Identification of the potential active site of the septal peptidoglycan polymerase FtsW.

SEDS (Shape, Elongation, Division and Sporulation) proteins are widely conserved peptidoglycan (PG) glycosyltransferases that form complexes with class B penicillin-binding proteins (bPBPs, with transpeptidase activity) to synthesize PG during bacterial cell growth and division. Because of their crucial roles in bacterial morphogenesis, SEDS proteins are one of the most promising targets for the development of new antibiotics. However, how SEDS proteins recognize their substrate lipid II, the building block of the PG layer, and polymerize it into glycan strands is still not clear. In this study, we isolated and characterized dominant-negative alleles of FtsW, a SEDS protein critical for septal PG synthesis during bacterial cytokinesis. Interestingly, most of the dominant-negative FtsW mutations reside in extracellular loops that are highly conserved in the SEDS family. Moreover, these mutations are scattered around a central cavity in a modeled FtsW structure, which has been proposed to be the active site of SEDS proteins. Consistent with this, we found that these mutations blocked septal PG synthesis but did not affect FtsW localization to the division site, interaction with its partners nor its substrate lipid II. Taken together, these results suggest that the residues corresponding to the dominant-negative mutations likely constitute the active site of FtsW, which may aid in the design of FtsW inhibitors.

Development of a bispecific Nanobody anti-F17 fimbria as a potential therapeutic tool.

Pathogenic strains of Escherichia coli F17+ are associated with various intestinal and extra-intestinal pathologies, including diarrhea, and result in significant animal mortality. These infections rely on the expression of virulence factors, such as F17 fimbriae, for adhesion. F17 fimbriae form a protective layer on the surface of E. coli bacteria, consisting of a major structural subunit, F17A, and a minor functional subunit, F17G. Because of the evolution of bacterial resistance, conventional antibiotic treatments have limited efficacy. Therefore, there is a pressing need to develop novel therapeutic tools. In this study, we cloned and produced the F17G protein. We then immunized a camel with the purified F17G protein and constructed a VHH library consisting of 2 × 109 clones. The library was then screened against F17G protein using phage display technology. Through this process, we identified an anti-F17G nanobody that was subsequently linked, via a linker, to an anti-F17A nanobody, resulting in the creation of an effective bispecific nanobody. Comprehensive characterization of this bispecific nanobody demonstrated excellent production, specific binding capacity to both recombinant forms of the two F17 antigens and the E. coli F17+ strain, remarkable stability in camel serum, and superior resistance to pepsin protease. The successful generation of this bispecific nanobody with excellent production, specific binding capacity and stability highlights its potential as a valuable tool for fighting infections caused by pathogenic E. coli F17+ strain.

The bacterial cell division protein fragment EFtsN binds to and activates the major peptidoglycan synthase PBP1b.

Peptidoglycan (PG) is an essential constituent of the bacterial cell wall. During cell division, the machinery responsible for PG synthesis localizes mid-cell, at the septum, under the control of a multiprotein complex called the divisome. In Escherichia coli, septal PG synthesis and cell constriction rely on the accumulation of FtsN at the division site. Interestingly, a short sequence of FtsN (Leu75-Gln93, known as EFtsN) was shown to be essential and sufficient for its functioning in vivo, but what exactly this sequence is doing remained unknown. Here, we show that EFtsN binds specifically to the major PG synthase PBP1b and is sufficient to stimulate its biosynthetic glycosyltransferase (GTase) activity. We also report the crystal structure of PBP1b in complex with EFtsN, which demonstrates that EFtsN binds at the junction between the GTase and UB2H domains of PBP1b. Interestingly, mutations to two residues (R141A/R397A) within the EFtsN-binding pocket reduced the activation of PBP1b by FtsN but not by the lipoprotein LpoB. This mutant was unable to rescue the ΔponB-ponAts strain, which lacks PBP1b and has a thermosensitive PBP1a, at nonpermissive temperature and induced a mild cell-chaining phenotype and cell lysis. Altogether, the results show that EFtsN interacts with PBP1b and that this interaction plays a role in the activation of its GTase activity by FtsN, which may contribute to the overall septal PG synthesis and regulation during cell division.

