Peptidoglycan Polymer Research Articles

High-throughput transposon sequencing highlights the cell wall as an important barrier for osmotic stress in methicillin resistant Staphylococcus aureus and underlines a tailored response to different osmotic stressors.

Staphylococcus aureus is an opportunistic pathogen that can cause soft tissue infections but is also a frequent cause of foodborne illnesses. One contributing factor for this food association is its high salt tolerance allowing this organism to survive commonly used food preservation methods. How this resistance is mediated is poorly understood, particularly during long‐term exposure. In this study, we used transposon sequencing (TN‐seq) to understand how the responses to osmotic stressors differ. Our results revealed distinctly different long‐term responses to NaCl, KCl and sucrose stresses. In addition, we identified the DUF2538 domain containing gene SAUSA300_0957 (gene 957) as essential under salt stress. Interestingly, a 957 mutant was less susceptible to oxacillin and showed increased peptidoglycan crosslinking. The salt sensitivity phenotype could be suppressed by amino acid substitutions in the transglycosylase domain of the penicillin‐binding protein Pbp2, and these changes restored the peptidoglycan crosslinking to WT levels. These results indicate that increased crosslinking of the peptidoglycan polymer can be detrimental and highlight a critical role of the bacterial cell wall for osmotic stress resistance. This study will serve as a starting point for future research on osmotic stress response and help develop better strategies to tackle foodborne staphylococcal infections.

The C-Type Lysozyme from the upper Gastrointestinal Tract of Opisthocomus hoatzin, the Stinkbird.

Muramidases/lysozymes are important bio-molecules, which cleave the glycan backbone in the peptidoglycan polymer found in bacterial cell walls. The glycoside hydrolase (GH) family 22 C-type lysozyme, from the folivorous bird Opisthocomus hoazin (stinkbird), was expressed in Aspergillus oryzae, and a set of variants was produced. All variants were enzymatically active, including those designed to probe key differences between the Hoatzin enzyme and Hen Egg White lysozyme. Four variants showed improved thermostability at pH 4.7, compared to the wild type. The X-ray structure of the enzyme was determined in the apo form and in complex with chitin oligomers. Bioinformatic analysis of avian GH22 amino acid sequences showed that they separate out into three distinct subgroups (chicken-like birds, sea birds and other birds). The Hoatzin is found in the “other birds” group and we propose that this represents a new cluster of avian upper-gut enzymes.

Peptidoglycan Muropeptides: Release, Perception, and Functions as Signaling Molecules.

Peptidoglycan (PG) is an essential molecule for the survival of bacteria, and thus, its biosynthesis and remodeling have always been in the spotlight when it comes to the development of antibiotics. The peptidoglycan polymer provides a protective function in bacteria, but at the same time is continuously subjected to editing activities that in some cases lead to the release of peptidoglycan fragments (i.e., muropeptides) to the environment. Several soluble muropeptides have been reported to work as signaling molecules. In this review, we summarize the mechanisms involved in muropeptide release (PG breakdown and PG recycling) and describe the known PG-receptor proteins responsible for PG sensing. Furthermore, we overview the role of muropeptides as signaling molecules, focusing on the microbial responses and their functions in the host beyond their immunostimulatory activity.

Ten-membered rings as key interaction motifs in folding of desmuramyl di-, tri-, and tetrapeptides

Muropeptides are fragments of polymeric peptidoglycan, unique constituent of bacterial cell walls, acting as immunostimulators (adjuvants). The smallest biologically active unit of peptidoglycan is N-acetylmuramyl-L-alanyl-D-isoglutamine. In this paper, the synthesis and conformational analysis of desmuramyl peptides, derivatives lacking the N-acetylmuramyl moiety, is described. Systematic conformational analysis (bottom-up approach) of peptides in zwitterionic and non-zwitterionic forms of corresponding desmuramyl di-, tri-, and tetrapeptides was performed and results were compared with experimental data obtained by NMR study. Ten-membered ring (C10, N1bH⋯O3) formed between isoGln amide group and C=O of AdGly was found to be a key interaction that constitutes the central structural spine and initiates β-turn-like folding in our model peptide systems.

The gram-negative bacterial periplasm: Size matters.

