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Microbiology Spectrum Aug 2023Although the immunomodulatory potency of bacterial membrane vesicles (MVs) is widely acknowledged, their interactions with host cells and the underlying signaling...
Characterization of the Inflammatory Response Evoked by Bacterial Membrane Vesicles in Intestinal Cells Reveals an RIPK2-Dependent Activation by Enterotoxigenic Escherichia coli Vesicles.
Although the immunomodulatory potency of bacterial membrane vesicles (MVs) is widely acknowledged, their interactions with host cells and the underlying signaling pathways have not been well studied. Herein, we provide a comparative analysis of the proinflammatory cytokine profile secreted by human intestinal epithelial cells exposed to MVs derived from 32 gut bacteria. In general, outer membrane vesicles (OMVs) from Gram-negative bacteria induced a stronger proinflammatory response than MVs from Gram-positive bacteria. However, the quality and quantity of cytokine induction varied between MVs from different species, highlighting their unique immunomodulatory properties. OMVs from enterotoxigenic Escherichia coli (ETEC) were among those showing the strongest proinflammatory potency. In depth analyses revealed that the immunomodulatory activity of ETEC OMVs relies on a so far unprecedented two-step mechanism, including their internalization into host cells followed by intracellular recognition. First, OMVs are efficiently taken up by intestinal epithelial cells, which mainly depends on caveolin-mediated endocytosis as well as the presence of the outer membrane porins OmpA and OmpF on the MVs. Second, lipopolysaccharide (LPS) delivered by OMVs is intracellularly recognized by novel caspase- and RIPK2-dependent pathways. This recognition likely occurs via detection of the lipid A moiety as ETEC OMVs with underacylated LPS exhibited reduced proinflammatory potency but similar uptake dynamics compared to OMVs derived from wild-type (WT) ETEC. Intracellular recognition of ETEC OMVs in intestinal epithelial cells is pivotal for the proinflammatory response as inhibition of OMV uptake also abolished cytokine induction. The study signifies the importance of OMV internalization by host cells to exercise their immunomodulatory activities. The release of membrane vesicles from the bacterial cell surface is highly conserved among most bacterial species, including outer membrane vesicles (OMVs) from Gram-negative bacteria as well as vesicles liberated from the cytoplasmic membrane of Gram-positive bacteria. It is becoming increasingly evident that these multifactorial spheres, carrying membranous, periplasmic, and even cytosolic content, contribute to intra- and interspecies communication. In particular, gut microbiota and the host engage in a myriad of immunogenic and metabolic interactions. This study highlights the individual immunomodulatory activities of bacterial membrane vesicles from different enteric species and provides new mechanistic insights into the recognition of ETEC OMVs by human intestinal epithelial cells.
Topics: Humans; Enterotoxigenic Escherichia coli; Lipopolysaccharides; Intestines; Bacteria; Cytokines; Bacterial Outer Membrane Proteins; Receptor-Interacting Protein Serine-Threonine Kinase 2
PubMed: 37306596
DOI: 10.1128/spectrum.01115-23 -
Applied and Environmental Microbiology Jan 2024In bacteria, the second messenger cyclic di-GMP (c-di-GMP) is synthesized and degraded by multiple diguanylate cyclases (DGCs) and phosphodiesterases. A high level of...
