Lytic transglycosylases: bacterial space-making autolysins

Lytic transglycosylases are an important class of bacterial enzymes that act on peptidoglycan with the same substrate specificity as lysozyme. Unlike the latter enzymes, however, the lytic transglycosylases are not hydrolases but instead cleave the glycosidic linkage between N-actetylmuramoyl and N-acetylglucosaminyl residues with the concomitant formation of a 1,6-anydromuramoyl product. They are ubiquitous in bacteria which produce a compliment of different forms that are responsible for creating space within the peptidoglycan sacculus for its biosynthesis and recycling, cell division, and the insertion of cell-envelope spanning structures, such as flagella and secretion systems. As well, the lytic transglyosylases may have a role in pathogenesis of some bacterial species. Given their important role in bacterial cell wall metabolism, the lytic transglycosylases may present an attractive new target for the development of broad-spectrum antibiotics.     

Endopeptidase-Mediated Beta Lactam Tolerance

In many bacteria, inhibition of cell wall synthesis leads to cell death and lysis. The pathways and enzymes that mediate cell lysis after exposure to cell wall-acting antibiotics (e.g. beta lactams) are incompletely understood, but the activities of enzymes that degrade the cell wall (‘autolysins’) are thought to be critical. Here, we report that Vibrio cholerae, the cholera pathogen, is tolerant to antibiotics targeting cell wall synthesis. In response to a wide variety of cell wall- acting antibiotics, this pathogen loses its rod shape, indicative of cell wall degradation, and becomes spherical. Genetic analyses revealed that paradoxically, V. cholerae survival via sphere formation required the activity of D,D endopeptidases, enzymes that cleave the cell wall. Other autolysins proved dispensable for this process. Our findings suggest the enzymes that mediate cell wall degradation are critical for determining bacterial cell fate - sphere formation vs. lysis – after treatment with antibiotics that target cell wall synthesis.     

肽聚糖的生物合成及其调控机制研究进展

肽聚糖(peptidoglycan)是细菌细胞壁的重要组成部分,对于维持细胞形态、大小及存活至关重要;同时,肽聚糖是众多常用抗生素的作用靶点。在细菌的正常生长过程中,肽聚糖不断地合成和水解,为了保证细胞壁的完整性,肽聚糖生物合成过程必然受到严谨的时空调控。肽聚糖的生物合成及其调控机制是微生物学中重要的基础研究之一,近年来国内外研究团队在该领域取得了突破性研究进展。基于此,本文综述了肽聚糖的从头合成和循环再利用过程,并重点阐述了肽聚糖合成关键酶——肽聚糖合酶及其调控机制的最新研究进展。最后,本文对未来需要加强研究的方向进行了展望。     

Classification of bacterial cell wall hydrolysases and their potentials as novel alternatives to antibiotics – a response to the letter of Biziulevicius and Kazlauskaite

This is a response to the letter of Biziulevicius and Kazlauskaite, who made a few valuable comments on our recent paper published on the Journal of Applied Microbiology regarding the classification and potential clinic applications as alternative antibiotics of bacterial cell wall hydrolases (BCWH). We confirm that it is of significant practical and theoretical relevance to categorize BCWH according to their sources owing to their strikingly different antibacterial specificities, which are of critical importance to their potential clinic applications. Among the three groups of lytic enzymes, i.e. lysozymes, microlysins and virolysins, virolysins seem to be the most promising alternative antibiotics as a result of a few unique characteristics of this group of lytic enzymes.     

Staphylococcus aureus Survives with a Minimal Peptidoglycan Synthesis Machine but Sacrifices Virulence and Antibiotic Resistance

