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BioRxiv : the Preprint Server For... Nov 2023Biofilm formation and surface attachment in multiple Alphaproteobacteria is driven by unipolar polysaccharide (UPP) adhesins. The pathogen produces a UPP adhesin, which...
Biofilm formation and surface attachment in multiple Alphaproteobacteria is driven by unipolar polysaccharide (UPP) adhesins. The pathogen produces a UPP adhesin, which is regulated by the intracellular second messenger cyclic diguanylate monophosphate (cdGMP). Prior studies revealed that DcpA, a diguanylate cyclase-phosphodiesterase (DGC-PDE), is crucial in control of UPP production and surface attachment. DcpA is regulated by PruR, a protein with distant similarity to enzymatic domains known to coordinate the molybdopterin cofactor (MoCo). Pterins are bicyclic nitrogen-rich compounds, several of which are formed via a non-essential branch of the folate biosynthesis pathway, distinct from MoCo. The pterin-binding protein PruR controls DcpA activity, fostering cdGMP breakdown and dampening its synthesis. Pterins are excreted and we report here that PruR associates with these metabolites in the periplasm, promoting interaction with the DcpA periplasmic domain. The pteridine reductase PruA, which reduces specific dihydro-pterin molecules to their tetrahydro forms, imparts control over DcpA activity through PruR. Tetrahydromonapterin preferentially associates with PruR relative to other related pterins, and the PruR-DcpA interaction is decreased in a mutant. PruR and DcpA are encoded in an operon that is conserved amongst multiple Proteobacteria including mammalian pathogens. Crystal structures reveal that PruR and several orthologs adopt a conserved fold, with a pterin-specific binding cleft that coordinates the bicyclic pterin ring. These findings define a new pterin-responsive regulatory mechanism that controls biofilm formation and related cdGMP-dependent phenotypes in and is found in multiple additional bacterial pathogens.
PubMed: 38014264
DOI: 10.1101/2023.11.18.567607 -
Frontiers in Immunology 2023a gram-negative rod-shaped bacterium in the family, infect humans, causing serious illnesses such as urinary tract infection, cystitis, biliary tract infection,...
a gram-negative rod-shaped bacterium in the family, infect humans, causing serious illnesses such as urinary tract infection, cystitis, biliary tract infection, pneumonia, meningitis, hemolytic uremic syndrome, and death. Initially treatable with penicillin, antibiotic misuse led to evolving resistance, including resistance to colistin, a last-resort drug. With no licensed vaccine, the study aimed to design a multi-epitope vaccine against . The study started with the retrieval of the complete proteome of all known strains and proceeded to filter the surface exposed virulent proteins. Seventeen virulent proteins (4 extracellular, 4 outer membranes, 9 periplasmic) with desirable physicochemical properties were identified from the complete proteome of known strains. Further, these proteins were processed for B-cell and T-cell epitope mapping. Obtained epitopes were evaluated for antigenicity, allergenicity, solubility, MHC-binding, and toxicity and the filtered epitopes were fused by specific linkers and an adjuvant into a vaccine construct. Structure of the vaccine candidate was predicted and refined resulting in 78.1% amino acids in allowed regions and VERIFY3D score of 81%. Vaccine construct was docked with TLR-4, MHC-I, and MHC-II, showing binding energies of -1040.8 kcal/mol, -871.4 kcal/mol, and -1154.6 kcal/mol and maximum interactions. Further, molecular dynamic simulation of the docked complexes was carried out resulting in a significant stable nature of the docked complexes (high B-factor and deformability values, lower Eigen and high variance values) in terms of intermolecular binding conformation and interactions. The vaccine was also reported to stimulate a variety of immunological pathways after administration. In short, the designed vaccine revealed promising predictions about its immune protective potential against infections however experimental validation is needed to validate the results.
Topics: Humans; Proteome; Molecular Docking Simulation; Epitopes, T-Lymphocyte; Bacterial Vaccines; Membrane Proteins
PubMed: 38143752
DOI: 10.3389/fimmu.2023.1332378 -
International Journal of Biochemistry... 2023The diverse nature of carbohydrate structures and linkages requires a variety of enzymes responsible for sugar degradation. The periplasmic protein encoded by the gene...
BACKGROUND
The diverse nature of carbohydrate structures and linkages requires a variety of enzymes responsible for sugar degradation. The periplasmic protein encoded by the gene has been assigned to glycoside hydrolase family 3 and is predicted to function as a β-glucosidase.
OBJECTIVES
We investigated the catalytic properties of the protein BglX and identified two functionally important amino acid residues.
METHODS
The gene was cloned into a pET20b(+) vector, and three mutants, D111N, D287G, and E293Q, were generated using site-directed mutagenesis. Kinetic studies were performed on the wild-type and mutant enzymes.
