-
BioRxiv : the Preprint Server For... Mar 2024Discovering new bacterial signaling pathways offers unique antibiotic strategies. Here, through an unbiased resistance screen of 3,884 gene knockout strains, we...
Discovering new bacterial signaling pathways offers unique antibiotic strategies. Here, through an unbiased resistance screen of 3,884 gene knockout strains, we uncovered a previously unknown non-lytic bactericidal mechanism that sequentially couples three transporters and downstream transcription to lethally suppress respiration of the highly virulent strain PA14 - one of three species on the WHO's 'Priority 1: Critical' list. By targeting outer membrane YaiW, cationic lacritin peptide 'N-104' translocates into the periplasm where it ligates outer loops 4 and 2 of the inner membrane transporters FeoB and PotH, respectively, to suppress both ferrous iron and polyamine uptake. This broadly shuts down transcription of many biofilm-associated genes, including ferrous iron-dependent TauD and ExbB1. The mechanism is innate to the surface of the eye and is enhanced by synergistic coupling with thrombin peptide GKY20. This is the first example of an inhibitor of multiple bacterial transporters.
PubMed: 38464199
DOI: 10.1101/2024.03.01.582947 -
The Journal of Chemical Physics Oct 2023Metal-reducing bacteria have adapted the ability to respire extracellular solid surfaces instead of soluble oxidants. This process requires an electron transport pathway...
Metal-reducing bacteria have adapted the ability to respire extracellular solid surfaces instead of soluble oxidants. This process requires an electron transport pathway that spans from the inner membrane, across the periplasm, through the outer membrane, and to an external surface. Multiheme cytochromes are the primary machinery for moving electrons through this pathway. Recent studies show that the chiral-induced spin selectivity (CISS) effect is observable in some of these proteins extracted from the model metal-reducing bacteria, Shewanella oneidensis MR-1. It was hypothesized that the CISS effect facilitates efficient electron transport in these proteins by coupling electron velocity to spin, thus reducing the probability of backscattering. However, these studies focused exclusively on the cell surface electron conduits, and thus, CISS has not been investigated in upstream electron transfer components such as the membrane-associated MtrA, or periplasmic proteins such as small tetraheme cytochrome (STC). By using conductive probe atomic force microscopy measurements of protein monolayers adsorbed onto ferromagnetic substrates, we show that electron transport is spin selective in both MtrA and STC. Moreover, we have determined the spin polarization of MtrA to be ∼77% and STC to be ∼35%. This disparity in spin polarizations could indicate that spin selectivity is length dependent in heme proteins, given that MtrA is approximately two times longer than STC. Most significantly, our study indicates that spin-dependent interactions affect the entire extracellular electron transport pathway.
Topics: Electron Transport; Oxidation-Reduction; Electrons; Periplasm; Metals; Bacteria; Bacterial Proteins; Bacterial Outer Membrane Proteins
PubMed: 37811828
DOI: 10.1063/5.0154211 -
IScience Oct 2023The mechanism by which a bacterial cell senses external nutrients remains largely unknown. In this study, we identified a bacterial cell sensing system for polycyclic...
The mechanism by which a bacterial cell senses external nutrients remains largely unknown. In this study, we identified a bacterial cell sensing system for polycyclic aromatic hydrocarbons (PAHs) in a common marine PAH-using bacterium, . It consists of an outer membrane receptor (PahS) and a periplasmic protein (PahP) in combination with a two-component sensing system (TCS) that ensures a rapid response to PAH occurrence by directly controlling serial reactions including chemotactic sensing and movement, PAH uptake and intracellular PAH metabolism. PahS protrudes from the cell and acts as a PAH sensor, transducing the PAH signal across the outer membrane to its periplasmic partner PahP, which in turn transduces the PAH signal across the periplasm to a specialized TCS. This sensing system plays a critical role in sensing and promoting the metabolism of PAHs, which can be scavenged by various hydrocarbon-degrading bacteria.
PubMed: 37841585
DOI: 10.1016/j.isci.2023.107912 -
The Journal of Biological Chemistry Nov 2023Enzymatic modifications of bacterial exopolysaccharides enhance immune evasion and persistence during infection. In the Gram-negative opportunistic pathogen Pseudomonas...
Enzymatic modifications of bacterial exopolysaccharides enhance immune evasion and persistence during infection. In the Gram-negative opportunistic pathogen Pseudomonas aeruginosa, acetylation of alginate reduces opsonic killing by phagocytes and improves reactive oxygen species scavenging. Although it is well known that alginate acetylation in P. aeruginosa requires AlgI, AlgJ, AlgF, and AlgX, how these proteins coordinate polymer modification at a molecular level remains unclear. Here, we describe the structural characterization of AlgF and its protein interaction network. We characterize direct interactions between AlgF and both AlgJ and AlgX in vitro and demonstrate an association between AlgF and AlgX, as well as AlgJ and AlgI, in P. aeruginosa. We determine that AlgF does not exhibit acetylesterase activity and is unable to bind to polymannuronate in vitro. Therefore, we propose that AlgF functions to mediate protein-protein interactions between alginate acetylation enzymes, forming the periplasmic AlgJFXK (AlgJ-AlgF-AlgX-AlgK) acetylation and export complex required for robust biofilm formation.
