Molecular Bases Determining Daptomycin Resistance-Mediated Resensitization to ß-Lactams (Seesaw Effect) in Methicillin-Resistant Staphylococcus aureus.

Antimicrobial resistance is recognized as one of the principal threats to public health worldwide, yet the problem is increasing. Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) strains are among the most difficult to treat in clinical settings due to the resistance of MRSA to nearly all available antibiotics. The cyclic anionic lipopeptide antibiotic daptomycin (DAP) is the clinical mainstay of anti-MRSA therapy. The decreased susceptibility to DAP (DAP resistance [DAPr]) reported in MRSA is frequently accompanied by a paradoxical decrease in ß-lactam resistance, a process known as the "seesaw effect." Despite the observed discordance in resistance phenotypes, the combination of DAP and ß-lactams has been proven to be clinically effective for the prevention and treatment of infections due to DAPr MRSA strains. However, the mechanisms underlying the interactions between DAP and ß-lactams are largely unknown. In the study described here, we studied the role of mprF with DAP-induced mutations in ß-lactam sensitization and its involvement in the effective killing by the DAP-oxacillin (OXA) combination. DAP-OXA-mediated effects resulted in cell wall perturbations, including changes in peptidoglycan insertion, penicillin-binding protein 2 (PBP 2) delocalization, and reduced membrane amounts of PBP 2a, despite the increased transcription of mecA through mec regulatory elements. We have found that the VraSR sensor-regulator is a key component of DAP resistance, triggering mutated mprF-mediated cell membrane (CM) modifications that result in impairment of PrsA location and chaperone functions, both of which are essential for PBP 2a maturation, the key determinant of ß-lactam resistance. These observations provide for the first time evidence that synergistic effects between DAP and ß-lactams involve PrsA posttranscriptional regulation of CM-associated PBP 2a.

Different walls for rods and balls: the diversity of peptidoglycan.

Peptidoglycan performs the essential role of resisting turgor in the cell walls of most bacteria. It determines cell shape, and its biosynthesis is the target for many important antibiotics. The fundamental chemical building blocks of peptidoglycan are conserved: repeating disaccharides cross-linked by peptides. However, these blocks come in many varieties and can be assembled in different ways. So beyond the fundamental similarity, prodigious chemical, organizational and architectural diversity is revealed. Here, we track the evolution of our current understanding of peptidoglycan and underpinning technical and methodological developments. The origin and function of chemical diversity is discussed with respect to some well-studied example species. We then explore how this chemistry is manifested in elegant and complex peptidoglycan organization and how this is interpreted in different and sometimes controversial architectural models. We contend that emerging technology brings about the possibility of achieving a complete understanding of peptidoglycan chemistry, through architecture, to the way in which diverse species and populations of cells meet the challenges of maintaining viability and growth within their environmental niches, by exploiting the bioengineering versatility of peptidoglycan.

Staphylococcus aureus†DivIB is a peptidoglycan-binding protein that is required for a morphological checkpoint in cell division.

Bacterial cell division is a fundamental process that requires the coordinated actions of a number of proteins which form a complex macromolecular machine known as the divisome. The membrane-spanning proteins DivIB and its orthologue FtsQ are crucial divisome components in Gram-positive and Gram-negative bacteria respectively. However, the role of almost all of the integral division proteins, including DivIB, still remains largely unknown. Here we show that the extracellular domain of DivIB is able to bind peptidoglycan and have mapped the binding to its ß subdomain. Conditional mutational studies show that divIB is essential for Staphylococcus aureus growth, while phenotypic analyses following depletion of DivIB results in a block in the completion, but not initiation, of septum formation. Localisation studies suggest that DivIB only transiently localises to the division site and may mark previous sites of septation. We propose that DivIB is required for a molecular checkpoint during division to ensure the correct assembly of the divisome at midcell and to prevent hydrolytic growth of the cell in the absence of a completed septum.

