Identification of a family of peptidoglycan transpeptidases reveals that Clostridioides difficile requires noncanonical cross-links for viability.

Most bacteria are surrounded by a cell wall that contains peptidoglycan (PG), a large polymer composed of glycan strands held together by short peptide cross-links. There are two major types of cross-links, termed 4-3 and 3-3 based on the amino acids involved. 4-3 cross-links are created by penicillin-binding proteins, while 3-3 cross-links are created by L,D-transpeptidases (LDTs). In most bacteria, the predominant mode of cross-linking is 4-3, and these cross-links are essential for viability, while 3-3 cross-links comprise only a minor fraction and are not essential. However, in the opportunistic intestinal pathogen Clostridioides difficile, about 70% of the cross-links are 3-3. We show here that 3-3 cross-links and LDTs are essential for viability in C. difficile. We also show that C. difficile has five LDTs, three with a YkuD catalytic domain as in all previously known LDTs and two with a VanW catalytic domain, whose function was until now unknown. The five LDTs exhibit extensive functional redundancy. VanW domain proteins are found in many gram-positive bacteria but scarce in other lineages. We tested seven non-C. difficile VanW domain proteins and confirmed LDT activity in three cases. In summary, our findings uncover a previously unrecognized family of PG cross-linking enzymes, assign a catalytic function to VanW domains, and demonstrate that 3-3 cross-linking is essential for viability in C. difficile, the first time this has been shown in any bacterial species. The essentiality of LDTs in C. difficile makes them potential targets for antibiotics that kill C. difficile selectively.

Genetic analysis of the septal peptidoglycan synthase FtsWI complex supports a conserved activation mechanism for SEDS-bPBP complexes.

SEDS family peptidoglycan (PG) glycosyltransferases, RodA and FtsW, require their cognate transpeptidases PBP2 and FtsI (class B penicillin binding proteins) to synthesize PG along the cell cylinder and at the septum, respectively. The activities of these SEDS-bPBPs complexes are tightly regulated to ensure proper cell elongation and division. In Escherichia coli FtsN switches FtsA and FtsQLB to the active forms that synergize to stimulate FtsWI, but the exact mechanism is not well understood. Previously, we isolated an activation mutation in ftsW (M269I) that allows cell division with reduced FtsN function. To try to understand the basis for activation we isolated additional substitutions at this position and found that only the original substitution produced an active mutant whereas drastic changes resulted in an inactive mutant. In another approach we isolated suppressors of an inactive FtsL mutant and obtained FtsWE289G and FtsIK211I and found they bypassed FtsN. Epistatic analysis of these mutations and others confirmed that the FtsN-triggered activation signal goes from FtsQLB to FtsI to FtsW. Mapping these mutations, as well as others affecting the activity of FtsWI, on the RodA-PBP2 structure revealed they are located at the interaction interface between the extracellular loop 4 (ECL4) of FtsW and the pedestal domain of FtsI (PBP3). This supports a model in which the interaction between the ECL4 of SEDS proteins and the pedestal domain of their cognate bPBPs plays a critical role in the activation mechanism.

Photoaffinity labeling of benzophenone-containing salicylanilide compounds to give an insight into the mechanism in disrupting peptidoglycan formation.

A series of salicylanilide compounds was previously identified as antibacterial agents that inhibit the peptidoglycan formation. To find the exact binding mode, we synthesized a benzophenone-containing salicylanilide compound (1) and used it as a photoaffinity probe to label Acinetobacter baumannii penicillin-binding protein (PBP1b). After incubation and photo-irradiation, the labeled protein was subjected to trypsin digestion, dialysis enrichment, LC-ESI-MS/MS analysis, and Mascot search to reveal an octadecapeptide sequence 364RQLRTEYQESDLTNQGLR381 that was labeled at E372. Our molecular docking experiments suggest a hydrophobic pocket surrounded by R367 and E372 is the binding site of salicylanilide 1. The pocket lies in between the transglycosylase and transpeptidase domains, thus binding of salicylanilide 1 can block the propagation pathway to disrupt the growth of peptidoglycan chain.

