Bacterial Cell Wall Peptidoglycan Research Articles

Structure of a peptidoglycan-related polysaccharide from Providencia alcalifaciens O45

A polysaccharide was isolated from the opportunistic human pathogen Providencia alcalifaciens O45:H26 by extraction with aqueous phenol and studied by sugar and methylation analyses along with (1)H and (13)C NMR spectroscopy, including two-dimensional ROESY and H-detected (1)H,(13)C HSQC experiments. The polysaccharide contains N-acetylglucosamine and N-acetylmuramic acid (D-GlcpNAc3Rlac) amidated with L-alanine and has the following structure: →4)-β-D-GlcpNAc-(1→4)-β-D-GlcpNAc3(Rlac-L-Ala)-(1→. The polysaccharide possesses a remarkable structural similarity to the bacterial cell wall peptidoglycan. It is not unique to the strain studied but is common to strains of at least four P. alcalifaciens O-serogroups (O3, O24, O38, and O45). No evidence was obtained that the polysaccharide is associated with the LPS, and hence it might represent a bacterial capsule component.

Characterization of Colicin M and its Orthologs Targeting Bacterial Cell Wall Peptidoglycan Biosynthesis

For a long time, colicin M was known for killing susceptible Escherichia coli cells by interfering with cell wall peptidoglycan biosynthesis, but its precise mode of action was only recently elucidated: this bacterial toxin was demonstrated to be an enzyme that catalyzes the specific degradation of peptidoglycan lipid intermediate II, thereby provoking the arrest of peptidoglycan synthesis and cell lysis. The discovery of this activity renewed the interest in this colicin and opened the way for biochemical and structural analyses of this new class of enzyme (phosphoesterase). The identification of a few orthologs produced by pathogenic strains of Pseudomonas further enlarged the field of investigation. The present article aims at reviewing recently acquired knowledge on the biology of this small family of bacteriocins.

Neisseria gonorrhoeae Virulence Factor NG1686 Is a Bifunctional M23B Family Metallopeptidase That Influences Resistance to Hydrogen Peroxide and Colony Morphology

Symptomatic gonococcal infection, caused exclusively by the human-specific pathogen Neisseria gonorrhoeae (the gonococcus), is characterized by the influx of polymorphonuclear leukocytes (PMNs) to the site of infection. Although PMNs possess a potent antimicrobial arsenal comprising both oxidative and non-oxidative killing mechanisms, gonococci survive this interaction, suggesting that the gonococcus has evolved many defenses against PMN killing. We previously identified the NG1686 protein as a gonococcal virulence factor that protects against both non-oxidative PMN-mediated killing and oxidative killing by hydrogen peroxide. In this work, we show that deletion of ng1686 affects gonococcal colony morphology but not cell morphology and that overexpression of ng1686 does not confer enhanced survival to hydrogen peroxide on gonococci. NG1686 contains M23B endopeptidase active sites found in proteins that cleave bacterial cell wall peptidoglycan. Strains of N. gonorrhoeae expressing mutant NG1686 proteins with substitutions in many, but not all, conserved metallopeptidase active sites recapitulated the hydrogen peroxide sensitivity and altered colony morphology of the Δng1686 mutant strain. We showed that purified NG1686 protein degrades peptidoglycan in vitro and that mutations in many conserved active site residues abolished its degradative activity. Finally, we demonstrated that NG1686 possesses both dd-carboxypeptidase and endopeptidase activities. We conclude that the NG1686 protein is a M23B peptidase with dual activities that targets the cell wall to affect colony morphology and resistance to hydrogen peroxide and PMN-mediated killing.

