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Proceedings of the National Academy of... May 2024Cell surface glycans are major drivers of antigenic diversity in bacteria. The biochemistry and molecular biology underpinning their synthesis are important in...
Cell surface glycans are major drivers of antigenic diversity in bacteria. The biochemistry and molecular biology underpinning their synthesis are important in understanding host-pathogen interactions and for vaccine development with emerging chemoenzymatic and glycoengineering approaches. Structural diversity in glycostructures arises from the action of glycosyltransferases (GTs) that use an immense catalog of activated sugar donors to build the repeating unit and modifying enzymes that add further heterogeneity. Classical Leloir GTs incorporate α- or β-linked sugars by inverting or retaining mechanisms, depending on the nucleotide sugar donor. In contrast, the mechanism of known ribofuranosyltransferases is confined to β-linkages, so the existence of α-linked ribofuranose in some glycans dictates an alternative strategy. Here, we use O1 and O2 lipopolysaccharide O antigens as prototypes to describe a widespread, versatile pathway for incorporating side-chain α-linked pentofuranoses by extracytoplasmic postpolymerization glycosylation. The pathway requires a polyprenyl phosphoribose synthase to generate a lipid-linked donor, a MATE-family flippase to transport the donor to the periplasm, and a GT-C type GT (founding the GT136 family) that performs the final glycosylation reaction. The characterized system shares similarities, but also fundamental differences, with both cell wall arabinan biosynthesis in mycobacteria, and periplasmic glucosylation of O antigens first discovered in and . The participation of auxiliary epimerases allows the diversification of incorporated pentofuranoses. The results offer insight into a broad concept in microbial glycobiology and provide prototype systems and bioinformatic guides that facilitate discovery of further examples from diverse species, some in currently unknown glycans.
Topics: Glycosyltransferases; Glycosylation; Citrobacter; O Antigens; Polysaccharides; Bacterial Proteins; Polysaccharides, Bacterial
PubMed: 38748580
DOI: 10.1073/pnas.2402554121 -
BioRxiv : the Preprint Server For... Apr 2024Cell growth in mycobacteria involves cell wall expansion that is restricted to the cell poles. The DivIVA homolog Wag31 is required for this process, but the molecular...
Cell growth in mycobacteria involves cell wall expansion that is restricted to the cell poles. The DivIVA homolog Wag31 is required for this process, but the molecular mechanism and protein partners of Wag31 have not been described. In this study of , we identify a connection between and trehalose monomycolate (TMM) transporter in a suppressor screen, and show that Wag31 and polar regulator PlrA are required for MmpL3's polar localization. In addition, the localization of PlrA and MmpL3 are responsive to nutrient and energy deprivation and inhibition of peptidoglycan metabolism. We show that inhibition of MmpL3 causes delocalized cell wall metabolism, but does not delocalize MmpL3 itself. We found that cells with an MmpL3 C-terminal truncation, which is defective for localization, have only minor defects in polar growth, but are impaired in their ability to downregulate cell wall metabolism under stress. Our work suggests that, in addition to its established function in TMM transport, MmpL3 has a second function in regulating global cell wall metabolism in response to stress. Our data are consistent with a model in which the presence of TMMs in the periplasm stimulates polar elongation, and in which the connection between Wag31, PlrA and the C-terminus of MmpL3 is involved in detecting and responding to stress in order to coordinate synthesis of the different layers of the mycobacterial cell wall in changing conditions.
PubMed: 38746181
DOI: 10.1101/2024.04.29.591792 -
Nature Communications May 2024Microbial Ni homeostasis underpins the virulence of several clinical pathogens. Ni is an essential cofactor in urease and [NiFe]-hydrogenases involved in colonization...
