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Microbiology (Reading, England) May 2024Endolysins are bacteriophage (or phage)-encoded enzymes that catalyse the peptidoglycan breakdown in the bacterial cell wall. The exogenous action of recombinant phage...
Endolysins are bacteriophage (or phage)-encoded enzymes that catalyse the peptidoglycan breakdown in the bacterial cell wall. The exogenous action of recombinant phage endolysins against Gram-positive organisms has been extensively studied. However, the outer membrane acts as a physical barrier when considering the use of recombinant endolysins to combat Gram-negative bacteria. This study aimed to evaluate the antimicrobial activity of the SAR-endolysin LysKpV475 against Gram-negative bacteria as single or combined therapies, using an outer membrane permeabilizer (polymyxin B) and a phage, free or immobilized in a pullulan matrix. In the first step, the endolysin LysKpV475 in solution, alone and combined with polymyxin B, was tested and against ten Gram-negative bacteria, including highly virulent strains and multidrug-resistant isolates. In the second step, the lyophilized LysKpV475 endolysin was combined with the phage phSE-5 and investigated, free or immobilized in a pullulan matrix, against subsp. serovar Typhimurium ATCC 13311. The bacteriostatic action of purified LysKpV475 varied between 8.125 μg ml against ATCC 27853, 16.25 μg ml against . Typhimurium ATCC 13311, and 32.50 μg ml against ATCC BAA-2146 and P2224. LysKpV475 showed bactericidal activity only for ATCC 27853 (32.50 μg ml) and P2307 (65.00 μg ml) at the tested concentrations. The effect of the LysKpV475 combined with polymyxin B increased against ATCC BAA-2146 [fractional inhibitory concentration index (FICI) 0.34; a value lower than 1.0 indicates an additive/combined effect] and . Typhimurium ATCC 13311 (FICI 0.93). A synergistic effect against . Typhimurium was also observed when the lyophilized LysKpV475 at ⅔ MIC was combined with the phage phSE-5 (m.o.i. of 100). The lyophilized LysKpV475 immobilized in a pullulan matrix maintained a significant S reduction of 2 logs after 6 h of treatment. These results demonstrate the potential of SAR-endolysins, alone or in combination with other treatments, in the free form or immobilized in solid matrices, which paves the way for their application in different areas, such as in biocontrol at the food processing stage, biosanitation of food contact surfaces and biopreservation of processed food in active food packing.
Topics: Endopeptidases; Polymyxin B; Anti-Bacterial Agents; Salmonella Phages; Glucans; Animals; Microbial Sensitivity Tests; Gram-Negative Bacteria; Mice; Salmonella typhimurium; Bacteriophages; Viral Proteins
PubMed: 38739436
DOI: 10.1099/mic.0.001462 -
International Journal of Molecular... May 2024C-type lectins in organisms play an important role in the process of innate immunity. In this study, a C-type lectin belonging to the DC-SIGN class of was identified....
C-type lectins in organisms play an important role in the process of innate immunity. In this study, a C-type lectin belonging to the DC-SIGN class of was identified. MsDC-SIGN is classified as a type II transmembrane protein. The extracellular segment of MsDC-SIGN possesses a coiled-coil region and a carbohydrate recognition domain (CRD). The key amino acid motifs of the extracellular CRD of MsDC-SIGN in Ca-binding site 2 were EPN (Glu-Pro-Asn) and WYD (Trp-Tyr-Asp). MsDC-SIGN-CRD can bind to four pathogen-associated molecular patterns (PAMPs), including lipopolysaccharide (LPS), glucan, peptidoglycan (PGN), and mannan. Moreover, it can also bind to Gram-positive, Gram-negative bacteria, and fungi. Its CRD can agglutinate microbes and displays D-mannose and D-galactose binding specificity. MsDC-SIGN was distributed in seven tissues of the largemouth bass, among which the highest expression was observed in the liver, followed by the spleen and intestine. Additionally, MsDC-SIGN was present on the membrane of . leukocytes, thereby augmenting the phagocytic activity against bacteria. In a subsequent investigation, the expression patterns of the MsDC-SIGN gene and key genes associated with the TLR signaling pathway (TLR4, NF-κB, and IL10) exhibited an up-regulated expression response to the stimulation of . Furthermore, through RNA interference of MsDC-SIGN, the expression level of the DC-SIGN signaling pathway-related gene (RAF1) and key genes associated with the TLR signaling pathway (TLR4, NF-κB, and IL10) was decreased. Therefore, MsDC-SIGN plays a pivotal role in the immune defense against . by modulating the TLR signaling pathway.