Virtual screening of phenylsulfonamido-3-morpholinopropan-2-yl dihydrogen phosphate derivatives as novel inhibitors of MurC–MurF ligases from Mycobacterium leprae

Multi-drug resistance capacity for Mycobacterium leprae demands the profound need for developing new multi-targeted anti-leprosy drugs. Mur ligases (MurC, MurD, MurE, and MurF) involved in biosynthesis of bacterial cell wall peptidoglycan are the best known and validated targets for antibacterial therapy. Transition-state analogs, such as phosphonates, phosphinates, and sulfonamides have good inhibitory activity toward any one or two of these Mur ligases. With an objective of designing a better inhibitor targeting all of these four Mur ligases, we developed phenylsulfonamido-3-morpholinopropan-2-yl dihydrogen phosphate derivatives as multi-targeted small molecule inhibitors for Mur ligases and evaluated using virtual screening studies. The results suggested the 1-(3-acetyl phenyl sulfonamide)-3-morpholino propan-2-yl dihydrogen phosphate as a novel multiple inhibitor for M. leprae MurC–MurF ligases.

Peptidoglycan as Nod1 ligand; fragment structures in the environment, chemical synthesis, and their innate immunostimulation

Covering: up to 2011. This review focuses on the recent revealing of the immunostimulatory bacterial cell wall peptidoglycan (PGN) fragments as Nod1 ligands, especially a newly developed chemical synthesis of the partial structures, fragment structures in the environment and bacterial supernatant, and the immunostimulatory activities of the Nod1 ligands.

N-Acetylglucosamine Functions in Cell Signaling

The amino sugar N-acetylglucosamine (GlcNAc) is well known for the important structural roles that it plays at the cell surface. It is a key component of bacterial cell wall peptidoglycan, fungal cell wall chitin, and the extracellular matrix of animal cells. Interestingly, recent studies have also identified new roles for GlcNAc in cell signaling. For example, GlcNAc stimulates the human fungal pathogen Candida albicans to undergo changes in morphogenesis and expression of virulence genes. Pathogenic E. coli responds to GlcNAc by altering the expression of fimbriae and CURLI fibers that promote biofilm formation and GlcNAc stimulates soil bacteria to undergo changes in morphogenesis and production of antibiotics. Studies with animal cells have revealed that GlcNAc influences cell signaling through the posttranslational modification of proteins by glycosylation. O-linked attachment of GlcNAc to Ser and Thr residues regulates a variety of intracellular proteins, including transcription factors such as NFκB, c-myc, and p53. In addition, the specificity of Notch family receptors for different ligands is altered by GlcNAc attachment to fucose residues in the extracellular domain. GlcNAc also impacts signal transduction by altering the degree of branching of N-linked glycans, which influences cell surface signaling proteins. These emerging roles of GlcNAc as an activator and mediator of cellular signaling in fungi, animals, and bacteria will be the focus of this paper.

Chapter Eight - Fe(II)-Dependent, Uridine-5′-Monophosphate α-Ketoglutarate Dioxygenases in the Synthesis of 5′-Modified Nucleosides

Several nucleoside antibiotics from various actinomycetes contain a high-carbon sugar nucleoside that is putatively derived via C-5'-modification of the canonical nucleoside. Two prominent examples are the 5'-C-carbamoyluridine- and 5'-C-glycyluridine-containing nucleosides, both families of which were discovered using screens aimed at finding inhibitors of bacterial translocase I involved in the assembly of the bacterial peptidoglycan cell wall. A shared open reading frame was identified whose gene product is similar to enzymes of the nonheme, Fe(II)-, and α-ketoglutarate-dependent dioxygenases. The enzyme LipL from the biosynthetic pathway for A-90289, a 5'-C-glycyluridine-containing nucleoside, was functionally characterized as an UMP:α-ketoglutarate dioxygenase, providing the enzymatic imperative for the generation of a nucleoside-5'-aldehdye that serves as a downstream substrate for an aldol or aldol-type reaction leading to the high-carbon sugar scaffold. The functional assignment of LipL and the homologous enzymes-including bioinformatic analysis, iron detection and quantification, and assay development for biochemical characterization-is presented herein.

Structural features and kinetic characterization of alanine racemase from Staphylococcus aureus (Mu50).

