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Bioconjugate Chemistry Apr 2024The role of the intestinal microbiota in host health is increasingly revealed in its contributions to disease states. The host-microbiome interaction is multifactorial...
The role of the intestinal microbiota in host health is increasingly revealed in its contributions to disease states. The host-microbiome interaction is multifactorial and dynamic. One of the factors that has recently been strongly associated with host physiological responses is peptidoglycan from bacterial cell walls. Peptidoglycan from gut commensal bacteria activates peptidoglycan sensors in human cells, including the nucleotide-binding oligomerization domain-containing protein 2. When present in the gastrointestinal tract, both the polymeric form (sacculi) and depolymerized fragments can modulate host physiology, including checkpoint anticancer therapy efficacy, body temperature and appetite, and postnatal growth. To utilize this growing area of biology toward therapeutic prescriptions, it will be critical to directly analyze a key feature of the host-microbiome interaction from living hosts in a reproducible and noninvasive way. Here we show that metabolically labeled peptidoglycan/sacculi can be readily isolated from fecal samples collected from both mice and humans. Analysis of fecal samples provided a noninvasive route to probe the gut commensal community including the metabolic synchronicity with the host circadian clock. Together, these results pave the way for noninvasive diagnostic tools to interrogate the causal nature of peptidoglycan in host health and disease.
Topics: Humans; Animals; Mice; Peptidoglycan; Gastrointestinal Microbiome; Microbiota; Bacteria
PubMed: 38591251
DOI: 10.1021/acs.bioconjchem.4c00007 -
Research Square Mar 2024Due to envelope differences between Gram-positive and Gram-negative bacteria, engineering precision bactericidal contractile nanomachines requires atomic-level...
Due to envelope differences between Gram-positive and Gram-negative bacteria, engineering precision bactericidal contractile nanomachines requires atomic-level understanding of their structures; however, only those killing a Gram-negative bacterium are currently known. Here, we report the atomic structures of an engineered diffocin, a contractile syringe-like molecular machine that kills the Gram-positive bacterium . Captured in one pre-contraction and two post-contraction states, each structure fashions six proteins in the bacteria-targeting baseplate, two proteins in the energy-storing trunk, and a collar protein linking the sheath with the membrane-penetrating tube. Compared to contractile machines targeting Gram-negative bacteria, major differences reside in the baseplate and contraction magnitude, consistent with differences between their targeted envelopes. The multifunctional hub-hydrolase protein connects the tube and baseplate and is positioned to degrade peptidoglycan during penetration. The full-length tape measure protein forms a coiled-coil helix bundle homotrimer spanning the entire length of the diffocin. Our study offers mechanical insights and principles for designing potent protein-based precision antibiotics.
PubMed: 38586031
DOI: 10.21203/rs.3.rs-4007122/v1 -
Antibiotic's target site affects the potentiation of inhibition and inactivation by electroporation.Frontiers in Microbiology 2024Antibiotic resistance represents a growing global threat, and thus the motivation to develop novel and combined methods of bacterial inactivation is increasing....
INTRODUCTION
Antibiotic resistance represents a growing global threat, and thus the motivation to develop novel and combined methods of bacterial inactivation is increasing. Electroporation is a technique in which electric pulses of sufficient strength are applied to permeabilize cells, including bacteria. Combining antibacterials with electroporation is a promising strategy to potentiate their bactericidal and bacteriostatic effectiveness. This approach has already proved useful for increasing bacterial inactivation, yet most studies so far have mainly focused on the maximal achievable effects, and less on the underlying mechanisms. We recently demonstrated that in the Gram-negative (G-) bacterium electroporation potentiates antibacterials targeting the peptidoglycan wall more than those with intracellular targets. However, in Gram-positive (G+) bacteria, the wall is directly accessible from the outside, and thus the dependence of potentiation on the antibacterial's target may be rather different. Here, we compare the inactivation and growth inhibition of the G+ bacterium for two antibiotics with different modes of action: ampicillin (inhibits cell-wall synthesis) and tetracycline (inhibits intracellular protein synthesis).
METHODS
We used antibiotic concentrations ranging from 0 to 30 × MIC (minimum inhibitory concentration that we predetermined for each antibiotic), a single 1-ms electric pulse with an amplitude from 0 to 20 kV/cm, and post-pulse pre-dilution incubation of 24 h or 1 h.
RESULTS
Electroporation increased the inhibition and inactivation efficiency of both antibiotics, but this was more pronounced for tetracycline, with statistical significance mostly limited to 24-h incubation. In general, both inhibition and inactivation grew stronger with increasing antibiotic concentration and electric field amplitude.
DISCUSSION
Our results indicate that electroporation potentiates inactivation of G+ bacteria to a larger extent for antibiotics that inhibit intracellular processes and require transport into the cytoplasm, and to a smaller extent for antibiotics that inhibit cell-wall synthesis. This is the inverse of the relation observed in G- bacteria, and can be explained by the difference in the envelope structure: in G- bacteria the outer membrane must be breached for wall-inhibiting antibiotics to access their target, whereas in G+ bacteria the wall is inherently accessible from the outside and permeabilization does not affect this access.