SEDS proteins are a widespread family of bacterial cell wall polymerases.

Elongation of rod-shaped bacteria is mediated by a dynamic peptidoglycan-synthetizing machinery called the Rod complex. Here we report that, in Bacillus subtilis, this complex is functional in the absence of all known peptidoglycan polymerases. Cells lacking these enzymes survive by inducing an envelope stress response that increases the expression of RodA, a widely conserved core component of the Rod complex. RodA is a member of the SEDS (shape, elongation, division and sporulation) family of proteins, which have essential but ill-defined roles in cell wall biogenesis during growth, division and sporulation. Our genetic and biochemical analyses indicate that SEDS proteins constitute a family of peptidoglycan polymerases. Thus, B. subtilis and probably most bacteria use two distinct classes of polymerase to synthesize their exoskeleton. Our findings indicate that SEDS family proteins are core cell wall synthases of the cell elongation and division machinery, and represent attractive targets for antibiotic development.

Substrate and Stereochemical Control of Peptidoglycan Cross-Linking by Transpeptidation by Escherichia coli PBP1B.

Penicillin binding proteins (PBPs) catalyzing transpeptidation reactions that stabilize the peptidoglycan component of the bacterial cell wall are the targets of ß-lactams, the most clinically successful antibiotics to date. However, PBP-transpeptidation enzymology has evaded detailed analysis, because of the historical unavailability of kinetically competent assays with physiologically relevant substrates and the previously unappreciated contribution of protein cofactors to PBP activity. By re-engineering peptidoglycan synthesis, we have constructed a continuous spectrophotometric assay for transpeptidation of native or near native peptidoglycan precursors and fragments by Escherichia coli PBP1B, allowing us to (a) identify recognition elements of transpeptidase substrates, (b) reveal a novel mechanism of stereochemical editing within peptidoglycan transpeptidation, (c) assess the impact of peptidoglycan substrates on ß-lactam targeting of transpeptidation, and (d) demonstrate that both substrates have to be bound before transpeptidation occurs. The results allow characterization of high molecular weight PBPs as enzymes and not merely the targets of ß-lactam acylation.

Induced conformational changes activate the peptidoglycan synthase PBP1B.

Bacteria surround their cytoplasmic membrane with an essential, stress-bearing peptidoglycan (PG) layer consisting of glycan chains linked by short peptides into a mesh-like structure. Growing and dividing cells expand their PG layer using inner-membrane anchored PG synthases, including Penicillin-binding proteins (PBPs), which participate in dynamic protein complexes to facilitate cell wall growth. In Escherichia coli, and presumably other Gram-negative bacteria, growth of the mainly single layered PG is regulated by outer membrane-anchored lipoproteins. The lipoprotein LpoB is required to activate PBP1B, which is a major, bi-functional PG synthase with glycan chain polymerising (glycosyltransferase) and peptide cross-linking (transpeptidase) activities. In this work we show how the binding of LpoB to the regulatory UB2H domain of PBP1B activates both activities. Binding induces structural changes in the UB2H domain, which transduce to the two catalytic domains by distinct allosteric pathways. We also show how an additional regulator protein, CpoB, is able to selectively modulate the TPase activation by LpoB without interfering with GTase activation.

Structural Insights into Inhibition of Escherichia coli Penicillin-binding Protein 1B.

In Escherichia coli, the peptidoglycan cell wall is synthesized by bifunctional penicillin-binding proteins such as PBP1b that have both transpeptidase and transglycosylase activities. The PBP1b transpeptidase domain is a major target of ß-lactams, and therefore it is important to attain a detailed understanding of its inhibition. The peptidoglycan glycosyltransferase domain of PBP1b is also considered an excellent antibiotic target yet is not exploited by any clinically approved antibacterials. Herein, we adapt a pyrophosphate sensor assay to monitor PBP1b-catalyzed glycosyltransfer and present an improved crystallographic model for inhibition of the PBP1b glycosyltransferase domain by the potent substrate analog moenomycin. We elucidate the structure of a previously disordered region in the glycosyltransferase active site and discuss its implications with regards to peptidoglycan polymerization. Furthermore, we solve the crystal structures of E. coli PBP1b bound to multiple different ß-lactams in the transpeptidase active site and complement these data with gel-based competition assays to provide a detailed structural understanding of its inhibition. Taken together, these biochemical and structural data allow us to propose new insights into inhibition of both enzymatic domains in PBP1b.