Gram-negative bacteria are surrounded by two membrane bilayers separated by a space termed the periplasm. The periplasm is a multipurpose compartment separate from the cytoplasm whose distinct reducing environment allows more efficient and diverse mechanisms of protein oxidation, folding, and quality control. The periplasm also contains structural elements and important environmental sensing modules, and it allows complex nanomachines to span the cell envelope. Recent work indicates that the size or intermembrane distance of the periplasm is controlled by periplasmic lipoproteins that anchor the outer membrane to the periplasmic peptidoglycan polymer. This periplasm intermembrane distance is critical for sensing outer membrane damage and dictates length of the flagellar periplasmic rotor, which controls motility. These exciting results resolve longstanding debates about whether the periplasmic distance has a biological function and raise the possibility that the mechanisms for maintenance of periplasmic size could be exploited for antibiotic development.

Channel-forming activity of nisin in two mercury-supported biomimetic membranes

Nisin, the most prominent lantibiotic used as a food preservative, due to its high potency against certain Gram-positive bacteria, was investigated in mercury-supported lipid monolayers and bilayers, with the distal monolayer consisting of a dioleoylphosphatidylcholine (DOPC) or dioleoylphosphatidylserine (DOPS) monolayer. The voltage-gated behavior of the cyclic voltammogram (CV) of nisin at DPTL/DOPC tBLM in a pH5.4 unbuffered solution of 0.1M KCl is converted into an ohmic behavior upon addition of the glucopeptide CSF114(Glc), under favorable experimental conditions. This indicates that nisin targets Gram-positive bacteria mainly at the NHAc-substituted α-d-glucose ring, which is present both in the peptidoglycan polymer forming their cell walls and in CSF114(Glc). While the β-d-glucose and β-d-galactose rings of the glycosphingolipid GM1 are ineffective, addition of α-d-glucose, which interacts attractively with the GM1 sugar chain, has a moderate but significant enhancing effect on the nisin CV. This suggests a synergic effect of α-d-glucose and NHAc substitution in creating a target for nisin attack. Conversely, CSF114(Glc) has no appreciable effect on the DOPS distal monolayer at both pH values 5.4 and 6.8 and does not induce ohmic behavior. More precisely, at pH6.8 the DOPS polar heads are negatively charged and recruit the positively charged nisin molecules, preventing their penetration into the hydrocarbon tail region. This penetration becomes possible at pH5.4, where the DOPS distal monolayer is neutral, allowing the formation of an ion channel that yields a voltage-gated CV.

The disruptive effect of lysozyme on the bacterial cell wall explored by an in-silico structural outlook.

The bacterial cell wall, a structural unit of peptidoglycan polymer comprised of glycan strands consisting of a repeating disaccharide motif [N-acetylglucosamine (NAG) and N-acetylmuramylpentapeptide (NAM pentapeptide)], encases bacteria and provides structural integrity and protection. Lysozymes are enzymes that break down the bacterial cell wall and disrupt the bacterial life cycle by cleaving the linkage between the NAG and NAM carbohydrates. Lab exercises focused on the effects of lysozyme on the bacterial cell wall are frequently incorporated in biochemistry classes designed for undergraduate students in diverse fields as biology, microbiology, chemistry, agronomy, medicine, and veterinary medicine. Such exercises typically do not include structural data. We describe here a sequence of computer tasks designed to illustrate and reinforce both physiological and structural concepts involved in lysozyme effects on the bacterial cell-wall structure. This lab class usually lasts 3.5 hours. First, the instructor presents introductory concepts of the bacterial cell wall and the effect of lysozyme on its structure. Then, students are taught to use computer modeling to visualize the three-dimensional structure of a lysozyme in complex with bacterial cell-wall fragments. Finally, the lysozyme inhibitory effect on a bacterial culture is optionally proposed as a simple microbiological assay. The computer lab exercises described here give students a realistic understanding of the disruptive effect of lysozymes on the bacterial cell wall, a crucial component in bacterial survival. © 2017 by The International Union of Biochemistry and Molecular Biology, 46(1):83-90, 2018.

In vitro characterization of the antivirulence target of Gram-positive pathogens, peptidoglycan O-acetyltransferase A (OatA).