In bacteria, the second messenger cyclic di-GMP (c-di-GMP) is synthesized and degraded by multiple diguanylate cyclases (DGCs) and phosphodiesterases. A high level of c-di-GMP induces biofilm formation and represses motility. WspR, a hybrid response regulator DGC, produces c-di-GMP when it is phosphorylated. FlhF, a signal recognition particle-type GTPase, is initially localized to the cell poles and is indispensable for polar flagellar localization in . In this study, we report that deletion of affected biofilm formation and the c-di-GMP level in . Phenotypic analysis of a knockout mutant revealed increased biofilm formation, wrinkled colonies on Congo red agar, and an elevated c-di-GMP level compared to the wild-type strain, PAO1. Yeast and bacterial two-hybrid systems showed that FlhF binds to the response regulator HsbR, and HsbR binds to WspR. Deletion of or in the Δ background abolished the phenotype of Δ. In addition, confocal microscopy demonstrated that WspR-GFP was distributed throughout the cytoplasm and formed a visible cluster at one cell pole in PAO1 and Δ, but it was mainly distributed as visible clusters at the lateral side of the periplasm and with visible clusters at both cell poles in Δ. These findings suggest that FlhF influences the subcellular cluster and localization of WspR and negatively modulates WspR DGC activity in a manner dependent on HsbR. Together, our findings demonstrate a novel mechanism for FlhF modulating the lifestyle transition between motility and biofilm via HsbR to regulate the DGC activity of WspR.IMPORTANCECyclic di-GMP (c-di-GMP) is a second messenger that controls flagellum biosynthesis, adhesion, virulence, motility, exopolysaccharide production, and biofilm formation in bacteria. Recent research has shown that distinct diguanylate cyclases (DGCs) or phosphodiesterases (PDEs) produce highly specific outputs. Some DGCs and PDEs contribute to the total global c-di-GMP concentration, but others only affect local c-di-GMP in a microenvironment. However, the underlying mechanisms are unclear. Here, we report that FlhF affects the localization and DGC activity of WspR via HsbR and is implicated in local c-di-GMP signaling in . This study establishes the link between the c-di-GMP signaling system and the flagellar localization and provides insight for understanding the complex regulatory network of c-di-GMP signaling.
Topics: Pseudomonas aeruginosa; Escherichia coli Proteins; Cyclic GMP; Biofilms; Phosphorus-Oxygen Lyases; Phosphoric Diester Hydrolases; Bacterial Proteins; Gene Expression Regulation, Bacterial; Diethylstilbestrol
PubMed: 38112425
DOI: 10.1128/aem.01548-23 -
Protein Expression and Purification Jul 2024E. coli plays a substantial role in recombinant protein production. Its importance increased with the discovery of recombinant DNA technology and the subsequent... (Review)
Review
E. coli plays a substantial role in recombinant protein production. Its importance increased with the discovery of recombinant DNA technology and the subsequent production of the first recombinant insulin in E. coli. E. coli is a widely used and cost-effective host to produce recombinant proteins. It is also noteworthy that a significant portion of the approved therapeutic proteins have been produced in this organism. Despite these advantages, it has some disadvantages, such as toxicity and lack of eukaryotic post-translational modifications that can lead to the production of misfolded, insoluble, or dysfunctional proteins. This study focused on the challenges and engineering approaches for improved expression and solubility in recombinant protein production in E. coli. In this context, solution strategies such as strain and vector selection, codon usage, mRNA stability, expression conditions, translocation to the periplasmic region and addition of fusion tags in E. coli were discussed.
Topics: Escherichia coli; Recombinant Proteins; Humans; Solubility; Genetic Vectors
PubMed: 38479588
DOI: 10.1016/j.pep.2024.106463 -
BioRxiv : the Preprint Server For... Aug 2023Bacterial growth and division rely on intricate regulation of morphogenetic complexes to remodel the cell envelope without compromising envelope integrity. Significant...
Bacterial growth and division rely on intricate regulation of morphogenetic complexes to remodel the cell envelope without compromising envelope integrity. Significant progress has been made in recent years towards understanding the regulation of cell wall metabolic enzymes. However, other cell envelope components play a role in morphogenesis as well. Components required to maintain osmotic homeostasis are among these understudied envelope-associated enzymes that may contribute to cell morphology. A primary factor required to protect envelope integrity in low osmolarity environments is OpgH, the synthase of osmoregulated periplasmic glucans (OPGs). Here, we demonstrate that OpgH is essential in the α-proteobacterium . Unexpectedly, depletion of OpgH results in striking asymmetric bulging and cell lysis, accompanied by misregulation of cell wall insertion and mislocalization of morphogenetic complexes. The enzymatic activity of OpgH is required for normal cell morphology as production of an OpgH mutant that disrupts a conserved glycosyltransferase motif phenocopies the depletion. Our data establish a surprising function for an OpgH homolog in morphogenesis and reveal an essential role of OpgH in maintaining proper cell morphology during normal growth and division in .
