Gram-positive Bacterial Cell Wall Research Articles

A CTL − Lys immune function maintains insect metamorphosis by preventing gut bacterial dysbiosis and limiting opportunistic infections

BackgroundGut bacteria are beneficial to the host, many of which must be passed on to host offspring. During metamorphosis, the midgut of holometabolous insects undergoes histolysis and remodeling, and thus risks losing gut bacteria. Strategies employed by holometabolous insects to minimize this risk are obscure. How gut bacteria affect host insects after entering the hemocoel and causing opportunistic infections remains largely elusive.ResultsWe used holometabolous Helicoverpa armigera as a model and found low Lactobacillus load, high level of a C-type lectin (CTL) gene CD209 antigen-like protein 2 (CD209) and its downstream lysozyme 1 (Lys1) in the midgut of the wandering stage. CD209 or Lys1 depletion increased the load of midgut Lactobacillus, which further translocate to the hemocoel. In particular, CD209 or Lys1 depletion, injection of Lactobacillus plantarum, or translocation of midgut L. plantarum into the hemocoel suppressed 20-hydroxyecdysone (20E) signaling and delayed pupariation. Injection of L. plantarum decreased triacylglycerol and cholesterol storage, which may result in insufficient energy and 20E available for pupariation. Further, Lysine-type peptidoglycan, the major component of gram-positive bacterial cell wall, contributed to delayed pupariation and decreased levels of triacylglycerols, cholesterols, and 20E, in both H. armigera and Drosophila melanogaster.ConclusionsA mechanism by which (Lactobacillus-induced) opportunistic infections delay insect metamorphosis was found, namely by disturbing the homeostasis of lipid metabolism and reducing 20E production. Moreover, the immune function of CTL − Lys was characterized for insect metamorphosis by maintaining gut homeostasis and limiting the opportunistic infections.

Cryo-EM analysis of S. aureus TarL, a polymerase in wall teichoic acid biogenesis central to virulence and antibiotic resistance.

Wall teichoic acid (WTA), a covalent adduct of Gram-positive bacterial cell wall peptidoglycan, contributes directly to virulence and antibiotic resistance in pathogenic species. Polymerization of the Staphylococcus aureus WTA ribitol-phosphate chain is catalyzed by TarL, a member of the largely uncharacterized TagF-like family of membrane-associated enzymes. We report the cryo-electron microscopy structure of TarL, showing a tetramer that forms an extensive membrane-binding platform of monotopic helices. TarL is composed of an amino-terminal immunoglobulin-like domain and a carboxyl-terminal glycosyltransferase-B domain for ribitol-phosphate polymerization. The active site of the latter is complexed to donor substrate cytidine diphosphate-ribitol, providing mechanistic insights into the catalyzed phosphotransfer reaction. Furthermore, the active site is surrounded by electropositive residues that serve to retain the lipid-linked acceptor for polymerization. Our data advance general insight into the architecture and membrane association of the still poorly characterized monotopic membrane protein class and present molecular details of ribitol-phosphate polymerization that may aid in the design of new antimicrobials.

Lysozyme Peptides as a Novel Nutra-Preservative to Control Some Food Poisoning and Food Spoilage Microorganisms.

Foodborne illnesses and microbial food contamination are crucial concerns and still issues of great worldwide concern. Additionally, the serious health hazards associated with the use of chemical preservatives in food technology. Lysozyme (Lz) is an active protein against Gram-positive bacterial cell wall through its muramidase lytic activity; however, several authors could identify some antimicrobial peptides derived from Lz that have an exaggerated and broad-spectrum antibacterial activity. Therefore, a lysozyme peptides preparation (LzP) is developed to broaden the Lz spectrum. In this work, we investigated the potential efficacy of LzP as a novel Nutra-preservative (food origin) agent against some pathogenic and spoilage bacteria. Our results showed that LzP demonstrated only 11% of the lysozyme lytic activity. However, LzP exhibited strong antibacterial activity against Escherichia coli, Salmonella enteritidis, and Pseudomonas species, while Salmonella typhi and Aeromonas hydrophila exhibited slight resistance. Despite the lowest LzP concentration (0.1%) employed, it performs stronger antibacterial activity than weak organic acids (0.3%). Interestingly, the synergistic multi-component formulation (LzP, glycine, and citric acid) could inhibit 6 log10 cfu/ml of E. coli survival growth. The effect of heat treatment on LzP showed a decrease in its antibacterial activity at 5 and 67% by boiling at 100 °C/30 min, and autoclaving at 121 °C/15 min; respectively. On the other hand, LzP acquired stable antibacterial activity at different pH values (4-7). In conclusion, LzP would be an innovative, natural, and food origin preservative to control the growth of food poisoning and spoilage bacteria in food instead chemical one.

