Cell Wall Synthase Research Articles

MacP bypass variants of Streptococcus pneumoniae PBP2a suggest a conserved mechanism for the activation of bifunctional cell wall synthases.

Class A penicillin-binding proteins (aPBPs) play critical roles in bacterial cell wall biogenesis. As the targets of penicillin, they are among the most important drug targets in history. Although the biochemical activities of these enzymes have been well studied, little is known about how they are regulated in cells to control when and where peptidoglycan is made. In this report, we isolate variants of the Streptococcus pneumoniae enzyme PBP2a that function in cells without MacP, a partner normally required for its activity. The amino acid substitutions activate the cell wall synthase activity of PBP2a, and their location in a model structure suggests an activation mechanism for this enzyme that is shared with aPBPs from distantly related organisms with distinct activators.

A cell wall synthase accelerates plasma membrane partitioning in mycobacteria.

Lateral partitioning of proteins and lipids shapes membrane function. In model membranes, partitioning can be influenced both by bilayer-intrinsic factors like molecular composition and by bilayer-extrinsic factors such as interactions with other membranes and solid supports. While cellular membranes can departition in response to bilayer-intrinsic or -extrinsic disruptions, the mechanisms by which they partition de novo are largely unknown. The plasma membrane of Mycobacterium smegmatis spatially and biochemically departitions in response to the fluidizing agent benzyl alcohol, then repartitions upon fluidizer washout. By screening for mutants that are sensitive to benzyl alcohol, we show that the bifunctional cell wall synthase PonA2 promotes membrane partitioning and cell growth during recovery from benzyl alcohol exposure. PonA2's role in membrane repartitioning and regrowth depends solely on its conserved transglycosylase domain. Active cell wall polymerization promotes de novo membrane partitioning and the completed cell wall polymer helps to maintain membrane partitioning. Our work highlights the complexity of membrane-cell wall interactions and establishes a facile model system for departitioning and repartitioning cellular membranes.

Conformational changes in the essential E. coli septal cell wall synthesis complex suggest an activation mechanism

The bacterial divisome is a macromolecular machine composed of more than 30 proteins that controls cell wall constriction during division. Here, we present a model of the structure and dynamics of the core complex of the E. coli divisome, supported by a combination of structure prediction, molecular dynamics simulation, single-molecule imaging, and mutagenesis. We focus on the septal cell wall synthase complex formed by FtsW and FtsI, and its regulators FtsQ, FtsL, FtsB, and FtsN. The results indicate extensive interactions in four regions in the periplasmic domains of the complex. FtsQ, FtsL, and FtsB support FtsI in an extended conformation, with the FtsI transpeptidase domain lifted away from the membrane through interactions among the C-terminal domains. FtsN binds between FtsI and FtsL in a region rich in residues with superfission (activating) and dominant negative (inhibitory) mutations. Mutagenesis experiments and simulations suggest that the essential domain of FtsN links FtsI and FtsL together, potentially modulating interactions between the anchor-loop of FtsI and the putative catalytic cavity of FtsW, thus suggesting a mechanism of how FtsN activates the cell wall synthesis activities of FtsW and FtsI.

ERV14 receptor impacts mycelial growth via its interactions with cell wall synthase and transporters in Aspergillus niger.

Efficient protein secretion is closely correlated with vesicle sorting and packaging, especially with cargo receptor-mediated selective transport for ER exit. Even though Aspergillus niger is considered an industrially natural host for protein production due to its exceptional secretion capacity, the trafficking mechanism in the early secretory pathway remains a black box for us to explore. Here, we identified and characterized all putative ER cargo receptors of the three families in A. niger. We successfully constructed overexpression and deletion strains of each receptor and compared the colony morphology and protein secretion status of each strain. Among them, the deletion of Erv14 severely inhibited mycelial growth and secretion of extracellular proteins such as glucoamylase. To gain a comprehensive understanding of the proteins associated with Erv14, we developed a high-throughput method by combining yeast two-hybrid (Y2H) with next-generation sequencing (NGS) technology. We found Erv14 specifically interacted with transporters. Following further validation of the quantitative membrane proteome, we determined that Erv14 was associated with the transport of proteins involved in processes such as cell wall synthesis, lipid metabolism, and organic substrate metabolism.

Integrated Multi-omics, Virtual Screening and Molecular Docking Analysis of Methicillin-Resistant Staphylococcus aureus USA300 for the Identification of Potential Therapeutic Targets: An In-Silico Approach.