Staphylococcus aureus is an opportunistic Gram-positive bacterium which causes a wide variety of diseases ranging from minor skin infections to potentially fatal conditions such as pneumonia, meningitis and septicaemia. The pathogen is a leading cause of nosocomial acquired infections, a problem that is exacerbated by the existence of methicillin- and glycopeptide antibiotic-resistant strains which can be challenging to treat. Alanine racemase (Alr) is a pyridoxal-5'-phosphate-dependent enzyme which catalyzes reversible racemization between enantiomers of alanine. As D-alanine is an essential component of the bacterial cell-wall peptidoglycan, inhibition of Alr is lethal to prokaryotes. Additionally, while ubiquitous amongst bacteria, this enzyme is absent in humans and most eukaryotes, making it an excellent antibiotic drug target. The crystal structure of S. aureus alanine racemase (Alr(Sas)), the sequence of which corresponds to that from the highly antibiotic-resistant Mu50 strain, has been solved to 2.15 Å resolution. Comparison of the Alr(Sas) structure with those of various alanine racemases demonstrates a conserved overall fold, with the enzyme sharing most similarity to those from other Gram-positive bacteria. Structural examination indicates that the active-site binding pocket, dimer interface and active-site entryway of the enzyme are potential targets for structure-aided inhibitor design. Kinetic constants were calculated in this study and are reported here. The potential for a disulfide bond in this structure is noted. This structural and biochemical information provides a template for future structure-based drug-development efforts targeting Alr(Sas).

Sortase A Substrate Specificity in GBS Pilus 2a Cell Wall Anchoring

Streptococcus agalactiae, also referred to as Group B Streptococcus (GBS), is one of the most common causes of life-threatening bacterial infections in infants. In recent years cell surface pili have been identified in several Gram-positive bacteria, including GBS, as important virulence factors and promising vaccine candidates. In GBS, three structurally distinct types of pili have been discovered (pilus 1, 2a and 2b), whose structural subunits are assembled in high-molecular weight polymers by specific class C sortases. In addition, the highly conserved housekeeping sortase A (SrtA), whose main role is to link surface proteins to bacterial cell wall peptidoglycan by a transpeptidation reaction, is also involved in pili cell wall anchoring in many bacteria. Through in vivo mutagenesis, we demonstrate that the LPXTG sorting signal of the minor ancillary protein (AP2) is essential for pilus 2a anchoring. We successfully produced a highly purified recombinant SrtA (SrtAΔN40) able to specifically hydrolyze the sorting signal of pilus 2a minor ancillary protein (AP2-2a) and catalyze in vitro the transpeptidation reaction between peptidoglycan analogues and the LPXTG motif, using both synthetic fluorescent peptides and recombinant proteins. By contrast, SrtAΔN40 does not catalyze the transpeptidation reaction with substrate-peptides mimicking sorting signals of the other pilus 2a subunits (the backbone protein and the major ancillary protein). Thus, our results add further insight into the proposed model of GBS pilus 2a assembly, in which SrtA is required for pili cell wall covalent attachment, acting exclusively on the minor accessory pilin, representing the terminal subunit located at the base of the pilus.

Muramyl dipeptide and its derivatives: peptide adjuvant in immunological disorders and cancer therapy.

Muramyl dipeptide (MDP) is a synthetic immunoreactive peptide consisting of N-acetyl muramic acid attached to a short amino acid chain of L-Ala-D-isoGln. It was first identified in bacterial cell wall peptidoglycan as an active component in Freund's complete adjuvant. In the cell, MDP is detected by NOD2, a cytoplasmic receptor belonging to the human innate immune system. NOD2 mutations are frequently observed in patients with Crohn's disease, an autoimmune disorder, suggesting the significance of the MDP-NOD2 pathway in activating immunity. For this reason, structural modifications of MDP and its derivatives have been extensively studied in an attempt to increase adjuvant activity and boost the immune response effectively for clinical use in the treatment of cancer and other diseases. This review summarizes the synthetic chemistry of MDP and its derivatives and discusses their pharmacological action and stereoselective synthesis.