Many important cellular processes are performed by molecular machines, composed of multiple proteins that physically interact to execute biological functions. An example is the bacterial peptidoglycan (PG) synthesis machine, responsible for the synthesis of the main component of the cell wall and the target of many contemporary antibiotics. One approach for the identification of essential components of a cellular machine involves the determination of its minimal protein composition. Staphylococcus aureus is a Gram-positive pathogen, renowned for its resistance to many commonly used antibiotics and prevalence in hospitals. Its genome encodes a low number of proteins with PG synthesis activity (9 proteins), when compared to other model organisms, and is therefore a good model for the study of a minimal PG synthesis machine. We deleted seven of the nine genes encoding PG synthesis enzymes from the S. aureus genome without affecting normal growth or cell morphology, generating a strain capable of PG biosynthesis catalyzed only by two penicillin-binding proteins, PBP1 and the bi-functional PBP2. However, multiple PBPs are important in clinically relevant environments, as bacteria with a minimal PG synthesis machinery became highly susceptible to cell wall-targeting antibiotics, host lytic enzymes and displayed impaired virulence in a Drosophila infection model which is dependent on the presence of specific peptidoglycan receptor proteins, namely PGRP-SA. The fact that S. aureus can grow and divide with only two active PG synthesizing enzymes shows that most of these enzymes are redundant in vitro and identifies the minimal PG synthesis machinery of S. aureus. However a complex molecular machine is important in environments other than in vitro growth as the expendable PG synthesis enzymes play an important role in the pathogenicity and antibiotic resistance of S. aureus.     

Don’t let sleeping dogmas lie: new views of peptidoglycan synthesis and its regulation

Bacterial cell wall synthesis is the target for some of our most powerful antibiotics and has thus been the subject of intense research focus for more than 50 years. Surprisingly, we still lack a fundamental understanding of how bacteria build, maintain and expand their cell wall. Due to technical limitations, directly testing hypotheses about the coordination and biochemistry of cell wall synthesis enzymes or architecture has been challenging, and interpretation of data has therefore often relied on circumstantial evidence and implicit assumptions. A number of recent papers have exploited new technologies, like single molecule tracking and real‐time, high resolution temporal mapping of cell wall synthesis processes, to address fundamental questions of bacterial cell wall biogenesis. The results have challenged established dogmas and it is therefore timely to integrate new data and old observations into a new model of cell wall biogenesis in rod‐shaped bacteria.     

Taking aim on bacterial pathogens: from phage therapy to enzybiotics

The bactericidal activity of bacteriophages has been used to treat human infections for years as an alternative or a complement to antibiotic therapy. Nowadays, endolysins (phage-encoded enzymes that break down bacterial peptidoglycan at the terminal stage of the phage reproduction cycle) have been used successfully to control antibiotic-resistant pathogenic bacteria in animal models. Their cell wall binding domains target the enzymes to their substrate, and their corresponding catalytic domains are able to cleave bonds in the peptidoglycan network. Recent research has not only revealed the surprising rich structural catalytic diversity of these murein hydrolases but has also yielded insights into their modular organization, their three-dimensional structures, and their mechanism of recognition of bacterial cell wall. These results allow endolysins to be considered as effective antimicrobials with potentially important applications in medicine and biotechnology.     

Constructing and deconstructing the bacterial cell wall

The history of modern medicine cannot be written apart from the history of the antibiotics. Antibiotics are cytotoxic secondary metabolites that are isolated from Nature. The antibacterial antibiotics disproportionately target bacterial protein structure that is distinct from eukaryotic protein structure, notably within the ribosome and within the pathways for bacterial cell‐wall biosynthesis (for which there is not a eukaryotic counterpart). This review focuses on a pre‐eminent class of antibiotics—the β‐lactams, exemplified by the penicillins and cephalosporins—from the perspective of the evolving mechanisms for bacterial resistance. The mechanism of action of the β‐lactams is bacterial cell‐wall destruction. In the monoderm (single membrane, Gram‐positive staining) pathogen Staphylococcus aureus the dominant resistance mechanism is expression of a β‐lactam‐unreactive transpeptidase enzyme that functions in cell‐wall construction. In the diderm (dual membrane, Gram‐negative staining) pathogen Pseudomonas aeruginosa a dominant resistance mechanism (among several) is expression of a hydrolytic enzyme that destroys the critical β‐lactam ring of the antibiotic. The key sensing mechanism used by P. aeruginosa is monitoring the molecular difference between cell‐wall construction and cell‐wall deconstruction. In both bacteria, the resistance pathways are manifested only when the bacteria detect the presence of β‐lactams. This review summarizes how the β‐lactams are sensed and how the resistance mechanisms are manifested, with the expectation that preventing these processes will be critical to future chemotherapeutic control of multidrug resistant bacteria.     