Determination of the Crystal Structure of the Cell Wall-Anchored Proteins and Pilins.

Surface proteins and pili (or pilus) anchored on the Gram-positive bacterial cell wall play a vital role in adhesion, colonization, biofilm formation, and immunomodulation. The pilus consists of building blocks called pilins or pilus subunits. The surface proteins and pilins share some common sequences and structural features. They contain an N-terminal signal sequence and the C-terminal cell wall sorting region, enabling their transportation across the membrane and covalent attachment to the bacterial cell wall, respectively. The transpeptidase enzymes called sortases facilitate the covalent links between the pilins during the pilus assembly and between surface proteins or basal subunits of pili and peptidoglycan-bridge during the cell wall anchoring. Thus, elucidating three-dimensional structures for the surface proteins and pilins at the atomic level is essential for understanding the mechanism of adhesion, pilus assembly, and host interaction. This chapter aims to provide a general protocol for crystal structure determination of surface proteins and pilins anchored on the Gram-positive bacterial cell wall and substrates for sortases. The protocol involves the production of recombinant protein, crystallization, and structure determination by X-ray crystallography technique.

Evaluation of protein extraction methods to improve meta-proteomics analysis of treated wastewater biofilms.

Metaproteomics can be used to study functionally active biofilm-based bacterial populations in reclaimed water distribution systems, which in turn result in bacterial regrowth that impacts the water quality. However, existing protein extraction methods have differences in their protein recovery and have not been evaluated for their efficacies in reclaimed water biofilm samples. In this study, we first evaluated six different protein extraction methods with diverse chemical and physical properties on a mixture of bacterial cell culture. Based on a weighting scores-based evaluation, the extraction protocols in order of decreasing performance are listed as B-PER>RIPA>PreOmics>SDS>AllPrep>Urea. The highest four optimal methods on cell culture were further tested against treated wastewater non-chlorinated and chlorinated effluent biofilms. In terms of protein yield, our findings showed that RIPA performed the best; however, the highest number of proteins were extracted from SDS and PreOmics. Furthermore, SDS and PreOmics worked best to rupture gram-positive and gram-negative bacterial cell walls. Considering the five evaluation factors, PreOmics obtained highest weighted score, indicating its potential effectiveness in extracting proteins from biofilms. This study provides the first insight into evaluating protein extraction methods to facilitate metaproteomics for complex reclaimed water matrices.

Differentiating interactions of antimicrobials with Gram-negative and Gram-positive bacterial cell walls using molecular dynamics simulations.

Developing molecular models to capture the complex physicochemical architecture of the bacterial cell wall and to study the interaction with antibacterial molecules is an important aspect of assessing and developing novel antimicrobial molecules. We carried out molecular dynamics simulations using an atomistic model of peptidoglycan to represent the architecture for Gram-positive S. aureus. The model is developed to capture various structural features of the Staphylococcal cell wall, such as the peptide orientation, area per disaccharide, glycan length distribution, cross-linking, and pore size. A comparison of the cell wall density and electrostatic potentials is made with a previously developed cell wall model of Gram-negative bacteria, E. coli, and properties for both single and multilayered structures of the Staphylococcal cell wall are studied. We investigated the interactions of the antimicrobial peptide melittin with peptidoglycan structures. The depth of melittin binding to peptidoglycan is more pronounced in E. coli than in S. aureus, and consequently, melittin has greater contacts with glycan units of E. coli. Contacts of melittin with the amino acids of peptidoglycan are comparable across both the strains, and the D-Ala residues, which are sites for transpeptidation, show enhanced interactions with melittin. A low energetic barrier is observed for translocation of a naturally occurring antimicrobial thymol with the four-layered peptidoglycan model. The molecular model developed for Gram-positive peptidoglycan allows us to compare and contrast the cell wall penetrating properties with Gram-negative strains and assess for the first time binding and translocation of antimicrobial molecules for Gram-positive cell walls.

