Biochemical and crystallographic studies of L,D-transpeptidase 2 from Mycobacterium tuberculosis with its natural monomer substrate.

The essential L,D-transpeptidase of Mycobacterium tuberculosis (LdtMt2) catalyses the formation of 3 → 3 cross-links in cell wall peptidoglycan and is a target for development of antituberculosis therapeutics. Efforts to inhibit LdtMt2 have been hampered by lack of knowledge of how it binds its substrate. To address this gap, we optimised the isolation of natural disaccharide tetrapeptide monomers from the Corynebacterium jeikeium bacterial cell wall through overproduction of the peptidoglycan sacculus. The tetrapeptides were used in binding / turnover assays and biophysical studies on LdtMt2. We determined a crystal structure of wild-type LdtMt2 reacted with its natural substrate, the tetrapeptide monomer of the peptidoglycan layer. This structure shows formation of a thioester linking the catalytic cysteine and the donor substrate, reflecting an intermediate in the transpeptidase reaction; it informs on the mode of entrance of the donor substrate into the LdtMt2 active site. The results will be useful in design of LdtMt2 inhibitors, including those based on substrate binding interactions, a strategy successfully employed for other nucleophilic cysteine enzymes.

Atacama desert is a source of new Micromonospora strains: description of Micromonospora sicca sp. nov.

Several strains were isolated from subsurface soil of the Atacama Desert and were previously assigned to the Micromonospora genus. A polyphasic study was designed to determine the taxonomic affiliation of isolates 4G51T, 4G53, and 4G57. All the strains showed chemotaxonomic properties in line with their classification in the genus Micromonospora, including meso-diaminopimelic acid in the cell wall peptidoglycan, MK-9(H4) as major respiratory quinone, iso-C15:0 and iso-C16:0 as major fatty acids and diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylinositol as major polar lipids. The 16S rRNA gene sequences of strains 4G51T, 4G53, and 4G57 showed the highest similarity (97.9 %) with the type strain of Micromonospora costi CS1-12T, forming an independent branch in the phylogenetic gene tree. Their independent position was confirmed with genome phylogenies, being most closely related to the type strain of Micromonospora kangleipakensis. Digital DNA-DNA hybridization and average nucleotide identity analyses between the isolates and their closest phylogenomic neighbours confirmed that they should be assigned to a new species within the genus Micromonospora for which the name Micromonospora sicca sp. nov. (4G51T=PCM 3031T=LMG 30756T) is proposed.

Metabolic labeling of peptidoglycan enabled optical analysis of probiotic vitality.

The growing awareness of the health benefits associated with probiotics has led to an increasing interest in probiotic products. To develop probiotic functional foods that deliver health benefits, it is essential to characterize both probiotic viability (the ability to survive) and vitality (the ability to remain active and effective). However, traditional probiotic assays only provide limited information about their survival state. To gain a comprehensive understanding of probiotic states, a D-amino-acid-based metabolic labeling strategy was applied to quantitatively depict probiotic vitality. In this approach, probiotics were first metabolically incorporated with azido-modified D-lysine and then labeled with dibenzocyclooctyne-sulfo-Cy5 through click chemistry. This two-step labeling process provides a visual representation of the metabolic levels of probiotics as well as the bacterial membrane integrity. Besides, this method is capable of characterizing the influence of various environmental conditions, from manufacturing to oral administration, on probiotic vitality. With its rapid detection process and general applicability, this strategy has the potential to be widely implemented in the food industry for probiotic vitality evaluation.

Discovery and characterization of a novel LysinB from F2 sub-cluster mycobacteriophage RitSun.

