Three-Step Enzymatic Remodeling of Chitin into Bioactive Chitooligomers.

Here we describe a complex enzymatic approach to the efficient transformation of abundant waste chitin, a byproduct of the food industry, into valuable chitooligomers with a degree of polymerization (DP) ranging from 6 to 11. This method involves a three-step process: initial hydrolysis of chitin using engineered variants of a novel fungal chitinase from Talaromyces flavus to generate low-DP chitooligomers, followed by an extension to the desired DP using the high-yielding Y445N variant of ß-N-acetylhexosaminidase from Aspergillus oryzae, achieving yields of up to 57%. Subsequently, enzymatic deacetylation of chitooligomers with DP 6 and 7 was accomplished using peptidoglycan deacetylase from Bacillus subtilis BsPdaC. The innovative enzymatic procedure demonstrates a sustainable and feasible route for converting waste chitin into unavailable bioactive chitooligomers potentially applicable as natural pesticides in ecological and sustainable agriculture.

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.

Bacterial peptidoglycan signalling in microglia: Activation by MDP via the NF-κB/MAPK pathway.

Bacterial peptidoglycan (PGN) fragments are commonly studied in the context of bacterial infections. However, PGN fragments recently gained recognition as signalling molecules from the commensal gut microbiota in the healthy host. Here we focus on the minimal bioactive PGN motif muramyl dipeptide (MDP), found in both Gram-positive and Gram-negative commensal bacteria, which signals through the Nod2 receptor. MDP from the gut microbiota translocates to the brain and is associated with changes in neurodevelopment and behaviour, yet there is limited knowledge about the underlying mechanisms. In this study we demonstrate that physiologically relevant doses of MDP induce rapid changes in microglial gene expression and lead to cytokine and chemokine secretion. In immortalised microglial (IMG) cells, C-C Motif Chemokine Ligand 5 (CCL5/RANTES) expression is acutely sensitive to the lowest physiologically prevalent dose (0.1 µg/ml) of MDP. As CCL5 plays an important role in memory formation and synaptic plasticity, microglial CCL5 might be the missing link in elucidating MDP-induced alterations in synaptic gene expression. We observed that a higher physiological dose of MDP elevates the expression of cytokines TNF-α and IL-1ß, indicating a transition toward a pro-inflammatory phenotype in IMG cells, which was validated in primary microglial cultures. Furthermore, MDP induces the translocation of NF-κB subunit p65 into the nucleus, which is blocked by MAPK p38 inhibitor SB202190, suggesting that an interplay of both the NF-κB and MAPK pathways is responsible for the MDP-specific microglial phenotype. These findings underscore the significance of different MDP levels in shaping microglial function in the CNS and indicate MDP as a potential mediator for early inflammatory processes in the brain. It also positions microglia as an important target in the gut microbiota-brain-axis pathway through PGN signalling.

Evidence for archaeal metabolism of D-amino acids in the deep marine sediments.

The deep marine sediments represent a major repository of organic matter whilst hosting a great number of uncultivated microbes. Microbial metabolism plays a key role in the recycling of organic matter in the deep marine sediments. D-amino acids (DAAs) and DAA-containing muropeptides, an important group of organic matter in the deep marine sediments, are primarily derived from bacterial peptidoglycan decomposition. Archaea are abundant in the deep ocean microbiome, yet their role in DAA metabolism remains poorly studied. Here, we report bioinformatic investigation and enzymatic characterization of deep marine sedimentary archaea involved in DAA metabolism. Our analyses suggest that a variety of archaea, particularly the Candidatus Bathyarchaeota and the Candidatus Lokiarchaeaota, can metabolize DAAs. DAAs are converted into L-amino acids via amino acid racemases (Ala racemase, Asp racemase and broad substrate specificity amino acid racemase), and converted into α-keto acid via d-serine ammonia-lyase, whereas DAA-containing di-/tri-muropeptides can be hydrolyzed by peptidases (dipeptidase and D-aminopeptidase). Overall, this study reveals the identity and activity of deep marine sedimentary archaea involved in DAA metabolism, shedding light on the mineralization and biogeochemical cycling of DAAs in the deep marine sediments.

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.

Building the Bacterial Divisome at the Septum.