Molecular Modeling and Simulation of the Peptidoglycan Layer of Gram-Positive Bacteria Staphylococcus aureus.

The peptidoglycan (PG) layer is a vital component of the bacterial cell wall that protects the cell from rupturing due to internal pressure. Its ubiquity across the bacterial kingdom but not animals has made it the target of drug discovery efforts. The PG layer composed of cross-linked PG strands is porous enough to allow the diffusion of molecules through the PG mesh and into the cell. The lack of an accurate atomistic model of the PG mesh has limited the computational investigations of drug diffusion in Gram-positive bacteria, which lack the outer membrane but consist of a much thicker PG layer compared to Gram-negative bacteria. In this work, we built an atomistic model of the Staphylococcus aureus PG layer architecture with horizontally aligned PG strands and performed molecular dynamics simulations of the diffusion of curcumin molecules through the PG mesh. An accurate model of the Gram-positive bacterial cell wall may aid in developing novel antibiotics to tackle the threat posed by antibiotic resistance.

Mechanisms of Anergic Inflammatory Response in Nasopharyngeal Carcinoma Cells Despite Ubiquitous Constitutive NF-κB Activation.

Commensal microbes cross talk with their colonized mucosa. We show that microbes and their cell wall components induce an inflammatory response in cultured human mucosal cells derived from the nonmalignant nasopharyngeal epithelium (NNE) cells in vitro. NNE cells show significant induction of NF-κB with nuclear shuttling and inflammatory gene response when exposed to Gram-positive bacteria (streptococci) or peptidoglycan (PGN), a component of the Gram-positive bacterial cell wall. This response is abrogated in nasopharyngeal carcinoma (NPC)–derived cell lines. The inflammatory response induced by NF-κB signaling was blocked at two levels in the tumor-derived cells. We found that NF-κB was largely trapped in lipid droplets (LDs) in the cytoplasm of the NPC-derived cells, while the increased expression of lysine-specific histone demethylase 1 (LSD1, a repressive nuclear factor) reduces the response mediated by remaining NF-κB at the promoters responding to inflammatory stimuli. This refractory response in NPC cells might be a consequence of long-term exposure to microbes in vivo during carcinogenic progression. It may contribute to the decreased antitumor immune responses in NPC, among others despite heavy T-helper cell infiltration, and thus facilitate tumor progression.

A conserved signal-peptidase antagonist modulates membrane homeostasis of actinobacterial sortase critical for surface morphogenesis

Most Actinobacteria encode a small transmembrane protein, whose gene lies immediately downstream of the housekeeping sortase coding for a transpeptidase that anchors many extracellular proteins to the Gram-positive bacterial cell wall. Here, we uncover the hitherto unknown function of this class of conserved proteins, which we name SafA, as a topological modulator of sortase in the oral Actinobacterium Actinomyces oris. Genetic deletion of safA induces cleavage and excretion of the otherwise predominantly membrane-bound SrtA in wild-type cells. Strikingly, the safA mutant, although viable, exhibits severe abnormalities in cell morphology, pilus assembly, surface protein localization, and polymicrobial interactions-the phenotypes that are mirrored by srtA depletion. The pleiotropic defect of the safA mutant is rescued by ectopic expression of safA from not only A. oris, but also Corynebacterium diphtheriae or Corynebacterium matruchotii. Importantly, the SrtA N terminus harbors a tripartite-domain feature typical of a bacterial signal peptide, including a cleavage motif AXA, mutations in which prevent SrtA cleavage mediated by the signal peptidase LepB2. Bacterial two-hybrid analysis demonstrates that SafA and SrtA directly interact. This interaction involves a conserved motif FPW within the exoplasmic face of SafA, since mutations of this motif abrogate SafA-SrtA interaction and induce SrtA cleavage and excretion as observed in the safA mutant. Evidently, SafA is a membrane-imbedded antagonist of signal peptidase that safeguards and maintains membrane homeostasis of the housekeeping sortase SrtA, a central player of cell surface assembly.