Exploiting what phage have evolved to control gram-positive pathogens

In the billion years that bacteriophage (or phage) have existed together with bacteria the phage have evolved systems that may be exploited for our benefit. One of these is the lytic system used by the phage to release their progeny from an infected bacterium. Endolysins (or lysins) are highly evolved enzymes in the lytic system produced to cleave essential bonds in the bacterial cell wall peptidoglycan for progeny release. Small quantities of purified recombinant lysin added externally to gram-positive bacteria results in immediate lysis causing log-fold death of the target bacterium. Lysins have now been used successfully in a variety of animal models to control pathogenic antibiotic resistant bacteria found on mucosal surfaces and in infected tissues. The advantages over antibiotics are their specificity for the pathogen without disturbing the normal flora, the low chance of bacterial resistance, and their ability to kill colonizing pathogens on mucosal surfaces, a capacity previously unavailable. Lysins therefore, may be a much-needed anti-infective (or enzybiotic) in an age of mounting antibiotic resistance.

Changes in the repertoire of natural antibodies caused by immunization with bacterial antigens

The repertoire of natural anti-glycan antibodies in naïve chickens and in chickens immunized with bacteria Burkholderia mallei, Burkholderia pseudomallei, and Francisella tularensis as well as with peptides from an outer membrane protein of B. pseudomallei was studied. A relatively restricted pattern of natural antibodies (first of all IgY against bacterial cell wall peptidoglycan fragments, L-Rha, and core N-acetyllactosamine) shrank and, moreover, the level of detectable antibodies decreased as a result of immunization.

The nine peptidoglycan hydrolases genes in Lactobacillus helveticus are ubiquitous and early transcribed

Peptidoglycan hydrolases (PGHs) are bacterial enzymes that can hydrolyze the peptidoglycan in bacterial cell wall leading to autolysis. By releasing intracellular enzymes, autolysis of Lactobacillus helveticus has important applications in cheese ripening as its extent varied from strain to strain. Nine genes coding PGHs were previously annotated in the genome of the high autolytic strain L. helveticus DPC 4571. This study was conducted to evaluate the clone diversity of the nine PGHs genes within a collection of 24 L. helveticus strains, highly diverse in terms of origin, biotope and autolytic activity. Pulsed field gel electrophoresis was applied to assess the genomic diversity of the 24 strains. The presence or absence of nine PGHs genes was verified for all L. helveticus strains. Nucleotide and deduced amino acid sequence were compared for six relevant strains. Finally, gene expression was monitored by reverse transcription during growth and by zymogram for 12 strains. The nine PGHs genes are ubiquitous and transcripted early during growth. Zymograms were similar in terms of molecular size of the bands, but exhibited strain to strain variations in the number of bands revealing from 2 to 5 lytic bands per strain.

Dectin-1 and NOD2 mediate cathepsin activation in zymosan-induced arthritis in mice

Activation of pattern recognition receptors (PRR) may contribute to arthritis. Here, we elucidated the role of NOD2, a genetic cause of inflammatory arthritis, and several other PRR in a murine model of inflammatory arthritis. The roles of CR3, TLR2, MyD88, NOD1, NOD2, Dectin-1 and Dectin-2 were tested in vivo in arthritis elicited by intra-articular injections of zymosan, the fungal cell wall components curdlan, laminarin and mannan, and the bacterial cell wall peptidoglycan. Dectin-1, and to a lesser extent Dectin-2, contributed to arthritis. TLR2, MyD88 and CR3 played non-essential roles. Observations based on injection of curdlan, laminarin or mannan supported the dominant role of the Dectin-1 pathway in the joint. We demonstrated differential roles for NOD1 and NOD2 and identified NOD2 as a novel and essential mediator of zymosan-induced arthritis. Together, Dectin-1 and NOD2 are critical, sentinel receptors in the arthritogenic effects of zymosan. Our data identify a novel role for NOD2 during inflammatory responses within joints.