Potentialization of antibiotics by lytic enzymes]

Few lytic enzymes, specially papaine and lysozyme, acting on the membrane and cell wall structures facilitate effects of bacitracine, streptomycine and other antibiotics. Streptomycino resistant strains became sensibles to this antibiotic after contact with papaine and lysozyme. The results of tests in physiological suspensions concern only the lytic activity of enzymes. The results on nutrient medium concern together lytic, and antibiotic activities.     

Binding domains of Bacillus anthracis phage endolysins recognize cell culture age‐related features on the bacterial surface

Bacteriolytic enzymes often possess a C‐terminal binding domain that recognizes specific motifs on the bacterial surface and a catalytic domain that cleaves covalent linkages within the cell wall peptidoglycan. PlyPH, one such lytic enzyme of bacteriophage origin, has been reported to be highly effective against Bacillus anthracis, and can kill up to 99.99% of the viable bacteria. The bactericidal activity of this enzyme, however, appears to be strongly dependent on the age of the bacterial culture. Although highly bactericidal against cells in the early exponential phase, the enzyme is substantially less effective against stationary phase cells, thus limiting its application in real‐world settings. We hypothesized that the binding domain of PlyPH may differ in affinity to cells in different Bacillus growth stages and may be primarily responsible for the age‐restricted activity. We therefore employed an in silico approach to identify phage lysins differing in their specificity for the bacterial cell wall. Specifically we focused our attention on Plyβ, an enzyme with improved cell wall‐binding ability and age‐independent bactericidal activity. Although PlyPH and Plyβ have dissimilar binding domains, their catalytic domains are highly homologous. We characterized the biocatalytic mechanism of Plyβ by identifying the specific bonds cleaved within the cell wall peptidoglycan. Our results provide an example of the diversity of phage endolysins and the opportunity for these biocatalysts to be used for broad‐based protection from bacterial pathogens. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1487–1493, 2015     

Mechanism of action of antibiotics:some examples]

Antibiotics are very commonly used substances to eradicate bacterial infections by bacteriostatic or even bactericid effect. They act at a very specific stage (target), although other less important or secondary interactions can occur. We studied the interaction of three antibiotic families (beta-lactamins, aminosides, rifampicin) with bacterial cell. Penicillin disturbs the cell wall synthesis and more accurately the glycopeptide (or murein) formation, a substance giving rigidity or shape to bacteria. It acts in the late phase of murein-biosynthesis, when N-acetyl glucosamin -- N-acetyl muramic acid L ala -D glu M-DAP (L lys) -D ala -D ala are linked together by the peptide part, under the effect of several enzymes, particularly transpeptidase and DD-carboxy-peptidase. It would appear that beta-lactame-thiazolidine rings have a steric analogy with dipeptide D-alanyl D-alanine. The result would be that the enzyme would act on the antibiotic instead of peptide: the consequence would be inhibition of the peptidic link, giving an abnormal murein, and an incomplete cell wall i.e. fragile bacteria. Aminosides, particularly Streptomycin, link themselves to 30 S subunit of bacterial ribosome. In this case, it seems that it is a 3'OH function which reacts with lysine (from S 12 protein part of 30 S subunit). The consequence is an alteration in the RNA messager lecture, and a false traduction and consequently protein biosynthesis stops with a decrease of polyribosomes and of the formation of inert 70 S ribosome. Rifamycins, and particularly Rifampicin act by inhibition of RNA messager synthesis. One molecule of antibiotic links itself to one molecule of RNA messager : hydroxyl and cetone function in C1 Cs C21 C23 and "ansa" bridge link to beta subunit of RNA polymerase. This linkage gives a conformational change to the RNA polymerase-DNA complex, inhibiting the catalytic action of this enzyme, and consequently stopping RNA messager and protein synthesis. The study of the action mechanism of these antibiotics enables us to show the action specificity of these products in the bacteria. This specificity is more accurate when the target is not to be found in the eucaryotic cells : in this case the antibiotic may be considered as entirely atoxic. If the study of the action mechanism of antibiotics gives a better understanding of the use of these drugs, their action at a definite stage in bacterial metabolism is a valuable tool for scientists in their approach to cell functioning.     

