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Nature Communications Feb 2021The recently discovered lytic polysaccharide monooxygenases (LPMOs), which cleave polysaccharides by oxidation, have been associated with bacterial virulence, but...
The recently discovered lytic polysaccharide monooxygenases (LPMOs), which cleave polysaccharides by oxidation, have been associated with bacterial virulence, but supporting functional data is scarce. Here we show that CbpD, the LPMO of Pseudomonas aeruginosa, is a chitin-oxidizing virulence factor that promotes survival of the bacterium in human blood. The catalytic activity of CbpD was promoted by azurin and pyocyanin, two redox-active virulence factors also secreted by P. aeruginosa. Homology modeling, molecular dynamics simulations, and small angle X-ray scattering indicated that CbpD is a monomeric tri-modular enzyme with flexible linkers. Deletion of cbpD rendered P. aeruginosa unable to establish a lethal systemic infection, associated with enhanced bacterial clearance in vivo. CbpD-dependent survival of the wild-type bacterium was not attributable to dampening of pro-inflammatory responses by CbpD ex vivo or in vivo. Rather, we found that CbpD attenuates the terminal complement cascade in human serum. Studies with an active site mutant of CbpD indicated that catalytic activity is crucial for virulence function. Finally, profiling of the bacterial and splenic proteomes showed that the lack of this single enzyme resulted in substantial re-organization of the bacterial and host proteomes. LPMOs similar to CbpD occur in other pathogens and may have similar immune evasive functions.
Topics: Animals; Bacterial Proteins; Carrier Proteins; Cell Death; Complement System Proteins; Humans; Mice; Microbial Viability; Mixed Function Oxygenases; Oxidation-Reduction; Polysaccharides; Protein Domains; Proteome; Proteomics; Pseudomonas Infections; Pseudomonas aeruginosa; Substrate Specificity; Transcription, Genetic; Virulence; Virulence Factors
PubMed: 33623002
DOI: 10.1038/s41467-021-21473-0 -
ACS Physical Chemistry Au Jan 2023Tryptophan is one of few residues that participates in biological electron transfer reactions. Upon substitution of the native Cu center with Zn in the blue-copper...
Tryptophan is one of few residues that participates in biological electron transfer reactions. Upon substitution of the native Cu center with Zn in the blue-copper protein azurin, a long-lived tryptophan neutral radical can be photogenerated. We report the following quantum yield values for Zn-substituted azurin in the presence of the electron acceptor Cu(II)-azurin: formation of the tryptophan neutral radical (Φ), electron transfer (Φ), fluorescence (Φ), and phosphorescence (Φ), as well as the efficiency of proton transfer of the cation radical (Φ). Increasing the concentration of the electron acceptor increased Φ and Φ values and decreased Φ without affecting Φ. At all concentrations of the acceptor, the value of Φ was nearly unity. These observations indicate that the phosphorescent triplet state is the parent state of electron transfer and that nearly all electron transfer events lead to proton loss. Similar results regarding the parent state were obtained with a different electron acceptor, [Co(NH)Cl]; however, Stern-Volmer graphs revealed that [Co(NH)Cl] was a more effective phosphorescence quencher ( = 230 000 M) compared to Cu(II)-azurin ( = 88 000 M). Competition experiments in the presence of both [Co(NH)Cl] and Cu(II)-azurin suggested that [Co(NH)Cl] is the preferred electron acceptor. Implications of these results in terms of quenching mechanisms are discussed.
PubMed: 36718260
DOI: 10.1021/acsphyschemau.2c00042 -
Infection, Genetics and Evolution :... Aug 2023Gonorrhea is an urgent antimicrobial resistance threat and its therapeutic options are continuously getting restricted. Moreover, no vaccine has been approved against it...
Reverse vaccinology approaches to introduce promising immunogenic and drug targets against antibiotic-resistant Neisseria gonorrhoeae: Thinking outside the box in current prevention and treatment.