Direct chemical editing of Gram-positive bacterial cell walls via an enzyme-catalyzed oxidative coupling reaction.

Chemically manipulating bacterial surface structures, a cutting-edge research direction in the biomedical field, predominantly relies on metabolic labeling by now. However, this method may involve daunting precursor synthesis and only labels nascent surface structures. Here, we report a facile and rapid modification strategy based on a tyrosinase-catalyzed oxidative coupling reaction (TyOCR) for bacterial surface engineering. This strategy employs phenol-tagged small molecules and tyrosinase to initiate direct chemical modification of Gram-positive bacterial cell walls with high labeling efficiency, while Gram-negative bacteria are inert to this modification due to the hindrance of an outer membrane. By using the biotin‒avidin system, we further present the selective deposition of various materials, including photosensitizer, magnetic nanoparticle, and horseradish peroxidase, on Gram-positive bacterial surfaces, and realize the purification/isolation/enrichment and naked-eye detection of bacterial strains. This work demonstrates that TyOCR is a promising strategy for engineering live bacterial cells.

Structural and functional analysis of the D-alanyl carrier protein ligase DltA from Staphylococcus aureus Mu50.

D-Alanylation of the teichoic acids of the Gram-positive bacterial cell wall plays crucial roles in bacterial physiology and virulence. Deprivation of D-alanine from the teichoic acids of Staphylococcus aureus impairs biofilm and colony formation, induces autolysis and ultimately renders methicillin-resistant S.aureus highly susceptible to antimicrobial agents and host defense peptides. Hence, the D-alanylation pathway has emerged as a promising antibacterial target against drug-resistant S. aureus. D-Alanylation of teichoic acids is mediated via the action of four proteins encoded by the dlt operon, DltABCD, all four of which are essential for the process. In order to develop novel antimicrobial agents against S. aureus, the D-alanyl carrier protein ligase DltA, which is the first protein in the D-alanylation pathway, was focused on. Here, the crystal structure of DltA from the methicillin-resistant S. aureus strain Mu50 is presented, which reveals the unique molecular details of the catalytic center and the role of the P-loop. Kinetic analysis shows that the enantioselectivity of S.aureus DltA is much higher than that of DltA from other species. In the presence of DltC, the enzymatic activity of DltA is increased by an order of magnitude, suggesting a new exploitable binding pocket. This discovery may pave the way for a new generation of treatments for drug-resistant S. aureus.

Bacterial culture and immunohistochemical detection of bacteria and endotoxin in cats with suppurative cholangitis-cholangiohepatitis syndrome

To characterize the frequency and type of bacterial infection by culture- and immunohistochemical (IHC)-based methods and determine the impact of infection on clinical features and survival time in cats with suppurative cholangitis-cholangiohepatitis syndrome (S-CCHS). 168 client-owned cats with S-CCHS (cases). Clinical features, bacterial culture results, culture-inoculate sources, and survival details were recorded. Cases were subcategorized by comorbidity (extrahepatic bile duct obstruction, cholelithiasis, cholecystitis, ductal plate malformation, biopsy-confirmed inflammatory bowel disease, and biopsy-confirmed pancreatitis) or treatment by cholecystectomy or cholecystoenterostomy. Culture results, bacterial isolates, Gram-stain characteristics, and IHC staining were compared among comorbidities. Lipoteichoic acid IHC staining detected gram-positive bacterial cell wall components, and toll-like receptor expression IHC reflected pathologic endotoxin (gram-negative bacteria) exposure. Clinical features were similar among cases except for more frequent abdominal pain and lethargy in cats with positive culture results and pyrexia, abdominal pain, and hepatomegaly for cats with polymicrobial infections. Bacteria were cultured in 93 of 135 (69%) cats, with common isolates including Enterococcus spp and Escherichia coli. IHC staining was positive in 142 of 151 (94%) cats (lipoteichoic acid, 107/142 [75%]; toll-like receptor 4, 99/142 [70%]). With in-parallel interpretation of culture and IHC-based bacterial detection, 154 of 166 (93%) cats had bacterial infections (gram-positive, 118/154 [77%]; gram-negative, 111/154 [72%]; polymicrobial, 79/154 [51%]). Greater frequency of bacterial isolation occurred with combined tissue, bile, and crushed cholelith inoculates. Infection and gram-positive bacterial isolates were associated with significantly shorter long-term survival times. S-CCHS was associated with bacterial infection, pathologic endotoxin exposure, and frequent polymicrobial infection in cats. Combined tissue inoculates improved culture detection of associated bacteria.