RESULTS
Substrate specificity tests indicated that the BglX enzyme hydrolyzes β-glycosidic bonds in nitrophenyl-β-glycosides and demonstrates greater activity towards galactose-containing substrates compared to glucose derivatives. Monomeric glucose and galactose inhibit enzyme activity to a different degree in a substrate-dependent manner. In addition, BglX can hydrolyze lactose but not cellobiose, maltose, or laminarin. Subsequently, cells overexpressing active BglX have a growth advantage on minimal media supplemented with lactose as a carbon source. Mutation of D287 or D111 residues negatively affected the activity of BglX indicating their involvement in catalysis. Overexpression of BglX by cells did not increase biofilm formation.
CONCLUSIONS
The low activity towards glucose-containing substrates and significantly elevated activity towards galactosides suggests that β-glucosidase activity may not be the primary function of the BglX enzyme.
PubMed: 37736388
DOI: No ID Found -
IScience Jul 2023Cell-surface signaling (CSS) is a signal transfer system of Gram-negative bacteria that produces the activation of an extracytoplasmic function σ factor (σ) in the...
Cell-surface signaling (CSS) is a signal transfer system of Gram-negative bacteria that produces the activation of an extracytoplasmic function σ factor (σ) in the cytosol in response to an extracellular signal. Activation requires the regulated and sequential proteolysis of the σ-associated anti-σ factor, and the function of the Prc and RseP proteases. In this work, we have identified another protease that modulates CSS activity, namely the periplasmic carboxyl-terminal processing protease CtpA. CtpA functions upstream of Prc in the proteolytic cascade and seems to prevent the Prc-mediated proteolysis of the CSS anti-σ factor. Importantly, using zebrafish embryos and the A549 lung epithelial cell line as hosts, we show that mutants in the and proteases of the human pathogen are considerably attenuated in virulence while the mutation increases virulence likely by enhancing the production of membrane vesicles.
PubMed: 37534181
DOI: 10.1016/j.isci.2023.107216 -
The Journal of Biological Chemistry Nov 2023We evaluate cryoEM and crystal structures of two molecular machines that traffick heme and attach it to cytochrome c (cyt c), the second activity performed by a cyt c...
We evaluate cryoEM and crystal structures of two molecular machines that traffick heme and attach it to cytochrome c (cyt c), the second activity performed by a cyt c synthase. These integral membrane proteins, CcsBA and CcmF/H, both covalently attach heme to cyt c, but carry it out via different mechanisms. A CcsB-CcsA complex transports heme through a channel to its external active site, where it forms two thioethers between reduced (Fe) heme and CysXxxXxxCysHis in cyt c. The active site is formed by a periplasmic WWD sequence and two histidines (P-His1 and P-His2). We evaluate each proposed functional domain in CcsBA cryoEM densities, exploring their presence in other CcsB-CcsA proteins from a wide distribution of organisms (e.g., from Gram positive to Gram negative bacteria to chloroplasts.) Two conserved pockets, for the first and second cysteines of CXXCH, explain stereochemical heme attachment. In addition to other universal features, a conserved periplasmic beta stranded structure, called the beta cap, protects the active site when external heme is not present. Analysis of CcmF/H, here called an oxidoreductase and cyt c synthase, addresses mechanisms of heme access and attachment. We provide evidence that CcmF/H receives Fe heme from holoCcmE via a periplasmic entry point in CcmF, whereby heme is inserted directly into a conserved WWD/P-His domain from above. Evidence suggests that CcmF acts as a heme reductase, reducing holoCcmE (to Fe) through a transmembrane electron transfer conduit, which initiates a complicated series of events at the active site.
Topics: Biological Transport; Cytochromes c; Heme; Membrane Proteins; Helicobacter hepaticus; Bacterial Proteins
PubMed: 37827288
DOI: 10.1016/j.jbc.2023.105332 -
BioRxiv : the Preprint Server For... Aug 2023Active 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 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.
PubMed: 37609138
DOI: 10.1101/2023.08.11.552980 -
EcoSal Plus Dec 2023The O-antigen, a long polysaccharide that constitutes the distal part of the outer membrane-anchored lipopolysaccharide, is one of the critical components in the... (Review)
Review
The O-antigen, a long polysaccharide that constitutes the distal part of the outer membrane-anchored lipopolysaccharide, is one of the critical components in the protective outer membrane of Gram-negative bacteria. Most species produce one of the structurally diverse O-antigens, with nearly all the polysaccharide components having complex structures made by the Wzx/Wzy pathway. This pathway produces repeat-units of mostly 3-8 sugars on the cytosolic face of the cytoplasmic membrane that is translocated by Wzx flippase to the periplasmic face and polymerized by Wzy polymerase to give long-chain polysaccharides. The Wzy polymerase is a highly diverse integral membrane protein typically containing 10-14 transmembrane segments. Biochemical evidence confirmed that Wzy polymerase is the sole driver of polymerization, and recent progress also began to demystify its interacting partner, Wzz, shedding some light to speculate how the proteins may operate together during polysaccharide biogenesis. However, our knowledge of how the highly variable Wzy proteins work as part of the O-antigen processing machinery remains poor. Here, we discuss the progress to the current understanding of repeat-unit polymerization and propose an updated model to explain the formation of additional short chain O-antigen polymers found in the lipopolysaccharide of diverse Gram-negative species and their importance in the biosynthetic process.
