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The Journal of Physical Chemistry. B Mar 2024The cell envelope of Gram-negative bacteria is a crowded tripartite architecture that separates the cell interior from the external environment. Two membranes...
The cell envelope of Gram-negative bacteria is a crowded tripartite architecture that separates the cell interior from the external environment. Two membranes encapsulate the aqueous periplasm, which contains the cell wall. Little is known about the mechanisms via which antimicrobial peptides move through the periplasm from the outer membrane to their site of action, the inner membrane. We utilize all-atom molecular dynamics to study two antimicrobial peptides, polymyxins B1 and E, within models of the periplasm crowded to different extents. In a simple chemical environment, both PMB1 and PME bind irreversibly to the cell wall. The presence of specific macromolecules leads to competition with the polymyxins for cell wall interaction sites, resulting in polymyxin dissociation from the cell wall. Chemical complexity also impacts interactions between polymyxins and Braun's lipoprotein; thus, the interaction modes of lipoprotein antibiotics within the periplasm are dependent upon the nature of the other species present.
Topics: Escherichia coli; Periplasm; Molecular Dynamics Simulation; Lipopeptides; Polymyxins; Anti-Bacterial Agents; Lipoproteins
PubMed: 38457439
DOI: 10.1021/acs.jpcb.3c07985 -
ACS Catalysis Mar 2024Technologies to improve the applicability of artificial metalloenzymes (ArMs) are gaining considerable interest; one such approach is the immobilization of these...
Technologies to improve the applicability of artificial metalloenzymes (ArMs) are gaining considerable interest; one such approach is the immobilization of these biohybrid catalysts on support materials to enhance stability and enable their retention, recovery, and reuse. Here, we describe the immobilization of polyhistidine-tagged ArMs that allow the redox-controlled replacement of catalytic cofactors that have lost activity, e.g., due to poisoning or decomposition, on immobilized metal affinity chromatography resins. By using periplasmic siderophore-binding protein scaffolds that originate from thermophilic bacteria (CeuE and CeuE) in combination with a siderophore-linked imine reduction catalyst, reaction rates were achieved that are about 3.5 times faster than those previously obtained with CeuE, the analogous protein of . Upon immobilization, the CeuE-derived ArM showed a decrease in turnover frequency in the reduction of dehydrosalsolidine by 3.4-fold, while retaining enantioselectivity (36%) and showing improved stability that allowed repeat recovery and recycling cycles. Catalytic activity was preserved over the initial four cycles. In subsequent cycles, a gradual reduction of activity was evident. Once the initial activity decreased to around 40% of the initial activity (23rd recycling cycle), the redox-triggered artificial cofactor release permitted the subsequent recharging of the immobilized protein scaffold with fresh, active cofactor, thereby restoring the initial catalytic activity of the immobilized ArM and allowing its reuse for several more cycles. Furthermore, the ArM could be assembled directly from protein present in crude cell extracts, avoiding time-consuming and costly protein purification steps. Overall, this study demonstrates that the immobilization of redox-reversible ArMs facilitates their "catch-and-release" assembly and disassembly and the recycling of their components, improving their potential commercial viability and environmental footprint.
PubMed: 38449525
DOI: 10.1021/acscatal.3c05294 -
Microbial Physiology 2024C4-dicarboxylates (C4-DC) have emerged as significant growth substrates and signaling molecules for various Enterobacteriaceae during their colonization of mammalian...
INTRODUCTION
C4-dicarboxylates (C4-DC) have emerged as significant growth substrates and signaling molecules for various Enterobacteriaceae during their colonization of mammalian hosts. Particularly noteworthy is the essential role of fumarate respiration during colonization of pathogenic bacteria. To investigate the regulation of aerobic C4-DC metabolism, the study explored the transcriptional control of the main aerobic C4-DC transporter, dctA, under different carbohydrate conditions. In addition, mutants related to carbon catabolite repression (CCR) and C4-DC regulation (DcuS-DcuR) were examined to better understand the regulatory integration of aerobic C4-DC metabolism into CCR. For initial insight into posttranslational regulation, the interaction between the aerobic C4-DC transporter DctA and EIIAGlc from the glucose-specific phosphotransferase system was investigated.
METHODS
The expression of dctA was characterized in the presence of various carbohydrates and regulatory mutants affecting CCR. This was accomplished by fusing the dctA promoter (PdctA) to the lacZ reporter gene. Additionally, the interaction between DctA and EIIAGlc of the glucose-specific phosphotransferase system was examined in vivo using a bacterial two-hybrid system.