Microbial Ni homeostasis underpins the virulence of several clinical pathogens. Ni is an essential cofactor in urease and [NiFe]-hydrogenases involved in colonization and persistence. Many microbes produce metallophores to sequester metals necessary for their metabolism and starve competing neighboring organisms. The fungal metallophore aspergillomarasmine A (AMA) shows narrow specificity for Zn, Ni, and Co. Here, we show that this specificity allows AMA to block the uptake of Ni and attenuate bacterial Ni-dependent enzymes, offering a potential strategy for reducing virulence. Bacterial exposure to AMA perturbs H metabolism, ureolysis, struvite crystallization, and biofilm formation and shows efficacy in a Galleria mellonella animal infection model. The inhibition of Ni-dependent enzymes was aided by Zn which complexes with AMA and competes with the native nickelophore for the uptake of Ni. Biochemical analyses demonstrated high-affinity binding of AMA-metal complexes to NikA, the periplasmic substrate-binding protein of the Ni uptake system. Structural examination of NikA in complex with Ni-AMA revealed that the coordination geometry of Ni-AMA mimics the native ligand, Ni-(L-His), providing a structural basis for binding AMA-metal complexes. Structure-activity relationship studies of AMA identified regions of the molecule that improve NikA affinity and offer potential routes for further developing this compound as an anti-virulence agent.
Topics: Nickel; Animals; Virulence; Bacterial Proteins; Biofilms; Zinc; Moths; Urease; Biological Transport
PubMed: 38740750
DOI: 10.1038/s41467-024-48232-1 -
Chemosphere Jul 2024Titanate nanotubes (TNs) functionalized with CuS nanoparticles using the microwave-assisted hydrothermal method were characterized via XRD, Raman spectroscopy, UV-Vis...
Titanate nanotubes (TNs) functionalized with CuS nanoparticles using the microwave-assisted hydrothermal method were characterized via XRD, Raman spectroscopy, UV-Vis spectrophotometry, and N physisorption. The as-synthesized CuS/TNs had anatase as the main crystalline phase and the band-gap energy was in the visible region, 2.9 eV. The TNs were recrystallized on titania and functionalized with CuS, forming spherical bundles. SEM showed agglomerates of cauliflower-like semispherical particles. The antimicrobial photoactive assets were evaluated against the bacteria Staphylococcus aureus and Escherichia coli. Inhibition was clearly visible in S. aureus after the first 20 min of exposure to a 6-W LED irradiation lamp. The visible-light catalyzed completely and irreversibly the inactivation of S. aureus after 60 min, however, in the case of E. coli, this material only slightly disturbed its growth, which was recovered after 60 min. The successful result obtained with S. aureus can be explained by the fact that it lacks periplasmic superoxide dismutase (SOD) but has staphyloxanthin for external protection against ROS. However, the CuS/TN particles could release Cu ions, which got attached to bacterium structures or entered the cytoplasm; these events together with the generation of ROS under visible LED light helped inactivate quickly staphyloxanthin, thus inflicting permanent damage to the periplasmic membrane.
Topics: Staphylococcus aureus; Titanium; Escherichia coli; Copper; Light; Anti-Infective Agents; Nanotubes; Anti-Bacterial Agents
PubMed: 38735492
DOI: 10.1016/j.chemosphere.2024.142317 -
ACS Infectious Diseases Jun 2024The design of siderophore-antibiotic conjugates is a promising strategy to overcome drug resistance in negative bacteria. However, accumulating studies have shown that...
The design of siderophore-antibiotic conjugates is a promising strategy to overcome drug resistance in negative bacteria. However, accumulating studies have shown that only those antibiotics acting on the cell wall or cell membrane multiply their antibacterial effects when coupled with siderophores, while antibiotics acting on targets in the cytoplasm of bacteria do not show an obvious enhancement of their antibacterial effects when coupled with siderophores. To explore the causes of this phenomenon, we synthesized several conjugate probes using 3-hydroxypyridin-4(1)-ones as siderophores and replacing the antibiotic cargo with 5-carboxyfluorescein (5-FAM) or malachite green (MG) cargo. By monitoring changes in the fluorescence intensity of FAM conjugate in bacteria, the translocation of the conjugate across the outer membranes of Gram-negative pathogens was confirmed. Further, the use of the fluorogen activating protein(FAP)/MG system revealed that 3-hydroxypyridin-4(1)-one-MG conjugate was ultimately distributed mainly in the periplasm rather than being translocated into the cytosol of and PAO1. Additional mechanistic studies suggested that the uptake of the conjugate involved the siderophore-dependent iron transport pathway and the 3-hydroxypyridin-4(1)-ones siderophore receptor-dependent mechanism. Meanwhile, we demonstrated that the conjugation of 3-hydroxypyridin-4(1)-ones to the fluorescein 5-FAM can reduce the possibility of the conjugates crossing the membrane layers of mammalian Vero cells by passive diffusion, and the advantages of the mono-3-hydroxypyridin-4(1)-ones as a delivery vehicle in the design of conjugates compared to the tri-3-hydroxypyridin-4(1)-ones. Overall, this work reveals the localization rules of 3-hydroxypyridin-4(1)-ones as siderophores to deliver the cargo into Gram-negative bacteria. It provides a theoretical basis for the subsequent design of siderophore-antibiotic conjugates, especially based on 3-hydroxypyridin-4(1)-ones as siderophores.