The crystal structure of the cell division amidase AmiC reveals the fold of the AMIN domain, a new peptidoglycan binding domain.

Binary fission is the ultimate step of the prokaryotic cell cycle. In Gram-negative bacteria like Escherichia coli, this step implies the invagination of three biological layers (cytoplasmic membrane, peptidoglycan and outer membrane), biosynthesis of the new poles and eventually, daughter cells separation. The latter requires the coordinated action of the N-acetylmuramyl-L-alanine amidases AmiA/B/C and their LytM activators EnvC and NlpD to cleave the septal peptidoglycan. We present here the 2.5 Å crystal structure of AmiC which includes the first report of an AMIN domain structure, a ß-sandwich of two symmetrical four-stranded ß-sheets exposing highly conserved motifs on the two outer faces. We show that this N-terminal domain, involved in the localization of AmiC at the division site, is a new peptidoglycan-binding domain. The C-terminal catalytic domain shows an auto-inhibitory alpha helix obstructing the active site. AmiC lacking this helix exhibits by itself an activity comparable to that of the wild type AmiC activated by NlpD. We also demonstrate the interaction between AmiC and NlpD by microscale thermophoresis and confirm the importance of the active site blocking alpha helix in the regulation of the amidase activity.

Cooperativity of peptidoglycan synthases active in bacterial cell elongation.

Growth of the bacterial cell wall peptidoglycan sacculus requires the co-ordinated activities of peptidoglycan synthases, hydrolases and cell morphogenesis proteins, but the details of these interactions are largely unknown. We now show that the Escherichia coli peptidoglycan glycosyltrasferase-transpeptidase PBP1A interacts with the cell elongation-specific transpeptidase PBP2 in vitro and in the cell. Cells lacking PBP1A are thinner and initiate cell division later in the cell cycle. PBP1A localizes mainly to the cylindrical wall of the cell, supporting its role in cell elongation. Our in vitro peptidoglycan synthesis assays provide novel insights into the cooperativity of peptidoglycan synthases with different activities. PBP2 stimulates the glycosyltransferase activity of PBP1A, and PBP1A and PBP2 cooperate to attach newly synthesized peptidoglycan to sacculi. PBP2 has peptidoglycan transpeptidase activity in the presence of active PBP1A. Our data also provide a possible explanation for the depletion of lipid II precursors in penicillin-treated cells.

Nanobody-Based Sandwich Immunoassay for Pathogenic Escherichia coli F17 Strain Detection.

Rapid and specific detection of pathogenic bacteria in fecal samples is of critical importance for the diagnosis of neonatal diarrhea in veterinary clinics. Nanobodies are a promising tool for the treatment and diagnosis of infectious diseases due to their unique recognition properties. In this study, we report the design of a nanobody-based magnetofluorescent immunoassay for the sensitive detection of pathogenic Escherichia coli F17-positive strains (E. coli F17). For this, a camel was immunized with purified F17A protein from F17 fimbriae and a nanobody library was constructed by phage display. Two specific anti-F17A nanobodies (Nbs) were selected to design the bioassay. The first one (Nb1) was conjugated to magnetic beads (MBs) to form a complex capable of efficiently capturing the target bacteria. A second horseradish peroxidase (HRP)-conjugated nanobody (Nb4) was used for detection by oxidizing o-phenylenediamine (OPD) to fluorescent 2,3-diaminophenazine (DAP). Our results show that the immunoassay recognizes E. coli F17 with high specificity and sensitivity, with a detection limit of 1.8 CFU/mL in only 90 min. Furthermore, we showed that the immunoassay can be applied to fecal samples without pretreatment and remains stable for at least one month when stored at 4 °C.

Synthesis of modified peptidoglycan precursor analogues for the inhibition of glycosyltransferase.

The peptidoglycan glycosyltransferases (GTs) are essential enzymes that catalyze the polymerization of glycan chains of the bacterial cell wall from lipid II and thus constitute a validated antibacterial target. Their enzymatic cavity is composed of a donor site for the growing glycan chain (where the inhibitor moenomycin binds) and an acceptor site for lipid II substrate. In order to find lead inhibitors able to fill this large active site, we have synthesized a series of substrate analogues of lipid I and lipid II with variations in the lipid, the pyrophosphate, and the peptide moieties and evaluated their biological effect on the GT activity of E. coli PBP1b and their antibacterial potential. We found several compounds able to inhibit the GT activity in vitro and cause growth defect in Bacillus subtilis . The more active was C16-phosphoglycerate-MurNAc-(L-Ala-D-Glu)-GlcNAc, which also showed antibacterial activity. These molecules are promising leads for the design of new antibacterial GT inhibitors.