Topics: Acetylation; Alginates; Bacterial Proteins; Biofilms; Periplasm; Protein Processing, Post-Translational; Pseudomonas aeruginosa
PubMed: 37797696
DOI: 10.1016/j.jbc.2023.105314 -
Scientific Reports Nov 2023Periplasmic solute-binding proteins (SBPs) specific for chitooligosaccharides, (GlcNAc) (n = 2, 3, 4, 5 and 6), are involved in the uptake of chitinous nutrients and...
Periplasmic solute-binding proteins (SBPs) specific for chitooligosaccharides, (GlcNAc) (n = 2, 3, 4, 5 and 6), are involved in the uptake of chitinous nutrients and the negative control of chitin signal transduction in Vibrios. Most translocation processes by SBPs across the inner membrane have been explained thus far by two-domain open/closed mechanism. Here we propose three-domain mechanism of the (GlcNAc) translocation based on experiments using a recombinant VcCBP, SBP specific for (GlcNAc) from Vibrio cholerae. X-ray crystal structures of unliganded or (GlcNAc)-liganded VcCBP solved at 1.2-1.6 Å revealed three distinct domains, the Upper1, Upper2 and Lower domains for this protein. Molecular dynamics simulation indicated that the motions of the three domains are independent and that in the (GlcNAc)-liganded state the Upper2/Lower interface fluctuated more intensively, compared to the Upper1/Lower interface. The Upper1/Lower interface bound two GlcNAc residues tightly, while the Upper2/Lower interface appeared to loosen and release the bound sugar molecule. The three-domain mechanism proposed here was fully supported by binding data obtained by thermal unfolding experiments and ITC, and may be applicable to other translocation systems involving SBPs belonging to the same cluster.
Topics: Humans; Periplasmic Binding Proteins; Chitosan; Chitin; Carrier Proteins; Molecular Dynamics Simulation; Ligands; Translocation, Genetic; Crystallography, X-Ray
PubMed: 37996461
DOI: 10.1038/s41598-023-47253-y -
Biochemical and Biophysical Research... Nov 2023Klebsiella pneumoniae, a facultative anaerobe, relies on acquiring molybdenum to sustain growth in anaerobic conditions, a crucial factor for the pathogen to establish...
Klebsiella pneumoniae, a facultative anaerobe, relies on acquiring molybdenum to sustain growth in anaerobic conditions, a crucial factor for the pathogen to establish infections within host environments. Molybdenum plays a critical role in pathogenesis as it forms an essential component of cofactors for molybdoenzymes. K. pneumoniae utilizes the ABC (ATP-Binding-Cassette) transporter encoded by the modABC operon for uptake of the group VI elements molybdenum and tungsten. In this study, we determined the X-ray crystal structures of both the molybdenum-free and molybdenum-bound substrate-binding protein (SBP) ModA from Klebsiella pneumoniae to 2.00 Å and 1.77 Å resolution respectively. ModA crystallizes in the space group P222 with a single monomer in one asymmetric unit. The purified protein remained soluble and specifically bound molybdate and tungstate with K values of 6.3 nM and 5.2 nM, respectively. Tungstate competes with molybdate by binding to ModA, resulting in enhanced antimicrobial activity. These data provide a starting point for structural and functional analyses of molybdate transport in K. pneumoniae.
Topics: Molybdenum; Klebsiella pneumoniae; Bacterial Proteins; Periplasmic Binding Proteins; ATP-Binding Cassette Transporters; Protein Binding
PubMed: 37751633
DOI: 10.1016/j.bbrc.2023.09.055 -
FEBS Letters Jun 2024Modular assembly is a compelling pathway to create new proteins, a concept supported by protein engineering and millennia of evolution. Natural evolution provided a...
Modular assembly is a compelling pathway to create new proteins, a concept supported by protein engineering and millennia of evolution. Natural evolution provided a repository of building blocks, known as domains, which trace back to even shorter segments that underwent numerous 'copy-paste' processes culminating in the scaffolds we see today. Utilizing the subdomain-database Fuzzle, we constructed a fold-chimera by integrating a flavodoxin-like fragment into a periplasmic binding protein. This chimera is well-folded and a crystal structure reveals stable interfaces between the fragments. These findings demonstrate the adaptability of α/β-proteins and offer a stepping stone for optimization. By emphasizing the practicality of fragment databases, our work pioneers new pathways in protein engineering. Ultimately, the results substantiate the conjecture that periplasmic binding proteins originated from a flavodoxin-like ancestor.