X-ray Structure of Catenated Lytic Transglycosylase SltB1.

Formation of catenanes by proteins is rare, with few known examples. We report herein the X-ray structure of a catenane dimer of lytic transglycosylase SltB1 of Pseudomonas aeruginosa. The enzyme is soluble and exists in the periplasmic space, where it modifies the bacterial cell wall. The catenane dimer exhibits the protein monomers in a noncovalent chain-link arrangement, whereby a stretch of 51 amino acids (to become a loop and three helices) from one monomer threads through the central opening of the structure of the partner monomer. The protein folds after threading in a manner that leaves two helices (α1 and α2) as stoppers to impart stability to the dimer structure. The symmetric embrace by the two SltB1 molecules occludes both active sites entirely, an arrangement that is sustained by six electrostatic interactions between the two monomers. In light of the observation of these structural motifs in all members of Family 3 lytic transglycosylases, catenanes might be present for those enzymes, as well. The dimeric catenane might represent a regulated form of SltB1.

Crystal Structures of the SpoIID Lytic Transglycosylases Essential for Bacterial Sporulation.

Bacterial spores are the most resistant form of life known on Earth and represent a serious problem for (i) bioterrorism attack, (ii) horizontal transmission of microbial pathogens in the community, and (iii) persistence in patients and in a nosocomial environment. Stage II sporulation protein D (SpoIID) is a lytic transglycosylase (LT) essential for sporulation. The LT superfamily is a potential drug target because it is active in essential bacterial processes involving the peptidoglycan, which is unique to bacteria. However, the absence of structural information for the sporulation-specific LT enzymes has hindered mechanistic understanding of SpoIID. Here, we report the first crystal structures with and without ligands of the SpoIID family from two community relevant spore-forming pathogens, Bacillus anthracis and Clostridium difficile. The structures allow us to visualize the overall architecture, characterize the substrate recognition model, identify critical residues, and provide the structural basis for catalysis by this new family of enzymes.

The Predicted Lytic Transglycosylase HpaH from Xanthomonas campestris pv. vesicatoria Associates with the Type III Secretion System and Promotes Effector Protein Translocation.

The pathogenicity of the Gram-negative plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria depends on a type III secretion (T3S) system, which spans both bacterial membranes and translocates effector proteins into plant cells. The assembly of the T3S system presumably involves the predicted lytic transglycosylase (LT) HpaH, which is encoded adjacent to the T3S gene cluster. Bacterial LTs degrade peptidoglycan and often promote the formation of membrane-spanning macromolecular protein complexes. In the present study, we show that HpaH localizes to the bacterial periplasm and binds to peptidoglycan as well as to components of the T3S system, including the predicted periplasmic inner rod proteins HrpB1 and HrpB2 as well as the pilus protein HrpE. In vivo translocation assays revealed that HpaH promotes the translocation of various effector proteins and of early substrates of the T3S system, suggesting a general contribution of HpaH to type III-dependent protein export. Mutant studies and the analysis of reporter fusions showed that the N-terminal region of HpaH contributes to protein function and is proteolytically cleaved. The N-terminally truncated HpaH cleavage product is secreted into the extracellular milieu by a yet-unknown transport pathway, which is independent of the T3S system.

Structural basis for the transglycosylase activity of a GH57-type glycogen branching enzyme from Pyrococcus horikoshii.

