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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 -
BioRxiv : the Preprint Server For... Jul 2023FliL is an essential component of the flagellar machinery in some bacteria, but a conditional one in others. The conditional role is for optimal swarming in some...
FliL is an essential component of the flagellar machinery in some bacteria, but a conditional one in others. The conditional role is for optimal swarming in some bacteria. During swarming, physical forces associated with movement on a surface are expected to exert a higher load on the flagellum, requiring more motor torque to move. Bacterial physiology and morphology are also altered during swarming to cope with the challenges of surface navigation. FliL was reported to enhance motor output in several bacteria and observed to assemble as a ring around ion-conducting stators that power the motor. In this study we identify a common new function for FliL in diverse bacteria - and . During swarming, all these bacteria show increased cell speed and a skewed motor bias that suppresses cell tumbling. We demonstrate that these altered motor parameters, or 'motor remodeling', require FliL. Both swarming and motor remodeling can be restored in an mutant by complementation with genes from and , showing conservation of swarming-associated FliL function across phyla. In addition, we demonstrate that the strong interaction we reported earlier between FliL and the flagellar MS-ring protein FliF is confined to the RBM-3 domain of FliF that links the periplasmic rod to the cytoplasmic C-ring. This interaction may explain several phenotypes associated with the absence of FliL.
PubMed: 37503052
DOI: 10.1101/2023.07.14.549092 -
Frontiers in Cellular and Infection... 2024The genus , which colonizes mucosal surfaces, includes both commensal and pathogenic species that are exclusive to humans. The two pathogenic species are closely... (Review)
Review
The genus , which colonizes mucosal surfaces, includes both commensal and pathogenic species that are exclusive to humans. The two pathogenic species are closely related but cause quite different diseases, meningococcal sepsis and meningitis () and sexually transmitted gonorrhea ). Although obvious differences in bacterial niches and mechanisms for transmission exists, pathogenic have high levels of conservation at the levels of nucleotide sequences, gene content and synteny. Species of express broad-spectrum -linked protein glycosylation where the glycoproteins are largely transmembrane proteins or lipoproteins localized on the cell surface or in the periplasm. There are diverse functions among the identified glycoproteins, for example type IV biogenesis proteins, proteins involved in antimicrobial resistance, as well as surface proteins that have been suggested as vaccine candidates. The most abundant glycoprotein, PilE, is the major subunit of pili which are an important colonization factor. The glycans attached can vary extensively due to phase variation of protein glycosylation ( genes and polymorphic gene content. The exact roles of glycosylation in remains to be determined, but increasing evidence suggests that glycan variability can be a strategy to evade the human immune system. In addition, pathogenic and commensal appear to have significant glycosylation differences. Here, the current knowledge and implications of protein glycosylation genes, glycan diversity, glycoproteins and immunogenicity in pathogenic are summarized and discussed.
Topics: Humans; Bacterial Proteins; Glycoproteins; Glycosylation; Neisseria gonorrhoeae; Neisseria meningitidis; Polysaccharides; Meningitis, Meningococcal; Gonorrhea
PubMed: 38808060
DOI: 10.3389/fcimb.2024.1407863 -
Biological Chemistry Jan 2024Understanding how neuronal networks generate complex behavior is one of the major goals of Neuroscience. Neurotransmitter and Neuromodulators are crucial for information... (Review)
Review
Understanding how neuronal networks generate complex behavior is one of the major goals of Neuroscience. Neurotransmitter and Neuromodulators are crucial for information flow between neurons and understanding their dynamics is the key to unravel their role in behavior. To understand how the brain transmits information and how brain states arise, it is essential to visualize the dynamics of neurotransmitters, neuromodulators and neurochemicals. In the last five years, an increasing number of single-wavelength biosensors either based on periplasmic binding proteins (PBPs) or on G-protein-coupled receptors (GPCR) have been published that are able to detect neurotransmitter release and with high spatial and temporal resolution. Here we review and discuss recent progress in the development of these sensors, their limitations and future directions.