The escalating antibiotic resistance in mycobacterial species poses a significant threat globally, necessitating an urgent need to find alternative solutions. Bacteriophage-derived endolysins, which facilitate phage progeny release by attacking bacterial cell walls, present promising antibacterial candidates due to their rapid lytic action, high specificity and low risk of resistance development. In mycobacteria, owing to the complex, hydrophobic cell wall, mycobacteriophages usually synthesize two endolysins: LysinA, which hydrolyzes peptidoglycan; LysinB, which delinks mycolic acid-containing outer membrane and arabinogalactan, releasing free mycolic acid. In this study, we conducted domain analysis and functional characterization of a novel LysinB from RitSun, an F2 sub-cluster mycobacteriophage from our phage collection. Several key properties of RitSun LysinB make it an important antimycobacterial agent: its ability to lyse Mycobacterium smegmatis from without, a higher than previously reported specific activity of 1.36 U/mg and its inhibitory effect on biofilm formation. Given the impermeable nature of the mycobacterial cell envelope, dissecting RitSun LysinB at the molecular level to identify its cell wall-destabilizing sequence could be utilized to engineer other native lysins as fusion proteins, broadening their activity spectrum.

Endolysin CHAP domain-carbosilane metallodendrimer complexes with triple action on Gram-negative bacteria: Membrane destabilization, reactive oxygen species production and peptidoglycan degradation.

Bacterial resistance to antibiotics is a significant challenge that is associated with increased morbidity and mortality. Gram-negative bacteria are particularly problematic due to an outer membrane (OM). Current alternatives to antibiotics include antimicrobial peptides or proteins and multifunctional systems such as dendrimers. Antimicrobial proteins such as lysins can degrade the bacterial cell wall, whereas dendrimers can permeabilize the OM, enhancing the activity of endolysins against gram-negative bacteria. In this study, we present a three-stage action of endolysin combined with two different carbosilane (CBS) silver metallodendrimers, in which the periphery is modified with N-heterocyclic carbene (NHC) ligands coordinating a silver atom. The different NHC ligands contained hydrophobic methyl or N-donor pyridyl moieties. The effects of these endolysin/dendrimer combinations are based on OM permeabilization, peptidoglycan degradation, and reactive oxygen species production. The results showed that CBS possess a permeabilization effect (first action), significantly reduced bacterial growth at higher concentrations alone and in the presence of endolysin, increased ROS production (second action), and led to bacterial cell damage (third action). The complex formed between the CHAP domain of endolysin and a CBS silver metallodendrimer, with a triple mechanism of action, may represent an excellent alternative to other antimicrobials with only one resistance mechanism.

Atomic structures of a bacteriocin targeting Gram-positive bacteria.

Due to envelope differences between Gram-positive and Gram-negative bacteria, engineering precision bactericidal contractile nanomachines requires atomic-level understanding of their structures; however, only those killing Gram-negative bacteria are currently known. Here, we report the atomic structures of an engineered diffocin, a contractile syringe-like molecular machine that kills the Gram-positive bacterium Clostridioides difficile. Captured in one pre-contraction and two post-contraction states, each structure fashions six proteins in the bacteria-targeting baseplate, two proteins in the energy-storing trunk, and a collar linking the sheath with the membrane-penetrating tube. Compared to contractile machines targeting Gram-negative bacteria, major differences reside in the baseplate and contraction magnitude, consistent with target envelope differences. The multifunctional hub-hydrolase protein connects the tube and baseplate and is positioned to degrade peptidoglycan during penetration. The full-length tape measure protein forms a coiled-coil helix bundle homotrimer spanning the entire diffocin. Our study offers mechanical insights and principles for designing potent protein-based precision antibiotics.

Nanomechanics of CCN1-Mediated Staphylococcus aureus Phagocytosis.

Phagocytosis is an essential mechanism of the human immune system where pathogens are eliminated by immune cells. The CCN1 protein plays an important role in the phagocytosis of Staphylococcus aureus by favoring the bridging of the αVß3 integrin to the bacterial peptidoglycan (PG), through mechanical forces that remain unknown. Here, we employ single-molecule experiments to unravel the nanomechanics of the PG-CCN1-αVß3 ternary complex. While CCN1 binds αVß3 integrins with moderate force (∼60 pN), much higher binding strengths (up to ∼800 pN) are observed between CCN1 and PG. Notably, the strength of both CCN1-αVß3 and CCN1-PG bonds is dramatically enhanced by tensile loading, favoring a model in which mechanical stress induces the exposure of cryptic integrin binding sites in CCN1 and multivalent binding between CCN1 lectin sites and monosaccharides along the PG glycan chains.

PGFinder, an Open-Source Software for Peptidoglycomics: The Structural Analysis of Bacterial Peptidoglycan by LC-MS.