Impact of the ß-Lactam Resistance Modifier (-)-Epicatechin Gallate on the Non-Random Distribution of Phospholipids across the Cytoplasmic Membrane of Staphylococcus aureus.

The polyphenol (-)-epicatechin gallate (ECg) inserts into the cytoplasmic membrane (CM) of methicillin-resistant Staphylococcus aureus (MRSA) and reversibly abrogates resistance to ß-lactam antibiotics. ECg elicits an increase in MRSA cell size and induces thickened cell walls. As ECg partially delocalizes penicillin-binding protein PBP2 from the septal division site, reduces PBP2 and PBP2a complexation and induces CM remodelling, we examined the impact of ECg membrane intercalation on phospholipid distribution across the CM and determined if ECg affects the equatorial, orthogonal mode of division. The major phospholipids of the staphylococcal CM, lysylphosphatidylglycerol (LPG), phosphatidylglycerol (PG), and cardiolipin (CL), were distributed in highly asymmetric fashion; 95%-97% of LPG was associated with the inner leaflet whereas PG (~90%) and CL (~80%) were found predominantly in the outer leaflet. ECg elicited small, significant changes in LPG distribution. Atomic force microscopy established that ECg-exposed cells divided in similar fashion to control bacteria, with a thickened band of encircling peptidoglycan representing the most recent plane of cell division, less distinct ribs indicative of previous sites of orthogonal division and concentric rings and "knobbles" representing stages of peptidoglycan remodelling during the cell cycle. Preservation of staphylococcal membrane lipid asymmetry and mode of division in sequential orthogonal planes appear key features of ECg-induced stress.

The interplay between cell wall mechanical properties and the cell cycle in Staphylococcus aureus.

The nanoscale mechanical properties of live Staphylococcus aureus cells during different phases of growth were studied by atomic force microscopy. Indentation to different depths provided access to both local cell wall mechanical properties and whole-cell properties, including a component related to cell turgor pressure. Local cell wall properties were found to change in a characteristic manner throughout the division cycle. Splitting of the cell into two daughter cells followed a local softening of the cell wall along the division circumference, with the cell wall on either side of the division circumference becoming stiffer. Once exposed, the newly formed septum was found to be stiffer than the surrounding, older cell wall. Deeper indentations, which were affected by cell turgor pressure, did not show a change in stiffness throughout the division cycle, implying that enzymatic cell wall remodeling and local variations in wall properties are responsible for the evolution of cell shape through division.

A moonlighting role for LysM peptidoglycan binding domains underpins Enterococcus faecalis daughter cell separation.

Control of cell size and morphology is of paramount importance for bacterial fitness. In the opportunistic pathogen Enterococcus faecalis, the formation of diplococci and short cell chains facilitates innate immune evasion and dissemination in the host. Minimisation of cell chain size relies on the activity of a peptidoglycan hydrolase called AtlA, dedicated to septum cleavage. To prevent autolysis, AtlA activity is tightly controlled, both temporally and spatially. Here, we show that the restricted localization of AtlA at the septum occurs via an unexpected mechanism. We demonstrate that the C-terminal LysM domain that allows the enzyme to bind peptidoglycan is essential to target this enzyme to the septum inside the cell before its translocation across the membrane. We identify a membrane-bound cytoplasmic protein partner (called AdmA) involved in the recruitment of AtlA via its LysM domains. This work reveals a moonlighting role for LysM domains, and a mechanism evolved to restrict the subcellular localization of a potentially lethal autolysin to its site of action.

PGFinder, a novel analysis pipeline for the consistent, reproducible, and high-resolution structural analysis of bacterial peptidoglycans.

