Interactions of two enantiomers of a designer antimicrobial peptide with structural components of the bacterial cell envelope

Antimicrobial peptides (AMPs) have great potential in treating multi-drug resistant bacterial infections. The antimicrobial activity of d -enantiomers is significantly higher than l -enantiomers and sometimes selectively enhanced against Gram-positive bacteria. Unlike phospholipids in the bacterial plasma membrane, the role of other bacterial cell envelop components is often overlooked in the mode of action of AMPs. In this work, we explored the structural interactions between the main different structural components in Gram-negative/Gram-positive bacteria and the two enantiomers of a designer AMP, GL13K. We observed that both l -GL13K and d -GL13K formed self-assembled amyloid-like nanofibrils when the peptides interacted with lipopolysaccharide and lipoteichoic acid, components of the outer membrane of Gram-negative bacteria and cell wall of Gram-positive bacteria, respectively. Another cell wall component, peptidoglycan, showed strong interactions exclusively with d -GL13K and formed distinct laminar structures. This specific interaction between peptidoglycans and d -GL13K might contribute to the enhanced activity of d -GL13K against Gram-positive bacteria as they have a much thicker peptidoglycan layer than Gram-negative bacteria. A better understanding of the specific role of bacterial cell envelop components in the AMPs mechanism of action can guide the design of more effective Gram-selective AMPs.     

Gram-positive and Gram-negative bacteria elicit different patterns of pro-inflammatory cytokines in human monocytes

Pro-inflammatory cytokines secreted by tissue macrophages recruit polymorphonuclear leukocytes and evoke fever, cachexia and production of acute phase proteins. This study investigates whether Gram-positive and Gram-negative bacteria equally and efficiently trigger production of the pro-inflammatory cytokines IL-1 beta, IL-6, IL-8 and TNF-alpha in human monocytes. A range of aerobic and anaerobic Gram-positive and Gram-negative bacteria were killed by UV-light and added in different concentrations to human monocytes. Cytokines were measured in 24 h supernatants by ELISA. Gram-positive and Gram-negative bacteria were equally efficient inducers of IL-1 beta, but Gram-positive bacteria generated twice as much TNF-alpha as did Gram-negative bacteria (p<0.001 for 25 and 250 bacteria/cell). In contrast, Gram-negative bacteria induced at least twice as much IL-6 and IL-8 as did Gram-positive bacteria (p<0.001 for 2.5, 25 and 250 bacteria/cell). While the cytokine responses to LPS were similar to those induced by the corresponding amount of Gram-negative bacteria, the strong IL-1 beta and TNF-alpha responses to Gram-positive bacteria could not be induced by soluble peptidoglycan or lipotheicoic acid. The particular nature of the bacteria, thus seem to modify the response to Gram-positive bacterial components. The different cytokine profiles evoked by Gram-positive and Gram-negative bacteria might optimize clearance of bacteria that differ in cell wall structure.     

MD-2 enables Toll-like receptor 2 (TLR2)-mediated responses to lipopolysaccharide and enhances TLR2-mediated responses to Gram-positive and Gram-negative bacteria and their cell wall components

MD-2 is associated with Toll-like receptor 4 (TLR4) on the cell surface and enables TLR4 to respond to LPS. We tested whether MD-2 enhances or enables the responses of both TLR2 and TLR4 to Gram-negative and Gram-positive bacteria and their components. TLR2 without MD-2 did not efficiently respond to highly purified LPS and LPS partial structures. MD-2 enabled TLR2 to respond to nonactivating protein-free LPS, LPS mutants, or lipid A and enhanced TLR2-mediated responses to both Gram-negative and Gram-positive bacteria and their LPS, peptidoglycan, and lipoteichoic acid components. MD-2 enabled TLR4 to respond to a wide variety of LPS partial structures, Gram-negative bacteria, and Gram-positive lipoteichoic acid, but not to Gram-positive bacteria, peptidoglycan, and lipopeptide. MD-2 physically associated with TLR2, but this association was weaker than with TLR4. MD-2 enhanced expression of both TLR2 and TLR4, and TLR2 and TLR4 enhanced expression of MD-2. Thus, MD-2 enables both TLR4 and TLR2 to respond with high sensitivity to a broad range of LPS structures and to lipoteichoic acid, and, moreover, MD-2 enhances the responses of TLR2 to Gram-positive bacteria and peptidoglycan, to which the TLR4-MD-2 complex is unresponsive.     