Peptidoglycan is a major and essential component of the bacterial cell envelope that confers cell shape and provides protection against internal osmotic pressure. This complex macromolecule is made of glycan strands cross-linked by short peptides, and its structure is continually modified throughout growth via a process referred to as "remodeling." Peptidoglycan remodeling allows cells to grow, adapt to their environment, and release fragments that can act as signaling molecules during host-pathogen interactions. Preparing peptidoglycan samples for structural analysis first requires purification of the peptidoglycan sacculus, followed by its enzymatic digestion into disaccharide peptides (muropeptides). These muropeptides can then be characterized by liquid chromatography coupled mass spectrometry (LC-MS) and used to infer the structure of intact peptidoglycan sacculi. Due to the presence of unusual crosslinks, noncanonical amino acids, and amino sugars, the analysis of peptidoglycan LC-MS datasets cannot be handled by traditional proteomics software. In this chapter, we describe a protocol to perform the analysis of peptidoglycan LC-MS datasets using the open-source software PGFinder. We provide a step-by-step strategy to deconvolute data from various mass spectrometry instruments, generate muropeptide databases, perform a PGFinder search, and process the data output.

Synthesis of a Borrelia burgdorferi-Derived Muropeptide Standard Fragment Library.

The interplay between the human innate immune system and bacterial cell wall components is pivotal in understanding diseases such as Crohn's disease and Lyme arthritis. Lyme disease, caused by Borrelia burgdorferi, is the most prevalent tick-borne illness in the United States, with a substantial number of cases reported annually. While antibiotic treatments are generally effective, approximately 10% of Lyme disease cases develop persistent arthritis, suggesting a dysregulated host immune response. We have previously identified a link between the immunogenic B. burgdorferi peptidoglycan (PG) and Lyme arthritis and showed that this pathogen sheds significant amounts of PG fragments during growth. Here, we synthesize these PG fragments, including ornithine-containing monosaccharides and disaccharides, to mimic the unique composition of Borrelia cell walls, using reproducible and rigorous synthetic methods. This synthetic approach allows for the modular preparation of PG derivatives, providing a diverse library of well-defined fragments. These fragments will serve as valuable tools for investigating the role of PG-mediated innate immune response in Lyme disease and aid in the development of improved diagnostic methods and treatment strategies.

Structural analysis of the peptidoglycan DL-endopeptidase CwlO complexed with its inhibitory protein IseA.

Peptidoglycan DL-endopeptidases locally cleave the peptide stem of peptidoglycan in the bacterial cell wall. This process facilitates bacterial growth and division by loosening the rigid peptidoglycan layer. IseA binds to the active site of multiple DL-endopeptidases and inhibits excessive peptidoglycan degradation that leads to cell lysis. To better understand how IseA inhibits DL-endopeptidase activity, we determined the crystal structure of the peptidoglycan DL-endopeptidase CwlO/IseA complex and compared it with that of the peptidoglycan DL-endopeptidase LytE/IseA complex. Structural analyses showed significant differences between the hydrophobic pocket-binding residues of the DL-endopeptidases (F361 of CwlO and W237 of LytE). Additionally, binding assays showed that the F361 mutation of CwlO to the bulkier hydrophobic residue, tryptophan, increased its binding affinity for IseA, whereas mutation to alanine reduced the affinity. These analyses revealed that the hydrophobic pocket-binding residue of DL-endopeptidases determines IseA-binding affinity and is required for substrate-mimetic inhibition by IseA.

Postbiotics-peptidoglycan, lipoteichoic acid, exopolysaccharides, surface layer protein and pili proteins-Structure, activity in wounds and their delivery systems.