Many software solutions are available for proteomics and glycomics studies, but none are ideal for the structural analysis of peptidoglycan (PG), the essential and major component of bacterial cell envelopes. It icomprises glycan chains and peptide stems, both containing unusual amino acids and sugars. This has forced the field to rely on manual analysis approaches, which are time-consuming, labour-intensive, and prone to error. The lack of automated tools has hampered the ability to perform high-throughput analyses and prevented the adoption of a standard methodology. Here, we describe a novel tool called PGFinder for the analysis of PG structure and demonstrate that it represents a powerful tool to quantify PG fragments and discover novel structural features. Our analysis workflow, which relies on open-access tools, is a breakthrough towards a consistent and reproducible analysis of bacterial PGs. It represents a significant advance towards peptidoglycomics as a full-fledged discipline.

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.

Use of silver-based additives for the development of antibacterial functionality in Laser Sintered polyamide 12 parts.

Infectious diseases (exacerbated by antimicrobial resistance) cause death, loss of quality of life and economic burden globally. Materials with inherent antimicrobial properties offer the potential to reduce the spread of infection through transfer via surfaces or solutions, or to directly reduce microbial numbers in a host if used as implants. Additive Manufacturing (AM) techniques offer shorter supply chains, faster delivery, mass customisation and reduced unit costs, as well as highly complicated part geometries which are potentially harder to clean and sterilise. Here, we present a new approach to introducing antibacterial properties into AM, using Laser Sintering, by combining antimicrobial and base polymer powders prior to processing. We demonstrate that the mechanical properties of the resultant composite parts are similar to standard polymer parts and reveal the mode of the antibacterial activity. We show that antibacterial activity is modulated by the presence of obstructing compounds in different experimental media, which will inform appropriate use cases. We show that the material is not toxic to mammalian cells. This material could be quickly used for commercial products, and our approach could be adopted more generally to add new functionality to Laser Sintered parts.

Molecular imaging of glycan chains couples cell-wall polysaccharide architecture to bacterial cell morphology.

Biopolymer composite cell walls maintain cell shape and resist forces in plants, fungi and bacteria. Peptidoglycan, a crucial antibiotic target and immunomodulator, performs this role in bacteria. The textbook structural model of peptidoglycan is a highly ordered, crystalline material. Here we use atomic force microscopy (AFM) to image individual glycan chains in peptidoglycan from Escherichia coli in unprecedented detail. We quantify and map the extent to which chains are oriented in a similar direction (orientational order), showing it is much less ordered than previously depicted. Combining AFM with size exclusion chromatography, we reveal glycan chains up to 200 nm long. We show that altered cell shape is associated with substantial changes in peptidoglycan biophysical properties. Glycans from E. coli in its normal rod shape are long and circumferentially oriented, but when a spheroid shape is induced (chemically or genetically) glycans become short and disordered.

ELM: super-resolution analysis of wide-field images of fluorescent shell structures.

It is often necessary to precisely quantify the size of specimens in biological studies. When measuring feature size in fluorescence microscopy, significant biases can arise due to blurring of its edges if the feature is smaller than the diffraction limit of resolution. This problem is avoided if an equation describing the feature's entire image is fitted to its image data. In this paper we present open-source software, ELM, which uses this approach to measure the size of spheroidal or cylindrical fluorescent shells with a precision of around 10 nm. This has been used to measure coat protein locations in bacterial spores and cell wall diameter in vegetative bacilli, and may also be valuable in microbiological studies of algae, fungi and viruses. ELM is available for download at https://github.com/quantitativeimaging/ELM.

Heterogeneous localisation of membrane proteins in Staphylococcus aureus.

The bacterial cytoplasmic membrane is the interface between the cell and its environment, with multiple membrane proteins serving its many functions. However, how these proteins are organised to permit optimal physiological processes is largely unknown. Based on our initial findings that 2 phospholipid biosynthetic enzymes (PlsY and CdsA) localise heterogeneously in the membrane of the bacterium Staphylococcus aureus, we have analysed the localisation of other key membrane proteins. A range of protein fusions were constructed and used in conjunction with quantitative image analysis. Enzymes involved in phospholipid biosynthesis as well as the lipid raft marker FloT exhibited a heterogeneous localisation pattern. However, the secretion associated SecY protein, was more homogeneously distributed in the membrane. A FRET-based system also identified novel colocalisation between phospholipid biosynthesis enzymes and the respiratory protein CydB revealing a likely larger network of partners. PlsY localisation was found to be dose dependent but not to be affected by membrane lipid composition. Disruption of the activity of the essential cell division organiser FtsZ, using the inhibitor PC190723 led to loss of PlsY localisation, revealing a link to cell division and a possible role for FtsZ in functions not strictly associated with septum formation.