Protein sorting to the cell wall envelope of Gram-positive bacteria

The covalent anchoring of surface proteins to the cell wall envelope of Gram-positive bacteria occurs by a universal mechanism requiring sortases, extracellular transpeptidases that are positioned in the plasma membrane. Surface protein precursors are first initiated into the secretory pathway of Gram-positive bacteria via N-terminal signal peptides. C-terminal sorting signals of surface proteins, bearing an LPXTG motif or other recognition sequences, provide for sortase-mediated cleavage and acyl enzyme formation, a thioester linkage between the active site cysteine residue of sortase and the C-terminal carboxyl group of cleaved surface proteins. During cell wall anchoring, sortase acyl enzymes are resolved by the nucleophilic attack of peptidoglycan substrates, resulting in amide bond formation between the C-terminal end of surface proteins and peptidoglycan cross-bridges within the bacterial cell wall envelope. The genomes of Gram-positive bacteria encode multiple sortase genes. Recent evidence suggests that sortase enzymes catalyze protein anchoring reactions of multiple different substrate classes with different sorting signal motif sequences, protein linkage to unique cell wall anchor structures as well as protein polymerization leading to the formation of pili on the surface of Gram-positive bacteria.     

Protein secretion and surface display in Gram-positive bacteria

The cell wall peptidoglycan of Gram-positive bacteria functions as a surface organelle for the transport and assembly of proteins that interact with the environment, in particular, the tissues of an infected host. Signal peptide-bearing precursor proteins are secreted across the plasma membrane of Gram-positive bacteria. Some precursors carry C-terminal sorting signals with unique sequence motifs that are cleaved by sortase enzymes and linked to the cell wall peptidoglycan of vegetative forms or spores. The sorting signals of pilin precursors are cleaved by pilus-specific sortases, which generate covalent bonds between proteins leading to the assembly of fimbrial structures. Other precursors harbour surface (S)-layer homology domains (SLH), which fold into a three-pronged spindle structure and bind secondary cell wall polysaccharides, thereby associating with the surface of specific Gram-positive microbes. Type VII secretion is a non-canonical secretion pathway for WXG100 family proteins in mycobacteria. Gram-positive bacteria also secrete WXG100 proteins and carry unique genes that either contribute to discrete steps in secretion or represent distinctive substrates for protein transport reactions.     

Peptidoglycan turnover and recycling in Gram-positive bacteria

Bacterial cells are protected by an exoskeleton, the stabilizing and shape-maintaining cell wall, consisting of the complex macromolecule peptidoglycan. In view of its function, it could be assumed that the cell wall is a static structure. In truth, however, it is steadily broken down by peptidoglycan-cleaving enzymes during cell growth. In this process, named cell wall turnover, in one generation up to half of the preexisting peptidoglycan of a bacterial cell is released from the wall. This would result in a massive loss of cell material, if turnover products were not be taken up and recovered. Indeed, in the Gram-negative model organism Escherichia coli, peptidoglycan recovery has been recognized as a complex pathway, named cell wall recycling. It involves about a dozen dedicated recycling enzymes that convey cell wall turnover products to peptidoglycan synthesis or energy pathways. Whether Gram-positive bacteria also recover their cell wall is currently questioned. Given the much larger portion of peptidoglycan in the cell wall of Gram-positive bacteria, however, recovery of the wall material would provide an even greater benefit in these organisms compared to Gram-negatives. Consistently, in many Gram-positives, orthologs of recycling enzymes were identified, indicating that the cell wall may also be recycled in these organisms. This mini-review provides a compilation of information about cell wall turnover and recycling in Gram-positive bacteria during cell growth and division, including recent findings relating to muropeptide recovery in Bacillus subtilis and Clostridium acetobutylicum from our group. Furthermore, the impact of cell wall turnover and recycling on biotechnological processes is discussed.     