Myeloid liver kinase B1 contributes to lung inflammation induced by lipoteichoic acid but not by viable Streptococcus pneumoniae

BackgroundLiver kinase B1 (Lkb1, gene name Stk11) functions as a tumor suppressor in cancer. Myeloid cell Lkb1 potentiates lung inflammation induced by the Gram-negative bacterial cell wall component lipopolysaccharide and in host defense during Gram-negative pneumonia. Here, we sought to investigate the role of myeloid Lkb1 in lung inflammation elicited by the Gram-positive bacterial cell wall component lipoteichoic acid (LTA) and during pneumonia caused by the Gram-positive respiratory pathogen Streptococcus pneumoniae (Spneu).MethodsAlveolar and bone marrow derived macrophages (AMs, BMDMs) harvested from myeloid-specific Lkb1 deficient (Stk11-ΔM) and littermate control mice were stimulated with LTA or Spneu in vitro. Stk11-ΔM and control mice were challenged via the airways with LTA or infected with Spneu in vivo.ResultsLkb1 deficient AMs and BMDMs produced less tumor necrosis factor (TNF)α upon activation by LTA or Spneu. During LTA-induced lung inflammation, Stk11-ΔM mice had reduced numbers of AMs in the lungs, as well as diminished cytokine release and neutrophil recruitment into the airways. During pneumonia induced by either encapsulated or non-encapsulated Spneu, Stk11-ΔM and control mice had comparable bacterial loads and inflammatory responses in the lung, with the exception of lower TNFα levels in Stk11-ΔM mice after infection with the non-encapsulated strain.ConclusionMyeloid Lkb1 contributes to LTA-induced lung inflammation, but is not important for host defense during pneumococcal pneumonia.

Insight into the molecular basis of substrate recognition by the wall teichoic acid glycosyltransferase TagA

Wall teichoic acid (WTA) polymers are covalently affixed to the Gram-positive bacterial cell wall and have important functions in cell elongation, cell morphology, biofilm formation, and β-lactam antibiotic resistance. The first committed step in WTA biosynthesis is catalyzed by the TagA glycosyltransferase (also called TarA), a peripheral membrane protein that produces the conserved linkage unit, which joins WTA to the cell wall peptidoglycan. TagA contains a conserved GT26 core domain followed by a C-terminal polypeptide tail that is important for catalysis and membrane binding. Here, we report the crystal structure of the Thermoanaerobacter italicus TagA enzyme bound to UDP-N-acetyl-d-mannosamine, revealing the molecular basis of substrate binding. Native MS experiments support the model that only monomeric TagA is enzymatically active and that it is stabilized by membrane binding. Molecular dynamics simulations and enzyme activity measurements indicate that the C-terminal polypeptide tail facilitates catalysis by encapsulating the UDP-N-acetyl-d-mannosamine substrate, presenting three highly conserved arginine residues to the active site that are important for catalysis (R214, R221, and R224). From these data, we present a mechanistic model of catalysis that ascribes functions for these residues. This work could facilitate the development of new antimicrobial compounds that disrupt WTA biosynthesis in pathogenic bacteria.