The O-acetylation of the essential cell wall polymer peptidoglycan occurs in most Gram-positive bacterial pathogens, including species of Staphylococcus, Streptococcus and Enterococcus. This modification to peptidoglycan protects these pathogens from the lytic action of the lysozymes of innate immunity systems and, as such, is recognized as a virulence factor. The key enzyme involved, peptidoglycan O-acetyltransferase A (OatA) represents a particular challenge to biochemical study since it is a membrane associated protein whose substrate is the insoluble peptidoglycan cell wall polymer. OatA is predicted to be bimodular, being comprised of an N-terminal integral membrane domain linked to a C-terminal extracytoplasmic domain. We present herein the first biochemical and kinetic characterization of the C-terminal catalytic domain of OatA from two important human pathogens, Staphylococcus aureus and Streptococcus pneumoniae. Using both pseudosubstrates and novel biosynthetically-prepared peptidoglycan polymers, we characterized distinct substrate specificities for the two enzymes. In addition, the high resolution crystal structure of the C-terminal domain reveals an SGNH/GDSL-like hydrolase fold with a catalytic triad of amino acids but with a non-canonical oxyanion hole structure. Site-specific replacements confirmed the identity of the catalytic and oxyanion hole residues. A model is presented for the O-acetylation of peptidoglycan whereby the translocation of acetyl groups from a cytoplasmic source across the cytoplasmic membrane is catalyzed by the N-terminal domain of OatA for their transfer to peptidoglycan by its C-terminal domain. This study on the structure-function relationship of OatA provides a molecular and mechanistic understanding of this bacterial resistance mechanism opening the prospect for novel chemotherapeutic exploration to enhance innate immunity protection against Gram-positive pathogens.

Synthesis of novel muramic acid derivatives and their interaction with lysozyme: Action of lysozyme revisited.

The interaction of lysozyme with the N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) unit of peptidoglycan (PGN) polymer of the bacterial cell wall is of immense importance to understand the mechanism of lysozyme on PGN. The synthesis of three novel NAM derivatives containing fused oxazinone ring to the NAM moiety has been achieved. The synthesized compounds were evaluated for their potential as a glycomimetic acceptor of lysozyme using different biophysical and computational methods such as 1H NMR, STD NMR, DOSY and Molecular docking. Novel modified muramic acid derivatives have been synthesized in excellent yield containing fused cyclooxazine ring embedded on the muramic acid moiety using a newly developed hydrazinolysis reaction condition. From various biophysical studies, it has been established that the compound containing endo modified muramic acid moiety (compound 1) shows significant binding property for the lysozyme while the other isomer (compound 2) did not bind to the lysozyme. The catalytic residues Glu35 and Asp52 were found to be in the close proximity for the active molecule which justifies the selectivity of this molecule in conjunction to lysozyme enzymatic activity.

The Absence of a Mature Cell Wall Sacculus in Stable Listeria monocytogenes L-Form Cells Is Independent of Peptidoglycan Synthesis.

L-forms are cell wall-deficient variants of otherwise walled bacteria that maintain the ability to survive and proliferate in absence of the surrounding peptidoglycan sacculus. While transient or unstable L-forms can revert to the walled state and may still rely on residual peptidoglycan synthesis for multiplication, stable L-forms cannot revert to the walled form and are believed to propagate in the complete absence of peptidoglycan. L-forms are increasingly studied as a fundamental biological model system for cell wall synthesis. Here, we show that a stable L-form of the intracellular pathogen Listeria monocytogenes features a surprisingly intact peptidoglycan synthesis pathway including glycosyl transfer, in spite of the accumulation of multiple mutations during prolonged passage in the cell wall-deficient state. Microscopic and biochemical analysis revealed the presence of peptidoglycan precursors and functional glycosyl transferases, resulting in the formation of peptidoglycan polymers but without the synthesis of a mature cell wall sacculus. In conclusion, we found that stable, non-reverting L-forms, which do not require active PG synthesis for proliferation, may still continue to produce aberrant peptidoglycan.

A balancing act times two: sensing and regulating cell envelope homeostasis in Bacillus subtilis.