A Forward Chemical Screen Identifies Antibiotic Adjuvants in Escherichia coli

Multi-drug-resistant infections caused by Gram-negative pathogens are rapidly increasing, highlighting the need for new chemotherapies. Unlike Gram-positive bacteria, where many different chemical classes of antibiotics show efficacy, Gram-negatives are intrinsically insensitive to many antimicrobials including the macrolides, rifamycins, and aminocoumarins, despite intracellular targets that are susceptible to these drugs. The basis for this insensitivity is the presence of the impermeant outer membrane of Gram-negative bacteria in addition to the expression of pumps and porins that reduce intracellular concentrations of many molecules. Compounds that sensitize Gram-negative cells to "Gram-positive antibiotics", antibiotic adjuvants, offer an orthogonal approach to addressing the crisis of multi-drug-resistant Gram-negative pathogens. We performed a forward chemical genetic screen of 30,000 small molecules designed to identify such antibiotic adjuvants of the aminocoumarin antibiotic novobiocin in Escherichia coli. Four compounds from this screen were shown to be synergistic with novobiocin including inhibitors of the bacterial cytoskeleton protein MreB, cell wall biosynthesis enzymes, and DNA synthesis. All of these molecules were associated with altered cell shape and small molecule permeability, suggesting a unifying mechanism for these antibiotic adjuvants. The potential exists to expand this approach as a means to develop novel combination therapies for the treatment of infections caused by Gram-negative pathogens.     

Bacteriophage lytic enzymes: novel anti-infectives

Bacteriophage lytic enzymes, or lysins, are highly evolved molecules produced by bacterial viruses (bacteriophage) to digest the bacterial cell wall for bacteriophage progeny release. Small quantities of purified recombinant lysin added to gram-positive bacteria causes immediate lysis resulting in log-fold death of the target bacterium. Lysins have now been used successfully in animal models to control pathogenic antibiotic resistant bacteria found on mucosal surfaces and in blood. The advantages over antibiotics are their specificity for the pathogen without disturbing the normal flora, the low chance of bacterial resistance to lysins and their ability to kill colonizing pathogens on mucosal surfaces, capabilities that were previously unavailable. Thus, lysins could be an effective anti-infective in an age of mounting antibiotic resistance.     

Structural Basis for Catalysis by the Mono- and Dimetalated Forms of the dapE-Encoded N-succinyl-l,l-Diaminopimelic Acid Desuccinylase

Biosynthesis of lysine and meso-diaminopimelic acid in bacteria provides essential components for protein synthesis and construction of the bacterial peptidoglycan cell wall. The dapE operon enzymes synthesize both meso-diaminopimelic acid and lysine and, therefore, represent potential targets for novel antibacterials. The dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase functions in a late step of the pathway and converts N-succinyl-l,l-diaminopimelic acid to l,l-diaminopimelic acid and succinate. Deletion of the dapE gene is lethal to Helicobacter pylori and Mycobacterium smegmatis, indicating that DapE's are essential for cell growth and proliferation. Since there are no similar pathways in humans, inhibitors that target DapE may have selective toxicity against only bacteria. A major limitation in developing antimicrobial agents that target DapE has been the lack of structural information. Herein, we report the high-resolution X-ray crystal structures of the DapE from Haemophilus influenzae with one and two zinc ions bound in the active site, respectively. These two forms show different activity. Based on these newly determined structures, we propose a revised catalytic mechanism of peptide bond cleavage by DapE enzymes. These structures provide important insight into catalytic mechanism of DapE enzymes as well as a structural foundation that is critical for the rational design of DapE inhibitors.     

Quantifying enzymatic lysis: estimating the combined effects of chemistry, physiology and physics

The number of microbial pathogens resistant to antibiotics continues to increase even as the rate of discovery and approval of new antibiotic therapeutics steadily decreases. Many researchers have begun to investigate the therapeutic potential of naturally occurring lytic enzymes as an alternative to traditional antibiotics. However, direct characterization of lytic enzymes using techniques based on synthetic substrates is often difficult because lytic enzymes bind to the complex superstructure of intact cell walls. Here we present a new standard for the analysis of lytic enzymes based on turbidity assays which allow us to probe the dynamics of lysis without preparing a synthetic substrate. The challenge in the analysis of these assays is to infer the microscopic details of lysis from macroscopic turbidity data. We propose a model of enzymatic lysis that integrates the chemistry responsible for bond cleavage with the physical mechanisms leading to cell wall failure. We then present a solution to an inverse problem in which we estimate reaction rate constants and the heterogeneous susceptibility to lysis among target cells. We validate our model given simulated and experimental turbidity assays. The ability to estimate reaction rate constants for lytic enzymes will facilitate their biochemical characterization and development as antimicrobial therapeutics.     

