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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 -
International Journal of Molecular... Feb 2024To delve into the structure-function relationship of transmembrane proteins (TMPs), robust protocols are needed to produce them in a pure, stable, and functional state.... (Review)
Review
To delve into the structure-function relationship of transmembrane proteins (TMPs), robust protocols are needed to produce them in a pure, stable, and functional state. Among all hosts that express heterologous TMPs, has the lowest cost and fastest turnover. However, many of the TMPs expressed in are misfolded. Several strategies have been developed to either direct the foreign TMPs to 's membrane or retain them in a cytosolic soluble form to overcome this deficiency. Here, we summarize protein engineering methods to produce chimera constructs of the desired TMPs fused to either a signal peptide or precursor maltose binding protein (pMBP) to direct the entire construct to the periplasm, therefore depositing the fused TMP in the plasma membrane. We further describe strategies to produce TMPs in soluble form by utilizing N-terminally fused MBP without a signal peptide. Depending on its N- or C-terminus location, a fusion to apolipoprotein AI can either direct the TMP to the membrane or shield the hydrophobic regions of the TMP, maintaining the soluble form. Strategies to produce G-protein-coupled receptors, TMPs of , HIV-1 Vpu, and other TMPs are discussed. This knowledge could increase the scope of TMPs' expression in .
Topics: Escherichia coli; Membrane Proteins; Cell Membrane; Escherichia coli Proteins; Protein Sorting Signals; Maltose-Binding Proteins; Recombinant Fusion Proteins
PubMed: 38397029
DOI: 10.3390/ijms25042354 -
International Journal of Molecular... Feb 2024Currently, viable antibiotics available to mitigate infections caused by drug-resistant Gram-negative bacteria are highly limited. Thanatin, a 21-residue-long...
Currently, viable antibiotics available to mitigate infections caused by drug-resistant Gram-negative bacteria are highly limited. Thanatin, a 21-residue-long insect-derived antimicrobial peptide (AMP), is a promising lead molecule for the potential development of novel antibiotics. Thanatin is extremely potent, particularly against the Enterobacter group of Gram-negative pathogens, e.g., and . As a mode of action, cationic thanatin efficiently permeabilizes the LPS-outer membrane and binds to the periplasmic protein LptA to inhibit outer membrane biogenesis. Here, we have utilized N-terminal truncated 16- and 14-residue peptide fragments of thanatin and investigated structure, activity, and selectivity with correlating modes of action. A designed 16-residue peptide containing D-Lys (dk) named VF16 (V1PIIYCNRRT-dk-KCQRF16) demonstrated killing activity in Gram-negative bacteria. The VF16 peptide did not show any detectable toxicity to the HEK 293T cell line and kidney cell line Hep G2. As a mode of action, VF16 interacted with LPS, permeabilizing the outer membrane and binding to LptA with high affinity. Atomic-resolution structures of VF16 in complex with LPS revealed cationic and aromatic surfaces involved in outer membrane interactions and permeabilization. Further, analyses of an inactive 14-residue native thanatin peptide (IM14: IIYCNRRTGKCQRM) delineated the requirement of the β-sheet structure in activity and target interactions. Taken together, this work would pave the way for the designing of short analogs of thanatin-based antimicrobials.
Topics: Lipopolysaccharides; Antimicrobial Peptides; Escherichia coli; Antimicrobial Cationic Peptides; Anti-Bacterial Agents; Gram-Negative Bacteria; Microbial Sensitivity Tests; Carrier Proteins; Escherichia coli Proteins
PubMed: 38396798
DOI: 10.3390/ijms25042122 -
International Journal of Molecular... Feb 2024Lipopolysaccharides (LPSs) are major components of the outer membranes of Gram-negative bacteria. In this work, the structure of the O-polysaccharide of T1Kr02 was...
Lipopolysaccharides (LPSs) are major components of the outer membranes of Gram-negative bacteria. In this work, the structure of the O-polysaccharide of T1Kr02 was identified by nuclear magnetic resonance (NMR), and the physical-chemical properties and biological activity of LPS were also investigated. The NMR analysis showed that the O-polysaccharide has the following structure: →2)-β-d-Fuc-(1→3)-β-d-Fuc-(1→. The structure of the periplasmic glucan coextracted with LPS was established by NMR spectroscopy and chemical methods: →2)-β-d-Glc-(1→. Non-stoichiometric modifications were identified in both polysaccharides: 50% of d-fucofuranose residues at position 3 were O-acetylated, and 15% of d-Glc residues at position 6 were linked with succinate. This is the first report of a polysaccharide containing both d-fucopyranose and d-fucofuranose residues. The fatty acid analysis of the LPS showed the prevalence of 3-hydroxytetradecanoic, hexadecenoic, octadecenoic, lactobacillic, and 27-hydroxyoctacosanoic acids. The dynamic light scattering demonstrated that LPS (in an aqueous solution) formed supramolecular particles with a size of 72.2 nm and a zeta-potential of -21.5 mV. The LPS solution (10 mkg/mL) promoted the growth of potato microplants under in vitro conditions. Thus, LPS of T1Kr02 can be recommended as a promoter for plants and as a source of biotechnological production of d-fucose.