PubMed: 38585700
DOI: 10.3389/fmicb.2024.1331714 -
International Journal of Biological... May 2024Natural product bulgecin A potentiates the activity of β-lactam antibiotics by inhibition of three lytic transglycosylases in Pseudomonas aeruginosa, of which MltD is...
Natural product bulgecin A potentiates the activity of β-lactam antibiotics by inhibition of three lytic transglycosylases in Pseudomonas aeruginosa, of which MltD is one. MltD exhibits both endolytic and exolytic reactions in the turnover of the cell-wall peptidoglycan and tolerates the presence or absence of stem peptides in its substrates. The present study reveals structural features of the multimodular MltD, presenting a catalytic module and four cell-wall-binding LysM modules that account for these attributes. Three X-ray structures are reported herein for MltD that disclose one unpredicted LysM module tightly attached to the catalytic domain, whereas the other LysM modules are mobile, and connected to the catalytic domain through long flexible linkers. The formation of crystals depended on the presence of bulgecin A. The expansive active-site cleft is highlighted by the insertion of a helical region, a hallmark of the family 1D of lytic transglycosylases, which was mapped out in a ternary complex of MltD:bulgecinA:chitotetraose, revealing at the minimum the presence of eight subsites (from -4 to +4, with the seat of reaction at subsites -1 and + 1) for binding of sugars of the substrate for the endolytic reaction. The mechanism of the exolytic reaction is revealed in one of the structures, showing how the substrate's terminal anhydro-NAM moiety could be sequestered at subsite +2. Our results provide the structural insight for both the endolytic and exolytic activities of MltD during cell-wall-turnover events.
Topics: Pseudomonas aeruginosa; Catalytic Domain; Models, Molecular; Glycosyltransferases; Biological Products; Crystallography, X-Ray; Bacterial Proteins; Cell Wall; Substrate Specificity
PubMed: 38583835
DOI: 10.1016/j.ijbiomac.2024.131420 -
Cell Reports Apr 2024Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are alarmingly common, and treatment is confined to last-line antibiotics. Vancomycin is the...
Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are alarmingly common, and treatment is confined to last-line antibiotics. Vancomycin is the treatment of choice for MRSA bacteremia, and treatment failure is often associated with vancomycin-intermediate S. aureus isolates. The regulatory 3' UTR of the vigR mRNA contributes to vancomycin tolerance and upregulates the autolysin IsaA. Using MS2-affinity purification coupled with RNA sequencing, we find that the vigR 3' UTR also regulates dapE, a succinyl-diaminopimelate desuccinylase required for lysine and peptidoglycan synthesis, suggesting a broader role in controlling cell wall metabolism and vancomycin tolerance. Deletion of the 3' UTR increased virulence, while the isaA mutant is completely attenuated in a wax moth larvae model. Sequence and structural analyses of vigR indicated that the 3' UTR has expanded through the acquisition of Staphylococcus aureus repeat insertions that contribute sequence for the isaA interaction seed and may functionalize the 3' UTR.
Topics: Animals; 3' Untranslated Regions; Anti-Bacterial Agents; Bacterial Proteins; Base Sequence; Gene Expression Regulation, Bacterial; Methicillin-Resistant Staphylococcus aureus; Moths; Staphylococcal Infections; Staphylococcus aureus; Vancomycin; Virulence
PubMed: 38583155
DOI: 10.1016/j.celrep.2024.114082 -
Cell Reports Apr 2024Mitochondrial dysfunction critically contributes to many major human diseases. The impact of specific gut microbial metabolites on mitochondrial functions of animals and...
Mitochondrial dysfunction critically contributes to many major human diseases. The impact of specific gut microbial metabolites on mitochondrial functions of animals and the underlying mechanisms remain to be uncovered. Here, we report a profound role of bacterial peptidoglycan muropeptides in promoting mitochondrial functions in multiple mammalian models. Muropeptide addition to human intestinal epithelial cells (IECs) leads to increased oxidative respiration and ATP production and decreased oxidative stress. Strikingly, muropeptide treatment recovers mitochondrial structure and functions and inhibits several pathological phenotypes of fibroblast cells derived from patients with mitochondrial disease. In mice, muropeptides accumulate in mitochondria of IECs and promote small intestinal homeostasis and nutrient absorption by modulating energy metabolism. Muropeptides directly bind to ATP synthase, stabilize the complex, and promote its enzymatic activity in vitro, supporting the hypothesis that muropeptides promote mitochondria homeostasis at least in part by acting as ATP synthase agonists. This study reveals a potential treatment for human mitochondrial diseases.
Topics: Animals; Humans; Mitochondria; Oxidative Phosphorylation; Mice; Oxidative Stress; Peptidoglycan; Mice, Inbred C57BL; Adenosine Triphosphate
PubMed: 38583150
DOI: 10.1016/j.celrep.2024.114067 -
ELife Apr 2024An enzyme that remodels the cell wall of helps these gut bacteria to divide and generate peptide fragments that enhance the immune response against cancer.