RESULTS
The dctA promoter region contains a class I cAMP-CRP-binding site at position -81.5 and a DcuR-binding site at position -105.5. DcuR, the response regulator of the C4-DC-activated DcuS-DcuR two-component system, and cAMP-CRP stimulate dctA expression. The expression of dctA is subject to the influence of various carbohydrates via cAMP-CRP, which differently modulate cAMP levels. Here we show that EIIAGlc of the glucose-specific phosphotransferase system strongly interacts with DctA, potentially resulting in the exclusion of C4-DCs when preferred carbon substrates, such as sugars, are present. In contrast to the classical inducer exclusion known for lactose permease LacY, inhibition of C4-DC uptake into the cytoplasm affects only its role as a substrate, but not as an inducer since DcuS detects C4-DCs in the periplasmic space ("substrate exclusion"). The work shows an interplay between cAMP-CRP and the DcuS-DcuR regulatory system for the regulation of dctA at both transcriptional and posttranslational levels.
CONCLUSION
The study highlights a hierarchical interplay between global (cAMP-CRP) and specific (DcuS-DcuR) regulation of dctA at the transcriptional and posttranslational levels. The integration of global and specific transcriptional regulation of dctA, along with the influence of EIIAGlc on DctA, fine-tunes C4-DC catabolism in response to the availability of other preferred carbon sources. It attributes DctA a central role in the control of aerobic C4-DC catabolism and suggests a new role to EIIAGlc on transporters (control of substrate uptake by substrate exclusion).
Topics: Aerobiosis; Carbon; Catabolite Repression; Cyclic AMP; Cyclic AMP Receptor Protein; Dicarboxylic Acid Transporters; DNA-Binding Proteins; Escherichia coli; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Phosphoenolpyruvate Sugar Phosphotransferase System; Promoter Regions, Genetic; Protein Kinases; Signal Transduction; Succinic Acid; Transcription Factors
PubMed: 38432210
DOI: 10.1159/000538095 -
Biotechnology For Biofuels and... Feb 2024Bacterial cellulose (BC) is a biocompatible material with unique mechanical properties, thus holding a significant industrial potential. Despite many acetic acid...
BACKGROUND
Bacterial cellulose (BC) is a biocompatible material with unique mechanical properties, thus holding a significant industrial potential. Despite many acetic acid bacteria (AAB) being BC overproducers, cost-effective production remains a challenge. The role of pyrroloquinoline quinone (PQQ)-dependent membrane dehydrogenases (mDH) is crucial in the metabolism of AAB since it links substrate incomplete oxidation in the periplasm to energy generation. Specifically, glucose oxidation to gluconic acid substantially lowers environmental pH and hinders BC production. Conversely, ethanol supplementation is known to enhance BC yields in Komagataeibacter spp. by promoting efficient glucose utilization.
RESULTS
K. sucrofermentans ATCC 700178 was engineered, knocking out the four PQQ-mDHs, to assess their impact on BC production. The strain KS003, lacking PQQ-dependent glucose dehydrogenase (PQQ-GDH), did not produce gluconic acid and exhibited a 5.77-fold increase in BC production with glucose as the sole carbon source, and a 2.26-fold increase under optimal ethanol supplementation conditions. In contrast, the strain KS004, deficient in the PQQ-dependent alcohol dehydrogenase (PQQ-ADH), showed no significant change in BC yield in the single carbon source experiment but showed a restrained benefit from ethanol supplementation.
CONCLUSIONS
The results underscore the critical influence of PQQ-GDH and PQQ-ADH and clarify the effect of ethanol supplementation on BC production in K. sucrofermentans ATCC 700178. This study provides a foundation for further metabolic pathway optimization, emphasizing the importance of diauxic ethanol metabolism for high BC production.
PubMed: 38424558
DOI: 10.1186/s13068-024-02482-9 -
PLoS Genetics Feb 2024Peptidoglycan (PG) is a protective sac-like exoskeleton present in most bacterial cell walls. It is a large, covalently crosslinked mesh-like polymer made up of many...