Bacterial cell wall homeostasis is an intricately coordinated process that ensures that envelope integrity is maintained during cell growth and division, but can also adequately respond to growth-limiting conditions such as phosphate starvation. In Bacillus subtilis, biosynthesis of the two major cell wall components, peptidoglycan and anionic polymers, is controlled by a pair of paralogous two-component systems, WalRK and PhoPR respectively. Favorable growth conditions allow for a fast rate of cell wall biosynthesis (WalRK-ON) and the incorporation of the phosphate-containing anionic polymer teichoic acids (PhoPR-OFF). In contrast, growth-restricted cells under phosphate-limiting conditions reduce the incorporation of peptidoglycan building blocks (WalRK-OFF) and switch from the phosphate-containing teichoic acids to the phosphate-free anionic polymer teichuronic acid (PhoPR-ON). Botella et al. (2014) deepen our knowledge on the PhoPR system by identifying one signal that is perceived by its histidine kinase PhoR. In fast-growing cells, intracellular intermediates of teichoic acid biosynthesis are sensed by the cytoplasmic Per-Arnt-Sim domain as an indicator of favorable conditions, thereby inhibiting the autokinase activity of PhoR and keeping the system inactive. Depletion of teichoic acid building blocks under phosphate-limiting conditions relieves this inhibition, activates PhoPR-dependent signal transduction and hence the switch to teichuronic acid biosynthesis.

Mechanism of action of peptidoglycan O-acetyltransferase B involves a Ser-His-Asp catalytic triad.

The O-acetylation of the essential cell wall polymer peptidoglycan is essential in many bacteria for their integrity and survival, and it is catalyzed by peptidoglycan O-acetlytransferase B (PatB). Using PatB from Neisseria gonorrhoeae as the model, we have shown previously that the enzyme has specificity for polymeric muropeptides that possess tri- and tetrapeptide stems and that rates of reaction increase with increasing degrees of polymerization. Here, we present the catalytic mechanism of action of PatB, the first to be described for an O-acetyltransferase of any bacterial exopolysaccharide. The influence of pH on PatB activity was investigated, and pKa values of 6.4-6.45 and 6.25-6.35 for the enzyme-substrate complex (kcat vs pH) and the free enzyme (kcat·KM(-1) vs pH), respectively, were determined for the respective cosubstrates. The enzyme is partially inactivated by sulfonyl fluorides but not by EDTA, suggesting the participation of a serine residue in its catalytic mechanism. Alignment of the known and hypothetical PatB amino acid sequences identified Ser133, Asp302, and His305 as three invariant amino acid residues that could potentially serve as a catalytic triad. Replacement of Asp302 with Ala resulted in an enzyme with less than 20% residual activity, whereas activity was barely detectable with (His305 → Ala)PatB and (Ser133 → Ala)PatB was totally inactive. The reaction intermediate of the transferase reaction involving acetyl- and propionyl-acyl donors was trapped on both the wild-type and (Asp302 → Ala) enzymes and LC-MS/MS analysis of tryptic peptides identified Ser133 as the catalytic nucleophile. A transacetylase mechanism is proposed based on the mechanism of action of serine esterases.

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.

Structural and binding properties of the PASTA domain of PonA2, a key penicillin binding protein from Mycobacterium tuberculosis

PonA2 is one of the two class A penicillin binding proteins of Mycobacterium tuberculosis, the etiologic agent of tuberculosis. It plays a complex role in mycobacterial physiology and is spotted as a promising target for inhibitors. PonA2 is involved in adaptation of M. tuberculosis to dormancy, an ability which has been attributed to the presence in its sequence of a C-terminal PASTA domain. Since PASTA modules are typically considered as β-lactam antibiotic binding domains, we determined the solution structure of the PASTA domain from PonA2 and analyzed its binding properties versus a plethora of potential binders, including the β-lactam antibiotics, two typical muropeptide mimics, and polymeric peptidoglycan. We show that, despite a high structural similarity with other PASTA domains, the PASTA domain of PonA2 displays different binding properties, as it is not able to bind muropeptides, or β-lactams, or polymeric peptidoglycan. These results indicate that the role of PASTA domains cannot be generalized, as their specific binding properties strongly depend on surface residues, which are widely variable.