Topics: Animals; Aeromonas hydrophila; Bass; Cell Adhesion Molecules; Fish Diseases; Fish Proteins; Gram-Negative Bacterial Infections; Immunity, Innate; Lectins, C-Type; Pathogen-Associated Molecular Pattern Molecules; Receptors, Cell Surface; Signal Transduction; Toll-Like Receptors
PubMed: 38732232
DOI: 10.3390/ijms25095013 -
International Journal of Molecular... Apr 2024Insects possess an effective immune system, which has been extensively characterized in several model species, revealing a plethora of conserved genes involved in...
Insects possess an effective immune system, which has been extensively characterized in several model species, revealing a plethora of conserved genes involved in recognition, signaling, and responses to pathogens and parasites. However, some taxonomic groups, characterized by peculiar trophic niches, such as plant-sap feeders, which are often important pests of crops and forestry ecosystems, have been largely overlooked regarding their immune gene repertoire. Here we annotated the immune genes of soft scale insects (Hemiptera: Coccidae) for which omics data are publicly available. By using immune genes of aphids and to query the genome of , as well as the transcriptomes of and sp., we highlight the lack of peptidoglycan recognition proteins, galectins, thaumatins, and antimicrobial peptides in Coccidae. This work contributes to expanding our knowledge about the evolutionary trajectories of immune genes and offers a list of promising candidates for developing new control strategies based on the suppression of pests' immunity through RNAi technologies.
Topics: Animals; Hemiptera; Insect Proteins; Transcriptome; Phylogeny; Antimicrobial Peptides; Galectins; Carrier Proteins
PubMed: 38732132
DOI: 10.3390/ijms25094922 -
The Journal of Biological Chemistry Jun 2024FtsZ, the tubulin homolog essential for bacterial cell division, assembles as the Z-ring at the division site, and directs peptidoglycan synthesis by treadmilling. It is...
FtsZ, the tubulin homolog essential for bacterial cell division, assembles as the Z-ring at the division site, and directs peptidoglycan synthesis by treadmilling. It is unclear how FtsZ achieves kinetic polarity that drives treadmilling. To obtain insights into fundamental features of FtsZ assembly dynamics independent of peptidoglycan synthesis, we carried out structural and biochemical characterization of FtsZ from the cell wall-less bacteria, Spiroplasma melliferum (SmFtsZ). Interestingly the structures of SmFtsZ, bound to GDP and GMPPNP respectively, were captured as domain swapped dimers. SmFtsZ was found to be a slower GTPase with a higher critical concentration (CC) compared to Escherichia coli FtsZ (EcFtsZ). In FtsZs, a conformational switch from R-state (close) to T-state (open) favors polymerization. We identified that Phe224, located at the interdomain cleft of SmFtsZ, is crucial for R- to T-state transition. SmFtsZ exhibited higher GTPase activity and lower CC, whereas the corresponding EcFtsZ resulted in cell division defects in E. coli. Our results demonstrate that relative rotation of the domains is a rate-limiting step of polymerization. Our structural analysis suggests that the rotation is plausibly triggered upon addition of a GTP-bound monomer to the filament through interaction of the preformed N-terminal domain (NTD). Hence, addition of monomers to the NTD-exposed end of filament is slower in comparison to the C-terminal domain (CTD) end, thus explaining kinetic polarity. In summary, the study highlights the importance of interdomain interactions and conformational changes in regulating FtsZ assembly dynamics.
Topics: Cytoskeletal Proteins; Bacterial Proteins; Escherichia coli; Protein Domains; Protein Multimerization; Guanosine Diphosphate; GTP Phosphohydrolases; Cell Division
PubMed: 38718863
DOI: 10.1016/j.jbc.2024.107336 -
Current Opinion in Microbiology Jun 2024Bacteria surround themselves with complex cell envelopes to maintain their integrity and protect against external insults. The envelope of Gram-negative organisms is... (Review)
Review
Bacteria surround themselves with complex cell envelopes to maintain their integrity and protect against external insults. The envelope of Gram-negative organisms is multilayered, with two membranes sandwiching the periplasmic space that contains the peptidoglycan cell wall. Understanding how this complicated surface architecture is assembled during cell growth and division is a major fundamental problem in microbiology. Additionally, because the envelope is an important antibiotic target and determinant of intrinsic antibiotic resistance, understanding the mechanisms governing its assembly is relevant to therapeutic development. In the last several decades, most of the factors required to build the Gram-negative envelope have been identified. However, surprisingly, little is known about how the biogenesis of the different cell surface layers is co-ordinated. Here, we provide an overview of recent work that is beginning to uncover the links connecting the different envelope biosynthetic pathways and assembly machines to ensure uniform envelope growth.