Chronic wound healing is a pressing global public health concern. Abuse and drug resistance of antibiotics are the key problems in the treatment of chronic wounds at present. Postbiotics are a novel promising strategy. Previous studies have reported that postbiotics have a wide range of biological activities including antimicrobial, immunomodulatory, antioxidant and anti-inflammatory abilities. However, several aspects related to these postbiotic activities remain unexplored or poorly known. Therefore, this work aims to outline general aspects and emerging trends in the use of postbiotics for wound healing, such as the production, characterization, biological activities and delivery strategies of postbiotics. In this review, a comprehensive overview of the physiological activities and structures of postbiotic biomolecules that contribute to wound healing is provided, such as peptidoglycan, lipoteichoic acid, bacteriocins, exopolysaccharides, surface layer proteins, pili proteins, and secretory proteins (p40 and p75 proteins). Considering the presence of readily degradable components in postbiotics, potential natural polymer delivery materials and delivery systems are emphasized, followed by the potential applications and commercialization prospects of postbiotics. These findings suggest that the treatment of chronic wounds with postbiotic ingredients will help provide new insights into wound healing and better guidance for the development of postbiotic products.

Interdomain flexibility and putative active site was revealed by crystal structure of MltG from Acinetobacter baumannii.

MltG, positioned within the inner membrane of bacteria, functions as a lytic transglycosylase (LT) essential for integrating into the cell wall by cleaving the newly synthesized glycan strand, emphasizing its critical involvement in bacterial cell wall biosynthesis and remodeling. Current study reported the first structure of MltG family of LT. We have elucidated the structure of MltG from Acinetobacter baumannii (abMltG), a formidable superbug renowned for its remarkable antibiotic resistance. Our structural and biochemical investigations unveiled the presence of a flexible peptidoglycan (PG)-binding domain (PGD) within MltG family, which exists as a monomer in solution. Furthermore, we delineated the putative active site of abMltG via a combination of structural analysis and sequence comparison. This discovery enhances our comprehension of the transglycosylation process mediated by the MltG family, offering insights that could inform the development of novel antibiotics tailored to combat A. baumannii.

Lytic transglycosylase Slt of Pseudomonas aeruginosa as a periplasmic hub protein.

Peptidoglycan is a major constituent of the bacterial cell wall. Its integrity as a polymeric edifice is critical for bacterial survival and, as such, it is a preeminent target for antibiotics. The peptidoglycan is a dynamic crosslinked polymer that undergoes constant biosynthesis and turnover. The soluble lytic transglycosylase (Slt) of Pseudomonas aeruginosa is a periplasmic enzyme involved in this dynamic turnover. Using amber-codon-suppression methodology in live bacteria, we incorporated a fluorescent chromophore into the structure of Slt. Fluorescent microscopy shows that Slt populates the length of the periplasmic space and concentrates at the sites of septation in daughter cells. This concentration persists after separation of the cells. Amber-codon-suppression methodology was also used to incorporate a photoaffinity amino acid for the capture of partner proteins. Mass-spectrometry-based proteomics identified 12 partners for Slt in vivo. These proteomics experiments were complemented with in vitro pulldown analyses. Twenty additional partners were identified. We cloned the genes and purified to homogeneity 22 identified partners. Biophysical characterization confirmed all as bona fide Slt binders. The identities of the protein partners of Slt span disparate periplasmic protein families, inclusive of several proteins known to be present in the divisome. Notable periplasmic partners (KD < 0.5 µM) include PBPs (PBP1a, KD = 0.07 µM; PBP5 = 0.4 µM); other lytic transglycosylases (SltB2, KD = 0.09 µM; RlpA, KD = 0.4 µM); a type VI secretion system effector (Tse5, KD = 0.3 µM); and a regulatory protease for alginate biosynthesis (AlgO, KD < 0.4 µM). In light of the functional breadth of its interactome, Slt is conceptualized as a hub protein within the periplasm.

Potent and specific antibiotic combination therapy against Clostridioides difficile.

Keratinicyclins and keratinimicins are recently discovered glycopeptide antibiotics. Keratinimicins show broad-spectrum activity against Gram-positive bacteria, while keratinicyclins form a new chemotype by virtue of an unusual oxazolidinone moiety and exhibit specific antibiosis against Clostridioides difficile. Here we report the mechanism of action of keratinicyclin B (KCB). We find that steric constraints preclude KCB from binding peptidoglycan termini. Instead, KCB inhibits C. difficile growth by binding wall teichoic acids (WTAs) and interfering with cell wall remodeling. A computational model, guided by biochemical studies, provides an image of the interaction of KCB with C. difficile WTAs and shows that the same H-bonding framework used by glycopeptide antibiotics to bind peptidoglycan termini is used by KCB for interacting with WTAs. Analysis of KCB in combination with vancomycin (VAN) shows highly synergistic and specific antimicrobial activity, and that nanomolar combinations of the two drugs are sufficient for complete growth inhibition of C. difficile, while leaving common commensal strains unaffected.