Topics: Lipopolysaccharides; Fucose; O Antigens; Bacteria; Ochrobactrum
PubMed: 38396650
DOI: 10.3390/ijms25041970 -
Applied Microbiology and Biotechnology Feb 2024Bacterial outer membrane vesicles (OMVs) are nanosized spheroidal particles shed by gram-negative bacteria that contain biomolecules derived from the periplasmic space,... (Review)
Review
Bacterial outer membrane vesicles (OMVs) are nanosized spheroidal particles shed by gram-negative bacteria that contain biomolecules derived from the periplasmic space, the bacterial outer membrane, and possibly other compartments. OMVs can be purified from bacterial culture supernatants, and by genetically manipulating the bacterial cells that produce them, they can be engineered to harbor cargoes and/or display molecules of interest on their surfaces including antigens that are immunogenic in mammals. Since OMV bilayer-embedded components presumably maintain their native structures, OMVs may represent highly useful tools for generating antibodies to bacterial outer membrane targets. OMVs have historically been utilized as vaccines or vaccine constituents. Antibodies that target bacterial surfaces are increasingly being explored as antimicrobial agents either in unmodified form or as targeting moieties for bactericidal compounds. Here, we review the properties of OMVs, their use as immunogens, and their ability to elicit antibody responses against bacterial antigens. We highlight antigens from bacterial pathogens that have been successfully targeted using antibodies derived from OMV-based immunization and describe opportunities and limitations for OMVs as a platform for antimicrobial antibody development. KEY POINTS: • Outer membrane vesicles (OMVs) of gram-negative bacteria bear cell-surface molecules • OMV immunization allows rapid antibody (Ab) isolation to bacterial membrane targets • Review and analysis of OMV-based immunogens for antimicrobial Ab development.
Topics: Animals; Antigens, Bacterial; Bacterial Outer Membrane Proteins; Antibodies; Gram-Negative Bacteria; Anti-Infective Agents; Antibodies, Bacterial; Bacterial Vaccines; Mammals
PubMed: 38396192
DOI: 10.1007/s00253-024-13033-5 -
Marine Drugs Jan 2024Biofilm is accountable for nosocomial infections and chronic illness, making it a serious economic and public health problem. , thanks to its ability to form biofilm and...
Biofilm is accountable for nosocomial infections and chronic illness, making it a serious economic and public health problem. , thanks to its ability to form biofilm and colonize biomaterials, represents the most frequent causative agent involved in biofilm-associated infections of medical devices. Therefore, the research of new molecules able to interfere with biofilm formation has a remarkable interest. In the present work, the attention was focused on sp. TAE6080, an Antarctic marine bacterium able to produce and secrete an effective antibiofilm compound. The molecule responsible for this activity was purified by an activity-guided approach and identified by LC-MS/MS. Results indicated the active protein was a periplasmic protein similar to the PAO1 azurin, named cold-azurin. The cold-azurin was recombinantly produced in and purified. The recombinant protein was able to impair attachment to the polystyrene surface and effectively prevent biofilm formation.
Topics: Pseudomonas; Azurin; Anti-Bacterial Agents; Antarctic Regions; Escherichia coli; Chromatography, Liquid; Tandem Mass Spectrometry; Biofilms; Pseudomonas aeruginosa; Staphylococcus epidermidis
PubMed: 38393032
DOI: 10.3390/md22020061 -
Frontiers in Microbiology 2024The tendency of the periplasmic redox proteins in acidophiles to have more positive redox potentials () than their homologous counterparts in neutrophiles suggests an...
Electrochemical and structural characterization of recombinant respiratory proteins of the acidophilic iron oxidizer sp. PN-J47-F6 suggests adaptations to the acidic pH at protein level.