Peptidoglycan (PG) is a protective sac-like exoskeleton present in most bacterial cell walls. It is a large, covalently crosslinked mesh-like polymer made up of many glycan strands cross-bridged to each other by short peptide chains. Because PG forms a continuous mesh around the bacterial cytoplasmic membrane, opening the mesh is critical to generate space for the incorporation of new material during its expansion. In Escherichia coli, the 'space-making activity' is known to be achieved by cleavage of crosslinks between the glycan strands by a set of redundant PG endopeptidases whose absence leads to rapid lysis and cell death. Here, we demonstrate a hitherto unknown role of glycan strand cleavage in cell wall expansion in E. coli. We find that overexpression of a membrane-bound lytic transglycosylase, MltD that cuts the glycan polymers of the PG sacculus rescues the cell lysis caused by the absence of essential crosslink-specific endopeptidases, MepS, MepM and MepH. We find that cellular MltD levels are stringently controlled by two independent regulatory pathways; at the step of post-translational stability by a periplasmic adaptor-protease complex, NlpI-Prc, and post-transcriptionally by RpoS, a stationary-phase specific sigma factor. Further detailed genetic and biochemical analysis implicated a role for MltD in cleaving the nascent uncrosslinked glycan strands generated during the expansion of PG. Overall, our results show that the combined activity of PG endopeptidases and lytic transglycosylases is necessary for successful expansion of the cell wall during growth of a bacterium.
Topics: Escherichia coli; Peptidoglycan; Escherichia coli Proteins; Endopeptidases; Cell Wall; Bacteria; Lipoproteins
PubMed: 38422114
DOI: 10.1371/journal.pgen.1011161 -
BMC Microbiology Feb 2024Carbapenems represent the first line treatment of serious infections caused by drug-resistant Klebsiella pneumoniae. Carbapenem-resistant K. pneumoniae (CRKP) is one of...
BACKGROUND
Carbapenems represent the first line treatment of serious infections caused by drug-resistant Klebsiella pneumoniae. Carbapenem-resistant K. pneumoniae (CRKP) is one of the urgent threats to human health worldwide. The current study aims to evaluate the carbapenemase inhibitory potential of coumarin and to test its ability to restore meropenem activity against CRKP. Disk diffusion method was used to test the antimicrobial susceptibility of K. pneumoniae clinical isolates to various antibiotics. Carbapenemase genes (NDM-1, VIM-2, and OXA-9) were detected using PCR. The effect of sub-MIC of coumarin on CRKP isolates was performed using combined disk assay, enzyme inhibition assay, and checkerboard assay. In addition, qRT-PCR was used to estimate the coumarin effect on expression of carbapenemase genes. Molecular docking was used to confirm the interaction between coumarin and binding sites within three carbapenemases.
RESULTS
K. pneumoniae clinical isolates were found to be multi-drug resistant and showed high resistance to meropenem. All bacterial isolates harbor at least one carbapenemase-encoding gene. Coumarin significantly inhibited carbapenemases in the crude periplasmic extract of CRKP. The checkerboard assay indicated that coumarin-meropenem combination was synergistic exhibiting a fractional inhibitory concentration index ≤ 0.5. In addition, qRT-PCR results revealed that coumarin significantly decreased carbapenemase-genes expression. Molecular docking revealed that the binding energies of coumarin to NDM1, VIM-2, OXA-48 and OXA-9 showed a free binding energy of -7.8757, -7.1532, -6.2064 and - 7.4331 Kcal/mol, respectively.
CONCLUSION
Coumarin rendered CRKP sensitive to meropenem as evidenced by its inhibitory action on hydrolytic activity and expression of carbapenemases. The current findings suggest that coumarin could be a possible solution to overcome carbapenems resistance in CRKP.
Topics: Humans; Meropenem; Klebsiella pneumoniae; Molecular Docking Simulation; Bacterial Proteins; Anti-Bacterial Agents; beta-Lactamases; Carbapenems; Coumarins; Microbial Sensitivity Tests; Klebsiella Infections
PubMed: 38413891
DOI: 10.1186/s12866-024-03214-7 -
Journal of Hazardous Materials May 2024The utility of endophytic bacteria in Cadmium (Cd) remediation has gained significant attention due to their ability to alleviate metal-induced stress and enhance plant...