Molecular coordination of Staphylococcus aureus cell division.

The bacterial cell wall is essential for viability, but despite its ability to withstand internal turgor must remain dynamic to permit growth and division. Peptidoglycan is the major cell wall structural polymer, whose synthesis requires multiple interacting components. The human pathogen Staphylococcus aureus is a prolate spheroid that divides in three orthogonal planes. Here, we have integrated cellular morphology during division with molecular level resolution imaging of peptidoglycan synthesis and the components responsible. Synthesis occurs across the developing septal surface in a diffuse pattern, a necessity of the observed septal geometry, that is matched by variegated division component distribution. Synthesis continues after septal annulus completion, where the core division component FtsZ remains. The novel molecular level information requires re-evaluation of the growth and division processes leading to a new conceptual model, whereby the cell cycle is expedited by a set of functionally connected but not regularly distributed components.

Atomic Force Microscopy Analysis of Bacterial Cell Wall Peptidoglycan Architecture.

Atomic force microscopy (AFM) has been used extensively to characterize the surface structure and mechanical properties of bacterial cells. Extraction of the cell wall peptidoglycan sacculus enables AFM analysis exclusively of peptidoglycan architecture and mechanical properties, unobscured by other cell wall components. This has led to discoveries of new architectural features within the cell wall, and new insights into the level of long range order in peptidoglycan (Turner et al. Mol Microbiol 91:862-874, 2014). Such information has great relevance to the development of models of bacterial growth and division, where peptidoglycan structure is frequently invoked as a means of guiding the activities of the proteins that execute these processes.

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

UNLABELLED: 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.

Cell wall elongation mode in Gram-negative bacteria is determined by peptidoglycan architecture.

Cellular integrity and morphology of most bacteria is maintained by cell wall peptidoglycan, the target of antibiotics essential in modern healthcare. It consists of glycan strands, cross-linked by peptides, whose arrangement determines cell shape, prevents lysis due to turgor pressure and yet remains dynamic to allow insertion of new material, and hence growth. The cellular architecture and insertion pattern of peptidoglycan have remained elusive. Here we determine the peptidoglycan architecture and dynamics during growth in rod-shaped Gram-negative bacteria. Peptidoglycan is made up of circumferentially oriented bands of material interspersed with a more porous network. Super-resolution fluorescence microscopy reveals an unexpected discontinuous, patchy synthesis pattern. We present a consolidated model of growth via architecture-regulated insertion, where we propose only the more porous regions of the peptidoglycan network that are permissive for synthesis.

Peptidoglycan architecture can specify division planes in Staphylococcus aureus.

Division in Staphylococci occurs equatorially and on specific sequentially orthogonal planes in three dimensions, resulting, after incomplete cell separation, in the 'bunch of grapes' cluster organization that defines the genus. The shape of Staphylococci is principally maintained by peptidoglycan. In this study, we use Atomic Force Microscopy (AFM) and fluorescence microscopy with vancomycin labelling to examine purified peptidoglycan architecture and its dynamics in Staphylococcus aureus and correlate these with the cell cycle. At the presumptive septum, cells were found to form a large belt of peptidoglycan in the division plane before the centripetal formation of the septal disc; this often had a 'piecrust' texture. After division, the structures remain as orthogonal ribs, encoding the location of past division planes in the cell wall. We propose that this epigenetic information is used to enable S. aureus to divide in sequentially orthogonal planes, explaining how a spherical organism can maintain division plane localization with fidelity over many generations.