Topics: Gram-Negative Bacteria; Cell Wall; Peptidoglycan; Cell Membrane; Bacterial Proteins
PubMed: 38718542
DOI: 10.1016/j.mib.2024.102479 -
Frontiers in Cellular and Infection... 2024High-fat diets (HFDs), a prevailing daily dietary style worldwide, induce chronic low-grade inflammation in the central nervous system and peripheral tissues, promoting...
High-fat diets (HFDs), a prevailing daily dietary style worldwide, induce chronic low-grade inflammation in the central nervous system and peripheral tissues, promoting a variety of diseases including pathologies associated with neuroinflammation. However, the mechanisms linking HFDs to inflammation are not entirely clear. Here, using a HFD model, we explored the mechanism of HFD-induced inflammation in remote tissues. We found that HFDs activated the IMD/NFκB immune pathway in the head through remodeling of the commensal gut bacteria. Removal of gut microbiota abolished such HFD-induced remote inflammatory response. Further experiments revealed that HFDs significantly increased the abundance of in the gut, and the re-association of this bacterium was sufficient to elicit inflammatory response in remote tissues. Mechanistically, produced a greater amount of peptidoglycan (PGN), a well-defined microbial molecular pattern that enters the circulation and remotely activates an inflammatory response. Our results thus show that HFDs trigger inflammation mediated by a bacterial molecular pattern that elicits host immune response.
Topics: Animals; Acetobacter; Diet, High-Fat; Disease Models, Animal; Drosophila melanogaster; Drosophila Proteins; Gastrointestinal Microbiome; Inflammation; NF-kappa B; Peptidoglycan; Signal Transduction
PubMed: 38716198
DOI: 10.3389/fcimb.2024.1347716 -
Brain, Behavior, and Immunity Jul 2024Metabolites and compounds derived from gut-associated bacteria can modulate numerous physiological processes in the host, including immunity and behavior. Using a model...
Metabolites and compounds derived from gut-associated bacteria can modulate numerous physiological processes in the host, including immunity and behavior. Using a model of oral bacterial infection, we previously demonstrated that gut-derived peptidoglycan (PGN), an essential constituent of the bacterial cell envelope, influences female fruit fly egg-laying behavior by activating the NF-κB cascade in a subset of brain neurons. These findings underscore PGN as a potential mediator of communication between gut bacteria and the brain in Drosophila, prompting further investigation into its impact on all brain cells. Through high-resolution mass spectrometry, we now show that PGN fragments produced by gut bacteria can rapidly reach the central nervous system. In Addition, by employing a combination of whole-genome transcriptome analyses, comprehensive genetic assays, and reporter gene systems, we reveal that gut bacterial infection triggers a PGN dose-dependent NF-κB immune response in perineurial glia, forming the continuous outer cell layer of the blood-brain barrier. Furthermore, we demonstrate that persistent PGN-dependent NF-κB activation in perineurial glial cells correlates with a reduction in lifespan and early neurological decline. Overall, our findings establish gut-derived PGN as a critical mediator of the gut-immune-brain axis in Drosophila.
Topics: Animals; Peptidoglycan; NF-kappa B; Brain; Gastrointestinal Microbiome; Brain-Gut Axis; Female; Drosophila; Neuroglia; Drosophila melanogaster; Neurons; Blood-Brain Barrier; Drosophila Proteins
PubMed: 38710338
DOI: 10.1016/j.bbi.2024.05.009 -
Current Research in Microbial Sciences 2024Plasmids pNP40 and pUC11B encode two prevalent yet divergent conjugation systems, which have been characterized in detail recently. Here, we report the elucidation of...
Plasmids pNP40 and pUC11B encode two prevalent yet divergent conjugation systems, which have been characterized in detail recently. Here, we report the elucidation of the putative adhesins of the pNP40 and pUC11B conjugation systems, encoded by and , respectively. Despite their significant sequence divergence, TraAd and TrsAd represent the most conserved component between the pNP40- and the pUC11B-encoded conjugation systems and share similar peptidoglycan-hydrolase domains. Protein structure prediction using AlphaFold2 highlighted the structural similarities between their predicted domains, as well as the potential homo-dimeric state of both proteins. Expression of the putative surface adhesins resulted in a cell clumping phenotype not only among cells expressing these surface adhesins but also between adhesin-expressing and non-producing cells. Furthermore, mutant derivatives of plasmids pNP40 or pUC11B carrying a mutation in or , respectively, were shown to act as efficient donors provided the corresponding recipient expresses either or , thus demonstrating reciprocal complementarity of these proteins in conjugation systems.