Monofunctional transglycosylases are not essential for Staphylococcus aureus cell wall synthesis.

The polymerization of peptidoglycan is the result of two types of enzymatic activities: transglycosylation, the formation of linear glycan chains, and transpeptidation, the formation of peptide cross-bridges between the glycan strands. Staphylococcus aureus has four penicillin binding proteins (PBP1 to PBP4) with transpeptidation activity, one of which, PBP2, is a bifunctional enzyme that is also capable of catalyzing transglycosylation reactions. Additionally, two monofunctional transglycosylases have been reported in S. aureus: MGT, which has been shown to have in vitro transglycosylase activity, and a second putative transglycosylase, SgtA, identified only by sequence analysis. We have now shown that purified SgtA has in vitro transglycosylase activity and that both MGT and SgtA are not essential in S. aureus. However, in the absence of PBP2 transglycosylase activity, MGT but not SgtA becomes essential for cell viability. This indicates that S. aureus cells require one transglycosylase for survival, either PBP2 or MGT, both of which can act as the sole synthetic transglycosylase for cell wall synthesis. We have also shown that both MGT and SgtA interact with PBP2 and other enzymes involved in cell wall synthesis in a bacterial two-hybrid assay, suggesting that these enzymes may work in collaboration as part of a larger, as-yet-uncharacterized cell wall-synthetic complex.

Structural basis of protein phosphatase 1 regulation.

The coordinated and reciprocal action of serine/threonine (Ser/Thr) protein kinases and phosphatases produces transient phosphorylation, a fundamental regulatory mechanism for many biological processes. The human genome encodes a far greater number of Ser/Thr protein kinases than of phosphatases. Protein phosphatase 1 (PP1), in particular, is ubiquitously distributed and regulates a broad range of cellular functions, including glycogen metabolism, cell-cycle progression and muscle relaxation. PP1 has evolved effective catalytic machinery but lacks substrate specificity. Substrate specificity is conferred upon PP1 through interactions with a large number of regulatory subunits. The regulatory subunits are generally unrelated, but most possess the RVxF motif, a canonical PP1-binding sequence. Here we reveal the crystal structure at 2.7 A resolution of the complex between PP1 and a 34-kDa N-terminal domain of the myosin phosphatase targeting subunit MYPT1. MYPT1 is the protein that regulates PP1 function in smooth muscle relaxation. Structural elements amino- and carboxy-terminal to the RVxF motif of MYPT1 are positioned in a way that leads to a pronounced reshaping of the catalytic cleft of PP1, contributing to the increased myosin specificity of this complex. The structure has general implications for the control of PP1 activity by other regulatory subunits.

The penicillin-binding proteins: structure and role in peptidoglycan biosynthesis.

Penicillin-binding proteins (PBPs) have been scrutinized for over 40 years. Recent structural information on PBPs together with the ongoing long-term biochemical experimental investigations, and results from more recent techniques such as protein localization by green fluorescent protein-fusion immunofluorescence or double-hybrid assay, have brought our understanding of the last stages of the peptidoglycan biosynthesis to an outstanding level that allows a broad outlook on the properties of these enzymes. Details are emerging regarding the interaction between the peptidoglycan-synthesizing PBPs and the peptidoglycan, their mesh net-like product that surrounds and protects bacteria. This review focuses on the detailed structure of PBPs and their implication in peptidoglycan synthesis, maturation and recycling. An overview of the content in PBPs of some bacteria is provided with an emphasis on comparing the biochemical properties of homologous PBPs (orthologues) belonging to different bacteria.

Fluorescence anisotropy assays for high throughput screening of compounds binding to lipid II, PBP1b, FtsW and MurJ.