Topics: Protein Folding; Protein Engineering; Models, Molecular; Flavodoxin; Periplasmic Binding Proteins; Crystallography, X-Ray; Recombinant Fusion Proteins; Protein Domains
PubMed: 38508768
DOI: 10.1002/1873-3468.14856 -
Environmental Science & Technology Aug 2023Denitrification-driven Fe(II) oxidation is an important microbial metabolism that connects iron and nitrogen cycling in the environment. The formation of Fe(III)...
Denitrification-driven Fe(II) oxidation is an important microbial metabolism that connects iron and nitrogen cycling in the environment. The formation of Fe(III) minerals in the periplasmic space has a significant effect on microbial metabolism and electron transfer, but direct evidence of iron ions entering the periplasm and resulting in periplasmic mineral precipitation and electron conduction properties has yet to be conclusively determined. Here, we investigated the pathways and amounts of iron, with different valence states and morphologies, entering the periplasmic space of the denitrifier sp. JM-7 ( JM-7), and the possible effects on the electron transfer and the denitrifying ability. When consistently provided with Fe(II) ions (from siderite (FeCO)), the dissolved Fe(II) ions entered the periplasmic space and were oxidized to Fe(III), leading to the formation of a 25 nm thick crystalline goethite crust, which functioned as a semiconductor, accelerating the transfer of electrons from the intracellular to the extracellular matrix. This consequently doubled the denitrification rate and increased the electron transport capacity by 4-30 times (0.015-0.04 μA). However, as the Fe(II) concentration further increased to above 4 mM, the Fe(II) ions tended to preferentially nucleate, oxidize, and crystallize on the outer surface of JM-7, leading to the formation of a densely crystallized goethite layer, which significantly slowed down the metabolism of JM-7. In contrast to the Fe(II) conditions, regardless of the initial concentration of Fe(III), it was challenging for Fe(III) ions to form goethite in the periplasmic space. This work has shed light on the likely effects of iron on environmental microorganisms, improved our understanding of globally significant iron and nitrogen geochemical cycles in water, and expanded our ability to study and control these important processes.
Topics: Ferric Compounds; Periplasm; Water; Denitrification; Iron Compounds; Minerals; Iron; Oxidation-Reduction; Ferrous Compounds; Nitrogen
PubMed: 37467428
DOI: 10.1021/acs.est.3c02303 -
Microorganisms Jul 2023Prokaryotic extracellular vesicles (EVs) are vesicles that bud from the cell membrane and are secreted by bacteria and archaea. EV cargo in Gram-negative bacteria...
Prokaryotic extracellular vesicles (EVs) are vesicles that bud from the cell membrane and are secreted by bacteria and archaea. EV cargo in Gram-negative bacteria includes mostly periplasmic and outer membrane proteins. EVs are clinically important as their cargo can include toxins associated with bacterial virulence and toxicity; additionally, they have been proposed as efficient vaccine agents and as the ancestors of the eukaryotic endomembrane system. However, the mechanistic details behind EV cargo selection and release are still poorly understood. In this study, we have performed bioinformatics analysis of published data on EV proteomes from 38 species of bacteria and 4 archaea. Focusing on clusters of orthologous genes (COGs) and using the EggNOG mapper function, we have identified cargo proteins that are commonly found in EVs across species. We discuss the putative role of these prominent proteins in EV biogenesis and function. We also analyzed the published EV proteomes for conserved signal sequences and discuss the potential role of these signal sequences for EV cargo selection.
PubMed: 37630535
DOI: 10.3390/microorganisms11081977 -
Recent Patents on Biotechnology 2024Immune thrombocytopenic purpura (ITP) is an autoimmune disorder determined by immune-mediated platelet demolition and reduction of platelet production. Romiplostim is a... (Review)
Review
Immune thrombocytopenic purpura (ITP) is an autoimmune disorder determined by immune-mediated platelet demolition and reduction of platelet production. Romiplostim is a new thrombopoiesis motivating peptibody that binds and stimulates the human thrombopoietin receptor the patent of which was registered in 2008. It is used to treat thrombocytopenia in patients with chronic immune thrombocytopenic purpura. Romiplostim is a 60 kDa peptibody designed to inhibit cross-reacting immune responses. It consists of four high-affinity TPO-receptor binding domains for the Mpl receptor and one human IgG1 Fc domain. is a good host for the fabrication of recombinant proteins such as romiplostim. The expression of a gene intended in is dependent on many factors such as a protein's inherent ability to fold, mRNA's secondary structure, its solubility, its toxicity preferential codon use, and its need for post-translational modification (PTM). This review focuses on the structure, function, mechanism of action, and expressive approach to romiplostim in .
Topics: Humans; Purpura, Thrombocytopenic, Idiopathic; Escherichia coli; Patents as Topic; Blood Platelets; Thrombopoietin; Receptors, Fc; Recombinant Fusion Proteins
PubMed: 38282441
DOI: 10.2174/1872208317666230503094451