Effect of polyene antibiotics on bacterial protoplasts

The carbonyl-conjugated pentaenes flavofungin, nigrofungin and flavopentin exhibit considerable lytic activity toward Micrococcus lysodeikticus and Bacillus megaterium protoplasts. The antibiotics at concentrations of 5 to 14 microgram/ml cause lysis of 50% of the protoplasts within 15 min of their incubation. The antibiotics inhibit the activity of NADH oxidase and malate oxidase by 50% in the lysates of Micrococcus lysodeikticus and Bacillus megaterium protoplasts at concentrations of 30 to 50 microgram/ml; preincubation of the lysates with the antibiotics intensify the inhibiting action of the polyenes. Growth of the bacteria is inhibited when the minimal concentration of the polyenes is 75 to 100 microgram/ml. Interaction of the polyenes with bacterial membranes lacking sterols indicates that resistance of at least some bacteria to polyenes is caused by impermeability of the cell wall for these substances rather than by the absence of sterols in the membranes.     

噬菌体裂解酶——现状与未来

噬菌体裂解酶是一种由DNA噬菌体基因编码的高特异性酶, 可高效消化细菌细胞壁。革兰氏阳性菌噬菌体裂解酶的结构域相似, 裂解效率高, 与抗生素具协同抗菌作用, 且不易产生耐受性菌株, 抗体等体液因子对裂解酶的裂解活性影响小, 裂解酶作为一种潜在抗感染药物具有重要的研究价值。目前已建立了多种病原菌裂解酶应用的动物模型, 在防控耐药性病原菌感染上取得重要进展。本文就噬菌体裂解酶的抗菌作用进行综述。     

Complete lysis of glutamic acid-producing bacteria by the use of antibiotics which inhibit the biosynthesis of cell walls.

A novel method for obtaining complete lysis of coryneform bacteria has been devised. Bacteria in the late-exponential phase of growth are treated with antibiotics which inhibit biosynthesis of the cell wall. Organisms treated in this way show increased sensitivity to lysis by added lytic enzymes. The preparation of DNA from this group of organisms is thus greatly facilitated.     

Relationship between Cell Wall, Cytoplasmic Membrane, and Bacterial Motility

High-resolution electron microscopy of polarly flagellated bacteria revealed that their flagella originate at a circular, differentiated portion of the cytoplasmic membrane approximately 25 nm in diameter. The flagella also have discs attaching them to the cell wall. These attachment discs are extremely resistant to lytic damage and are firmly bound to the flagella. The cytoplasm beneath the flagellum contains a granulated basal body about 60 nm in diameter, and a specialized polar membrane. The existence of membrane-bound basal bodies is shown to be an artifact arising from adherence of cell wall and cytoplasmic membrane fragments to flagella in lysed preparations. Based on structures observed, a mechanism to explain bacterial flagellar movement is proposed. Flagella are considered to be anchored to the cell wall and activated by displacement of underlying cytoplasmic membrane to which they are also firmly attached. An explanation for the membrane displacement is given.     

Novel and Improved Crystal Structures of H. influenzae,E. coli and P. aeruginosa Penicillin-Binding Protein 3 (PBP3) and N. gonorrhoeae PBP2: Toward a Better Understanding of β-Lactam Target-Mediated Resistance

Even with the emergence of antibiotic resistance, penicillin and the wider family of β-lactams have remained the single most important family of antibiotics. The periplasmic/extra-cytoplasmic targets of penicillin are a family of enzymes with a highly conserved catalytic activity involved in the final stage of bacterial cell wall (peptidoglycan) biosynthesis. Named after their ability to bind penicillin, rather than their catalytic activity, these key targets are called penicillin-binding proteins (PBPs). Resistance is predominantly mediated by reducing the target drug concentration via β-lactamases; however, naturally transformable bacteria have also acquired target-mediated resistance by inter-species recombination. Here we focus on structural based interpretations of amino acid alterations associated with the emergence of resistance within clinical isolates and include new PBP3 structures along with new, and improved, PBP-β-lactam co-structures.