Disposable Peptidoglycan-Specific Biosensor for Noninvasive Real-Time Detection of Broad-Spectrum Gram-Positive Bacteria in Exhaled Breath Condensates.

Rapidly identifying and quantifying Gram-positive bacteria are crucial to diagnosing and treating bacterial lower respiratory tract infections (LRTIs). This work presents a field-deployable biosensor for detecting Gram-positive bacteria from exhaled breath condensates (EBCs) based on peptidoglycan recognition using an aptamer. Dielectrophoretic force is employed to enrich the bacteria in 10 s without additional equipment or steps. Concurrently, the measurement of the sensor's interfacial capacitance is coupled to quantify the bacteria during the enrichment process. By incorporation of a semiconductor condenser, the whole detection process, including EBC collection, takes about 3 min. This biosensor has a detection limit of 10 CFU/mL, a linear range of up to 105 CFU/mL and a selectivity of 1479:1. It is cost-effective and disposable due to its low cost. The sensor provides a nonstaining, culture-free and PCR-independent solution for noninvasive and real-time diagnosis of Gram-positive bacterial LRTIs.

Split fluorescent protein-mediated multimerization of cell wall binding domain for highly sensitive and selective bacterial detection.

Cell wall peptidoglycan binding domains (CBDs) of cell lytic enzymes, including bacteriocins, autolysins and bacteriophage endolysins, enable highly selective bacterial binding, and thus, have potential as biorecognition molecules for nondestructive bacterial detection. Here, a novel design for a self-complementing split fluorescent protein (FP) complex is proposed, where a multimeric FP chain fused with specific CBDs ((FP-CBD)n) is assembled inside the cell, to improve sensitivity by enhancing the signal generated upon Staphylococcus aureus or Bacillus anthracis binding. Flow cytometry shows enhanced fluorescence on the cell surface with increasing FP stoichiometry and surface plasmon resonance reveals nanomolar binding affinity to isolated peptidoglycan. The breadth of function of these complexes is demonstrated through the use of CBD modularity and the ability to attach enzymatic detection modalities. Horseradish peroxidase-coupled (FP-CBD)n complexes generate a catalytic amplification, with the degree of amplification increasing as a function of FP length, reaching a limit of detection (LOD) of 103 cells/droplet (approximately 0.1 ng S. aureus or B. anthracis) within 15 min on a polystyrene surface. These fusion proteins can be multiplexed for simultaneous detection. Multimeric split FP-CBD fusions enable use as a biorecognition molecule with enhanced signal for use in bacterial biosensing platforms.

Surface-Displayed Mannanolytic and Chitinolytic Enzymes Using Peptidoglycan Binding LysM Domains.

Using Lactiplantibacillus plantarum as a food-grade carrier to create non-GMO whole-cell biocatalysts is gaining popularity. This work evaluates the immobilization yield of a chitosanase (CsnA, 30 kDa) from Bacillus subtilis and a mannanase (ManB, 40 kDa) from B. licheniformis on the surface of L. plantarum WCFS1 using either a single LysM domain derived from the extracellular transglycosylase Lp_3014 or a double LysM domain derived from the muropeptidase Lp_2162. ManB and CsnA were fused with the LysM domains of Lp_3014 or Lp_2162, produced in Escherichia coli and anchored to the cell surface of L. plantarum. The localization of the recombinant proteins on the bacterial cell surface was successfully confirmed by Western blot and flow cytometry analysis. The highest immobilization yields (44-48%) and activities of mannanase and chitosanase on the displaying cell surface (812 and 508 U/g of dry cell weight, respectively) were obtained when using the double LysM domain of Lp_2162 as an anchor. The presence of manno-oligosaccharides or chito-oligosaccharides in the reaction mixtures containing appropriate substrates and ManB or CsnA-displaying cells was determined by high-performance anion exchange chromatography. This study indicated that non-GMO Lactiplantibacillus chitosanase- and mannanase-displaying cells could be used to produce potentially prebiotic oligosaccharides.