Across living organisms, division is necessary for cell survival and passing heritable information to the next generation. For this reason, cell division is highly conserved among eukaryotes and prokaryotes. Among the most highly conserved cell division proteins in eukaryotes are tubulin and actin. Tubulin polymerizes to form microtubules, which assemble into cytoskeletal structures in eukaryotes, such as the mitotic spindle that pulls chromatids apart during mitosis. Actin polymerizes to form a morphological framework for the eukaryotic cell, or cytoskeleton, that undergoes reorganization during mitosis. In prokaryotes, two of the most highly conserved cell division proteins are the tubulin homolog FtsZ and the actin homolog FtsA. In this chapter, the functions of the essential bacterial cell division proteins FtsZ and FtsA and their roles in assembly of the divisome at the septum, the site of cell division, will be discussed. In most bacteria, including Escherichia coli, the tubulin homolog FtsZ polymerizes at midcell, and this step is crucial for recruitment of many other proteins to the division site. For this reason, both FtsZ abundance and polymerization are tightly regulated by a variety of proteins. The actin-like FtsA protein polymerizes and tethers FtsZ polymers to the cytoplasmic membrane. Additionally, FtsA interacts with later stage cell division proteins, which are essential for division and for building the new cell wall at the septum. Recent studies have investigated how actin-like polymerization of FtsA on the lipid membrane may impact division, and we will discuss this and other ways that division in bacteria is regulated through FtsZ and FtsA.

Bactericidal effectors of the Stenotrophomonas maltophilia type IV secretion system: functional definition of the nuclease TfdA and structural determination of TfcB.

Stenotrophomonas maltophilia expresses a type IV protein secretion system (T4SS) that promotes contact-dependent killing of other bacteria and does so partly by secreting the effector TfcB. Here, we report the structure of TfcB, comprising an N-terminal domain similar to the catalytic domain of glycosyl hydrolase (GH-19) chitinases and a C-terminal domain for recognition and translocation by the T4SS. Utilizing a two-hybrid assay to measure effector interactions with the T4SS coupling protein VirD4, we documented the existence of five more T4SS substrates. One of these was protein 20845, an annotated nuclease. A S. maltophilia mutant lacking the gene for 20845 was impaired for killing Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Moreover, the cloned 20845 gene conferred robust toxicity, with the recombinant E. coli being rescued when 20845 was co-expressed with its cognate immunity protein. The 20845 effector was an 899 amino-acid protein, comprised of a GHH-nuclease domain in its N-terminus, a large central region of indeterminant function, and a C-terminus for secretion. Engineered variants of the 20845 gene that had mutations in the predicted catalytic site did not impede E. coli, indicating that the antibacterial effect of 20845 involves its nuclease activity. Using flow cytometry with DNA staining, we determined that 20845, but not its mutant variants, confers a loss in DNA content of target bacteria. Database searches revealed that uncharacterized homologs of 20845 occur within a range of bacteria. These data indicate that the S. maltophilia T4SS promotes interbacterial competition through the action of multiple toxic effectors, including a potent, novel DNase.IMPORTANCEStenotrophomonas maltophilia is a multi-drug-resistant, Gram-negative bacterium that is an emerging pathogen of humans. Patients with cystic fibrosis are particularly susceptible to S. maltophilia infection. In hospital water systems and various types of infections, S. maltophilia co-exists with other bacteria, including other pathogens such as Pseudomonas aeruginosa. We previously demonstrated that S. maltophilia has a functional VirB/D4 type VI protein secretion system (T4SS) that promotes contact-dependent killing of other bacteria. Since most work on antibacterial systems involves the type VI secretion system, this observation remains noteworthy. Moreover, S. maltophilia currently stands alone as a model for a human pathogen expressing an antibacterial T4SS. Using biochemical, genetic, and cell biological approaches, we now report both the discovery of a novel antibacterial nuclease (TfdA) and the first structural determination of a bactericidal T4SS effector (TfcB).

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.

Protective effect of melatonin on imidacloprid-induced pyroptosis and ferroptosis by mediating peptidoglycan in the gut of the common carp (Cyprinus carpio).