Bacterial cell wall assembly: still an attractive antibacterial target

The development of new antibacterial agents to combat worsening antibiotic resistance is still a priority area in anti-infectives research, but in the post-genomic era it has been more difficult than expected to identify new lead compounds from high-throughput screening, and very challenging to obtain antibacterial activity for lead compounds. Bacterial cell-wall peptidoglycan biosynthesis is a well-established target for antibacterial chemotherapy, and recent developments enable the entire biosynthetic pathway to be reconstituted for detailed biochemical study and high-throughput inhibitor screening. This review article discusses recent developments in the availability of peptidoglycan biosynthetic intermediates, the identification of lead compounds for both the earlier cytoplasmic steps and the later lipid-linked steps, and the application of new methods such as structure-based drug design, phage display and surface science.

Sarcoidosis and Autoinflammation

Sporadic early-onset sarcoidosis (EOS) and familial Blau syndrome (BS) form a distinct set of autoinflammatory diseases, both of which onset in infancy and show a clinical triad of dermatitis, arthritis and uveitis histologically composed of noncaseating epithelioid cell granuloma. The responsible gene for EOS/BS is NOD2, encoding an intracellular receptor for muramyl dipeptide (MDP), the common component of bacterial cell wall peptidoglycan. The gain-of-function NOD2 mutations with MDP-independent basal NF-κB activation cause EOS/BS, while its loss-of-function mutations with impaired MDP-dependent NF-κB activation are associated with Crohn's disease and a part of sarcoidosis. As the result of analyzing genotype-phenotype relationship of Japanese EOS/BS cases, somewhat positive correlation was recognized between mutant NOD2-causing basal NF-κB activation and clinical severity, especially of ocular complications. Role of the basal NF-κB activation in monocytes has also been suggested by an inhibitory effect of thalidomide on ex vivo giant cell formation from EOS/BS patients' monocytes. Analysis of mutant NOD2-introduced human monocytic THP-1 cells, as well as the patients' samples, would provide further detailed molecular mechanisms of EOS/BS pathogenesis, in which a single point missense mutation causes a distinct pathological change forming epithelioid cell granuloma.

NOD2/CARD15 Mutations Correlate With Severe Pouchitis After Ileal Pouch-Anal Anastomosis

Pouchitis and Crohn's-like complications can plague patients after IPAA. NOD2 is an intracellular sensor for bacterial cell wall peptidoglycan. NOD2 mutations compromise host response to enteric bacteria and are increased in Crohn's disease. We hypothesize that IPAA patients with complications (Crohn's disease-like/pouchitis) have a higher rate of NOD2 mutations compared with asymptomatic IPAA patients. Patients were retrospectively subclassified into the following groups: 1) IPAA with Crohn's-like complications (n = 28, perianal fistula, pouch inlet stricture/upstream small-bowel disease, or biopsies showing granulomata) occurring at least 6 months after ileostomy closure; 2) IPAA with mild pouchitis (n = 33, ≤3 episodes/y for 2 consecutive years); 3) IPAA with severe pouchitis (n = 9, ≥4 episodes/y for 2 consecutive years or need for continuous antibiotics); 4) IPAA without complications or pouchitis (n = 37); 5) patients with Crohn's disease with colitis undergoing total proctocolectomy/ileostomy (n = 11); and 6) healthy controls (n = 269). The 3 NOD2 single-nucleotide polymorphism mutations (rs2066844, rs2066845, and rs2066847) previously identified as associated with Crohn's disease were genotyped using polymerase chain reaction. Groups were compared by use of χ with Yates continuity correction. NOD2 mutations were found in 8.5% of healthy controls. NOD2 mutations were significantly higher in the severe pouchitis group (67%) compared with both asymptomatic IPAA (5.4%, P < .001) and IPAA with Crohn's disease-like complications (14.3%, P = .008) groups. 1) Asymptomatic IPAA patients have a low incidence of NOD2 mutations not significantly different from patients with mild pouchitis or healthy controls. 2) Patients with severe pouchitis had the highest incidence of NOD2 mutations, suggesting that this group may have a compromised host defense mechanism to enteric bacteria. 3) Patients with Crohn's-like complications after IPAA have a significantly lower incidence of NOD2 mutations than patients with severe pouchitis, suggesting a different genetic makeup in these 2 patient groups. Preoperative assessment of NOD2 in the equivocal IPAA candidate may predict severe pouchitis and might assist in preoperative surgical decision making.