Glycogen branching enzyme (GBE) catalyzes the formation of α-1,6-branching points during glycogenesis by cleaving α-1,4 bonds and making new α-1,6 bonds. Most GBEs belong to the glycoside hydrolase 13 family (GH13), but new GBEs in the GH57 family have been isolated from Archaea. Here, we determined the crystal structure of a GH57 GBE from the hyperthermophilic archaeon Pyrococcus horikoshii (PhGBE) at a resolution of 2.3 Å. PhGBE exhibits both α-1,6-branching activity and endo-α-1,4 hydrolytic activity. PhGBE has a central (ß/α)7-barrel domain that contains an embedded helix domain and an α-helix-rich C-terminal domain. The active-site cleft is located at the interface of the central and C-terminal domains. Amino acid substitution at Trp22, which is separate from the catalytic nucleophilic residue, abolished both enzymatic activities, indicating that Trp22 might be responsible for substrate recognition. We also observed that shortening of the flexible loop near the catalytic residue changed branched chain lengths of the reaction products with increased hydrolytic activity. Taken together, our findings propose a molecular mechanism for how GH57 GBEs exhibit the two activities and where the substrate binds the enzyme.

Renew or die: The molecular mechanisms of peptidoglycan recycling and antibiotic resistance in Gram-negative pathogens.

Antimicrobial resistance is one of the most serious health threats. Cell-wall remodeling processes are tightly regulated to warrant bacterial survival and in some cases are directly linked to antibiotic resistance. Remodeling produces cell-wall fragments that are recycled but can also act as messengers for bacterial communication, as effector molecules in immune response and as signaling molecules triggering antibiotic resistance. This review is intended to provide state-of-the-art information about the molecular mechanisms governing this process and gather structural information of the different macromolecular machineries involved in peptidoglycan recycling in Gram-negative bacteria. The growing body of literature on the 3D structures of the corresponding macromolecules reveals an extraordinary complexity. Considering the increasing incidence and widespread emergence of Gram-negative multidrug-resistant pathogens in clinics, structural information on the main actors of the recycling process paves the way for designing novel antibiotics disrupting cellular communication in the recycling-resistance pathway.

Controlling Autolysis During Flagella Insertion in Gram-Negative Bacteria.

The flagellum is an important macromolecular machine for many pathogenic bacteria. It is a hetero-oligomeric structure comprised of three major sub-structures: basal body, hook and thin helical filament. An important step during flagellum assembly is the localized and controlled degradation of the peptidoglycan sacculus to allow for the insertion of the rod as well as to facilitate anchoring for proper motor function. The peptidoglycan lysis events require specialized lytic enzymes, ß-N-acetylglucosaminidases and lytic transglycosylases, which differ in flagellated proteobacteria. Due to their autolytic activity, these enzymes need to be controlled in order to prevent cellular lysis. This review summarizes are current understanding of the peptidoglycan lysis events required for flagellum assembly and motility with a main focus on Gram-negative bacteria.

Outer-membrane lipoprotein LpoB spans the periplasm to stimulate the peptidoglycan synthase PBP1B.

Bacteria surround their cytoplasmic membrane with an essential, stress-bearing peptidoglycan (PG) layer. Growing and dividing cells expand their PG layer by using membrane-anchored PG synthases, which are guided by dynamic cytoskeletal elements. In Escherichia coli, growth of the mainly single-layered PG is also regulated by outer membrane-anchored lipoproteins. The lipoprotein LpoB is required for the activation of penicillin-binding protein (PBP) 1B, which is a major, bifunctional PG synthase with glycan chain polymerizing (glycosyltransferase) and peptide cross-linking (transpeptidase) activities. Here, we report the structure of LpoB, determined by NMR spectroscopy, showing an N-terminal, 54-aa-long flexible stretch followed by a globular domain with similarity to the N-terminal domain of the prevalent periplasmic protein TolB. We have identified the interaction interface between the globular domain of LpoB and the noncatalytic UvrB domain 2 homolog domain of PBP1B and modeled the complex. Amino acid exchanges within this interface weaken the PBP1B-LpoB interaction, decrease the PBP1B stimulation in vitro, and impair its function in vivo. On the contrary, the N-terminal flexible stretch of LpoB is required to stimulate PBP1B in vivo, but is dispensable in vitro. This supports a model in which LpoB spans the periplasm to interact with PBP1B and stimulate PG synthesis.