Imidacloprid (IMI) is a contaminant widespread in surface water, causing serious intestinal damage in the common carp. Melatonin (MT), an endogenous indoleamine hormone, plays a crucial role in mitigating pesticide-induced toxicity. Our previous research has demonstrated that MT effectively reduces the production of intestinal microbial-derived signal peptidoglycan (PGN) induced by IMI, thereby alleviating intestinal tight junction injuries in the common carp. In this study, we performed a transcriptomic analysis to explore the effect of MT on the IMI exposure-induced gut damage of the common carp. The results elucidated that the ferroptosis, mitogen-activated protein kinases (MAPKs), and nucleotide oligomerization domain (NOD)-like signaling pathways were significantly associated with IMI exposure and MT treatment. Meanwhile, the exposure to IMI resulted in the formation of pyroptotic bodies and distinct morphological features of ferroptosis, both mitigated with the addition of MT. Immunofluorescence double staining demonstrated that MT abolished the elevated expression of NOD-like receptor thermal protein domain associated protein 3 (NLRP3) and Gasdermin D (GSDMD) induced by IMI, as well as reduced expression of ferritin heavy chains (FTH) and glutathione peroxidase 4 (GPX4) in gut tissues. Subsequently, we found that the exposure to IMI or PGN enhanced the expression of toll-like receptors (TLR) 2 (a direct recognition receptor of PGN) triggering the P38MAPK signaling pathway, thereby aggravating the process of pyroptosis and ferroptosis of cell models. The addition of MT or SB203580 (a P38MAPK inhibitor) significantly reduced pyroptotic cells, and also decreased iron accumulation. Consequently, these results indicate that MT alleviates IMI-induced pyroptosis and ferroptosis in the gut of the common carp through the PGN/TLR2/P38MAPK pathway.

The role of a novel secretory peptidoglycan recognition protein with antibacterial ability from the Chinese Oak Silkworm Antheraea pernyi in humoral immunity.

Peptidoglycan recognition proteins (PGRPs) are a family of pattern recognition receptors that play a critical role in the immune response of invertebrates and vertebrates. Herein, the short ApPGRP-D gene was cloned from the model lepidopteran Antheraea pernyi. Quantitative PCR (qPCR) confirmed that ApPGRP-D is an immune-related protein and that the expression of ApPGRP-D can be induced by microorganisms. ApPGRP-D is a broad-spectrum pattern recognition protein that activates the prophenoloxidase cascade activation system and promotes the agglutination of microbial cells. Likely due to its amidase activity, ApPGRP-D can inhibit the growth of E. coli and S. aureus. In addition, we demonstrated for the first time that zinc ions, as important metal coenzymes, could promote multiple functions of ApPGRP-D but not its amidase activity.

Cleavage of an engulfment peptidoglycan hydrolase by a sporulation signature protease in Clostridioides difficile.

In the model organism Bacillus subtilis, a signaling protease produced in the forespore, SpoIVB, is essential for the activation of the sigma factor σK, which is produced in the mother cell as an inactive pro-protein, pro-σK. SpoIVB has a second function essential to sporulation, most likely during cortex synthesis. The cortex is composed of peptidoglycan (PG) and is essential for the spore's heat resistance and dormancy. Surprisingly, the genome of the intestinal pathogen Clostridioides difficile, in which σK is produced without a pro-sequence, encodes two SpoIVB paralogs, SpoIVB1 and SpoIVB2. Here, we show that spoIVB1 is dispensable for sporulation, while a spoIVB2 in-frame deletion mutant fails to produce heat-resistant spores. The spoIVB2 mutant enters sporulation, undergoes asymmetric division, and completes engulfment of the forespore by the mother cell but fails to synthesize the spore cortex. We show that SpoIIP, a PG hydrolase and part of the engulfasome, the machinery essential for engulfment, is cleaved by SpoIVB2 into an inactive form. Within the engulfasome, the SpoIIP amidase activity generates the substrates for the SpoIID lytic transglycosylase. Thus, following engulfment completion, the cleavage and inactivation of SpoIIP by SpoIVB2 curtails the engulfasome hydrolytic activity, at a time when synthesis of the spore cortex peptidoglycan begins. SpoIVB2 is also required for normal late gene expression in the forespore by a currently unknown mechanism. Together, these observations suggest a role for SpoIVB2 in coordinating late morphological and gene expression events between the forespore and the mother cell.

Lytic transglycosylase repertoire diversity enables intrinsic antibiotic resistance and daughter cell separation in Escherichia coli under acidic stress.