Lipid II precursor and its processing by a flippase and peptidoglycan polymerases are considered key hot spot targets for antibiotics. We have developed a fluorescent anisotropy (FA) assay using a unique and versatile probe (fluorescent lipid II) and monitored direct binding between lipid II and interacting proteins (PBP1b, FtsW and MurJ), as well as between lipid II and interacting antibiotics (vancomycin, nisin, ramoplanin and a small molecule). Competition experiments performed using unlabelled lipid II, four lipid II-binding antibiotics and moenomycin demonstrate that the assay can detect compounds interacting with lipid II or the proteins. These results provide a proof-of-concept for the use of this assay in a high-throughput screening of compounds against all these targets. In addition, the assay constitutes a powerful tool in the study of the mode of action of compounds that interfere with these processes. Interestingly, FA assay with lipid II probe has the advantage over moenomycin based probe to potentially identify compounds that interfere with both donor and acceptor sites of the aPBPs GTase as well as compounds that bind to lipid II. In addition, this assay would allow the screening of compounds against SEDS proteins and MurJ which do not interact with moenomycin.

Squalamine and Aminosterol Mimics Inhibit the Peptidoglycan Glycosyltransferase Activity of PBP1b.

Peptidoglycan (PG) is an essential polymer of the bacterial cell wall and a major antibacterial target. Its synthesis requires glycosyltransferase (GTase) and transpeptidase enzymes that, respectively, catalyze glycan chain elongation and their cross-linking to form the protective sacculus of the bacterial cell. The GTase domain of bifunctional penicillin-binding proteins (PBPs) of class A, such as Escherichia coli PBP1b, belong to the GTase 51 family. These enzymes play an essential role in PG synthesis, and their specific inhibition by moenomycin was shown to lead to bacterial cell death. In this work, we report that the aminosterol squalamine and mimic compounds present an unexpected mode of action consisting in the inhibition of the GTase activity of the model enzyme PBP1b. In addition, selected compounds were able to specifically displace the lipid II from the active site in a fluorescence anisotropy assay, suggesting that they act as competitive inhibitors.

SPOR Proteins Are Required for Functionality of Class A Penicillin-Binding Proteins in Escherichia coli.

Sporulation-related repeat (SPOR) domains are present in many bacterial cell envelope proteins and are known to bind peptidoglycan. Escherichia coli contains four SPOR proteins, DamX, DedD, FtsN, and RlpA, of which FtsN is essential for septal peptidoglycan synthesis. DamX and DedD may also play a role in cell division, based on mild cell division defects observed in strains lacking these SPOR domain proteins. Here, we show by nuclear magnetic resonance (NMR) spectroscopy that the periplasmic part of DedD consists of a disordered region followed by a canonical SPOR domain with a structure similar to that of the SPOR domains of FtsN, DamX, and RlpA. The absence of DamX or DedD decreases the functionality of the bifunctional transglycosylase-transpeptidase penicillin-binding protein 1B (PBP1B). DamX and DedD interact with PBP1B and stimulate its glycosyltransferase activity, and DamX also stimulates the transpeptidase activity. DedD also binds to PBP1A and stimulates its glycosyltransferase activity. Our data support a direct role of DamX and DedD in enhancing the activity of PBP1B and PBP1A, presumably during the synthesis of the cell division septum.IMPORTANCEEscherichia coli has four SPOR proteins that bind peptidoglycan, of which FtsN is essential for cell division. DamX and DedD are suggested to have semiredundant functions in cell division based on genetic evidence. Here, we solved the structure of the SPOR domain of DedD, and we show that both DamX and DedD interact with and stimulate the synthetic activity of the peptidoglycan synthases PBP1A and PBP1B, suggesting that these class A PBP enzymes act in concert with peptidoglycan-binding proteins during cell division.

Regulation of the Peptidoglycan Polymerase Activity of PBP1b by Antagonist Actions of the Core Divisome Proteins FtsBLQ and FtsN.