Staphylococcus aureus infection is a leading cause of mortality and morbidity in community, hospital and live-stock sectors, especially with the widespread emergence of methicillin-resistant S. aureus (MRSA) strains. To identify new drug molecules to treat MRSA patients, we have undertaken to search essential proteins that are indispensable for their survival but non-homologous to human host proteins. The current study utilizes a subtractive genome and proteome approach to screen the possible therapeutic targets against S. aureus USA300. Bacterial essential genes are obtained from the DEG database and are compared to avoid cross-reactivity with human host genes. In silico analysis shows 198 proteins that may be considered as therapeutic candidates. Depending on their sub-cellular localization, proteins are grouped as either vaccine or drug targets or both. Extracellular proteins such as cell division proteins (Q2FZ91, Q2FZ95), penicillin-binding proteins (Q2FZ94, Q2FYI0) of the bacterial cell wall, phosphoglucomutase (Q2FE11) and lipoteichoic acid synthase (Q2FIS2) are considered as vaccine targets, and their epitopes have been mapped. Altogether, 53 drug targets are identified, which have shown similarity with the drug targets available in the DrugBank database. Predicted drug targets belong to the common metabolic pathways of MRSA, such as fatty acid biosynthesis, folate biosynthesis, peptidoglycan biosynthesis, ribosome, etc. Protein–protein interaction analysis emphasizing peptidoglycan biosynthesis reveals the connection between penicillin-binding proteins, mur-family proteins and FemXAB proteins. In this study, staphylococcal FemA protein (P0A0A5) is subjected to structure-based virtual screening for the drug repurposing approach. There are 20 residues missing in the crystal structure of FemA, and 12 of these residues are located at the catalytic site. The missing residues are modelled, and stereochemistry is checked. FDA approved drugs available in the DrugBank database have been used in virtual screening with FemA in search of potential repurposed molecules. This approach provides us with 10 drugs that may be used in the treatment of methicillin-resistant staphylococcal mediated diseases. AutoDock 4.2 is used for in silico screening and shows a comparable inhibition constant (Ki) for all 10 FDA-approved drugs towards FemA. Most of these drugs are used in the treatment of various cancers, migraines and leukaemia. Protein–drug interaction analysis shows that the drugs mostly interact with hydrophobic residues of FemA. Moreover, Tyr328 and Lys383 contribute largely to hydrogen bondings during interactions. All interacting amino acids that bind to the drugs are part of the active site cavity of FemA.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10989-021-10287-9.

The LpoA activator is required to stimulate the peptidoglycan polymerase activity of its cognate cell wall synthase PBP1a

A cell wall made of the heteropolymer peptidoglycan (PG) surrounds most bacterial cells. This essential surface layer is required to prevent lysis from internal osmotic pressure. The class A penicillin-binding proteins (aPBPs) play key roles in building the PG network. These bifunctional enzymes possess both PG glycosyltransferase (PGT) and transpeptidase (TP) activity to polymerize the wall glycans and cross-link them, respectively. In Escherichia coli and other gram-negative bacteria, aPBP function is dependent on outer membrane lipoproteins. The lipoprotein LpoA activates PBP1a and LpoB promotes PBP1b activity. In a purified system, the major effect of LpoA on PBP1a is TP stimulation. However, the relevance of this activation to the cellular function of LpoA has remained unclear. To better understand why PBP1a requires LpoA for its activity in cells, we identified variants of PBP1a from E. coli and Pseudomonas aeruginosa that function in the absence of the lipoprotein. The changes resulting in LpoA bypass map to the PGT domain and the linker region between the two catalytic domains. Purification of the E. coli variants showed that they are hyperactivated for PGT but not TP activity. Furthermore, in vivo analysis found that LpoA is necessary for the glycan synthesis activity of PBP1a in cells. Thus, our results reveal that LpoA exerts a much greater control over the cellular activity of PBP1a than previously appreciated. It not only modulates PG cross-linking but is also required for its cognate synthase to make PG glycans in the first place.

Global phenotypic profiling identifies a conserved actinobacterial cofactor for a bifunctional PBP-type cell wall synthase.

Members of the Corynebacterineae suborder of Actinobacteria have a unique cell surface architecture and, unlike most well-studied bacteria, grow by tip-extension. To investigate the distinct morphogenic mechanisms shared by these organisms, we performed a genome-wide phenotypic profiling analysis using Corynebacterium glutamicum as a model. A high-density transposon mutagenized library was challenged with a panel of antibiotics and other stresses. The fitness of mutants in each gene under each condition was then assessed by transposon-sequencing. Clustering of the resulting phenotypic fingerprints revealed a role for several genes of previously unknown function in surface biogenesis. Further analysis identified CofA (Cgp_0016) as an interaction partner of the peptidoglycan synthase PBP1a that promotes its stable accumulation at sites of polar growth. The related Mycobacterium tuberculosis proteins were also found to interact, highlighting the utility of our dataset for uncovering conserved principles of morphogenesis for this clinically relevant bacterial suborder.