Topics: Brain; Receptors, G-Protein-Coupled; Neurons; Coloring Agents; Neurotransmitter Agents; Biosensing Techniques
PubMed: 37246368
DOI: 10.1515/hsz-2023-0175 -
The Journal of Biological Chemistry Mar 2024The bacterial envelope is an essential compartment involved in metabolism and metabolites transport, virulence, and stress defense. Its roles become more evident when... (Review)
Review
The bacterial envelope is an essential compartment involved in metabolism and metabolites transport, virulence, and stress defense. Its roles become more evident when homeostasis is challenged during host-pathogen interactions. In particular, the presence of free radical groups and excess copper in the periplasm causes noxious reactions, such as sulfhydryl group oxidation leading to enzymatic inactivation and protein denaturation. In response to this, canonical and accessory oxidoreductase systems are induced, performing quality control of thiol groups, and therefore contributing to restoring homeostasis and preserving survival under these conditions. Here, we examine recent advances in the characterization of the Dsb-like, Salmonella-specific Scs system. This system includes the ScsC/ScsB pair of Cu-binding proteins with thiol-oxidoreductase activity, an alternative ScsB-partner, the membrane-linked ScsD, and a likely associated protein, ScsA, with a role in peroxide resistance. We discuss the acquisition of the scsABCD locus and its integration into a global regulatory pathway directing envelope response to Cu stress during the evolution of pathogens that also harbor the canonical Dsb systems. The evidence suggests that the canonical Dsb systems cannot satisfy the extra demands that the host-pathogen interface imposes to preserve functional thiol groups. This resulted in the acquisition of the Scs system by Salmonella. We propose that the ScsABCD complex evolved to connect Cu and redox stress responses in this pathogen as well as in other bacterial pathogens.
Topics: Bacterial Proteins; Copper; Homeostasis; Oxidation-Reduction; Oxidoreductases; Salmonella; Sulfhydryl Compounds; Carrier Proteins
PubMed: 38309504
DOI: 10.1016/j.jbc.2024.105710 -
Nature Communications Jan 2024Active nutrient uptake is fundamental for survival and pathogenicity of Gram-negative bacteria, which operate a multi-protein Ton system to transport essential nutrients...
Active nutrient uptake is fundamental for survival and pathogenicity of Gram-negative bacteria, which operate a multi-protein Ton system to transport essential nutrients like metals and vitamins. This system harnesses the proton motive force at the inner membrane to energize the import through the outer membrane, but the mechanism of energy transfer remains enigmatic. Here, we study the periplasmic domain of ExbD, a crucial component of the proton channel of the Ton system. We show that this domain is a dynamic dimer switching between two conformations representing the proton channel's open and closed states. By in vivo phenotypic assays we demonstrate that this conformational switch is essential for the nutrient uptake by bacteria. The open state of ExbD triggers a disorder to order transition of TonB, enabling TonB to supply energy to the nutrient transporter. We also reveal the anchoring role of the peptidoglycan layer in this mechanism. Herein, we propose a mechanistic model for the Ton system, emphasizing ExbD duality and the pivotal catalytic role of peptidoglycan. Sequence analysis suggests that this mechanism is conserved in other systems energizing gliding motility and membrane integrity. Our study fills important gaps in understanding bacterial motor mechanism and proposes novel antibacterial strategies.
Topics: Peptidoglycan; Protons; Cell Wall; Nutrients; Bacteria
PubMed: 38184686
DOI: 10.1038/s41467-023-44606-z -
Plants (Basel, Switzerland) Jul 2023Since Darwin's "Power of Movement in Plants" the precise mechanism of oscillatory plant growth remains elusive. Hence the search continues for the hypothetical growth... (Review)
Review
Since Darwin's "Power of Movement in Plants" the precise mechanism of oscillatory plant growth remains elusive. Hence the search continues for the hypothetical growth oscillator that regulates a huge range of growth phenomena ranging from circumnutation to pollen tube tip growth and stomatal movements. Oscillators are essentially simple devices with few components. A universal growth oscillator with only four major components became apparent recently with the discovery of a missing component, notably arabinogalactan glycoproteins (AGPs) that store dynamic Ca at the cell surface. Demonstrably, auxin-activated proton pumps, AGPs, Ca channels, and auxin efflux "PIN" proteins, embedded in the plasma membrane, combine to generate cytosolic Ca oscillations that ultimately regulate oscillatory growth: Hechtian adhesion of the plasma membrane to the cell wall and auxin-activated proton pumps trigger the release of dynamic Ca stored in periplasmic AGP monolayers. These four major components represent a molecular PINball machine a strong visual metaphor that also recognises auxin efflux "PIN" proteins as an essential component. Proton "pinballs" dissociate Ca ions bound by paired glucuronic acid residues of AGP glycomodules, hence reassessing the role of proton pumps. It shifts the prevalent paradigm away from the recalcitrant "acid growth" theory that proposes direct action on cell wall properties, with an alternative explanation that connects proton pumps to Ca signalling with dynamic Ca storage by AGPs, auxin transport by auxin-efflux PIN proteins and Ca channels. The extensive Ca signalling literature of plants ignores arabinogalactan proteins (AGPs). Such scepticism leads us to reconsider the validity of the universal growth oscillator proposed here with some exceptions that involve marine plants and perhaps the most complex stress test, stomatal regulation.