Biosynthetic Mechanisms and Biological Significance of Glycerol Phosphate-Containing Glycan in Mammals.

Bacteria contain glycerol phosphate (GroP)-containing glycans, which are important constituents of cell-surface glycopolymers such as the teichoic acids of Gram-positive bacterial cell walls. These glycopolymers comprising GroP play crucial roles in bacterial physiology and virulence. Recently, the first identification of a GroP-containing glycan in mammals was reported as a variant form of O-mannosyl glycan on α-dystroglycan (α-DG). However, the biological significance of such GroP modification remains largely unknown. In this review, we provide an overview of this new discovery of GroP-containing glycan in mammals and then outline the recent progress in elucidating the biosynthetic mechanisms of GroP-containing glycans on α-DG. In addition, we discuss the potential biological role of GroP modification along with the challenges and prospects for further research. The progress in this newly identified glycan modification will provide insights into the phylogenetic implications of glycan.

Intrinsic Antibacterial and Conductive Hydrogels Based on the Distinct Bactericidal Effect of Polyaniline for Infected Chronic Wound Healing.

Most chronic wounds suffer from infections, and their treatment is challenging. The usage of antibiotics may lead to bacterial resistance and adverse side effects. Positively charged substances have shown promise, but their applications are usually limited by certain cytotoxicity or complex synthesis. Doped polyaniline that carries a high density of positive charges would be a promising candidate due to its good biocompatibility and easy availability, but its interaction with bacteria has not been elucidated. Herein, the distinct bactericidal effect of polyaniline against Gram-positive bacteria has been verified. The antibacterial activity may result from the specific interaction with lipoteichoic acid to destroy the Gram-positive bacterial cell wall. Polyaniline and a macromolecular dopant (sulfonated hyaluronic acid) are used to construct a flexible hydrogel with skin-mimic electrical conductivity. The in vivo results demonstrate that electrical stimulation (ES) through this hydrogel is superior to ES via separated electrodes (the ES strategy used clinically) for promoting infected chronic wound healing.

Bruton's Tyrosine Kinase-Mediated Signaling in Myeloid Cells Is Required for Protective Innate Immunity During Pneumococcal Pneumonia.

Bruton’s tyrosine kinase (Btk) is a cytoplasmic kinase expressed in B cells and myeloid cells. It is essential for B cell development and natural antibody-mediated host defense against bacteria in humans and mice, but little is known about the role of Btk in innate host defense in vivo. Previous studies have indicated that lack of (natural) antibodies is paramount for impaired host defense against Streptococcus (S.) pneumoniae in patients and mice with a deficiency in functional Btk. In the present study, we re-examined the role of Btk in B cells and myeloid cells during pneumococcal pneumonia and sepsis in mice. The antibacterial defense of Btk-/- mice was severely impaired during pneumococcal pneumosepsis and restoration of natural antibody production in Btk-/- mice by transgenic expression of Btk specifically in B cells did not suffice to protect against infection. Btk-/- mice with reinforced Btk expression in MhcII+ cells, including B cells, dendritic cells and macrophages, showed improved antibacterial defense as compared to Btk-/- mice. Bacterial outgrowth in Lysmcre-Btkfl/Y mice was unaltered despite a reduced capacity of Btk-deficient alveolar macrophages to respond to pneumococci. Mrp8cre-Btkfl/Y mice with a neutrophil specific paucity in Btk expression, however, demonstrated impaired antibacterial defense. Neutrophils of Mrp8cre-Btkfl/Y mice displayed reduced release of granule content after pulmonary installation of lipoteichoic acid, a gram-positive bacterial cell wall component relevant for pneumococci. Moreover, Btk deficient neutrophils showed impaired degranulation and phagocytosis upon incubation with pneumococci ex vivo. Taken together, the results of our study indicate that besides regulating B cell-mediated immunity, Btk is critical for regulation of myeloid cell-mediated, and particularly neutrophil-mediated, innate host defense against S. pneumoniae in vivo.