Peptidoglycan (PG) is an important architectural element that imparts physical toughness and rigidity to the bacterial envelope. It is also a dynamic structure that undergoes continuous turnover or autolysis. Escherichia coli possesses redundant PG degradation enzymes responsible for PG turnover; however, the advantage afforded by the existence of numerous PG degradation enzymes remains incompletely understood. In this study, we elucidated the physiological roles of MltE and MltC, members of the lytic transglycosylase (LTG) family that catalyze the cleavage of glycosidic bonds between disaccharide subunits within PG strands. MltE and MltC are acidic LTGs that exhibit increased enzymatic activity and protein levels under acidic pH conditions, respectively, and deletion of these two LTGs results in a pronounced growth defect at acidic pH. Furthermore, inactivation of these two LTGs induces increased susceptibility at acidic pH against various antibiotics, particularly vancomycin, which seems to be partially caused by elevated membrane permeability. Intriguingly, inactivation of these LTGs induces a chaining morphology, indicative of daughter cell separation defects, only under acidic pH conditions. Simultaneous deletion of PG amidases, known contributors to daughter cell separation, exacerbates the chaining phenotype at acidic pH. This suggests that the two LTGs may participate in the cleavage of glycan strands between daughter cells under acidic pH conditions. Collectively, our findings highlight the role of LTG repertoire diversity in facilitating bacterial survival and antibiotic resistance under stressful conditions.

Utilization of Peptidoglycans from Lactic Acid Bacterial Cell Walls for the Mitigation of Acrylamide and 5-Hydroxymethylfurfural.

Acrylamide (AA) and 5-hydroxymethylfurfural (HMF), which are potentially carcinogenic to humans, are often produced during the hot processing of foods. This study first used a molecular docking model to simulate the binding behavior of four lactic acid bacteria peptidoglycans (PGNs) to AA/HMF, and the binding rate of LAB-based PGNs to AA/HMF was evaluated in vitro. In silico results show that interaction energy is the driving force responsible for the adsorption of LAB-derived PGNs to AA/HMF. In vitro results showed that the PGN of B. lactis B1-04 bound the most AA (28.7%) and HMF (48.0%), followed by L. acidophilus NCFM, B. breve CICC 6079, and L. plantarum CICC 22135. Moreover, an AA/HMF-bound layer on the cell surface of B. lactis B1-04 was observed via AFM and SEM due to adsorption. XPS analysis indicated the removal rate of AA/HMF by selected strains was positively correlated with the proportion of C-O, C=O, and N-H groups of PGNs. The atoms O1, O2, O3, O4, N1, N2, N3, H1, and H2 are involved in the adsorption of LAB-based PGNs to AA/HMF. Thus, the PGNs derived from these four Lactobacillus strains can be regarded as natural adsorbents for the binding of AA/HMF.

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.

SpoIIQ-dependent localization of SpoIIE contributes to septal stability and compartmentalization during the engulfment stage of Bacillus subtilis sporulation.

During spore development in bacteria, a polar septum separates two transcriptionally distinct cellular compartments, the mother cell and the forespore. The conserved serine phosphatase SpoIIE is known for its critical role in the formation of this septum and activation of compartment-specific transcription in the forespore. Signaling between the mother cell and forespore then leads to activation of mother cell transcription and a phagocytic-like process called engulfment, which involves dramatic remodeling of the septum and requires a balance between peptidoglycan synthesis and hydrolysis to ensure septal stability and compartmentalization. Using Bacillus subtilis, we identify an additional role for SpoIIE in maintaining septal stability and compartmentalization at the onset of engulfment. This role for SpoIIE is mediated by SpoIIQ, which anchors SpoIIE in the engulfing membrane. A SpoIIQ mutant (SpoIIQ Y28A) that fails to anchor SpoIIE, results in septal instability and miscompartmentalization during septal peptidoglycan hydrolysis, when other septal stabilization factors are absent. Our data support a model whereby SpoIIE and its interactions with the peptidoglycan synthetic machinery contribute to the stabilization of the asymmetric septum early in engulfment, thereby ensuring compartmentalization during spore development.IMPORTANCEBacterial sporulation is a complex process involving a vast array of proteins. Some of these proteins are absolutely critical and regulate key points in the developmental process. Once such protein is SpoIIE, known for its role in the formation of the polar septum, a hallmark of the early stages of sporulation, and activation of the first sporulation-specific sigma factor, σF, in the developing spore. Interestingly, SpoIIE has been shown to interact with SpoIIQ, an important σF-regulated protein that functions during the engulfment stage. However, the significance of this interaction has remained unclear. Here, we unveil the importance of the SpoIIQ-SpoIIE interaction and identify a role for SpoIIE in the stabilization of the polar septum and maintenance of compartmentalization at the onset of engulfment. In this way, we demonstrate that key sporulation proteins, like SpoIIQ and SpoIIE, function in multiple processes during spore development.

A potential space-making role in cell wall biogenesis for SltB1and DacB revealed by a beta-lactamase induction phenotype in Pseudomonas aeruginosa.