PubMed: 37447091
DOI: 10.3390/plants12132531 -
Proteome Science Sep 2023Pseudomonas aeruginosa is well known for its intrinsic ability to resist a wide range of antibiotics, thus complicates treatment. Thus, understanding the response of the...
BACKGROUND
Pseudomonas aeruginosa is well known for its intrinsic ability to resist a wide range of antibiotics, thus complicates treatment. Thus, understanding the response of the pathogen to antibiotics is important for developing new therapies. In this study, proteomic response of P. aeruginosa to the commonly used anti-pseudomonas antibiotics, ceftazidime (Caz) and meropenem (Mem) was investigated.
METHODS
P. aeruginosa ATCC 9027, an antibiotic-susceptible strain, was exposed to sub-MIC values of antibiotics either Caz or Mem for 14 days to obtain E1 strains and then cultured in antibiotic-free environments for 10 days to obtain E2 strains. Proteomes of the initial and E1, E2 strains were identified and comparatively analyzed using isobaric tags for relative and absolute quantitation (iTRAQ) in cooperation with nano LC-MS/MS. Noted up and down-regulated proteins were confirmed with quantitative reverse transcriptase PCR (qRT-PCR).
RESULTS
Overall, 1039 and 1041 proteins were identified in Caz and Mem-exposed strains, respectively. Upon antibiotic exposure, there were 7-10% up-regulated (Caz: 71, Mem: 85) and down-regulated (Caz: 106, Mem: 69) proteins (1.5-fold change cut-off). For both Caz and Mem, the DEPs were primarily the ones involved in metabolic process, membrane, virulence, protein synthesis, and antibiotic resistance in which proteins involved in antibiotics resistance tended to be up-regulated while proteins involved in protein synthesis and metabolic process were down-regulated. Noted proteins included beta-lactamase AmpC which was up-regulated and OprD which was down-regulated in both the antibiotic-exposed strains. Besides, biofilm formation related proteins TssC1 and Hcp1 in Caz- exposed strains and the membrane/ periplasmic proteins Azu and PagL in Mem-exposed strains were found significantly down-regulated. qRT-PCR results confirmed the expression change of AmpC, Hcp1 and OprD proteins.
CONCLUSION
Exposure of Pseudomonas aeruginosa to sub-MIC values of Caz and Mem resulted in around 10% change in its proteome. Not only proteins with confirmed roles in antibiotic resistance mechanisms changed their expression but also virulence- associated proteins. Both Caz and Mem response involved up-regulation of AmpC and down-regulation of OprD. While TssC1 and Hcp1 were responsible for Caz response, Azu and PagL were more likely involved in Mem response.
PubMed: 37770917
DOI: 10.1186/s12953-023-00217-6 -
Microbial Cell Factories Jun 2024Recombinant peptide production in Escherichia coli provides a sustainable alternative to environmentally harmful and size-limited chemical synthesis. However, in-vivo...
BACKGROUND
Recombinant peptide production in Escherichia coli provides a sustainable alternative to environmentally harmful and size-limited chemical synthesis. However, in-vivo production of disulfide-bonded peptides at high yields remains challenging, due to degradation by host proteases/peptidases and the necessity of translocation into the periplasmic space for disulfide bond formation.
