peptidoglycan is tens of nanometres thick, generally portrayed as a homogeneous structure that provides mechanical strength4,5,6. Here we applied atomic force microscopy7,8,9,10,11,12 to interrogate the morphologically distinct Staphylococcus aureus and Bacillus subtilis species, using live cells and purified peptidoglycan. The mature surface of live cells is characterized by a landscape of large (up to 60 nm in diameter), deep (up to 23 nm) pores constituting a disordered gel of peptidoglycan. The inner peptidoglycan surface, consisting of more nascent material, is much denser, with glycan strand spacing typically less than 7 nm. The inner surface architecture is location dependent; the cylinder of B. subtilis has dense circumferential orientation, while in S. aureus and division septa for both species, peptidoglycan is dense but randomly oriented. Revealing the molecular architecture of the cell envelope frames our understanding of its mechanical properties and role as the environmental interface13,14, providing information complementary to traditional structural biology approaches

Correlative super-resolution optical and atomic force microscopy reveals relationships between bacterial cell wall architecture and synthesis in Bacillus subtilis

Understanding how bacteria grow and divide requires insight into both the molecular-level dynamics of ultrastructure and the chemistry of the constituent components. Atomic force microscopy (AFM) can provide near molecular resolution images of biological systems but typically provides limited chemical information. Conversely, while super-resolution optical microscopy allows localization of particular molecules and chemistries, information on the molecular context is difficult to obtain. Here, we combine these approaches into STORMForce (stochastic optical reconstruction with atomic force microscopy) and the complementary SIMForce (structured illumination with atomic force microscopy), to map the synthesis of the bacterial cell wall structural macromolecule, peptidoglycan, during growth and division in the rod-shaped bacterium Bacillus subtilis. Using “clickable” d-amino acid incorporation, we fluorescently label and spatially localize a short and controlled period of peptidoglycan synthesis and correlate this information with high-resolution AFM of the resulting architecture. During division, septal synthesis occurs across its developing surface, suggesting a two-stage process with incorporation at the leading edge and with considerable in-filling behind. During growth, the elongation of the rod occurs through bands of synthesis, spaced by ∼300 nm, and corresponds to denser regions of the internal cell wall as revealed by AFM. Combining super-resolution optics and AFM can provide insights into the synthesis processes that produce the complex architectures of bacterial structural biopolymers

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

Insights into the structure and nanomechanics of a quatsome membrane by force spectroscopy measurements and molecular simulations

Quatsomes (QS) are unilamellar nanovesicles constituted by quaternary ammonium surfactants and sterols in defined molar ratios. Unlike conventional liposomes, QS are stable upon long storage such as for several years, they show outstanding vesicle-to-vesicle homogeneity regarding size and lamellarity, and they have the structural and physicochemical requirements to be a potential platform for site-specific delivery of hydrophilic and lipophilic molecules. Knowing in detail the structure and mechanical properties of the QS membrane is of great importance for the design of deformable and flexible nanovesicle alternatives, highly pursued in nanomedicine applications such as the transdermal administration route. In this work, we report the first study on the detailed structure of the cholesterol : CTAB QS membrane at the nanoscale, using atomic force microscopy (AFM) and spectroscopy (AFM-FS) in a controlled liquid environment (ionic medium and temperature) to assess the topography of supported QS membranes (SQMs) and to evaluate the local membrane mechanics. We further perform molecular dynamics (MD) simulations to provide an atomistic interpretation of the obtained results. Our results are direct evidence of the bilayer nature of the QS membrane, with characteristics of a fluid-like membrane, compact and homogeneous in composition, and with structural and mechanical properties that depend on the surrounding environment. We show how ions alter the lateral packing, modifying the membrane mechanics. We observe that according to the ionic environment and temperature, different domains may coexist in the QS membranes, ascribed to variations in molecular tilt angles. Our results indicate that QS membrane properties may be easily tuned by altering the lateral interactions with either different environmental ions or counterions