An enzyme that remodels the cell wall of helps these gut bacteria to divide and generate peptide fragments that enhance the immune response against cancer.
PubMed: 38578679
DOI: 10.7554/eLife.97277 -
Heliyon Apr 2024Probiotics have been applied to a wide range of bacteria, causing gastrointestinal and vaginal infections. However, probiotics generally possess limited antimicrobial...
Probiotics have been applied to a wide range of bacteria, causing gastrointestinal and vaginal infections. However, probiotics generally possess limited antimicrobial spectra and are primarily utilized as dietary supplements. Recognizing the need for more versatile probiotics, this study focuses on isolating and characterizing strains suitable for antibiotic replacement. Among these strains, sp. SNUL2, derived from a traditional fermented food in Korea (i.e., Sikhae), emerged as a promising candidate. The correlation between optical density at 600 nm and colony-forming units was verified and applied in subsequent experiments. To assess the therapeutic potential of probiotics, antibacterial tests were conducted using a microplate reader to evaluate the inhibition of 60 bacterial strains (including common foodborne pathogens) induced by sp. SNUL2 cell-free supernatant (CFS). The results confirmed its broad-spectrum antibacterial properties compared to previously known probiotics. Furthermore, enzymatic treatment with proteinases (trypsin and pepsin) and a time-kill assay were conducted to elucidate the nature of the antibacterial substance in sp. SNUL2 CFS. Through sequential chromatography involving gel filtration and ion-exchange chromatography, specific fractions with enhanced antibacterial properties were identified. LC-MS/MS analysis of the secretome fraction revealed the presence of various proteins from the C39 family, peptidoglycan endopeptidases, and N-acetylmuramoyl-l-alanine amidase domain-containing protein precursors. Hence, the combined action of these proteins may contribute to sp. SNUL2's broad antimicrobial activity.
PubMed: 38576583
DOI: 10.1016/j.heliyon.2024.e28481 -
Current Opinion in Microbiology Jun 2024The governing principles and suites of genes for lateral elongation or incorporation of new cell wall material along the length of a rod-shaped cell are well described.... (Review)
Review
The governing principles and suites of genes for lateral elongation or incorporation of new cell wall material along the length of a rod-shaped cell are well described. In contrast, relatively little is known about unipolar elongation or incorporation of peptidoglycan at one end of the rod. Recent work in three related model systems of unipolar growth (Agrobacterium tumefaciens, Brucella abortus, and Sinorhizobium meliloti) has clearly established that unipolar growth in the Hyphomicrobiales order relies on a set of genes distinct from the canonical elongasome. Polar incorporation of envelope components relies on homologous proteins shared by the Hyphomicrobiales, reviewed here. Ongoing and future work will reveal how unipolar growth is integrated into the alphaproteobacterial cell cycle and coordinated with other processes such as chromosome segregation and cell division.
Topics: Brucella abortus; Sinorhizobium meliloti; Bacterial Proteins; Agrobacterium tumefaciens; Cell Wall; Peptidoglycan; Cell Division
PubMed: 38569420
DOI: 10.1016/j.mib.2024.102470 -
PloS One 2024Outer membrane vesicles (OMVs) are produced by Gram-negative bacteria and deliver microbial molecules to distant target cells in a host. OMVs secreted by probiotic...
Outer membrane vesicles (OMVs) are produced by Gram-negative bacteria and deliver microbial molecules to distant target cells in a host. OMVs secreted by probiotic probiotic strain Escherichia coli Nissle 1917 (EcN) have been reported to induce an immune response. In this study, we aimed to increase the OMV production of EcN. The double gene knockout of mlaE and nlpI was conducted in EcN because the ΔmlaEΔnlpI of experimental strain E. coli K12 showed the highest OMV production in our previous report. The ΔmlaEΔnlpI of EcN showed approximately 8 times higher OMV production compared with the parental (wild-type) strain. Quick-freeze, deep-etch replica electron microscopy revealed that plasmolysis occurred in the elongated ΔmlaEΔnlpI cells and the peptidoglycan (PG) had numerous holes. While these phenomena are similar to the findings for the ΔmlaEΔnlpI of K12, there were more PG holes in the ΔmlaEΔnlpI of EcN than the K12 strain, which were observed not only at the tip of the long axis but also in the whole PG structure. Further analysis clarified that the viability of ΔmlaEΔnlpI of EcN decreased compared with that of the wild-type. Although the amount of PG in ΔmlaEΔnlpI cells was about half of that in wild-type, the components of amino acids in PG did not change in ΔmlaEΔnlpI. Although the viability decreased compared to the wild-type, the ΔmlaEΔnlpI grew in normal culture conditions. The hypervesiculation strain constructed here is expected to be used as an enhanced probiotic strain.
Topics: Escherichia coli; Escherichia coli Proteins; Cell Wall; Probiotics
PubMed: 38564580
DOI: 10.1371/journal.pone.0301613