The utility of endophytic bacteria in Cadmium (Cd) remediation has gained significant attention due to their ability to alleviate metal-induced stress and enhance plant growth. Here, we investigate C. metallidurans CML2, an endophytic bacterial strain prevalent in rice, showing resilience against 2400 mg/L of Cd(II). We conducted an in-depth integrated morphological and transcriptomic analysis illustrating the multifarious mechanisms CML2 employs to combat Cd, including the formation of biofilm and CdO nanoparticles, upregulation of genes involved in periplasmic immobilization, and the utilization of RND efflux pumps to extract excess Cd ions. Beyond Cd, CML2 exhibited robust tolerance to an array of heavy metals, including Mn, Se, Ni, Cu, and Hg, demonstrating effective Cd(II) removal capacity. Furthermore, CML2 has exhibited plant growth-promoting properties through the production of indole-3-acetic acid (IAA) at 0.93 mg/L, soluble phosphorus compounds at 1.11 mg/L, and siderophores at 22.67%. Supportively, pot experiments indicated an increase in root lengths and a decrease in Cd bioaccumulation in rice seedlings inoculated with CML2, consequently reducing Cd translocation rates from 43% to 31%. These findings not only contribute to the understanding of Cd resistance mechanisms in C. metallidurans, but also underscore CML2's promising application in Cd remediation within rice farming ecosystems.
Topics: Cadmium; Oryza; Ecosystem; Biodegradation, Environmental; Metals, Heavy; Cupriavidus; Soil Pollutants; Plant Roots; Soil
PubMed: 38412644
DOI: 10.1016/j.jhazmat.2024.133846 -
Antimicrobial Agents and Chemotherapy Apr 2024Increasing evidence supports the repositioning of beta-lactams for tuberculosis (TB) therapy, but further research on their interaction with conventional anti-TB agents...
Increasing evidence supports the repositioning of beta-lactams for tuberculosis (TB) therapy, but further research on their interaction with conventional anti-TB agents is still warranted. Moreover, the complex cell envelope of () may pose an additional obstacle to beta-lactam diffusion. In this context, we aimed to identify synergies between beta-lactams and anti-TB drugs ethambutol (EMB) and isoniazid (INH) by assessing antimicrobial effects, intracellular activity, and immune responses. Checkerboard assays with H37Rv and eight clinical isolates, including four drug-resistant strains, exposed that only treatments containing EMB and beta-lactams achieved synergistic effects. Meanwhile, the standard EMB and INH association failed to produce any synergy. In -infected THP-1 macrophages, combinations of EMB with increasing meropenem (MEM) concentrations consistently displayed superior killing activities over the individual antibiotics. Flow cytometry with BODIPY FL vancomycin, which binds directly to the peptidoglycan (PG), confirmed an increased exposure of this layer after co-treatment. This was reinforced by the high IL-1β secretion levels found in infected macrophages after incubation with MEM concentrations above 5 mg/L, indicating an exposure of the host innate response sensors to pathogen-associated molecular patterns in the PG. Our findings show that the proposed impaired access of beta-lactams to periplasmic transpeptidases is counteracted by concomitant administration with EMB. The efficiency of this combination may be attributed to the synchronized inhibition of arabinogalactan and PG synthesis, two key cell wall components. Given that beta-lactams exhibit a time-dependent bactericidal activity, a more effective pathogen recognition and killing prompted by this association may be highly beneficial to optimize TB regimens containing carbapenems.IMPORTANCEAddressing drug-resistant tuberculosis with existing therapies is challenging and the treatment success rate is lower when compared to drug-susceptible infection. This study demonstrates that pairing beta-lactams with ethambutol (EMB) significantly improves their efficacy against (). The presence of EMB enhances beta-lactam access through the cell wall, which may translate into a prolonged contact between the drug and its targets at a concentration that effectively kills the pathogen. Importantly, we showed that the effects of the EMB and meropenem (MEM)/clavulanate combination were maintained intracellularly. These results are of high significance considering that the time above the minimum inhibitory concentration is the main determinant of beta-lactam efficacy. Moreover, a correlation was established between incubation with higher MEM concentrations during macrophage infection and increased IL-1β secretion. This finding unveils a previously overlooked aspect of carbapenem repurposing against tuberculosis, as certain strains suppress the secretion of this key pro-inflammatory cytokine to evade host surveillance.
Topics: Humans; Ethambutol; Mycobacterium tuberculosis; Meropenem; Clavulanic Acid; Antitubercular Agents; Tuberculosis, Multidrug-Resistant; Tuberculosis; Carbapenems; beta-Lactams; Microbial Sensitivity Tests
PubMed: 38411952
DOI: 10.1128/aac.01586-23 -
MicrobiologyOpen Apr 2024Prevotella intermedia, a Gram-negative bacterium from the Bacteroidota phylum, is associated with periodontitis. Other species within this phylum are known to possess...