Topics: O Antigens; Bacterial Proteins; Lipopolysaccharides; Polysaccharides, Bacterial; Gram-Negative Bacteria
PubMed: 36622162
DOI: 10.1128/ecosalplus.esp-0020-2022 -
Frontiers in Cellular and Infection... 2023is a World Health Organization priority pathogen and a significant clinical concern for infections of the respiratory and urinary tracts due to widespread and...
is a World Health Organization priority pathogen and a significant clinical concern for infections of the respiratory and urinary tracts due to widespread and increasing resistance to antimicrobials. In the absence of a vaccine, there is an urgent need to identify novel targets for therapeutic development. Bacterial pathogens, including , require the -block metal ion zinc as an essential micronutrient, which serves as a cofactor for ~6% of the proteome. During infection, zinc acquisition necessitates the use of high affinity uptake systems to overcome niche-specific zinc limitation and host-mediated nutritional immunity. Here, we report the identification of ZnuCBA and ZniCBA, two ATP-binding cassette permeases that are highly conserved in species and contribute to AJ218 zinc homeostasis, and the high-resolution structure of the zinc-recruiting solute-binding protein ZniA. The Znu and Zni permeases appear functionally redundant with abrogation of both systems required to reduce zinc accumulation. Disruption of both systems also exerted pleiotropic effects on the homeostasis of other -block elements. Zinc limitation perturbed cell morphology and compromised resistance to stressors, such as salt and oxidative stress. The mutant strain lacking both systems showed significantly impaired virulence in acute lung infection models, highlighting the necessity of zinc acquisition in the virulence and pathogenicity of .
Topics: Klebsiella pneumoniae; Zinc; Virulence; Klebsiella; Membrane Transport Proteins
PubMed: 38249299
DOI: 10.3389/fcimb.2023.1322973 -
BioRxiv : the Preprint Server For... Jul 2023Bacteroidota are abundant members of the human gut microbiota that shape the enteric landscape by modulating host immunity and degrading dietary- and host-derived...
Bacteroidota are abundant members of the human gut microbiota that shape the enteric landscape by modulating host immunity and degrading dietary- and host-derived glycans. These processes are at least partially mediated by O uter M embrane V esicles (OMVs). In this work, we developed a high-throughput screen to identify genes required for OMV biogenesis and its regulation in ( ). Our screening led us to the identification of a novel family of D ual M embrane-spanning A nti-sigma factors (Dma), which regulate OMV biogenesis in . We employed molecular and multiomic analyses to demonstrate that deletion of Dma1, the founding member of the Dma family, results in hypervesiculation by modulating the expression of NigD1, which belongs to a family of uncharacterized proteins found throughout Bacteroidota. Dma1 has an unprecedented domain organization: it contains a C-terminal β-barrel embedded in the OM; its N-terminal domain interacts with its cognate sigma factor in the cytoplasm, and both domains are tethered via an intrinsically disordered region that traverses the periplasm. Phylogenetic analyses reveal that the Dma family is a unique feature of Bacteroidota. This study provides the first mechanistic insights into the regulation of OMV biogenesis in human gut bacteria.
PubMed: 37503209
DOI: 10.1101/2023.07.13.548920 -
Communications Biology Aug 2023Lipopolysaccharides such as the enterobacterial common antigen are important components of the enterobacterial cell envelope that act as a protective barrier against the...
Lipopolysaccharides such as the enterobacterial common antigen are important components of the enterobacterial cell envelope that act as a protective barrier against the environment and are often polymerized by the inner membrane bound Wzy-dependent pathway. By employing cryo-electron microscopy we show that WzzE, the co-polymerase component of this pathway that is responsible for the length modulation of the enterobacterial common antigen, is octameric with alternating up-down conformations of its L4 loops. The alternating up-down nature of these essential loops, located at the top of the periplasmic bell, are modulated by clashing helical faces between adjacent protomers that flank the L4 loops around the octameric periplasmic bell. This alternating arrangement and a highly negatively charged binding face create a dynamic environment in which the polysaccharide chain is extended, and suggest a ratchet-type mechanism for polysaccharide elongation.
Topics: Escherichia coli Proteins; Cryoelectron Microscopy; Polysaccharides, Bacterial; Lipopolysaccharides; Cell Membrane
PubMed: 37532793
DOI: 10.1038/s42003-023-05157-7