Modulation of the Lytic Activity of the Dedicated Autolysin for Flagellum Formation SltF by Flagellar Rod Proteins FlgB and FlgF.

UNLABELLED: SltF was identified previously as an autolysin required for the assembly of flagella in the alphaproteobacteria, but the nature of its peptidoglycan lytic activity remained unknown. Sequence alignment analyses suggest that it could function as either a muramidase, lytic transglycosylase, or ß-N-acetylglucosaminidase. Recombinant SltF from Rhodobacter sphaeroides was purified to apparent homogeneity, and it was demonstrated to function as a lytic transglycosylase based on enzymatic assays involving mass spectrometric analyses. Circular dichroism (CD) analysis determined that it is composed of 83.4% α-structure and 1.48% ß-structure and thus is similar to family 1A lytic transglycosylases. However, alignment of apparent SltF homologs identified in the genome database defined a new subfamily of the family 1 lytic transglycosylases. SltF was demonstrated to be endo-acting, cleaving within chains of peptidoglycan, with optimal activity at pH 7.0. Its activity is modulated by two flagellar rod proteins, FlgB and FlgF: FlgB both stabilizes and stimulates SltF activity, while FlgF inhibits it. Invariant Glu57 was confirmed as the sole catalytic acid/base residue of SltF. IMPORTANCE: The bacterial flagellum is comprised of a basal body, hook, and helical filament, which are connected by a rod structure. With a diameter of approximately 4 nm, the rod is larger than the estimated pore size within the peptidoglycan sacculus, and hence its insertion requires the localized and controlled lysis of this essential cell wall component. In many beta- and gammaproteobacteria, this lysis is catalyzed by the ß-N-acetylglucosaminidase domain of FlgJ. However, FlgJ of the alphaproteobacteria lacks this activity and instead it recruits a separate enzyme, SltF, for this purpose. In this study, we demonstrate that SltF functions as a newly identified class of lytic transglycosylases and that its autolytic activity is uniquely modulated by two rod proteins, FlgB and FlgF.

Site-Specific Immobilization of the Peptidoglycan Synthase PBP1B on a Surface Plasmon Resonance Chip Surface.

Surface plasmon resonance (SPR) is one of the most powerful label-free methods to determine the kinetic parameters of molecular interactions in real time and in a highly sensitive way. Penicillin-binding proteins (PBPs) are peptidoglycan synthesis enzymes present in most bacteria. Established protocols to analyze interactions of PBPs by SPR involve immobilization to an ampicillin-coated chip surface (a ß-lactam antibiotic mimicking its substrate), thereby forming a covalent complex with the PBPs transpeptidase (TP) active site. However, PBP interactions measured with a substrate-bound TP domain potentially affect interactions near the TPase active site. Furthermore, in vivo PBPs are anchored in the inner membrane by an N-terminal transmembrane helix, and hence immobilization at the C-terminal TPase domain gives an orientation contrary to the in vivo situation. We designed a new procedure: immobilization of PBP by copper-free click chemistry at an azide incorporated in the N terminus. In a proof-of-principle study, we immobilized Escherichia coli PBP1B on an SPR chip surface and used this for the analysis of the well-characterized interaction of PBP1B with LpoB. The site-specific incorporation of the azide affords control over protein orientation, thereby resulting in a homogeneous immobilization on the chip surface. This method can be used to study topology-dependent interactions of any (membrane) protein.

One-pot protection-glycosylation reactions for synthesis of lipid†II analogues.