The tendency of the periplasmic redox proteins in acidophiles to have more positive redox potentials () than their homologous counterparts in neutrophiles suggests an adaptation to acidic pH at protein level, since thermodynamics of electron transfer processes are also affected by acidic pH. Since this conclusion is mainly based on the electrochemical characterization of redox proteins from extreme acidophiles of the genus , we aimed to characterize three recombinant redox proteins of the more moderate acidophile sp. PN-J47-F6. We applied protein film voltammetry and linear sweep voltammetry coupled to UV/Vis spectroscopy to characterize the redox behavior of HiPIP-41, CytC-18, and CytC-78, respectively. The -values of HiPIP-41 (571 ± 16 mV), CytC-18 (276 ± 8 mV, 416 ± 2 mV), and CytC-78 (308 ± 7 mV, 399 ± 7 mV) were indeed more positive than those of homologous redox proteins in neutrophiles. Moreover, our findings suggest that the adaptation of redox proteins with respect to their occurs more gradually in response to the pH, since there are also differences between moderate and more extreme acidophiles. In order to address structure function correlations in these redox proteins with respect to structural features affecting the , we conducted a comparative structural analysis of the -derived redox proteins and homologs of spp. and neutrophilic proteobacteria. Hydrophobic contacts in the redox cofactor binding pockets resulting in a low solvent accessibility appear to be the major factor contributing to the more positive in acidophile-derived redox proteins. While additional cysteines in HiPIPs of acidophiles might increase the effective shielding of the [4Fe-4S]-cofactor, the tight shielding of the heme centers in acidophile-derived cytochromes is achieved by a drastic increase in hydrophobic contacts (. Cyc), and by a larger fraction of aromatic residues in the binding pockets (CytC-18, CytC-78).
PubMed: 38384274
DOI: 10.3389/fmicb.2024.1357152 -
Molecular Biology of the Cell Apr 2024The bacterial cell wall is a meshwork of crosslinked peptidoglycan strands, with a thickness of up to 50 nm in Firmicutes. Little is known about how proteins move...
The bacterial cell wall is a meshwork of crosslinked peptidoglycan strands, with a thickness of up to 50 nm in Firmicutes. Little is known about how proteins move through the cell wall to find sites of enzymatic activity. Cell wall synthesis for cell elongation involves the integration of new peptidoglycan strands by integral membrane proteins, as well as the degradation of existing strands by so-called autolysins, soluble proteins that are secreted through the cell membrane. Autolysins comprise different classes of proteases and glucanases and mostly contain cell-wall binding domains in addition to their catalytic domain. We have studied dynamics of autolysins LytC, a major endopeptidase required for lateral cell wall growth, and LytF, a peptidase acting at the newly formed division site in order to achieve separation of daughter cells. We show that both proteins, fused to moxVenus are present as three distinct populations of different diffusion constants. The fastest population is compatible with free diffusion in a crowded liquid environment, that is similar to that of cytosolic enzymes, likely reflecting autolysins diffusing through the periplasm. The medium mobile fraction can be explained by constrained motion through a polymeric substance, indicating mobility of autolysins through the wall similar to that of DNA-binding proteins within the nucleoid. The slow-mobile fraction are most likely autolysins bound to their specific substrate sites. We show that LytF is more static during exponential phase, while LytC appears to be more active during the transition to stationary phase. Both autolysins became more static in backgrounds lacking redundant other autolysins, suggesting stochastic competition for binding sites. On the other hand, lack of inhibitor IseA or autolysin CwlS lead to an altered preference for polar localization of LytF within the cell wall, revealing that inhibitors and autolysins also affect each other's pattern of localization, in addition to their activity.
Topics: N-Acetylmuramoyl-L-alanine Amidase; Carrier Proteins; Bacillus subtilis; Peptidoglycan; Cell Wall; Endopeptidases; Bacterial Proteins
PubMed: 38381561
DOI: 10.1091/mbc.E23-10-0387 -
MicroLife 2024Pyoverdin is a water-soluble metal-chelator synthesized by members of the genus and used for the acquisition of insoluble ferric iron. Although freely diffusible in...
Pyoverdin is a water-soluble metal-chelator synthesized by members of the genus and used for the acquisition of insoluble ferric iron. Although freely diffusible in aqueous environments, preferential dissemination of pyoverdin among adjacent cells, fine-tuning of intracellular siderophore concentrations, and fitness advantages to pyoverdin-producing versus nonproducing cells, indicate control of location and release. Here, using time-lapse fluorescence microscopy to track single cells in growing microcolonies of SBW25, we show accumulation of pyoverdin at cell poles. Accumulation occurs on cessation of cell growth, is achieved by cross-feeding in pyoverdin-nonproducing mutants and is reversible. Moreover, accumulation coincides with localization of a fluorescent periplasmic reporter, suggesting that pyoverdin accumulation at cell poles is part of the general cellular response to starvation. Compatible with this conclusion is absence of non-accumulating phenotypes in a range of pyoverdin mutants. Analysis of the performance of pyoverdin-producing and nonproducing cells under conditions promoting polar accumulation shows an advantage to accumulation on resumption of growth after stress. Examination of pyoverdin polar accumulation in a multispecies community and in a range of laboratory and natural species of , including PAO1 and KT2440, confirms that the phenotype is characteristic of .
PubMed: 38370141
DOI: 10.1093/femsml/uqae001