An Essential Regulator of Bacterial Division Links FtsZ to Cell Wall Synthase Activation

Bacterial growth and division require insertion of new peptidoglycan (PG) into the existing cell wall by PG synthase enzymes. Emerging evidence suggests that many PG synthases require activation to function; however, it is unclear how activation of division-specific PG synthases occurs. The FtsZ cytoskeleton has been implicated as a regulator of PG synthesis during division, but the mechanisms through which it acts are unknown. Here, we show that FzlA, an FtsZ-binding protein and essential regulator of constriction in Caulobacter crescentus, helps link FtsZ to PG synthesis to promote division. We find that hyperactive mutants of the PG synthases FtsW and FtsI specifically render fzlA, but not other division genes, non-essential. However, FzlA is still required to maintain proper constriction rate and efficiency in a hyperactive PG synthase background. Intriguingly, loss of fzlA in the presence of hyperactivated FtsWI causes cells to rotate about the division plane during constriction and sensitizes cells to cell-wall-specific antibiotics. We demonstrate that FzlA-dependent signaling to division-specific PG synthesis is conserved in another α-proteobacterium, Agrobacterium tumefaciens. These data establish that FzlA helps link FtsZ to cell wall remodeling and is required for signaling to both activate and spatially orient PG synthesis during division. Overall, our findings support the paradigm that activation of SEDS-PBP PG synthases is a broadly conserved requirement for bacterial morphogenesis.

Defects in intracellular trafficking of fungal cell wall synthases lead to aberrant host immune recognition.

The human fungal pathogen, Cryptococcus neoformans, dramatically alters its cell wall, both in size and composition, upon entering the host. This cell wall remodeling is essential for host immune avoidance by this pathogen. In a genetic screen for mutants with changes in their cell wall, we identified a novel protein, Mar1, that controls cell wall organization and immune evasion. Through phenotypic studies of a loss-of-function strain, we have demonstrated that the mar1Δ mutant has an aberrant cell surface and a defect in polysaccharide capsule attachment, resulting in attenuated virulence. Furthermore, the mar1Δ mutant displays increased staining for exposed cell wall chitin and chitosan when the cells are grown in host-like tissue culture conditions. However, HPLC analysis of whole cell walls and RT-PCR analysis of cell wall synthase genes demonstrated that this increased chitin exposure is likely due to decreased levels of glucans and mannans in the outer cell wall layers. We observed that the Mar1 protein differentially localizes to cellular membranes in a condition dependent manner, and we have further shown that the mar1Δ mutant displays defects in intracellular trafficking, resulting in a mislocalization of the β-glucan synthase catalytic subunit, Fks1. These cell surface changes influence the host-pathogen interaction, resulting in increased macrophage activation to microbial challenge in vitro. We established that several host innate immune signaling proteins are required for the observed macrophage activation, including the Card9 and MyD88 adaptor proteins, as well as the Dectin-1 and TLR2 pattern recognition receptors. These studies explore novel mechanisms by which a microbial pathogen regulates its cell surface in response to the host, as well as how dysregulation of this adaptive response leads to defective immune avoidance.

Phosphorylation-dependent activation of the cell wall synthase PBP2a in Streptococcus pneumoniae by MacP

Most bacterial cells are surrounded by an essential cell wall composed of the net-like heteropolymer peptidoglycan (PG). Growth and division of bacteria are intimately linked to the expansion of the PG meshwork and the construction of a cell wall septum that separates the nascent daughter cells. Class A penicillin-binding proteins (aPBPs) are a major family of PG synthases that build the wall matrix. Given their central role in cell wall assembly and importance as drug targets, surprisingly little is known about how the activity of aPBPs is controlled to properly coordinate cell growth and division. Here, we report the identification of MacP (SPD_0876) as a membrane-anchored cofactor of PBP2a, an aPBP synthase of the Gram-positive pathogen Streptococcus pneumoniae We show that MacP localizes to the division site of S. pneumoniae, forms a complex with PBP2a, and is required for the in vivo activity of the synthase. Importantly, MacP was also found to be a substrate for the kinase StkP, a global cell cycle regulator. Although StkP has been implicated in controlling the balance between the elongation and septation modes of cell wall synthesis, none of its substrates are known to modulate PG synthetic activity. Here we show that a phosphoablative substitution in MacP that blocks StkP-mediated phosphorylation prevents PBP2a activity without affecting the MacP-PBP2a interaction. Our results thus reveal a direct connection between PG synthase function and the control of cell morphogenesis by the StkP regulatory network.