Prevotella intermedia, a Gram-negative bacterium from the Bacteroidota phylum, is associated with periodontitis. Other species within this phylum are known to possess the general O-glycosylation system. The O-glycoproteome has been characterized in several species, including Tannerella forsythia, Porphyromonas gingivalis, and Flavobacterium johnsoniae. In our study, we used electron cryotomography (cryoET) and glycoproteomics to reveal the ultrastructure of P. intermedia and characterize its O-glycoproteome. Our cryoET analysis unveiled the ultrastructural details of the cell envelope and outer membrane vesicles (OMVs) of P. intermedia. We observed an electron-dense surface layer surrounding both cells and OMVs. The OMVs were often large (>200 nm) and presented two types, with lumens being either electron-dense or translucent. LC-MS/MS analyses of P. intermedia fractions led to the identification of 1655 proteins, which included 62 predicted T9SS cargo proteins. Within the glycoproteome, we identified 443 unique O-glycosylation sites within 224 glycoproteins. Interestingly, the O-glycosylation motif exhibited a broader range than reported in other species, with O-glycosylation found at D(S/T)(A/I/L/M/T/V/S/C/G/F/N/E/Q/D/P). We identified a single O-glycan with a delta mass of 1531.48 Da. Its sequence was determined by MS2 and MS3 analyses using both collision-induced dissociation and high-energy collisional dissociation fragmentation modes. After partial deglycosylation with trifluoromethanesulfonic acid, the O-glycan sequence was confirmed to be dHex-dHex-HexNAc (HPO -C H O )-dHex-Hex-HexA-Hex(dHex). Bioinformatic analyses predicted the localization of O-glycoproteins, with 73 periplasmic proteins, 53 inner membrane proteins, 52 lipoproteins, 26 outer membrane proteins, and 14 proteins secreted by the T9SS.
Topics: Glycosylation; Prevotella intermedia; Chromatography, Liquid; Tandem Mass Spectrometry; Glycoproteins; Membrane Proteins; Proteome; Polysaccharides
PubMed: 38409911
DOI: 10.1002/mbo3.1401 -
Applied Microbiology and Biotechnology Feb 2024Pasteurella multocida is an important bacterial pathogen that can cause diseases in both animals and humans. Its elevated morbidity and mortality rates in animals result...
Pasteurella multocida is an important bacterial pathogen that can cause diseases in both animals and humans. Its elevated morbidity and mortality rates in animals result in substantial economic repercussions within the livestock industry. The prevention of diseases caused by P. multocida through immunization is impeded by the absence of a safe and effective vaccine. Outer membrane vesicles (OMVs) secreted from the outer membrane of Gram-negative bacteria are spherical vesicular structures that encompass an array of periplasmic components in conjunction with a diverse assortment of lipids and proteins. These vesicles can induce antibacterial immune responses within the host. P. multocida has been shown to produce OMVs. Nonetheless, the precise characteristics and immunomodulatory functions of P. multocida OMVs have not been fully elucidated. In this study, OMVs were isolated from P. multocida using an ultrafiltration concentration technique, and their morphology, protein constitution, and immunomodulatory properties in RAW264.7 cells were studied. Transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) revealed that the OMVs exhibited typical spherical and bilayered lipid vesicular architecture, exhibiting an average diameter of approximately 147.5 nm. The yield of OMVs was 2.6 × 10 particles/mL. Proteomic analysis revealed a high abundance of membrane-associated proteins within P. multocida OMVs, with the capability to instigate the host's immune response. Furthermore, OMVs stimulated the proliferation and cellular uptake of macrophages and triggered the secretion of cytokines, such as TNF-ɑ, IL-1β, IL-6, IL-10, and TGF-β1. Consequently, our results indicated that OMVs from P. multocida could directly interact with macrophages and regulate their immune function in vitro. These results supported the prospective applicability of P. multocida OMVs as a platform in the context of vaccine development. KEY POINTS: • Preparation and characterization of P. multocida OMVs. • P. multocida OMVs possess a range of antigens and lipoproteins associated with the activation of the immune system. • P. multocida OMVs can activate the proliferation, internalization, and cytokine secretion of macrophages in vitro.
Topics: Animals; Humans; Pasteurella multocida; Prospective Studies; Proteomics; Macrophages; Periplasm
PubMed: 38407600
DOI: 10.1007/s00253-024-13060-2