PubMed: 38706493
DOI: 10.1016/j.crmicr.2024.100239 -
The Journal of Biological Chemistry Jun 2024During sporulation, Bacillus subtilis forms an asymmetric septum, dividing the cell into two compartments, a mother cell and a forespore. The site of asymmetric...
During sporulation, Bacillus subtilis forms an asymmetric septum, dividing the cell into two compartments, a mother cell and a forespore. The site of asymmetric septation is linked to the membrane where FtsZ and SpoIIE initiate the formation of the Z-ring and the E-ring, respectively. These rings then serve as a scaffold for the other cell division and peptidoglycan synthesizing proteins needed to build the septum. However, despite decades of research, not enough is known about how the asymmetric septation site is determined. Here, we identified and characterized the interaction between SpoIIE and RefZ. We show that these two proteins transiently colocalize during the early stages of asymmetric septum formation when RefZ localizes primarily from the mother cell side of the septum. We propose that these proteins and their interplay with the spatial organization of the chromosome play a role in controlling asymmetric septum positioning.
Topics: Bacillus subtilis; Bacterial Proteins; Spores, Bacterial; Cell Division; Cytoskeletal Proteins
PubMed: 38705388
DOI: 10.1016/j.jbc.2024.107339 -
Acta Biomaterialia Jun 2024Cascade-reaction containers generating reactive oxygen species (ROS) as an alternative for antibiotic-based strategies for bacterial infection control, require...
Cetyltrimethylammonium-chloride assisted in situ metabolic incorporation of nano-sized ROS-generating cascade-reaction containers in Gram-positive and Gram-negative peptidoglycan layers for the control of bacterially-induced sepsis.
Cascade-reaction containers generating reactive oxygen species (ROS) as an alternative for antibiotic-based strategies for bacterial infection control, require endogenous oxygen-sources and ROS-generation close to or preferably inside target bacteria. Here, this is achieved by cetyltrimethylammonium-chloride (CTAC) assisted in situ metabolic labeling and incorporation of mesoporous SiO-nanoparticles, dual-loaded with glucose-oxidase and FeO-nanoparticles as cascade-reaction containers, inside bacterial cell walls. First, azide-functionalized d-alanine (D-Ala-N) was inserted in cell wall peptidoglycan layers of growing Gram-positive pathogens. In Gram-negatives, this could only be achieved after outer lipid-membrane permeabilization, using a low concentration of CTAC. Low concentrations of CTAC had no adverse effect on in vitro blood clotting or hemolysis nor on the health of mice when blood-injected. Next, dibenzocyclooctyne-polyethylene-glycol modified, SiO-nanoparticles were in situ click-reacted with d-Ala-N in bacterial cell wall peptidoglycan layers. Herewith, a two-step cascade-reaction is facilitated inside bacteria, in which glucose-oxidase generates HO at endogenously-available glucose concentrations, while subsequently FeO-nanoparticles catalyze generation of •OH from the HO generated. Generation of •OH inside bacterial cell walls by dual-loaded mesoporous SiO-nanoparticles yielded more effective in vitro killing of both planktonic Gram-positive and Gram-negative bacteria suspended in 10 % plasma than SiO-nanoparticles solely loaded with glucose-oxidase. Gram-positive or Gram-negative bacterially induced sepsis in mice could be effectively treated by in situ pre-treatment with tail-vein injected CTAC and d-Ala-N, followed by injection of dual-loaded cascade-reaction containers without using antibiotics. This makes in situ metabolic incorporation of cascade-reaction containers as described attractive for further investigation with respect to the control of other types of infections comprising planktonic bacteria. STATEMENT OF SIGNIFICANCE: In situ metabolic-incorporation of cascade-reaction-containers loaded with glucose-oxidase and FeO nanoparticles into bacterial cell-wall peptidoglycan is described, yielding ROS-generation from endogenous glucose, non-antibiotically killing bacteria before ROS inactivates. Hitherto, only Gram-positives could be metabolically-labeled, because Gram-negatives possess two lipid-membranes. The outer membrane impedes direct access to the peptidoglycan. This problem was solved by outer-membrane permeabilization using a quaternary-ammonium compound. Several studies on metabolic-labeling perform crucial labeling steps during bacterial-culturing that in real-life should be part of a treatment. In situ metabolic-incorporation as described, can be applied in well-plates during in vitro experiments or in the body as during in vivo animal experiments. Surprisingly, metabolic-incorporation proceeded unhampered in blood and a murine, bacterially-induced sepsis could be well treated.
Topics: Animals; Peptidoglycan; Reactive Oxygen Species; Sepsis; Mice; Nanoparticles; Silicon Dioxide; Gram-Positive Bacteria; Gram-Negative Bacteria
PubMed: 38702010
DOI: 10.1016/j.actbio.2024.04.045