Superresolution and pulse-chase imaging reveal the role of vesicle transport in polar growth of fungal cells.

Polarized growth of filamentous fungi requires continuous transport of biomolecules to the hyphal tip. To this end, construction materials are packaged in vesicles and transported by motor proteins along microtubules and actin filaments. We have studied these processes with quantitative superresolution localization microscopy of live Aspergillus nidulans cells expressing the photoconvertible protein mEosFPthermo fused to the chitin synthase ChsB. ChsB is mainly located at the Spitzenkörper near the hyphal tip and produces chitin, a key component of the cell wall. We have visualized the pulsatory dynamics of the Spitzenkörper, reflecting vesicle accumulation before exocytosis and their subsequent fusion with the apical plasma membrane. Furthermore, high-speed pulse-chase imaging after photoconversion of mEosFPthermo in a tightly focused spot revealed that ChsB is transported with two different speeds from the cell body to the hyphal tip and vice versa. Comparative analysis using motor protein deletion mutants allowed us to assign the fast movements (7 to 10 μm s-1) to transport of secretory vesicles by kinesin-1, and the slower ones (2 to 7 μm s-1) to transport by kinesin-3 on early endosomes. Our results show how motor proteins ensure the supply of vesicles to the hyphal tip, where temporally regulated exocytosis results in stepwise tip extension.

Microbiology: The bacterial cell wall takes centre stage.

An unexpected function has been assigned to part of the molecular machinery that synthesizes the bacterial cell wall — a dramatic shift in our understanding that may have major implications for antibiotic development. See Article p.634 It has been generally accepted that the cell wall peptidoglycans of the bacterial exoskeleton are synthesized by penicillin binding proteins (PBPs) known as class A PBPs. Now, using genetic manipulation, phylogenetic analysis and functional experiments in Bacillus subtilis, David Rudner and colleagues have identified SEDS family proteins as the main peptidoglycan polymerases more broadly conserved than class A PBPs. Specifically in B. subtilis, they show that the SEDS protein RodA, a widely conserved component of the Rod complex involved in elongation of rod-shaped bacteria, acts with class B PBPs as the core cell wall synthase of the cell elongation and division machinery. The authors conclude that B. subtilis and probably most bacteria use two distinct classes of polymerases to synthesize their exoskeleton. This work also suggests that SEDS family proteins should be attractive targets for antibiotic development.

Modulation of Alternaria infectoria cell wall chitin and glucan synthesis by cell wall synthase inhibitors.

The present work reports the effects of caspofungin, a β-1,3-glucan synthase inhibitor, and nikkomycin Z, an inhibitor of chitin synthases, on two strains of Alternaria infectoria, a melanized fungus involved in opportunistic human infections and respiratory allergies. One of the strains tested, IMF006, bore phenotypic traits that conferred advantages in resisting antifungal treatment. First, the resting cell wall chitin content was higher and in response to caspofungin, the chitin level remained constant. In the other strain, IMF001, the chitin content increased upon caspofungin treatment to values similar to basal IMF006 levels. Moreover, upon caspofungin treatment, the FKS1 gene was upregulated in IMF006 and downregulated in IMF001. In addition, the resting β-glucan content was also different in both strains, with higher levels in IMF001 than in IMF006. However, this did not provide any advantage with respect to echinocandin resistance. We identified eight different chitin synthase genes and studied relative gene expression when the fungus was exposed to the antifungals under study. In both strains, exposure to caspofungin and nikkomycin Z led to modulation of the expression of class V and VII chitin synthase genes, suggesting its importance in the robustness of A. infectoria. The pattern of A. infectoria phagocytosis and activation of murine macrophages by spores was not affected by caspofungin. Monotherapy with nikkomycin Z and caspofungin provided only fungistatic inhibition, while a combination of both led to fungal cell lysis, revealing a strong synergistic action between the chitin synthase inhibitor and the β-glucan synthase inhibitor against this fungus.