Structural and Biochemical Analyses of Mycobacterium tuberculosis N-Acetylmuramyl-l-alanine Amidase Rv3717 Point to a Role in Peptidoglycan Fragment Recycling

Peptidoglycan hydrolases are key enzymes in bacterial cell wall homeostasis. Understanding the substrate specificity and biochemical activity of peptidoglycan hydrolases in Mycobacterium tuberculosis is of special interest as it can aid in the development of new cell wall targeting therapeutics. In this study, we report biochemical and structural characterization of the mycobacterial N-acetylmuramyl-L-alanine amidase, Rv3717. The crystal structure of Rv3717 in complex with a dipeptide product shows that, compared with previously characterized peptidoglycan amidases, the enzyme contains an extra disulfide-bonded β-hairpin adjacent to the active site. The structure of two intermediates in assembly reveal that Zn(2+) binding rearranges active site residues, and disulfide formation promotes folding of the β-hairpin. Although Zn(2+) is required for hydrolysis of muramyl dipeptide, disulfide oxidation is not required for activity on this substrate. The orientation of the product in the active site suggests a role for a conserved glutamate (Glu-200) in catalysis; mutation of this residue abolishes activity. The product binds at the head of a closed tunnel, and the enzyme showed no activity on polymerized peptidoglycan. These results point to a potential role for Rv3717 in peptidoglycan fragment recycling.

Physical Model of the Staphylococcus aureus Transpeptidase PBP2a in Complex with an Anti‐Methicillin‐Resistant Staphylococcus aureus Cephalosporin

Transpeptidases, also known as penicillin‐binding proteins (PBP), catalyze cross‐linking of peptidoglycan polymers during bacterial cell wall synthesis. The natural transpeptidase substrate is the D‐Ala‐ D‐Ala peptidoglycan side chain terminus. Beta‐lactam (β‐lactam) antibiotics, which include the monobactams, penicillins, cephalosporins and carbapenems, bind and irreversibly inhibit transpeptidases by mimicking the D‐Ala‐D‐Ala substrate, resulting in the inhibition of cell wall synthesis and ultimately bacterial cell growth. Overuse and misuse of β‐lactams has led to the generation of methicillin‐resistant Staphylococcus aureus (MRSA) isolates that have acquired an alternative transpeptidase, PBP2a, which is neither bound nor inhibited by β‐lactams. MRSA isolates are resistant to all β‐lactams, can be hospital‐ or community‐acquired, and are the cause of significant morbidity and mortality. Furthermore, they are often only susceptible to “last resort antibiotics”, such as vancomycin. Recently, two cephalosporins ‐ ceftobiprole and ceftaroline ‐ that bind and inhibit PBP2a have been developed. The Hostos‐Lincoln Academy SMART Team (Students Modeling A Research Topic) has generated a model of the PBP2a/ceftobiprole complex (PDB 4DKI) using 3D printing technology to illustrate the mechanism of action of ceftobiprole. Supported by a grant from the Camille and Henry Dreyfus Foundation

Solution Structure and Peptidoglycan Interaction of Rv0899, a Virulence Factor from Mycobacterium Tuberculosis

Rv0899 is a membrane protein encoded by an operon that is required for fast ammonia secretion, rapid pH neutralization and growth of M. tuberculosis in acidic environments. The gene is restricted to pathogenic mycobacteria and, thus, is an attractive candidate for the development of anti-tuberculosis chemotherapy. Rv0899 contains three independently structured domains. The N-terminus (Rv0899-M; residues 1-80) includes a membrane-anchoring sequence of 20 hydrophobic amino acids (residues 28-50) that is required for membrane translocation. The central region (Rv0899-B; residues 81-194) contains two consecutive repeats of the BON (Bacterial OsmY and Nodulation) domain (pfam04972), a conserved, putative lipid binding sequence that is found in some bacterial osmotic shock protection proteins. Finally, the C-terminus (Rv0899-C; residues 195-326) contains an OmpA-like domain (pfam00691), a periplasmic peptidoglycan-binding structure found in several types of bacterial membrane proteins. Using NMR spectroscopy, we determined the high-resolution structures of Rv0899-B and Rv0899-C. Rv0899-B does not form a transmembrane beta barrel and has an undocumented mixed alpha/beta fold. Furthermore, we found Rv0899-B has a specific zinc binding site and associates with lipid vesicles in a pH dependent manner. The structure of Rv0899-C adopts the characteristic OmpA-like family fold. It exhibits pH-dependent conformational dynamics, with significant disorder at neutral pH and a more ordered structure at acidic pH. Rv0899-C associates tightly with polymeric peptidoglycan isolated from M. tuberculosis and also associates with a soluble peptide intermediate of peptidoglycan biosynthesis. This enabled us to characterize the peptidoglycan binding site where five highly conserved Rv0899-C residues, including two key arginines, establish the specificity for DAP- but not Lys-type peptidoglycan. The three-dimensional structure of Rv0899 suggests alternative possible modes of membrane association and provides a molecular basis for the screening of small molecules with anti-TB potential.