PubMed: 37693447
DOI: 10.1101/2023.08.28.555136 -
Biophysical Reviews Oct 2023For the last decades, significant progress has been made in studying the biological functions of H-bond networks in membrane proteins, proton transporters, receptors,... (Review)
Review
For the last decades, significant progress has been made in studying the biological functions of H-bond networks in membrane proteins, proton transporters, receptors, and photosynthetic reaction centers. Increasing availability of the X-ray crystal and cryo-electron microscopy structures of photosynthetic complexes resolved with high atomic resolution provides a platform for their comparative analysis. It allows identifying structural factors that are ensuring the high quantum yield of the photochemical reactions and are responsible for the stability of the membrane complexes. The H-bond networks are known to be responsible for proton transport associated with electron transfer from the primary to the secondary quinone as well as in the processes of water oxidation in photosystem II. Participation of such networks in reactions proceeding on the periplasmic side of bacterial photosynthetic reaction centers is less studied. This review summarizes the current understanding of the role of H-bond networks on the donor side of photosynthetic reaction centers from purple bacteria. It is discussed that the networks may be involved in providing close association with mobile electron carriers, in light-induced proton transport, in regulation of the redox properties of bacteriochlorophyll cofactors, and in stabilization of the membrane protein structure at the interface of membrane and soluble phases.
PubMed: 37974998
DOI: 10.1007/s12551-023-01109-x -
Journal of Nanobiotechnology May 2024The outer membrane vesicles (OMVs) produced by Gram-negative bacteria can modulate the immune system and have great potentials for bacterial vaccine development.
BACKGROUND
The outer membrane vesicles (OMVs) produced by Gram-negative bacteria can modulate the immune system and have great potentials for bacterial vaccine development.
RESULTS
A highly active Acinetobacter baumannii phage lysin, LysP53, can stimulate the production of OMVs after interacting with A. baumannii, Escherichia coli, and Salmonella. The OMVs prepared by the lysin (LOMVs) from A. baumannii showed better homogeneity, higher protein yield, lower endotoxin content, and lower cytotoxicity compared to the naturally produced OMVs (nOMVs). The LOMVs contain a significantly higher number of cytoplasmic and cytoplasmic membrane proteins but a smaller number of periplasmic and extracellular proteins compared to nOMVs. Intramuscular immunization with either LOMVs or nOMVs three times provided robust protection against A. baumannii infections in both pneumonia and bacteremia mouse models. Intranasal immunization offered good protection in the pneumonia model but weaker protection (20-40%) in the bacteremia model. However, with a single immunization, LOMVs demonstrated better protection than the nOMVs in the pneumonia mouse model.
CONCLUSIONS
The novel lysin approach provides a superior choice compared to current methods for OMV production, especially for vaccine development.
Topics: Acinetobacter baumannii; Animals; Acinetobacter Infections; Mice; Bacteriophages; Female; Mice, Inbred BALB C; Bacterial Vaccines; Immunization; Extracellular Vesicles; Bacterial Outer Membrane; Bacterial Outer Membrane Proteins; Disease Models, Animal; Humans; Administration, Intranasal; Viral Proteins
PubMed: 38773507
DOI: 10.1186/s12951-024-02553-x -
MBio Oct 2023Exclusively in the Bacteroidetes phylum, most proteins exported across the inner membrane via the Sec system and released into the periplasm by type I signal peptidase...
Exclusively in the Bacteroidetes phylum, most proteins exported across the inner membrane via the Sec system and released into the periplasm by type I signal peptidase have N-terminal glutamine converted to pyroglutamate. The reaction is catalyzed by the periplasmic enzyme glutaminyl cyclase (QC), which is essential for the growth of and other periodontopathogens. Apparently, pyroglutamyl formation stabilizes extracytoplasmic proteins and/or protects them from proteolytic degradation in the periplasm. Given the role of as the keystone pathogen in periodontitis, QC is a promising target for the development of drugs to treat and/or prevent this highly prevalent chronic inflammatory disease leading to tooth loss and associated with severe systemic diseases.
Topics: Humans; Aminoacyltransferases; Periodontitis; Pyrrolidonecarboxylic Acid; Glutamine
PubMed: 37750700
DOI: 10.1128/mbio.00980-23 -
Frontiers in Cellular and Infection... 2024The hemin acquisition system is composed of an outer membrane TonB-dependent transporter that internalizes hemin into the periplasm, periplasmic hemin-binding proteins...