Pseudomonas aeruginosa encodes the beta-lactamase AmpC, which promotes resistance to beta-lactam antibiotics. Expression of ampC is induced by anhydro-muropeptides (AMPs) released from the peptidoglycan (PG) cell wall upon beta-lactam treatment. AmpC can also be induced via genetic inactivation of PG biogenesis factors such as the endopeptidase DacB that cleaves PG crosslinks. Mutants in dacB occur in beta-lactam-resistant clinical isolates of P. aeruginosa, but it has remained unclear why DacB inactivation promotes ampC induction. Similarly, the inactivation of lytic transglycosylase (LT) enzymes such as SltB1 that cut PG glycans has also been associated with ampC induction and beta-lactam resistance. Given that LT enzymes are capable of producing AMP products that serve as ampC inducers, this latter observation has been especially difficult to explain. Here, we show that ampC induction in sltB1 or dacB mutants requires another LT enzyme called MltG. In Escherichia coli, MltG has been implicated in the degradation of nascent PG strands produced upon beta-lactam treatment. Accordingly, in P. aeruginosa sltB1 and dacB mutants, we detected the MltG-dependent production of pentapeptide-containing AMP products that are signatures of nascent PG degradation. Our results therefore support a model in which SltB1 and DacB use their PG-cleaving activity to open space in the PG matrix for the insertion of new material. Thus, their inactivation mimics low-level beta-lactam treatment by reducing the efficiency of new PG insertion into the wall, causing the degradation of some nascent PG material by MltG to produce the ampC-inducing signal. IMPORTANCE: Inducible beta-lactamases like the ampC system of Pseudomonas aeruginosa are a common determinant of beta-lactam resistance among gram-negative bacteria. The regulation of ampC is elegantly tuned to detect defects in cell wall synthesis caused by beta-lactam drugs. Studies of mutations causing ampC induction in the absence of drug therefore promise to reveal new insights into the process of cell wall biogenesis in addition to aiding our understanding of how resistance to beta-lactam antibiotics arises in the clinic. In this study, the ampC induction phenotype for mutants lacking a glycan-cleaving enzyme or an enzyme that cuts cell wall crosslinks was used to uncover a potential role for these enzymes in making space in the wall matrix for the insertion of new material during cell growth.

Enhancing extracellular membrane vesicle productivity of Shewanella vesiculosa HM13, a prospective host for vesiculation-mediated protein secretion, by weakening outer membrane-peptidoglycan linkage.

Shewanella vesiculosa HM13, a psychrotrophic gram-negative bacterium isolated from the intestinal contents of horse mackerel, produces abundant extracellular membrane vesicles (EMVs) by budding the outer membrane. The EMVs of this bacterium carry a single major cargo protein, P49, of unknown function, which may be useful as a carrier for the secretory production of heterologous proteins as cargoes of EMVs. In this study, to increase the utility of S. vesiculosa HM13 as a host for EMV-mediated protein production, we improved its EMV productivity by weakening the linkage between the outer membrane and underlying peptidoglycan layer. In gram-negative bacteria, the outer membrane is connected to peptidoglycans predominantly through Braun's lipoprotein (Lpp), and the formation of this linkage is catalyzed by an l,d-transpeptidase (Ldt). We constructed gene-disrupted mutants of Lpp and Ldt and assessed their EMV productivity. The EMVs of the lpp- and ldt-disrupted mutants grown at 18 °C were evaluated using nanoparticle tracking analysis, and their morphologies were observed using transmission electron microscopy. As a result, an approximately 2.5-fold increase in EMV production was achieved, whereas the morphology of the EMVs of these mutants remained almost identical to that of the parent strain. In accordance with the increase in EMV production, the mutants secreted approximately 2-fold higher amounts of P49 than the parent strain into the culture broth as the EMV cargo. These findings will contribute to the development of an EMV-based secretory production system for heterologous proteins using S. vesiculosa HM13 as a host.

The MraY Inhibitor Muraymycin D2 and Its Derivatives Induce Enlarged Cells in Obligate Intracellular Chlamydia and Wolbachia and Break the Persistence Phenotype in Chlamydia.