LysM, a widely distributed protein motif for binding to (peptido)glycans

Bacteria retain certain proteins at their cell envelopes by attaching them in a non-covalent manner to peptidoglycan, using specific protein domains, such as the prominent LysM (Lysin Motif) domain. More than 4000 (Pfam PF01476) proteins of both prokaryotes and eukaryotes have been found to contain one or more Lysin Motifs. Notably, this collection contains not only truly secreted proteins, but also (outer-)membrane proteins, lipoproteins or proteins bound to the cell wall in a (non-)covalent manner. The motif typically ranges in length from 44 to 65 amino acid residues and binds to various types of peptidoglycan and chitin, most likely recognizing the N-acetylglucosamine moiety. Most bacterial LysM-containing proteins are peptidoglycan hydrolases with various cleavage specificities. Binding of certain LysM proteins to cells of Gram-positive bacteria has been shown to occur at specific sites, as binding elsewhere is hindered by the presence of other cell wall components such as lipoteichoic acids. Interestingly, LysM domains of certain plant kinases enable the plant to recognize its symbiotic bacteria or sense and induce resistance against fungi. This interaction is triggered by chitin-like compounds that are secreted by the symbiotic bacteria or released from fungi, demonstrating an important sensing function of LysMs.     

The biosynthesis and functionality of the cell-wall of lactic acid bacteria

The cell wall of lactic acid bacteria has the typical Gram-positive structure made of a thick, multilayered peptidoglycan sacculus decorated with proteins, teichoic acids and polysaccharides, and surrounded in some species by an outer shell of proteins packed in a paracrystalline layer (S-layer). Specific biochemical or genetic data on the biosynthesis pathways of the cell wall constituents are scarce in lactic acid bacteria, but together with genomics information they indicate close similarities with those described in Escherichia coli and Bacillus subtilis, with one notable exception regarding the peptidoglycan precursor. In several species or strains of enterococci and lactobacilli, the terminal D-alanine residue of the muramyl pentapeptide is replaced by D-lactate or D-serine, which entails resistance to the glycopeptide antibiotic vancomycin. Diverse physiological functions may be assigned to the cell wall, which contribute to the technological and health-related attribut es of lactic acid bacteria. For instance, phage receptor activity relates to the presence of specific substituents on teichoic acids and polysaccharides; resistance to stress (UV radiation, acidic pH) depends on genes involved in peptidoglycan and teichoic acid biosynthesis; autolysis is controlled by the degree of esterification of teichoic acids with D-alanine; mucosal immunostimulation may result from interactions between epithelial cells and peptidoglycan or teichoic acids.     

Cutting edge: recognition of Gram-positive bacterial cell wall components by the innate immune system occurs via Toll-like receptor 2.

Invasive infection with Gram-positive and Gram-negative bacteria often results in septic shock and death. The basis for the earliest steps in innate immune response to Gram-positive bacterial infection is poorly understood. The LPS component of the Gram-negative bacterial cell wall appears to activate cells via CD14 and Toll-like receptor (TLR) 2 and TLR4. We hypothesized that Gram-positive bacteria might also be recognized by TLRs. Heterologous expression of human TLR2, but not TLR4, in fibroblasts conferred responsiveness to Staphylococcus aureus and Streptococcus pneumoniae as evidenced by inducible translocation of NF-kappaB. CD14 coexpression synergistically enhanced TLR2-mediated activation. To determine which components of Gram-positive cell walls activate Toll proteins, we tested a soluble preparation of peptidoglycan prepared from S. aureus. Soluble peptidoglycan substituted for whole organisms. These data suggest that the similarity of clinical response to invasive infection by Gram-positive and Gram-negative bacteria is due to bacterial recognition via similar TLRs.     