INTRODUCTION
The hemin acquisition system is composed of an outer membrane TonB-dependent transporter that internalizes hemin into the periplasm, periplasmic hemin-binding proteins to shuttle hemin, an inner membrane transporter that transports hemin into the cytoplasm, and cytoplasmic heme oxygenase to release iron. Fur and HemP are two known regulators involved in the regulation of hemin acquisition. The hemin acquisition system of is poorly understood, with the exception of HemA as a TonB-dependent transporter for hemin uptake.
METHODS
Putative candidates responsible for hemin acquisition were selected via a homolog search and a whole-genome survey of . Operon verification was performed by reverse transcription-polymerase chain reaction. The involvement of candidate genes in hemin acquisition was assessed using an in-frame deletion mutant construct and iron utilization assays. The transcript levels of candidate genes were determined using quantitative polymerase chain reaction.
RESULTS
and operons were selected as candidates for hemin acquisition. Compared with the parental strain, and mutants displayed a defect in their ability to use hemin as the sole iron source for growth. However, hemin utilization by the and mutants was comparable to that of the parental strain. expression was repressed by Fur in iron-replete conditions and derepressed in iron-depleted conditions. HemP negatively regulated expression. Like , was repressed by Fur in iron-replete conditions; however, was moderately derepressed in response to iron-depleted stress and fully derepressed when hemin was present. Unlike and , the operon was constitutively expressed, regardless of the iron level or the presence of hemin, and Fur and HemP had no influence on its expression.
CONCLUSION
HemA, HemU, and TonB1 contribute to hemin acquisition in . Fur represses the expression of and in iron-replete conditions. expression is regulated by low iron levels, and HemP acts as a negative regulator of this regulatory circuit. expression is regulated by low iron and hemin levels in a -dependent manner.
Topics: Hemin; Stenotrophomonas maltophilia; Bacterial Proteins; Membrane Proteins; Iron
PubMed: 38596648
DOI: 10.3389/fcimb.2024.1380976 -
Annual Review of Biochemistry Apr 2024Almost all outer membrane proteins (OMPs) in Gram-negative bacteria contain a β-barrel domain that spans the outer membrane (OM). To reach the OM, OMPs must be... (Review)
Review
Almost all outer membrane proteins (OMPs) in Gram-negative bacteria contain a β-barrel domain that spans the outer membrane (OM). To reach the OM, OMPs must be translocated across the inner membrane by the Sec machinery, transported across the crowded periplasmic space through the assistance of molecular chaperones, and finally assembled (folded and inserted into the OM) by the β-barrel assembly machine. In this review, we discuss how considerable new insights into the contributions of these factors to OMP biogenesis have emerged in recent years through the development of novel experimental, computational, and predictive methods. In addition, we describe recent evidence that molecular machines that were thought to function independently might interact to form dynamic intermembrane supercomplexes. Finally, we discuss new results that suggest that OMPs are inserted primarily near the middle of the cell and packed into supramolecular structures (OMP islands) that are distributed throughout the OM.
PubMed: 38603556
DOI: 10.1146/annurev-biochem-030122-033754 -
Molecular Biotechnology Dec 2023As a member of the tumor necrosis factor (TNF) superfamily, the B-cell activating factor (BAFF) plays a crucial role in B-cell survival and differentiation....
As a member of the tumor necrosis factor (TNF) superfamily, the B-cell activating factor (BAFF) plays a crucial role in B-cell survival and differentiation. Overexpression of this protein has been closely linked to autoimmune disorders and some B-cell malignancies. Using monoclonal antibodies (mAbs) against the BAFF soluble domain appears to be a complementary treatment for some of these diseases. This study aimed to produce and develop a specific Nanobody (Nb), a variable camelid antibody domain, against the soluble domain of BAFF protein. After camel immunization with recombinant protein and preparing cDNA from total RNAs separated from camel lymphocytes, an Nb library was developed. Individual colonies capable of binding selectively to rBAFF were obtained by periplasmic-ELISA, sequenced, and expressed in a bacterial expression system. The specificity and affinity of selected Nb were determined and its target identification and functionality were evaluated using flow cytometry.
Topics: Animals; B-Cell Activating Factor; Camelus; Interleukin-4; B-Lymphocytes; Antibodies, Monoclonal
PubMed: 36906729
DOI: 10.1007/s12033-023-00700-7