Gonorrhea is an urgent antimicrobial resistance threat and its therapeutic options are continuously getting restricted. Moreover, no vaccine has been approved against it so far. Hence, the present study aimed to introduce novel immunogenic and drug targets against antibiotic-resistant Neisseria gonorrhoeae strains. In the first step, the core proteins of 79 complete genomes of N. gonorrhoeae were retrieved. Next, the surface-exposed proteins were evaluated from different aspects such as antigenicity, allergenicity, conservancy, and B-cell and T-cell epitopes to introduce promising immunogenic candidates. Then, the interactions with human Toll-like receptors (TLR-1, 2, and 4), and immunoreactivity to elicit humoral and cellular immune responses were simulated. On the other hand, to identify novel broad-spectrum drug targets, the cytoplasmic and essential proteins were detected. Then, the N. gonorrhoeae metabolome-specific proteins were compared to the drug targets of the DrugBank, and novel drug targets were retrieved. Finally, the protein data bank (PDB) file availability and prevalence among the ESKAPE group and common sexually transmitted infection (STI) agents were assessed. Our analyses resulted in the recognition of ten novel and putative immunogenic targets including murein transglycosylase A, PBP1A, Opa, NlpD, Azurin, MtrE, RmpM, LptD, NspA, and TamA. Moreover, four potential and broad-spectrum drug targets were identified including UMP kinase, GlyQ, HU family DNA-binding protein, and IF-1. Some of the shortlisted immunogenic and drug targets have confirmed roles in adhesion, immune evasion, and antibiotic resistance that can induce bactericidal antibodies. Other immunogenic and drug targets might be associated with the virulence of N. gonorrhoeae as well. Thus, further experimental studies and site-directed mutations are recommended to investigate the role of potential vaccine and drug targets in the pathogenesis of N. gonorrhoeae. It seems that the efforts for proposing novel vaccines and drug targets appear to be paving the way for a prevention-treatment strategy against this bacterium. Additionally, a combination of bactericidal monoclonal antibodies and antibiotics is a promising approach to curing N. gonorrhoeae.
Topics: Humans; Neisseria gonorrhoeae; Anti-Bacterial Agents; Vaccinology; Gonorrhea; Membrane Proteins
PubMed: 37225067
DOI: 10.1016/j.meegid.2023.105449 -
The FEBS Journal Jan 2021Cu-containing nitrite reductases that convert NO to NO are critical enzymes in nitrogen-based energy metabolism. Among organisms in the order Rhizobiales, we have...
Cu-containing nitrite reductases that convert NO to NO are critical enzymes in nitrogen-based energy metabolism. Among organisms in the order Rhizobiales, we have identified two copies of nirK, one encoding a new class of 4-domain CuNiR that has both cytochrome and cupredoxin domains fused at the N terminus and the other, a classical 2-domain CuNiR (Br NiR). We report the first enzymatic studies of a novel 4-domain CuNiR from Bradyrhizobium sp. ORS 375 (BrNiR), its genetically engineered 3- and 2-domain variants, and Br NiR revealing up to ~ 500-fold difference in catalytic efficiency in comparison with classical 2-domain CuNiRs. Contrary to the expectation that tethering would enhance electron delivery by restricting the conformational search by having a self-contained donor-acceptor system, we demonstrate that 4-domain BrNiR utilizes N-terminal tethering for downregulating enzymatic activity instead. Both Br NiR and an engineered 2-domain variant of BrNiR (Δ(Cytc-Cup) BrNiR) have 3 to 5% NiR activity compared to the well-characterized 2-domain CuNiRs from Alcaligenes xylosoxidans (AxNiR) and Achromobacter cycloclastes (AcNiR). Structural comparison of Δ(Cytc-Cup) BrNiR and Br NiR with classical 2-domain AxNiR and AcNiR reveals structural differences of the proton transfer pathway that could be responsible for the lowering of activity. Our study provides insights into unique structural and functional characteristics of naturally occurring 4-domain CuNiR and its engineered 3- and 2-domain variants. The reverse protein engineering approach utilized here has shed light onto the broader question of the evolution of transient encounter complexes and tethered electron transfer complexes. ENZYME: Copper-containing nitrite reductase (CuNiR) (EC 1.7.2.1). DATABASE: The atomic coordinate and structure factor of Δ(Cytc-Cup) BrNiR and Br NiR have been deposited in the Protein Data Bank (http://www.rcsb.org/) under the accession code 6THE and 6THF, respectively.
Topics: Achromobacter cycloclastes; Alcaligenes; Amino Acid Sequence; Azurin; Bacterial Proteins; Bradyrhizobium; Catalytic Domain; Cloning, Molecular; Copper; Crystallography, X-Ray; Cytochromes c; Electrons; Escherichia coli; Gene Expression; Genetic Vectors; Models, Molecular; Nitrite Reductases; Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Engineering; Protein Interaction Domains and Motifs; Protons; Recombinant Proteins; Reverse Genetics; Sequence Alignment; Sequence Homology, Amino Acid; Substrate Specificity
PubMed: 32255260
DOI: 10.1111/febs.15324 -
Cell Reports. Medicine May 2024Bacteria-based therapies are powerful strategies for cancer therapy, yet their clinical application is limited by a lack of tunable genetic switches to safely regulate...
