Search

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. [ABSTRACT FROM AUTHOR]

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. Author summary: SEDS (Shape, Elongation, Division and Sporulation) proteins are widely conserved peptidoglycan polymerases that play critical roles in cell elongation and cell division in rod-shaped bacteria. However, how they catalyze PG polymerization remains poorly understood. In this study, we isolated and characterized a set of dominant-negative mutations in the SEDS protein FtsW, which synthesizes septal peptidoglycan during cell division in most bacteria. Our results revealed that the dominant-negative mutations disrupt FtsW's ability to synthesize peptidoglycan, but do not affect its other activities, suggesting that the corresponding amino acids may constitute the active site of FtsW. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 β-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 β-lactams, other classes of molecules (non-β-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. [ABSTRACT FROM AUTHOR]

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 β-sandwich of two symmetrical four-stranded β-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. [ABSTRACT FROM AUTHOR]

Peptidoglycan (PG) is an essential net-like macromolecule that surrounds bacteria, gives them their shape, and protects them against their own high osmotic pressure. PG synthesis inhibition leads to bacterial cell lysis, making it an important target for many antibiotics. The final two reactions in PG synthesis are performed by penicillin-binding proteins (PBPs). Their glycosyltransferase (GT) activity uses the lipid II precursor to synthesize glycan chains and their transpeptidase (TP) activity catalyzes the cross-linking of two glycan chains via the peptide side chains. Inhibition of either of these two reactions leads to bacterial cell death. β-lactam antibiotics target the transpeptidation reaction while antibiotic therapy based on inhibition of the GTs remains to be developed. Ongoing research is trying to fill this gap by studying the interactions of GTs with inhibitors and substrate mimics and utilizing the latter as templates for the design of new antibiotics. In this review we present an updated overview on the GTs and describe the structure-activity relationship of recently developed synthetic ligands. [ABSTRACT FROM AUTHOR]

Cell wall biosynthesis is a key target for antibacterial drugs. The major constituent of the bacterial wall, peptidoglycan, is a netlike polymer responsible for the size and shape of the cell and for resisting osmotic pressure. It consists of glycan chains of repeating disaccharide units cross-linked through short peptide chains. Peptidoglycan assembly is catalyzed by the periplasmic domain of bifunctional class A penicillin-binding proteins. Cross-linking of the peptide chains is catalyzed by their transpeptidase module, which can be inhibited by the most widely used antibiotics, the β-lactams. In contrast, no drug in clinical use inhibits the polymerization of the glycan chains, catalyzed by their glycosyltransferase module, although it is an obvious target. We report here the purification of the ectodomain of the class A penicillin-binding protein 1a from Thermotoga maritima (Tm-1a*), expressed recombinantly in Escherichia coli. A detergent screen showed that detergents with shorter aliphatic chains were better solubilizers. Cyclohexyl-hexyl-β-d-maltoside-purified Tm-1a* was found to be monomeric and to have improved thermal stability. A miniaturized, multiwell continuous fluorescence assay of the glycosyltransferase activity was used to screen for optimal reaction conditions. Tm-1a* was active as a glycosyltransferase, catalyzing the formation of glycan chains up to 16 disaccharide units long. Our results emphasize the importance of the detergent in preparing a stable monomeric ectodomain of a class A penicillin-binding protein. Our assay could be used to screen collections of compounds for inhibitors of peptidoglycan glycosyltransferases that could serve as the basis for the development of novel antibiotics. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Myosin V is a double-headed molecular motor involved in organelle transport. Two distinctive features of this motor, processivity and the ability to take extended linear steps of ≈36 nm along the actin helical track, depend on its unusually long light chain-binding domain (LCBD). The LCBD of myosin V consists of six tandem IQ motifs, which constitute the binding sites for calmodulin (CaM) and CaM-like light chains. Here, we report the 2-Å resolution crystal structure of myosin light chain 1 (Mlc1p) bound to the IQ2-IQ3 fragment of Myo2p, a myosin V from Saccharomyces cerevisiae. This structure, combined with FRET distance measurements between probes in various CaM-IQ complexes, comparative sequence analysis, and the previously determined structures of Mlc1p-IQ2 and MLc1p-IQ4, allowed building a model of the LCBD of myosin V. The IQs of myosin V are distributed into three pairs. There appear to be specific cooperative interactions between light chains within each IQ pair, but little or no interaction between pairs, providing flexibility at their junctions. The second and third IQ pairs each present a light chain, whether CaM or a CaM-related molecule, bound in a noncanonical extended conformation in which the N-lobe does not interact with the IQ motif The resulting free N-lobes may engage in protein-protein interactions. The extended conformation is characteristic of the single IQ of myosin Vi and is common throughout the myosin superfamily. The model points to a prominent role of the LCBD in the function, regulation, and molecular interactions of myosin V. [ABSTRACT FROM AUTHOR]

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?Å 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. [ABSTRACT FROM AUTHOR]