The calcineurin-responsive transcription factor Crz1 is required for conidation, full virulence and DMI resistance in Penicillium digitatum

The Ca2+/calmodulin-dependent pathway is a critical signal transduction pathway that mediates various cellular responses in multiple organisms. Calcineurin acts through zinc finger transcription factor Crz1 in fungi. In the present study, we identified an ortholog of Crz1, PdCrz1, in Penicillium digitatum, the most important pathogen of postharvest citrus. To determine the function(s) of PdCrz1, a gene-disruption mutant was generated using an Agrobacterium tumefaciens-mediated transformation (ATMT) procedure. The PdCrz1 disruption (ΔPdCrz1) mutant was defective in conidiation, cell wall integrity and virulence, and was hypersensitivity to stresses caused by Ca2+, H2O2 or 14α-demethylation inhibitor (DMI) fungicides. Expressions of cell wall synthase genes (CHS2, CHS3 and FKS1) and P-type ATPase genes (PMR1 and PMC1) were defective in the ΔPdCrz1 mutant when compared with the wild type. Calcineurin inhibitors were shown to enhance the effect of imazalil on P. digitatum. In contrast, Ca2+ (≤0.2moll−1) could alleviate the antifungal activity by imazalil. These results indicate that PdCrz1 has important roles in conidiation, virulence and DMI resistance in P. digitatum. In addition, this study also indicates that specific calcium signaling pathway inhibitors could be used as adjuvants in the conventional DMI fungicides.

Cdc42 Explores the Cell Periphery for Mate Selection in Fission Yeast

How cells polarize in response to external cues is a fundamental biological problem. For mating, yeast cells orient growth toward the source of a pheromone gradient produced by cells of the opposite mating type. Polarized growth depends on the small GTPase Cdc42, a central eukaryotic polarity regulator that controls signaling, cytoskeleton polarization, and vesicle trafficking. However, the mechanisms of polarity establishment and mate selection in complex cellular environments are poorly understood. Here we show that, in fission yeast, low-level pheromone signaling promotes a novel polarization state, where active Cdc42, its GEF Scd1, and scaffold Scd2 form colocalizing dynamic zones that sample the periphery of the cell. Two direct Cdc42 effectors--actin cables marked by myosin V Myo52 and the exocyst complex labeled by Sec6 and Sec8--also dynamically colocalize with active Cdc42. However, these cells do not grow due to a block in the exocytosis of cell wall synthases Bgs1 and Bgs4. High-level pheromone stabilizes active Cdc42 zones and promotes cell wall synthase exocytosis and polarized growth. However, in the absence of prior low-level pheromone signaling, exploration fails, and cells polarize growth at cell poles by default. Consequently, these cells show altered partner choice, mating preferentially with sister rather than nonsister cells. Thus, Cdc42 exploration serves to orient growth for partner selection. This process may also promote genetic diversification.

Electrical Control of Cell Polarization in the Fission Yeast Schizosaccharomyces pombe

Electric signals surround tissues and cells and have been proposed to participate in directing cell polarity in processes such as development, wound healing, and host invasion [1, 2]. The application of exogenous electric fields (EFs) can direct cell polarization in cell types ranging from bacteria and fungi to neurons and neutrophils [3-7]. The mechanisms by which EFs modulate cell polarity, however, remain poorly understood. Here we introduce the fission yeast Schizosaccharomyces pombe as a model organism to elucidate the mechanisms underlying this process. In these rod-shaped cells, an exogenous EF reorients cell growth in a direction orthogonal to the field, producing cells with a bent morphology. A candidate genetic screen identifies conserved factors involved in this process: an integral membrane proton ATPase pma1p that regulates intracellular pH, the small GTPase cdc42p, and the formin for3p that assembles actin cables. Interestingly, mutants in these genes still respond to the EF but orient in a different direction, toward the anode. In addition, EFs also cause electrophoretic movement of cell wall synthase complex proteins toward the anode. These data suggest molecular models for how the EF reorients cell polarization by modulating intracellular pH and steering cell polarity factors in multiple directions.

Bactofilins, a ubiquitous class of cytoskeletal proteins mediating polar localization of a cell wall synthase in Caulobacter crescentus

The cytoskeleton has a key function in the temporal and spatial organization of both prokaryotic and eukaryotic cells. Here, we report the identification of a new class of polymer-forming proteins, termed bactofilins, that are widely conserved among bacteria. In Caulobacter crescentus, two bactofilin paralogues cooperate to form a sheet-like structure lining the cytoplasmic membrane in proximity of the stalked cell pole. These assemblies mediate polar localization of a peptidoglycan synthase involved in stalk morphogenesis, thus complementing the function of the actin-like cytoskeleton and the cell division machinery in the regulation of cell wall biogenesis. In other bacteria, bactofilins can establish rod-shaped filaments or associate with the cell division apparatus, indicating considerable structural and functional flexibility. Bactofilins polymerize spontaneously in the absence of additional cofactors in vitro, forming stable ribbon- or rod-like filament bundles. Our results suggest that these structures have evolved as an alternative to intermediate filaments, serving as versatile molecular scaffolds in a variety of cellular pathways.