Bacteria-based therapies are powerful strategies for cancer therapy, yet their clinical application is limited by a lack of tunable genetic switches to safely regulate the local expression and release of therapeutic cargoes. Rapid advances in remote-control technologies have enabled precise control of biological processes in time and space. We developed therapeutically active engineered bacteria mediated by a sono-activatable integrated gene circuit based on the thermosensitive transcriptional repressor TlpA. Through promoter engineering and ribosome binding site screening, we achieved ultrasound (US)-induced protein expression and secretion in engineered bacteria with minimal noise and high induction efficiency. Specifically, delivered either intratumorally or intravenously, engineered bacteria colonizing tumors suppressed tumor growth through US-irradiation-induced release of the apoptotic protein azurin and an immune checkpoint inhibitor, a nanobody targeting programmed death-ligand 1, in different tumor mouse models. Beyond developing safe and high-performance designer bacteria for tumor therapy, our study illustrates a sonogenetics-controlled therapeutic platform that can be harnessed for bacteria-based precision medicine.
Topics: Animals; Mice; Humans; Neoplasms; Disease Models, Animal; Cell Line, Tumor; Female; B7-H1 Antigen; Immune Checkpoint Inhibitors; Escherichia coli
PubMed: 38608697
DOI: 10.1016/j.xcrm.2024.101513 -
Iranian Journal of Basic Medical... 2023Colon cancer is well-known as a life-threatening disease. Since the current treatment modalities for this type of cancer are powerful yet face some limitations, finding...
OBJECTIVES
Colon cancer is well-known as a life-threatening disease. Since the current treatment modalities for this type of cancer are powerful yet face some limitations, finding novel treatments is required to achieve better outcomes with fewer side effects. Here we investigated the therapeutic potential of Azurin-p28 alone or along with iRGD (Ac-CRGDKGPDC-amide) as a tumor-penetrating peptide and 5-fluorouracil (5-FU) for colon cancer.
MATERIALS AND METHODS
Inhibitory effect of p28 with or without iRGD/5-FU was studied in CT26 and HT29, as well as the xenograft animal model of cancer. The effect of p28 alone or along with iRGD/5-FU on cell migration, apoptotic activity, and cell cycle of the cell lines was assessed. Level of the BAX and BCL2 genes, tumor suppressor genes [(p53 and collagen type-Iα1 (COL1A1), collagen type-Iα2 (COL1A2)] were assessed by quantitative RT-PCR.
RESULTS
These findings show that using p28 with or without iRGD and 5-FU raised the level of p53 and BAX but decreased BCL2, compared with control and 5-FU groups in tissues of the tumor, which result in raising the apoptosis.
CONCLUSION
It seems that p28 may be used as a new therapeutic approach in colon cancer therapy that can enhance the anti-tumor effect of 5-FU.
PubMed: 37396945
DOI: 10.22038/IJBMS.2023.68331.14913 -
Proceedings of the National Academy of... Jul 2022The formation of carbon-carbon bonds from prebiotic precursors such as carbon dioxide represents the foundation of all primordial life processes. In extant organisms,...
The formation of carbon-carbon bonds from prebiotic precursors such as carbon dioxide represents the foundation of all primordial life processes. In extant organisms, this reaction is carried out by the carbon monoxide dehydrogenase (CODH)/acetyl coenzyme A synthase (ACS) enzyme, which performs the cornerstone reaction in the ancient Wood-Ljungdahl metabolic pathway to synthesize the key biological metabolite, acetyl-CoA. Despite its significance, a fundamental understanding of this transformation is lacking, hampering efforts to harness analogous chemistry. To address these knowledge gaps, we have designed an artificial metalloenzyme within the azurin protein scaffold as a structural, functional, and mechanistic model of ACS. We demonstrate the intermediacy of the Ni species and requirement for ordered substrate binding in the bioorganometallic carbon-carbon bond-forming reaction from the one-carbon ACS substrates. The electronic and geometric structures of the nickel-acetyl intermediate have been characterized using time-resolved optical, electron paramagnetic resonance, and X-ray absorption spectroscopy in conjunction with quantum chemical calculations. Moreover, we demonstrate that the nickel-acetyl species is chemically competent for selective acyl transfer upon thiol addition to biosynthesize an activated thioester. Drawing an analogy to the native enzyme, a mechanism for thioester generation by this ACS model has been proposed. The fundamental insight into the enzymatic process provided by this rudimentary ACS model has implications for the evolution of primitive ACS-like proteins. Ultimately, these findings offer strategies for development of highly active catalysts for sustainable generation of liquid fuels from one-carbon substrates, with potential for broad applications across diverse fields ranging from energy storage to environmental remediation.
Topics: Aldehyde Oxidoreductases; Azurin; Catalysis; Esters; Models, Chemical; Multienzyme Complexes; Nickel; Origin of Life; Sulfur Compounds
PubMed: 35858422
DOI: 10.1073/pnas.2123022119 -
PLoS Pathogens Dec 2019The type VI secretion system (T6SS) is widely distributed in Gram-negative bacteria, whose function is known to translocate substrates to eukaryotic and prokaryotic...