Chlamydial infections and diseases caused by filarial nematodes are global health concerns. However, treatment presents challenges due to treatment failures potentially caused by persisting Chlamydia and long regimens against filarial infections accompanied by low compliance. A new treatment strategy could be the targeting of the reduced peptidoglycan structures involved in cell division in the obligate intracellular bacteria Chlamydia and Wolbachia, the latter being obligate endosymbionts supporting filarial development, growth, and survival. Here, cell culture experiments with C. trachomatis and Wolbachia showed that the nucleoside antibiotics muraymycin and carbacaprazamycin interfere with bacterial cell division and induce enlarged, aberrant cells resembling the penicillin-induced persistence phenotype in Chlamydia. Enzymatic inhibition experiments with purified C. pneumoniae MraY revealed that muraymycin derivatives abolish the synthesis of the peptidoglycan precursor lipid I. Comparative in silico analyses of chlamydial and wolbachial MraY with the corresponding well-characterized enzyme in Aquifex aeolicus revealed a high degree of conservation, providing evidence for a similar mode of inhibition. Muraymycin D2 treatment eradicated persisting non-dividing C. trachomatis cells from an established penicillin-induced persistent infection. This finding indicates that nucleoside antibiotics may have additional properties that can break bacterial persistence.

Commensal Bacillus pumilus SE5-Derived Peptidoglycan and Lipoteichoic Acid Showed Synergistic Effects in Improving Growth, Immunity, and Intestinal Health of Grouper (Epinephelus coioides).

Commensal-derived peptidoglycan (PG) or lipoteichoic acid (LTA) can improve the growth, immunity, and intestinal health of fish, but it is not clear whether the two components have synergistic effects. To clarify this, grouper (Epinephelus coioides) was fed basal diet (CG) or diets containing 1.0 × 108 CFU/g heat-inactivated SE5 (HIB), PG (21.30 mg/kg), LTA (6.70 mg/kg), mixture (PL1) of PG (10.65 mg/kg) and LTA (3.35 mg/kg), and mixture (PL2) of PG (21.30 mg/kg) and LTA (6.70 mg/kg). Improved growth performance and feed utilization were observed in groups PG, LTA, PL1, and PL2, and the optimum growth performance was recorded in group PL1. Furthermore, improved serum alkaline phosphatase (AKP) activity and immunoglobulin M (IgM) and complement C3 (C3) contents were observed in all treatments, and the AKP activity in group PL1 was significantly superior to that of groups PG and LTA. Although PG and LTA alone or in combination exert comparable effects on intestinal microbiota and physical structure, obviously enhanced intestinal protease activity was observed in group PL1. The combined efficacy of PL1 could further potentiate the immune response by modulating the nucleotide-binding oligomerization domain-containing protein 2 (NOD2) and upregulating the expression of antimicrobial peptides (epinecidin-1, hepcidin-1, and ß-defensin) as well as IgM. At the same time, group PL1 could further mitigate intestinal inflammation by downregulating pro-inflammatory cytokines and upregulating anti-inflammatory cytokines. In conclusion, probiotic B. pumilus SE5-derived PG and LTA mixture (10.65 mg/kg PG and 3.35 mg/kg LTA) exhibits better potential for improving the growth performance, intestinal health, and immune function compared to another mixture (21.30 mg/kg PG and 6.70 mg/kg LTA) and PG or LTA alone in grouper.

MmpL3, Wag31 and PlrA are involved in coordinating polar growth with peptidoglycan metabolism and nutrient availability.

Cell growth in mycobacteria involves cell wall expansion that is restricted to the cell poles. The DivIVA homolog Wag31 is required for this process, but the molecular mechanism and protein partners of Wag31 have not been described. In this study of Mycobacterium smegmatis, we identify a connection between wag31 and trehalose monomycolate (TMM) transporter mmpl3 in a suppressor screen, and show that Wag31 and polar regulator PlrA are required for MmpL3's polar localization. In addition, the localization of PlrA and MmpL3 are responsive to nutrient and energy deprivation and inhibition of peptidoglycan metabolism. We show that inhibition of MmpL3 causes delocalized cell wall metabolism, but does not delocalize MmpL3 itself. We found that cells with an MmpL3 C-terminal truncation, which is defective for localization, have only minor defects in polar growth, but are impaired in their ability to downregulate cell wall metabolism under stress. Our work suggests that, in addition to its established function in TMM transport, MmpL3 has a second function in regulating global cell wall metabolism in response to stress. Our data are consistent with a model in which the presence of TMMs in the periplasm stimulates polar elongation, and in which the connection between Wag31, PlrA and the C-terminus of MmpL3 is involved in detecting and responding to stress in order to coordinate synthesis of the different layers of the mycobacterial cell wall in changing conditions.