Peptidoglycan (PG) is an essential constituent of the bacterial cell wall. During cell division, PG synthesis localizes at midcell under the control of a multiprotein complex, the divisome, allowing the safe formation of two new cell poles and separation of daughter cells. Genetic studies in Escherichia coli pointed out that FtsBLQ and FtsN participate in the regulation of septal PG (sPG) synthesis; however, the underlying molecular mechanisms remained largely unknown. Here we show that FtsBLQ subcomplex directly interacts with the PG synthase PBP1b and with the subcomplex FtsW-PBP3, mainly via FtsW. Strikingly, we discovered that FtsBLQ inhibits the glycosyltransferase activity of PBP1b and that this inhibition was antagonized by the PBP1b activators FtsN and LpoB. The same results were obtained in the presence of FtsW-PBP3. Moreover, using a simple thioester substrate (S2d), we showed that FtsBLQ also inhibits the transpeptidase domain of PBP3 but not of PBP1b. As the glycosyltransferase and transpeptidase activities of PBP1b are coupled and PBP3 activity requires nascent PG substrate, the results suggest that PBP1b inhibition by FtsBLQ will block sPG synthesis by these enzymes, thus maintaining cell division as repressed until the maturation of the divisome is signaled by the accumulation of FtsN, which triggers sPG synthesis and the initiation of cell constriction. These results confirm that PBP1b plays an important role in E. coli cell division and shed light on the specific role of FtsN, which seems to counterbalance the inhibitory effect of FtsBLQ to restore PBP1b activity.IMPORTANCE Bacterial cell division is governed by a multiprotein complex called divisome, which facilitates a precise cell wall synthesis at midcell and daughter cell separation. Protein-protein interactions and activity studies using different combinations of the septum synthesis core of the divisome revealed that the glycosyltransferase activity of PBP1b is repressed by FtsBLQ and that the presence of FtsN or LpoB suppresses this inhibition. Moreover, FtsBLQ also inhibits the PBP3 activity on a thioester substrate. These results provide enzymatic evidence of the regulation of the peptidoglycan synthase PBP1b and PBP3 within the divisome. The results confirm that PBP1b plays an important role in E. coli cell division and shed light on the specific role of FtsN, which functions to relieve the repression on PBP1b by FtsBLQ and to initiate septal peptidoglycan synthesis.

The monofunctional glycosyltransferase of Escherichia coli localizes to the cell division site and interacts with penicillin-binding protein 3, FtsW, and FtsN.

The monofunctional peptidoglycan glycosyltransferase (MtgA) catalyzes glycan chain elongation of the bacterial cell wall. Here we show that MtgA localizes at the division site of Escherichia coli cells that are deficient in PBP1b and produce a thermosensitive PBP1a and is able to interact with three constituents of the divisome, PBP3, FtsW, and FtsN, suggesting that MtgA may play a role in peptidoglycan assembly during the cell cycle in collaboration with other proteins.

Peptidoglycan glycosyltransferase-ligand binding assay based on tryptophan fluorescence quenching.

Peptidoglycan glycosyltransferases (GTase) of family 51 are essential enzymes for the synthesis of the glycan chains of the bacterial cell wall. They are considered potential antibacterial target, but discovery of inhibitors was hampered so far by the lack of efficient and affordable screening assay. Here we used Staphylococcus aureus MtgA to introduce a single tryptophan reporter residue in selected positions flanking the substrates binding cavity of the protein. We selected a mutant (Y181W) that shows strong fluorescence quenching in the presence of moenomycin A and two lipid II analogs inhibitors. The assay provides a simple method to study GTase-ligand interactions and can be used as primary high throughput screening of GTase inhibitors without the need for lipid II substrate or reporter ligands.

Interplay between Penicillin-binding proteins and SEDS proteins promotes bacterial cell wall synthesis.

Bacteria utilize specialized multi-protein machineries to synthesize the essential peptidoglycan (PG) cell wall during growth and division. The divisome controls septal PG synthesis and separation of daughter cells. In E. coli, the lipid II transporter candidate FtsW is thought to work in concert with the PG synthases penicillin-binding proteins PBP3 and PBP1b. Yet, the exact molecular mechanisms of their function in complexes are largely unknown. We show that FtsW interacts with PBP1b and lipid II and that PBP1b, FtsW and PBP3 co-purify suggesting that they form a trimeric complex. We also show that the large loop between transmembrane helices 7 and 8 of FtsW is important for the interaction with PBP3. Moreover, we found that FtsW, but not the other flippase candidate MurJ, impairs lipid II polymerization and peptide cross-linking activities of PBP1b, and that PBP3 relieves these inhibitory effects. All together the results suggest that FtsW interacts with lipid II preventing its polymerization by PBP1b unless PBP3 is also present, indicating that PBP3 facilitates lipid II release and/or its transfer to PBP1b after transport across the cytoplasmic membrane. This tight regulatory mechanism is consistent with the cell's need to ensure appropriate use of the limited pool of lipid II.