The structure of a family GH25 lysozyme from Aspergillus fumigatus.

Lysins are important biomolecules which cleave the bacterial cell-wall polymer peptidoglycan. They are finding increasing commercial and medical application. In order to gain an insight into the mechanism by which these enzymes operate, the X-ray structure of a CAZy family GH25 ;lysozyme' from Aspergillus fumigatus was determined. This is the first fungal structure from the family and reveals a modified alpha/beta-barrel-like fold in which an eight-stranded beta-barrel is flanked by three alpha-helices. The active site lies toward the bottom of a negatively charged pocket and its layout has much in common with other solved members of the GH25 and related GH families. A conserved active-site DXE motif may be implicated in catalysis, lending further weight to the argument that this glycoside hydrolase family operates via a ;substrate-assisted' catalytic mechanism.

Staphylococcus aureus Stimulates Neutrophil Targeting Chemokine Expression in Keratinocytes through an Autocrine IL-1α Signaling Loop

Staphylococcus aureus is a significant human pathogen that can colonize the skin. Neutrophils are well known to be involved in clearance of the bacterium. This study focused on exploring the role that human keratinocytes have as first responders to bacterial challenges. IL-1alpha and IL-1beta increased mRNA production and protein secretion of the neutrophil chemotactic CXCL1, CXCL2, and IL-8 in keratinocytes. S. aureus and the bacterial cell wall components lipoteichoic acid (LTA) and peptidoglycan (PGN) induced similar expression profiles in a Toll-like receptor (TLR)-2-dependent manner. Interestingly, the S. aureus-induced mRNA levels peaked at later time points than those induced by IL-1. The S. aureus-activated chemokine production was preceded by significant IL-1alpha and IL-1beta secretion. Expression of IL-1alpha was significantly higher than that of IL-1beta. Inhibition of IL-1RI using neutralizing antibodies revealed that S. aureus-derived LTA and PGN-induced chemokine expression requires IL-1RI engagement. Surprisingly, we further found that chemokine secretion is dependent upon endocrine IL-1alpha, but not IL-1beta, signaling. Our data show that the innate immune response of keratinocytes is regulated differently than those of other cell types. This may represent a fail-safe system that protects the host against genetic variation and immune evasion mechanisms developed by pathogens.

Review: Mammalian peptidoglycan recognition proteins (PGRPs) in innate immunity

Peptidoglycan recognition proteins (PGRPs or PGLYRPs) are innate immunity proteins that are conserved from insects to mammals, recognize bacterial peptidoglycan, and function in antibacterial immunity and inflammation. Mammals have four PGRPs - PGLYRP1, PGLYRP2, PGLYRP3, and PGLYRP4. They are secreted proteins expressed in polymorphonuclear leukocytes (PGLYRP1), liver (PGLYRP2), or on body surfaces, mucous membranes, and in secretions (saliva, sweat) (PGLYRP3 and PGLYRP4). All PGRPs recognize bacterial peptidoglycan. Three PGRPs, PGLYRP1, PGLYRP3, and PGLYRP4 are directly bactericidal for both Gram-positive and Gram-negative bacteria and have no enzymatic activity, whereas PGLYRP2 is an N-acetylmuramoyl-L-alanine amidase that hydrolyzes bacterial cell wall peptidoglycan. Peptidoglycan recognition proteins influence host- pathogen interactions not only through their antibacterial or peptidoglycan-hydrolytic properties, but also through their pro-inflammatory and anti-inflammatory properties that are independent of their hydrolytic and antibacterial activities. The PGRPs likely play a role both in antibacterial defenses and several inflammatory diseases. They modulate local inflammatory responses in tissues (such as arthritic joints) and there is evidence for association of PGRPs with inflammatory diseases, such as psoriasis.