(2,6-Dichloro-4-methoxyphenyl)(2,4-dichlorophenyl)methyl trichloroacetimidate (3) and its polymer-supported reagent 4 can be successfully applied to a one-pot protection-glycosylation reaction to form the disaccharide derivative 7 d for the synthesis of lipid II analogues. The temporary protecting group or linker at the C-6 position and N-Troc protecting group of 7 d can be cleaved simultaneously through a reductive condition. Overall yields of syntheses of lipid II (1) and neryl-lipid II N(ε)-dansylthiourea are significantly improved by using the described methods.

Evidence for lytic transglycosylase and ß-N-acetylglucosaminidase activities located at the polyhydroxyalkanoates (PHAs) granules of Thermus thermophilus HB8.

The thermophilic bacterium Thermus thermophilus HB8 accumulates polyhydroxyalkanoates (PHAs) as intracellular granules used by cells as carbon and energy storage compounds. PHAs granules were isolated from cells grown in sodium gluconate (1.5 % w/v) as carbon source. Lytic activities are strongly associated and act to the PHAs granules proved with various methods. Specialized lytic trasglycosylases (LTGs) are muramidases capable of locally degrading the peptidoglycan (PG) meshwork of Gram negative bacteria. These enzymes cleave the ß-1,4-glycosidic linkages between the N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc) residues of PG. Lysozyme-like activity/-ies were detected using lysoplate assay. Chitinolytic activity/-ies, were detected as N-acetyl glucosaminidases (NAG) (E.C.3.2.1.5.52) hydrolyzing the synthetic substrate p-nitrophenyl-N-acetyl-ß-D-glucosaminide (pNP-GlcNAc) releasing pNP and GlcNAc. Using zymogram analysis two abundant LTGs were revealed hydrolyzing cell wall of Micrococcus lysodeikticus or purified PG incorporated as natural substrates, in SDS-PAGE and then renaturation. These proteins corresponded in a SDS-PAGE and Coomassie-stained gel in molecular mass of 110 and 32 kDa respectively, were analyzed by MALDI-MS (Matrix-assisted laser desorption/ionization-Mass Spectrometry). The 110 kDa protein was identified as an S-layer domain-containing protein [gi|336233805], while the 32 kDa similar to the hypothetical protein VDG1235_2196 (gi/254443957). Overall, the localization of PG hydrolases in PHAs granules appears to be involved to their biogenesis from membranes, and probably promoting septal PG splitting and daughter cell separation.

SAHBNET, an accessible surface-based elastic network: an application to membrane protein.

Molecular Dynamics is a method of choice for membrane simulations and the rising of coarse-grained forcefields has opened the way to longer simulations with reduced calculations times. Here, we present an elastic network, SAHBNET (Surface Accessibility Hydrogen-Bonds elastic NETwork), that will maintain the structure of soluble or membrane proteins based on the hydrogen bonds present in the atomistic structure and the proximity between buried residues. This network is applied on the coarse-grained beads defined by the MARTINI model, and was designed to be more physics-based than a simple elastic network. The SAHBNET model is evaluated against atomistic simulations, and compared with ELNEDYN models. The SAHBNET is then used to simulate two membrane proteins inserted in complex lipid bilayers. These bilayers are formed by self-assembly and the use of a modified version of the GROMACS tool genbox (which is accessible through the gcgs.gembloux.ulg.ac.be website). The results show that SAHBNET keeps the structure close to the atomistic one and is successfully used for the simulation of membrane proteins.

The catalytic domain of the germination-specific lytic transglycosylase SleB from Bacillus anthracis displays a unique active site topology.

Bacillus anthracis produces metabolically inactive spores. Germination of these spores requires germination-specific lytic enzymes (GSLEs) that degrade the unique cortex peptidoglycan to permit resumption of metabolic activity and outgrowth. We report the first crystal structure of the catalytic domain of a GSLE, SleB. The structure revealed a transglycosylase fold with unique active site topology and permitted identification of the catalytic glutamate residue. Moreover, the structure provided insights into the molecular basis for the specificity of the enzyme for muramic-δ-lactam-containing cortex peptidoglycan. The protein also contains a metal-binding site that is positioned directly at the entrance of the substrate-binding cleft.