Nods and 'intracellular' innate immunity

Innate immunity relies on the detection of microbial invaders by two distinct systems. One system comprises a family of membrane-bound receptors, termed the Toll-like receptors, while the other family, termed the nucleotide-binding site/leucine-rich repeat (NBS/LRR) proteins, consists of molecules that are found in the cytoplasmic compartment. These two detection systems recognize conserved molecular components of microbes including such structural motifs as lipopolysaccharide from the Gram-negative bacterial cell wall and peptidoglycan (PGN) found in the cell wall of both Gram-negative and Gram-positive bacteria. This review focuses on two members of the NBS/LRR family of proteins, Nod1 and Nod2. Recently, the microbial motifs sensed by these two molecules have been characterized. Both Nod1 and Nod2 recognize PGN, however, each requires distinct molecular motifs to attain sensing. Nod1 recognizes a naturally occurring muropeptide of PGN that presents a unique amino acid at its terminus called diaminopilemic acid (DAP). This amino acid is found mainly in the PGN of Gram-negative bacteria designating Nodl as a sensor of Gram-negative bacteria. In contrast, Nod2 can detect the minimal bioactive fragment of PGN, called muramyl dipeptide. Thus Nod2 is a general sensor of bacterial PGN. Since mutations in the gene encoding Nod2 were recently shown to be associated with the chronic inflammatory disease, Crohn's disease, these results are discussed in the context of how disrupting the interplay between host detection and bacterial aggression may lead to inflammatory diseases.     

Pili with strong attachments: Gram-positive bacteria do it differently

Bacteria attach to their appropriate environmental niche by using adhesins. To maximize their contact with the environment, adhesins are often present on the ends of long hairlike structures called pili. Recently, attention has focused on pili of Gram-positive bacteria because they may be vaccine candidates in important human pathogens. These pili differ from the well-studied pili of Gram-negative bacteria because their subunits are covalently linked, they do not require specific chaperones for assembly, and the tip protein (likely to be the adhesin) is not required to initiate formation of the pilus structure. In Gram-positive bacteria, the genes for pili occur in clusters, which may constitute mobile genetic elements. These clusters include the transpeptidase(s) of the sortase family that is/are required for polymerization of the subunit proteins. However, efficient covalent attachment of the completed pilus structure to the cell wall is accomplished, in cases where this has been studied, by the 'housekeeping' sortase, which is responsible for attachment to the peptidoglycan of most surface proteins containing cell wall sorting signals. This enzyme is encoded elsewhere on the genome. Because pili of Gram-positive bacteria have not been extensively investigated yet, we hope that this MicroReview will help to pinpoint the areas most in need of further study.     

Sortases and the Art of Anchoring Proteins to the Envelopes of Gram-Positive Bacteria

The cell wall envelopes of gram-positive bacteria represent a surface organelle that not only functions as a cytoskeletal element but also promotes interactions between bacteria and their environment. Cell wall peptidoglycan is covalently and noncovalently decorated with teichoic acids, polysaccharides, and proteins. The sum of these molecular decorations provides bacterial envelopes with species- and strain-specific properties that are ultimately responsible for bacterial virulence, interactions with host immune systems, and the development of disease symptoms or successful outcomes of infections. Surface proteins typically carry two topogenic sequences, i.e., N-terminal signal peptides and C-terminal sorting signals. Sortases catalyze a transpeptidation reaction by first cleaving a surface protein substrate at the cell wall sorting signal. The resulting acyl enzyme intermediates between sortases and their substrates are then resolved by the nucleophilic attack of amino groups, typically provided by the cell wall cross bridges of peptidoglycan precursors. The surface protein linked to peptidoglycan is then incorporated into the envelope and displayed on the microbial surface. This review focuses on the mechanisms of surface protein anchoring to the cell wall envelope by sortases and the role that these enzymes play in bacterial physiology and pathogenesis.     

Sortases and the art of anchoring proteins to the envelopes of gram-positive bacteria.

