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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 May 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.
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 -
Microbiology Spectrum Jun 2024Antimicrobial resistance poses a significant global threat, reaching dangerously high levels as reported by the World Health Organization. The emergence and rapid spread...
Antimicrobial resistance poses a significant global threat, reaching dangerously high levels as reported by the World Health Organization. The emergence and rapid spread of new resistance mechanisms, coupled with the absence of effective treatments in recent decades, have led to thousands of deaths annually from infections caused by drug-resistant microorganisms. Consequently, there is an urgent need for the development of new compounds capable of combating antibiotic-resistant bacteria. A promising class of molecules exhibiting potent bactericidal effects is peptidoglycan hydrolases. Previously, we cloned and characterized the biochemical properties of the M23 catalytic domain of the EnpA (EnpA) protein from . Unlike other enzymes within the M23 family, EnpA demonstrates broad specificity. However, its activity is constrained under low ionic strength conditions. In this study, we present the engineering of three chimeric enzymes comprising EnpA fused with three distinct SH3b cell wall-binding domains. These chimeras exhibit enhanced tolerance to environmental conditions and sustained activity in bovine and human serum. Furthermore, our findings demonstrate that the addition of SH3b domains influences the activity of the chimeric enzymes, thereby expanding their potential applications in combating antimicrobial resistance.IMPORTANCEThese studies demonstrate that the addition of the SH3b-binding domain to the EnpA results in generation of chimeras with a broader tolerance to ionic strength and pH values, enabling them to remain active over a wider range of conditions. Such approach offers a relatively straightforward method for obtaining antibacterial enzymes with tailored properties and emphasizes the potential for proteins' engineering with enhanced functionality, contributing to the ongoing efforts to address antimicrobial resistance effectively.
Topics: Osmolar Concentration; Enterococcus faecalis; Humans; Bacterial Proteins; Protein Engineering; Anti-Bacterial Agents; Animals; N-Acetylmuramoyl-L-alanine Amidase; Cattle; Recombinant Fusion Proteins; Cell Wall; Catalytic Domain; Drug Resistance, Bacterial
PubMed: 38695664
DOI: 10.1128/spectrum.03546-23 -
Antimicrobial Agents and Chemotherapy May 2024The dual β-lactam approach has been successfully applied to overcome target redundancy in nontuberculous mycobacteria. Surprisingly, this approach has not been...
The dual β-lactam approach has been successfully applied to overcome target redundancy in nontuberculous mycobacteria. Surprisingly, this approach has not been leveraged for despite the high conservation of peptidoglycan synthesis. Through a comprehensive screen of oral β-lactam pairs, we have discovered that cefuroxime strongly potentiates the bactericidal activity of tebipenem and sulopenem-advanced clinical candidates-and amoxicillin, at concentrations achieved clinically. β-lactam pairs thus have the potential to reduce TB treatment duration.
PubMed: 38690896
DOI: 10.1128/aac.00034-24 -
PLoS Biology Apr 2024Peptidoglycan (PG) and most surface glycopolymers and their modifications are built in the cytoplasm on the lipid carrier undecaprenyl phosphate (UndP). These...
Peptidoglycan (PG) and most surface glycopolymers and their modifications are built in the cytoplasm on the lipid carrier undecaprenyl phosphate (UndP). These lipid-linked precursors are then flipped across the membrane and polymerized or directly transferred to surface polymers, lipids, or proteins. Despite its essential role in envelope biogenesis, UndP is maintained at low levels in the cytoplasmic membrane. The mechanisms by which bacteria distribute this limited resource among competing pathways is currently unknown. Here, we report that the Bacillus subtilis transcription factor SigM and its membrane-anchored anti-sigma factor respond to UndP levels and prioritize its use for the synthesis of the only essential surface polymer, the cell wall. Antibiotics that target virtually every step in PG synthesis activate SigM-directed gene expression, confounding identification of the signal and the logic of this stress-response pathway. Through systematic analyses, we discovered 2 distinct responses to these antibiotics. Drugs that trap UndP, UndP-linked intermediates, or precursors trigger SigM release from the membrane in <2 min, rapidly activating transcription. By contrasts, antibiotics that inhibited cell wall synthesis without directly affecting UndP induce SigM more slowly. We show that activation in the latter case can be explained by the accumulation of UndP-linked wall teichoic acid precursors that cannot be transferred to the PG due to the block in its synthesis. Furthermore, we report that reduction in UndP synthesis rapidly induces SigM, while increasing UndP production can dampen the SigM response. Finally, we show that SigM becomes essential for viability when the availability of UndP is restricted. Altogether, our data support a model in which the SigM pathway functions to homeostatically control UndP usage. When UndP levels are sufficiently high, the anti-sigma factor complex holds SigM inactive. When levels of UndP are reduced, SigM activates genes that increase flux through the PG synthesis pathway, boost UndP recycling, and liberate the lipid carrier from nonessential surface polymer pathways. Analogous homeostatic pathways that prioritize UndP usage are likely to be common in bacteria.
Topics: Cell Wall; Bacillus subtilis; Signal Transduction; Bacterial Proteins; Peptidoglycan; Polyisoprenyl Phosphates; Anti-Bacterial Agents; Gene Expression Regulation, Bacterial; Cell Membrane
PubMed: 38683856
DOI: 10.1371/journal.pbio.3002589 -
The Journal of Biological Chemistry Apr 2024The biphasic assembly of Gram-positive pili begins with the covalent polymerization of distinct pilins catalyzed by a pilus-specific sortase, followed by the cell wall...
The biphasic assembly of Gram-positive pili begins with the covalent polymerization of distinct pilins catalyzed by a pilus-specific sortase, followed by the cell wall anchoring of the resulting polymers mediated by the housekeeping sortase. In Actinomyces oris, the pilus-specific sortase SrtC2 not only polymerizes FimA pilins to assemble type 2 fimbriae with CafA at the tip, but it can also act as the anchoring sortase, linking both FimA polymers and SrtC1-catalyzed FimP polymers (type 1 fimbriae) to peptidoglycan when the housekeeping sortase SrtA is inactive. To date, the structure-function determinants governing the unique substrate specificity and dual enzymatic activity of SrtC2 have not been illuminated. Here, we present the crystal structure of SrtC2 solved to 2.10-Å resolution. SrtC2 harbors a canonical sortase fold and a lid typical for class C sortases and additional features specific to SrtC2. Structural, biochemical, and mutational analyses of SrtC2 reveal that the extended lid of SrtC2 modulates its dual activity. Specifically, we demonstrate that the polymerizing activity of SrtC2 is still maintained by alanine-substitution, partial deletion, and replacement of the SrtC2 lid with the SrtC1 lid. Strikingly, pilus incorporation of CafA is significantly reduced by these mutations, leading to compromised polymicrobial interactions mediated by CafA. In a srtA mutant, the partial deletion of the SrtC2 lid reduces surface anchoring of FimP polymers, and the lid-swapping mutation enhances this process, while both mutations diminish surface anchoring of FimA pili. Evidently, the extended lid of SrtC2 enables the enzyme the cell wall-anchoring activity in a substrate-selective fashion.
PubMed: 38679328
DOI: 10.1016/j.jbc.2024.107329