The type VI secretion system (T6SS) is widely distributed in Gram-negative bacteria, whose function is known to translocate substrates to eukaryotic and prokaryotic target cells to cause host damage or as a weapon for interbacterial competition. Pseudomonas aeruginosa encodes three distinct T6SS clusters (H1-, H2-, and H3-T6SS). The H1-T6SS-dependent substrates have been identified and well characterized; however, only limited information is available for the H2- and H3-T6SSs since relatively fewer substrates for them have yet been established. Here, we obtained P. aeruginosa H2-T6SS-dependent secretomes and further characterized the H2-T6SS-dependent copper (Cu2+)-binding effector azurin (Azu). Our data showed that both azu and H2-T6SS were repressed by CueR and were induced by low concentrations of Cu2+. We also identified the Azu-interacting partner OprC, a Cu2+-specific TonB-dependent outer membrane transporter. Similar to H2-T6SS genes and azu, expression of oprC was directly regulated by CueR and was induced by low Cu2+. In addition, the Azu-OprC-mediated Cu2+ transport system is critical for P. aeruginosa cells in bacterial competition and virulence. Our findings provide insights for understanding the diverse functions of T6SSs and the role of metal ions for P. aeruginosa in bacteria-bacteria competition.
Topics: Animals; Bacterial Proteins; Copper; DNA-Binding Proteins; Mice; Pseudomonas Infections; Pseudomonas aeruginosa; Type VI Secretion Systems; Virulence
PubMed: 31790504
DOI: 10.1371/journal.ppat.1008198 -
Dalton Transactions (Cambridge, England... Oct 2020Synthesis and characterisation of a dithiadiaza chelator NSNS2A, as well as copper complexes thereof are reported in this paper. Solution structures of copper(i/ii)...
Synthesis and characterisation of a dithiadiaza chelator NSNS2A, as well as copper complexes thereof are reported in this paper. Solution structures of copper(i/ii) complexes were calculated using density functional theory (DFT) and validated by both NMR and EPR spectroscopy. DFT calculations revealed a switch in the orientation of tetragonal distortion upon protonation, which might be responsible for poor stability of the Cu(II)NSNS2A complex in aqueous media, whilst the same switch in tetragonal distortion was experimentally observed by changing the solvent. The chelator was radiolabeled with 64Cu and evaluated using PET/MRI in rats. Despite a favorable redox potential to stabilize the cuprous state in vivo, the 64Cu(II)NSNS2A complex showed suboptimal stability compared to its tetraazamacrocyclic analogue, 64Cu(TE2A), with a significant 64Cu uptake in the liver.
Topics: Animals; Aza Compounds; Azurin; Chelating Agents; Coordination Complexes; Copper Radioisotopes; Density Functional Theory; Electrochemical Techniques; Kidney; Liver; Macrocyclic Compounds; Magnetic Resonance Imaging; Male; Molecular Conformation; Oxidation-Reduction; Positron-Emission Tomography; Protein Binding; Radiopharmaceuticals; Rats, Wistar; Solvents; Staining and Labeling; Structure-Activity Relationship
PubMed: 32970072
DOI: 10.1039/d0dt02787a -
The Journal of Physical Chemistry. B Sep 2022The protein, azurin, has enabled the study of the tryptophan radical. Upon UV excitation of tyrosine-deficient apoazurin and in the presence of a Co(III) electron...
The protein, azurin, has enabled the study of the tryptophan radical. Upon UV excitation of tyrosine-deficient apoazurin and in the presence of a Co(III) electron acceptor, the neutral radical (W48•) is formed. The lifetime of W48• in apoazurin is 41 s, which is shorter than the lifetime of several hours in Zn-substituted azurin. Molecular dynamics simulations revealed enhanced fluctuations of apoazurin which likely destabilize W48•. The photophysics of W48 was investigated to probe the precursor state for ET. The phosphorescence intensity was eliminated in the presence of an electron acceptor while the fluorescence was unchanged; this quenching of the phosphorescence is attributed to ET. The kinetics associated with W48• were examined with a model that incorporates intersystem crossing, ET, deprotonation, and decay of the cation radical. The estimated rate constants for ET (6 × 10 s) and deprotonation (3 × 10 s) are in agreement with a photoinduced mechanism where W48• is derived from the triplet state. The triplet as the precursor state for ET was supported by photolysis of apoazurin with 280 nm in the absence and presence of triplet-absorbing 405 nm light. Absorption bands from the neutral radical were observed only in the presence of blue light.
Topics: Apoproteins; Azurin; Kinetics; Tryptophan
PubMed: 35977067
DOI: 10.1021/acs.jpcb.2c02441