RESULTS
In this study, we established an expression system for efficient and soluble production of disulfide-bonded peptides in the periplasm of E. coli. We chose model peptides with varying complexity (size, structure, number of disulfide bonds), namely parathyroid hormone 1-84, somatostatin 1-28, plectasin, and bovine pancreatic trypsin inhibitor (aprotinin). All peptides were expressed without and with the N-terminal, low molecular weight CASPON™ tag (4.1 kDa), with the expression cassette being integrated into the host genome. During BioLector™ cultivations at microliter scale, we found that most of our model peptides can only be sufficiently expressed in combination with the CASPON™ tag, otherwise expression was only weak or undetectable on SDS-PAGE. Undesired degradation by host proteases/peptidases was evident even with the CASPON™ tag. Therefore, we investigated whether degradation happened before or after translocation by expressing the peptides in combination with either a co- or post-translational signal sequence. Our results suggest that degradation predominantly happened after the translocation, as degradation fragments appeared to be identical independent of the signal sequence, and expression was not enhanced with the co-translational signal sequence. Lastly, we expressed all CASPON™-tagged peptides in two industry-relevant host strains during C-limited fed-batch cultivations in bioreactors. We found that the process performance was highly dependent on the peptide-host-combination. The titers that were reached varied between 0.6-2.6 g L, and exceeded previously published data in E. coli. Moreover, all peptides were shown by mass spectrometry to be expressed to completion, including full formation of disulfide bonds.
CONCLUSION
In this work, we demonstrated the potential of the CASPON™ technology as a highly efficient platform for the production of soluble peptides in the periplasm of E. coli. The titers we show here are unprecedented whenever parathyroid hormone, somatostatin, plectasin or bovine pancreatic trypsin inhibitor were produced in E. coli, thus making our proposed upstream platform favorable over previously published approaches and chemical synthesis.
Topics: Escherichia coli; Periplasm; Disulfides; Peptides; Recombinant Proteins; Aprotinin
PubMed: 38840157
DOI: 10.1186/s12934-024-02446-6 -
Frontiers in Immunology 2023Syphilis, a sexually transmitted infection caused by the spirochete (), is resurging globally. 's repertoire of outer membrane proteins (OMPs) includes BamA (β-barrel...
INTRODUCTION
Syphilis, a sexually transmitted infection caused by the spirochete (), is resurging globally. 's repertoire of outer membrane proteins (OMPs) includes BamA (β-barrel assembly machinery subunit A/TP0326), a bipartite protein consisting of a 16-stranded β-barrel with nine extracellular loops (ECLs) and five periplasmic POTRA (polypeptide transport-associated) domains. BamA ECL4 antisera promotes internalization of by rabbit peritoneal macrophages.
METHODS
Three overlapping BamA ECL4 peptides and a two-stage, phage display strategy, termed "Epivolve" (for epitope evolution) were employed to generate single-chain variable fragments (scFvs). Additionally, antisera generated by immunizing mice and rabbits with BamA ECL4 displayed by a thioredoxin scaffold (Trx). MAbs and antisera reactivities were evaluated by immunoblotting and ELISA. A comparison of murine and rabbit opsonophagocytosis assays was conducted to evaluate the functional ability of the Abs (, opsonization) and validate the mouse assay. Sera from -infected mice (MSS) and rabbits (IRS) were evaluated for ECL4-specific Abs using Trx and overlapping ECL4 peptides in immunoblotting and ELISA assays.
RESULTS
Each of the five mAbs demonstrated reactivity by immunoblotting and ELISA to nanogram amounts of Trx. One mAb, containing a unique amino acid sequence in both the light and heavy chains, showed activity in the murine opsonophagocytosis assay. Mice and rabbits hyperimmunized with Trx produced opsonic antisera that strongly recognized the ECL presented in a heterologous scaffold and overlapping ECL4 peptides, including S2. In contrast, Abs generated during infection of mice and rabbits poorly recognized the peptides, indicating that S2 contains a subdominant epitope.
DISCUSSION
Epivolve produced mAbs target subdominant opsonic epitopes in BamA ECL4, a top syphilis vaccine candidate. The murine opsonophagocytosis assay can serve as an alternative model to investigate the opsonic potential of vaccinogens. Detailed characterization of BamA ECL4-specific Abs provided a means to dissect Ab responses elicited by infection.
Topics: Mice; Animals; Rabbits; Treponema pallidum; Antibodies, Monoclonal; Syphilis; Immune Sera; Bacteriophages; Epitopes
PubMed: 37675118
DOI: 10.3389/fimmu.2023.1222267