The cell wall envelopes of gram-positive bacteria represent a surface organelle that not only functions as a cytoskeletal element but also promotes interactions between bacteria and their environment. Cell wall peptidoglycan is covalently and noncovalently decorated with teichoic acids, polysaccharides, and proteins. The sum of these molecular decorations provides bacterial envelopes with species- and strain-specific properties that are ultimately responsible for bacterial virulence, interactions with host immune systems, and the development of disease symptoms or successful outcomes of infections. Surface proteins typically carry two topogenic sequences, i.e., N-terminal signal peptides and C-terminal sorting signals. Sortases catalyze a transpeptidation reaction by first cleaving a surface protein substrate at the cell wall sorting signal. The resulting acyl enzyme intermediates between sortases and their substrates are then resolved by the nucleophilic attack of amino groups, typically provided by the cell wall cross bridges of peptidoglycan precursors. The surface protein linked to peptidoglycan is then incorporated into the envelope and displayed on the microbial surface. This review focuses on the mechanisms of surface protein anchoring to the cell wall envelope by sortases and the role that these enzymes play in bacterial physiology and pathogenesis.     

Cell wall structure and function in lactic acid bacteria

The cell wall of Gram-positive bacteria is a complex assemblage of glycopolymers and proteins. It consists of a thick peptidoglycan sacculus that surrounds the cytoplasmic membrane and that is decorated with teichoic acids, polysaccharides, and proteins. It plays a major role in bacterial physiology since it maintains cell shape and integrity during growth and division; in addition, it acts as the interface between the bacterium and its environment. Lactic acid bacteria (LAB) are traditionally and widely used to ferment food, and they are also the subject of more and more research because of their potential health-related benefits. It is now recognized that understanding the composition, structure, and properties of LAB cell walls is a crucial part of developing technological and health applications using these bacteria. In this review, we examine the different components of the Gram-positive cell wall: peptidoglycan, teichoic acids, polysaccharides, and proteins. We present recent findings regarding the structure and function of these complex compounds, results that have emerged thanks to the tandem development of structural analysis and whole genome sequencing. Although general structures and biosynthesis pathways are conserved among Gram-positive bacteria, studies have revealed that LAB cell walls demonstrate unique properties; these studies have yielded some notable, fundamental, and novel findings. Given the potential of this research to contribute to future applied strategies, in our discussion of the role played by cell wall components in LAB physiology, we pay special attention to the mechanisms controlling bacterial autolysis, bacterial sensitivity to bacteriophages and the mechanisms underlying interactions between probiotic bacteria and their hosts.     

分选酶的研究进展及其在生物技术中的应用

分选酶(sortase)普遍存在于革兰氏阳性细菌中,是一类膜结合的转肽酶,负责将表面蛋白共价结合到细胞壁的肽聚糖上。由于其独特的作用机制,分选酶在生物技术领域具有广阔的应用前景,可应用于革兰氏阳性菌的表面展示、蛋白质工程等。     

Anchor structure of cell wall surface proteins in Listeria monocytogenes

Many surface proteins of Gram-positive bacteria are anchored to the cell wall by a mechanism requiring a COOH-terminal sorting signal with a conserved LPXTG motif. In Staphylococcus aureus, surface proteins are cleaved between the threonine and the glycine of the LPXTG motif. The carboxyl of threonine is subsequently amide linked to the amino group of the pentaglycine cell wall crossbridge. Here we investigated the anchor structure of surface proteins in Listeria monocytogenes. A methionine and six histidines (MH(6)) were inserted upstream of the LPXTG motif of internalin A (InlA), a cell-wall-anchored surface protein of L. monocytogenes. The engineered protein InlA-MH(6)-Cws was found anchored in the bacterial cell wall. After peptidoglycan digestion with phage endolysin, InlA-MH(6)-Cws was purified by affinity chromatography. COOH-terminal peptides of InlA-MH(6)-Cws were obtained by cyanogen bromide cleavage followed by purification on a nickel-nitriloacetic acid column. Analysis of COOH-terminal peptides with Edman degradation and mass spectrometry revealed an amide linkage between the threonine of the cleaved LPXTG motif and the amino group of the m-diaminopimelic acid crossbridge within the listerial peptidoglycan. These results reveal that the cell wall anchoring of surface proteins in Gram-positive bacteria such as S. aureus and L. monocytogenes occurs by a universal mechanism.     