Novel Lytic Enzyme of Prophage Origin from Clostridium botulinum E3 Strain Alaska E43 with Bactericidal Activity against Clostridial Cells.

Clostridium botulinum is a Gram-positive, anaerobic, spore-forming bacterium capable of producing botulinum toxin and responsible for botulism of humans and animals. Phage-encoded enzymes called endolysins, which can lyse bacteria when exposed externally, have potential as agents to combat bacteria of the genus Clostridium. Bioinformatics analysis revealed in the genomes of several Clostridium species genes encoding putative N-acetylmuramoyl-l-alanine amidases with anti-clostridial potential. One such enzyme, designated as LysB (224-aa), from the prophage of C. botulinum E3 strain Alaska E43 was chosen for further analysis. The recombinant 27,726 Da protein was expressed and purified from E. coli Tuner(DE3) with a yield of 37.5 mg per 1 L of cell culture. Size-exclusion chromatography and analytical ultracentrifugation experiments showed that the protein is dimeric in solution. Bioinformatics analysis and results of site-directed mutagenesis studies imply that five residues, namely H25, Y54, H126, S132, and C134, form the catalytic center of the enzyme. Twelve other residues, namely M13, H43, N47, G48, W49, A50, L73, A75, H76, Q78, N81, and Y182, were predicted to be involved in anchoring the protein to the lipoteichoic acid, a significant component of the Gram-positive bacterial cell wall. The LysB enzyme demonstrated lytic activity against bacteria belonging to the genera Clostridium, Bacillus, Staphylococcus, and Deinococcus, but did not lyse Gram-negative bacteria. Optimal lytic activity of LysB occurred between pH 4.0 and 7.5 in the absence of NaCl. This work presents the first characterization of an endolysin derived from a C. botulinum Group II prophage, which can potentially be used to control this important pathogen.

Novel Biomimetic Human TLR2-Derived Peptides for Potential Targeting of Lipoteichoic Acid: An In Silico Assessment.

Antimicrobial resistance is one of the most significant threats to health and economy around the globe and has been compounded by the emergence of COVID-19, raising important consequences for antimicrobial resistance development. Contrary to conventional targeting approaches, the use of biomimetic application via nanoparticles for enhanced cellular targeting, cell penetration and localized antibiotic delivery has been highlighted as a superior approach to identify novel targeting ligands for combatting antimicrobial resistance. Gram-positive bacterial cell walls contain lipoteichoic acid (LTA), which binds specifically to Toll-like receptor 2 (TLR2) on human macrophages. This phenomenon has the potential to be exploited for the design of biomimetic peptides for antibacterial application. In this study, we have derived peptides from sequences present in human TLR2 that bind to LTA with high affinity. In silico approaches including molecular modelling, molecular docking, molecular dynamics, and thermodynamics have enabled the identification of these crucial binding amino acids, the design of four novel biomimetic TLR2-derived peptides and their LTA binding potential. The outcomes of this study have revealed that one of these novel peptides binds to LTA more strongly and stably than the other three peptides and has the potential to enhance LTA targeting and bacterial cell penetration.

Entropy-driven translocation of disordered proteins through the Gram-positive bacterial cell wall.

In Gram-positive bacteria, a thick cross-linked cell wall separates the membrane from the extracellular space. Some surface-exposed proteins, such as the Listeria monocytogenes actin nucleation-promoting factor ActA, remain associated with the bacterial membrane but somehow thread through tens of nanometres of cell wall to expose their amino terminus to the exterior. Here, we report that entropy enables the translocation of disordered transmembrane proteins through the Gram-positive cell wall. We build a physical model, which predicts that the entropic constraint imposed by a thin periplasm is sufficient to drive the translocation of an intrinsically disordered protein such as ActA across a porous barrier similar to a peptidoglycan cell wall. We experimentally validate our model and show that ActA translocation depends on the cell-envelope dimensions and disordered-protein length, and that translocation is reversible. We also show that disordered regions of eukaryotic proteins can translocate Gram-positive cell walls via entropy. We propose that entropic forces are sufficient to drive the translocation of specific proteins to the outer surface.