Glycosyltransferases and Transpeptidases/Penicillin-Binding Proteins: Valuable Targets for New Antibacterials.

Peptidoglycan (PG) is an essential macromolecular sacculus surrounding most bacteria. It is assembled by the glycosyltransferase (GT) and transpeptidase (TP) activities of multimodular penicillin-binding proteins (PBPs) within multiprotein complex machineries. Both activities are essential for the synthesis of a functional stress-bearing PG shell. Although good progress has been made in terms of the functional and structural understanding of GT, finding a clinically useful antibiotic against them has been challenging until now. In contrast, the TP/PBP module has been successfully targeted by ß-lactam derivatives, but the extensive use of these antibiotics has selected resistant bacterial strains that employ a wide variety of mechanisms to escape the lethal action of these antibiotics. In addition to traditional ß-lactams, other classes of molecules (non-ß-lactams) that inhibit PBPs are now emerging, opening new perspectives for tackling the resistance problem while taking advantage of these valuable targets, for which a wealth of structural and functional knowledge has been accumulated. The overall evidence shows that PBPs are part of multiprotein machineries whose activities are modulated by cofactors. Perturbation of these systems could lead to lethal effects. Developing screening strategies to take advantage of these mechanisms could lead to new inhibitors of PG assembly. In this paper, we present a general background on the GTs and TPs/PBPs, a survey of recent issues of bacterial resistance and a review of recent works describing new inhibitors of these enzymes.

Structure-Activity Relationships of Novel Tryptamine-Based Inhibitors of Bacterial Transglycosylase.

Penicillin-binding proteins represent well-established, validated, and still very promising targets for the design and development of new antibacterial agents. The transglycosylase domain of penicillin-binding proteins is especially important, as it catalyzes polymerization of glycan chains, using the peptidoglycan precursor lipid II as a substrate. On the basis of the previous discovery of a noncovalent small-molecule inhibitor of transglycosylase activity, we systematically explored the structure-activity relationships of these tryptamine-based inhibitors. The main aim was to reduce the nonspecific cytotoxic properties of the initial hit compound and concurrently to retain the mode of its inhibition. A focused library of tryptamine-based compounds was synthesized, characterized, and evaluated biochemically. The results presented here show the successful reduction of the nonspecific cytotoxicity, and the retention of the inhibition of transglycosylase enzymatic activity, as well as the ability of these compounds to bind to lipid II and to have antibacterial actions.

Positive cooperativity between acceptor and donor sites of the peptidoglycan glycosyltransferase.

The glycosyltransferases of family 51 (GT51) catalyze the polymerization of lipid II to form linear glycan chains, which, after cross linking by the transpeptidases, form the net-like peptidoglycan macromolecule. The essential function of the GT makes it an attractive antimicrobial target; therefore a better understanding of its function and its mechanism of interaction with substrates could help in the design and the development of new antibiotics. In this work, we have used a surface plasmon resonance Biacore(®) biosensor, based on an amine derivative of moenomycin A immobilized on a sensor chip surface, to investigate the mechanism of binding of substrate analogous inhibitors to the GT. Addition of increasing concentrations of moenomycin A to the Staphylococcus aureus MtgA led to reduced binding of the protein to the sensor chip as expected. Remarkably, in the presence of low concentrations of the most active disaccharide inhibitors, binding of MtgA to immobilized moenomycin A was found to increase; in contrast competition with moenomycin A occurred only at high concentrations. This finding suggests that at low concentrations, the lipid II analogs bind to the acceptor site and induce a cooperative binding of moenomycin A to the donor site. Our results constitute the first indication of the existence of a positive cooperativity between the acceptor and the donor sites of peptidoglycan GTs. In addition, our study indicates that a modification of two residues (L119N and F120S) within the hydrophobic region of MtgA can yield monodisperse forms of the protein with apparently no change in its secondary structure content, but this is at the expense of the enzyme function.