Thicket and Mesh: How the Outer Membrane Can Resist Tension Imposed by the Cell Wall.

The cell envelope of Gram-negative bacteria is composed of an outer membrane (OM) and an inner membrane (IM) and a peptidoglycan cell wall (CW) between them. Combined with Braun's lipoprotein (Lpp), which connects the OM and the CW, and numerous membrane proteins that exist in both OM and IM, the cell envelope creates a mechanically stable environment that resists various physical and chemical perturbations to the cell, including turgor pressure caused by the solute concentration difference between the cytoplasm of the cell and the extracellular environment. Previous computational studies have explored how individual components (OM, IM, and CW) can resist turgor pressure although combinations of them have been less well studied. To that end, we constructed multiple OM-CW systems, including the Lpp connections with the CW under increasing degrees of strain. The results show that the OM can effectively resist the tension imposed by the CW, shrinking by only 3-5% in area even when the CW is stretched to 2.5× its relaxed area. The area expansion modulus of the system increases with increasing CW strain, although the OM remains a significant contributor to the envelope's mechanical stability. Additionally, we find that when the protein TolC is embedded in the OM, its stiffness increases.

Probing Peptidoglycan Synthesis in the Gut Commensal Akkermansia Muciniphila with Bioorthogonal Chemical Reporters.

Our gut microbiota directly influences human physiology in health and disease. The myriad of surface glycoconjugates in both the bacterial cell envelope and our gut cells dominate the microbiota-host interface and play a critical role in host response and microbiota homeostasis. Among these, peptidoglycan is the basic glycan polymer offering the cell rigidity and a basis on which many other glycoconjugates are anchored. To directly study peptidoglycan in gut commensals and obtain the molecular insight required to understand their functional activities we need effective techniques like chemical probes to label peptidoglycan in live bacteria. Here we report a chemically guided approach to study peptidoglycan in a key mucin-degrading gut microbiota member of the Verrucomicrobia phylum, Akkermansia muciniphila. Two novel non-toxic tetrazine click-compatible peptidoglycan probes with either a cyclopropene or isonitrile handle allowed for the detection and imaging of peptidoglycan synthesis in this intestinal species.

Saxibacter everestensis gen. nov., sp. nov., A Novel Member of the Family Brevibacteriaceae, Isolated from the North Slope of Mount Everest.

We isolated and analyzed a novel, Gram-stain-positive, aerobic, rod-shaped, non-motile actinobacterium, designated as strain ZFBP1038T, from rock sampled on the north slope of Mount Everest. The growth requirements of this strain were 10-37 °C, pH 4-10, and 0-6% (w/v) NaCl. The sole respiratory quinone was MK-9, and the major fatty acids were anteiso-C15:0 and iso-C17:0. Peptidoglycan containing meso-diaminopimelic acid, ribose, and glucose were the major cell wall sugars, while polar lipids included diphosphatidyl glycerol, phosphatidyl glycerol, an unidentified phospholipid, and an unidentified glycolipid. A phylogenetic analysis based on 16S rRNA gene sequences showed that strain ZFBP1038T has the highest similarity with Spelaeicoccus albus DSM 26341 T (96.02%). ZFBP1038T formed a distinct monophyletic clade within the family Brevibacteriaceae and was distantly related to the genus Spelaeicoccus. The G + C content of strain ZFBP1038T was 63.65 mol% and the genome size was 4.05 Mb. Digital DNA-DNA hybridization, average nucleotide identity, and average amino acid identity values between the genomes of strain ZFBP1038T and representative reference strains were 19.3-25.2, 68.0-71.0, and 52.8-60.1%, respectively. Phylogenetic, phenotypic, and chemotaxonomic characteristics as well as comparative genome analyses suggested that strain ZFBP1038T represents a novel species of a new genus, for which the name Saxibacter gen. nov., sp. nov. was assigned with the type strain Saxibacter everestensis ZFBP1038T (= EE 014 T = GDMCC 1.3024 T = JCM 35335 T).