IQ motifs are widespread in nature. Mlc1p is a calmodulin-tike myosin tight chain that binds to IQ motifs of a class V myosin, Myo2p, and an IQGAP- related protein, Iqg1p, playing a rote in polarized growth and cytokinesis in Saccharomyces cerevisiae. The crystal structures of Mlc1p bound to IQ2 and IQ4 of Myo2p differ dramatically. When bound to IQ2, MIc1p adopts a compact conformation in which both the N- and C-lobes interact with the IQ motif. However, in the complex with IQ4, the N-lobe no longer interacts with the IQ motif, resulting in an extended conformation of Mlc1p. The two tight chain structures relate to two distinct subfamilies of IQ motifs, one of which does not interact with the N-lobes of calmodutin-like tight chains. The correlation between tight chain structure and IQ sequence is demonstrated further by sedimentation velocity analysis of complexes of Mlc1p with IQ motifs from Myo2p and Iqg1p. The resulting 'free' N-lobes of myosin tight chains in the extended conformation could mediate the formation of ternary complexes during protein localization and/or partner recruitment. [ABSTRACT FROM AUTHOR]

Mlc1p is a calmodulin-like protein from the budding yeast Saccharomyces cerevisiae, where it has been identified as a subunit of a class V myosin, Myo2p, and a binding partner of an IQGAP-like protein, Iqg1p. Through its interactions with these two proteins, Mlc1p plays a role in polarized growth and cytokinesis. Mlc1p has been crystallized in complexes with four different IQ target motifs from the neck region of Myo2p: IQ2, IQ3, IQ4 and IQ2-IQ3 (referred to as IQ2,3). Electron-density maps for two of the complexes (Mlc1p-IQ4 and Mlclp-IQ2,3) were obtained from multiple anomalous dispersion (MAD) experiments based on selenomethionine derivatives. The other two structures (Mlc1p-IQ2 and Mlc1p-IQ3) were determined by molecular replacement using the partially refined structure of Mlclp-IQ2,3 as a search model. [ABSTRACT FROM AUTHOR]

The penicillin-binding protein (PBP) 1b of Escherichia coli catalyses the assembly of lipid-transported N -acetyl glucosaminyl-β-1,4-N -acetylmuramoyl-l-alanyl-γ-d-glutamyl-(l)-meso -diaminopimelyl-(l)-d-alanyl-d-alanine disaccharide pentapeptide units into polymeric peptidoglycan. These units are phosphodiester linked, at C1 of muramic acid, to a C55 undecaprenyl carrier. PBP1b has been purified in the form of His tag (M46-N844) PBP1bγ. This derivative provides the host cell in which it is produced with a functional wall peptidoglycan. His tag (M46-N844) PBP1bγ possesses an amino-terminal hydrophobic segment, which serves as transmembrane spanner of the native PBP. This segment is linked, via an ≅ 100-amino-acid insert, to a D198-G435 glycosyl transferase module that possesses the five motifs characteristic of the PBPs of class A. In in vitro assays, the glycosyl transferase of the PBP catalyses the synthesis of linear glycan chains from the lipid carrier with an efficiency of ≅ 39 000 M-1 s-1 . Glu-233, of motif 1, is central to the catalysed reaction. It is proposed that the Glu-233 γ-COOH donates its proton to the oxygen atom of the scissile phosphoester bond of the lipid carrier, leading to the formation of an oxocarbonium cation, which then undergoes attack by the 4-OH group of a nucleophile N -acetylglucosamine. Asp-234 of motif 1 or Glu-290 of motif 3 could be involved in the stabilization of the oxocarbonium cation and the activation of the 4-OH group of the N -acetylglucosamine. In turn, Tyr-310 of motif 4 is an important component of the amino acid sequence-folding information. The glycosyl transferase module of PBP1b, the lysozymes and the lytic transglycosylase Slt70 have much the same catalytic machinery. They might be members of the same superfamily. The glycosyl transferase module is linked, via a short junction site, to the amino end of a Q447-N844 acyl transferase module, which... [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background:Lantibiotics are heat-stable peptides characterized by the presence of thioether amino acid lanthionine and methyllanthionine. They are capable to inhibit the growth of Gram-positive bacteria, including Listeria monocytogenes, Staphylococcus aureus or Bacillus cereus, the causative agents of food-borne diseases or nosocomial infections. Lantibiotic biosynthetic machinery is encoded by gene cluster composed by a structural gene that codes for a pre-lantibiotic peptide and other genes involved in pre-lantibiotic modifications, regulation, export and immunity. Methodology/Findings:Bacillus amyloliquefaciens GA1 was found to produce an antimicrobial peptide, named amylolysin, active on an array of Gram-positive bacteria, including methicillin resistant S. aureus. Genome characterization led to the identification of a putative lantibiotic gene cluster that comprises a structural gene (amlA) and genes involved in modification (amlM), transport (amlT), regulation (amlKR) and immunity (amlFE). Disruption of amlA led to loss of biological activity, confirming thus that the identified gene cluster is related to amylolysin synthesis. MALDI-TOF and LC-MS analysis on purified amylolysin demonstrated that this latter corresponds to a novel lantibiotic not described to date. The ability of amylolysin to interact invitro with the lipid II, the carrier of peptidoglycan monomers across the cytoplasmic membrane and the presence of a unique modification gene suggest that the identified peptide belongs to the group B lantibiotic. Amylolysin immunity seems to be driven by only two AmlF and AmlE proteins, which is uncommon within the Bacillus genus. Conclusion/Significance:Apart from mersacidin produced by Bacillus amyloliquefaciens strains Y2 and HIL Y-85,544728, reports on the synthesis of type B-lantibiotic in this species are scarce. This study reports on a genetic and structural characterization of another representative of the type B lantibiotic in B. amyloliquefaciens. [ABSTRACT FROM AUTHOR]

A correction to the article "The penicillin-binding proteins: structure and role in peptidoglycan biosynthesis" that was published in the 2008 issue is presented.