Sec-secretion and sortase-mediated anchoring of proteins in Gram-positive bacteria

Signal peptide-driven secretion of precursor proteins directs polypeptides across the plasma membrane of bacteria. Two pathways, Sec- and SRP-dependent, converge at the SecYEG translocon to thread unfolded precursor proteins across the membrane, whereas folded preproteins are routed via the Tat secretion pathway. Gram-positive bacteria lack an outer membrane and are surrounded by a rigid layer of peptidoglycan. Interactions with their environment are mediated by proteins that are retained in the cell wall, often through covalent attachment to the peptidoglycan. In this review, we describe the mechanisms for both Sec-dependent secretion and sortase-dependent assembly of proteins in the envelope of Gram-positive bacteria. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

Requirements of peptidoglycan structure that allow detection by the Drosophila Toll pathway

The Drosophila immune system is able to discriminate between classes of bacteria. Detection of Gram-positive bacteria involves a complex of two pattern recognition receptors: peptidoglycan recognition protein SA (PGRP-SA) and Gram-negative binding protein 1 (GNBP1). These activate the Toll signalling pathway. To define the cell wall components sensed by the host, we used highly purified peptidoglycan fragments of two principal Gram-positive bacterial pathogens Staphylococcus aureus and Streptococcus pneumoniae. We report that in both peptidoglycans, the minimal structure needed to activate the Toll pathway is a muropeptide dimer and that the free reducing end of the N-acetyl muramic acid residues of the muropeptides is essential for activity. Monomeric muropeptides were inactive and inhibitory in combination with dimers. Finally, peptidoglycan was degraded by the haemolymph of wild-type but not GNBP1 mutant flies. We suggest a model whereby GNBP1 is involved in the hydrolysis of Gram-positive peptidoglycan producing new glycan reducing ends, which are subsequently detected by PGRP-SA.     

Substrate Specificity and Kinetic Characterization of Peptidoglycan O-Acetyltransferase B from Neisseria gonorrhoeae

The O-acetylation of the essential cell wall polymer peptidoglycan is a major virulence factor identified in many bacteria, both Gram-positive and Gram-negative, including Staphylococcus aureus, Bacillus anthracis, Neisseria gonorrhoeae, and Neisseria meningitidis. With Gram-negative bacteria, the translocation of acetyl groups from the cytoplasm is performed by an integral membrane protein, PatA, for its transfer to peptidoglycan by O-acetyltransferase PatB, whereas a single bimodal membrane protein, OatA, appears to catalyze both reactions of the process in Gram-positive bacteria. Only phenotypic evidence existed in support of these pathways because no in vitro biochemical assay was available for their analysis, which reflected the complexities of investigating integral membrane proteins that act on a totally insoluble and heterogeneous substrate, such as peptidoglycan. In this study, we present the first biochemical and kinetic analysis of a peptidoglycan O-acetyltransferase using PatB from N. gonorrhoeae as the model system. The enzyme has specificity for muropeptides that possess tri- and tetrapeptide stems on muramyl residues. With chitooligosaccharides as substrates, rates of reaction increase with increasing degrees of polymerization to 5/6. This information will be valuable for the identification and development of peptidoglycan O-acetyltransferase inhibitors that could represent potential leads to novel classes of antibiotics.     

The interaction of macrophage receptors with bacterial ligands

Innate immune receptors play a key role in the early recognition of invading bacterial pathogens and initiate the crucial innate immune response. The diverse macrophage receptors recognise Gram-positive and Gram-negative bacteria via conserved structures on the bacterial surface and facilitate phagocytosis and/or signalling, providing the trigger for the adaptive immune response. These receptors include scavenger receptors, C-type lectins, integrins, Toll-like receptors and siglecs. The bacterial ligands generally recognised by these receptors range from lipopolysaccharides on Gram-negative bacteria to peptidoglycan and lipoteichoic acid on Gram-positive bacteria. However, emerging evidence indicates that bacterial proteins are also important ligands; for example, surface proteins from Neisseria meningitidis have been shown to be ligands for class A scavenger receptors. In addition, a group of cytosolic receptors, the NBS-LRR proteins, have been implicated in recognition of bacterial breakdown products. It is becoming increasingly apparent that macrophage receptors can act in conjunction with one another to deliver an appropriate response.