Molecular Structure and Peptidoglycan Recognition of Mycobacterium tuberculosis ArfA (Rv0899)

Mycobacterium tuberculosis ArfA (Rv0899) is a membrane protein encoded by an operon that is required for supporting bacterial growth in acidic environments. Its C-terminal domain (C domain) shares significant sequence homology with the OmpA-like family of peptidoglycan-binding domains, suggesting that its physiological function in acid stress protection may be related to its interaction with the mycobacterial cell wall. Previously, we showed that ArfA forms three independently structured modules, and we reported the structure of its central domain (B domain). Here, we describe the high-resolution structure and dynamics of the C domain, we identify ArfA as a peptidoglycan-binding protein and we elucidate the molecular basis for its specific recognition of diaminopimelate-type peptidoglycan. The C domain of ArfA adopts the characteristic fold of the OmpA-like family. It exhibits pH-dependent conformational dynamics (with significant heterogeneity at neutral pH and a more ordered structure at acidic pH), which could be related to its acid stress response. The C domain associates tightly with polymeric peptidoglycan isolated from M. tuberculosis and also associates with a soluble peptide intermediate of peptidoglycan biosynthesis. This enabled us to characterize the peptidoglycan binding site where five highly conserved ArfA residues, including two key arginines, establish the specificity for diaminopimelate- but not Lys-type peptidoglycan. ArfA is the first peptidoglycan-binding protein to be identified in M. tuberculosis. Its functions in acid stress protection and peptidoglycan binding suggest a link between the acid stress response and the physicochemical properties of the mycobacterial cell wall.

Cutting Edge: Primary Innate Immune Cells Respond Efficiently to Polymeric Peptidoglycan, but Not to Peptidoglycan Monomers

The cell wall of bacteria induces proinflammatory cytokines in monocytes and neutrophils in human blood. The nature of the stimulating component of bacterial cell walls is not well understood. We have previously shown polymeric peptidoglycan (PGN) has this activity, and the cytokine response requires PGN internalization and trafficking to lysosomes. In this study, we demonstrate that peptidoglycan monomers such as muramyl dipeptide and soluble peptidoglycan fail to induce robust cytokine production in immune cells, although they activate the nucleotide-binding oligomerization domain proteins in transfected cell models. We further show that lysosomal extracts from immune cells degrade intact peptidoglycan into simpler products and that the lysosomal digestion products activate the nucleotide-binding oligomerization domain proteins. We conclude that naive innate immune cells recognize PGN in its polymeric form rather than monomers such as muramyl dipeptide and require PGN lysosomal hydrolysis to respond. These findings offer new opportunities in the treatment of sepsis, especially sepsis arising from Gram-positive organisms.

Biochemical characterization of evasion from peptidoglycan recognition by Staphylococcus aureusd-alanylated wall teichoic acid in insect innate immunity

We recently reported that d-alanylation of Staphylococcus aureus wall teichoic acid (WTA) mitigates an induction of the Toll-mediated humoral response in Drosophila by interfering with peptidoglycan (PG) recognition by PG recognition protein-SA (PGRP-SA). Here, we investigated the mode of this interference by using an in vitro cell free system from larvae of the coleoptran insect Tenebrio molitor. WTA modification on PG had a potent inhibitory effect on PGRP-SA-mediated Toll proteolytic cascade activation, and the d-alanylation of WTA enhanced its inhibitory effect. Purified d-alanylated WTA released from PG lost its inhibitory action on both Toll cascade activation and PGRP-SA binding to insoluble PG. The inhibition of PGRP-SA binding to PG by d-alanylated WTA took place not only on polymeric PG but also on WTA-attached disaccharide units of monomeric PG. These results suggest that d-alanylation-mediated evasion requires the covalent bonding of d-alanylated WTA to PG, but not net-like cross-linking structure of PG.