Topics: Siderophores; Anti-Bacterial Agents; Pseudomonas aeruginosa; Gram-Negative Bacteria; Fluorescent Dyes; Escherichia coli; Pyridones; Pyridines; Animals; Fluorescein; Biological Transport; Microbial Sensitivity Tests
PubMed: 38725130
DOI: 10.1021/acsinfecdis.4c00287 -
Current Opinion in Microbiology Jun 2024Bacteria surround themselves with complex cell envelopes to maintain their integrity and protect against external insults. The envelope of Gram-negative organisms is... (Review)
Review
Bacteria surround themselves with complex cell envelopes to maintain their integrity and protect against external insults. The envelope of Gram-negative organisms is multilayered, with two membranes sandwiching the periplasmic space that contains the peptidoglycan cell wall. Understanding how this complicated surface architecture is assembled during cell growth and division is a major fundamental problem in microbiology. Additionally, because the envelope is an important antibiotic target and determinant of intrinsic antibiotic resistance, understanding the mechanisms governing its assembly is relevant to therapeutic development. In the last several decades, most of the factors required to build the Gram-negative envelope have been identified. However, surprisingly, little is known about how the biogenesis of the different cell surface layers is co-ordinated. Here, we provide an overview of recent work that is beginning to uncover the links connecting the different envelope biosynthetic pathways and assembly machines to ensure uniform envelope growth.
Topics: Gram-Negative Bacteria; Cell Wall; Peptidoglycan; Cell Membrane; Bacterial Proteins
PubMed: 38718542
DOI: 10.1016/j.mib.2024.102479 -
Biochemical and Biophysical Research... Jul 2024Sugar phosphates are potential sources of carbon and phosphate for bacteria. Despite that the process of internalization of Glucose-6-Phosphate (G6P) through plasma...
Sugar phosphates are potential sources of carbon and phosphate for bacteria. Despite that the process of internalization of Glucose-6-Phosphate (G6P) through plasma membrane remained elusive in several bacteria. VCA0625-27, made of periplasmic ligand binding protein (PLBP) VCA0625, an atypical monomeric permease VCA0626, and a cytosolic ATPase VCA0627, recently emerged as hexose-6-phosphate uptake system of Vibrio cholerae. Here we report high resolution crystal structure of VCA0625 in G6P bound state that largely resembles AfuA of Actinobacillus pleuropneumoniae. MD simulations on VCA0625 in apo and G6P bound states unraveled an 'open to close' and swinging bi-lobal motions, which are diminished upon G6P binding. Mutagenesis followed by biochemical assays on VCA0625 underscored that R34 works as gateway to bind G6P. Although VCA0627 binds ATP, it is ATPase deficient in the absence of VCA0625 and VCA0626, which is a signature phenomenon of type-I ABC importer. Further, modeling, docking and systematic sequence analysis allowed us to envisage the existence of similar atypical type-I G6P importer with fused monomeric permease in 27 other gram-negative bacteria.
Topics: Vibrio cholerae; Bacterial Proteins; Crystallography, X-Ray; Glucose-6-Phosphate; ATP-Binding Cassette Transporters; Molecular Dynamics Simulation; Protein Conformation; Models, Molecular; Protein Binding; Binding Sites
PubMed: 38704889
DOI: 10.1016/j.bbrc.2024.150030 -
Marine Pollution Bulletin Jun 2024The impact of heavy metal ions on the biodenitrification process remains unknown, which is the key to understand the nitrogen cycle in estuarine areas. Here,...
The impact of heavy metal ions on the biodenitrification process remains unknown, which is the key to understand the nitrogen cycle in estuarine areas. Here, denitrification rate and the abundance of five denitrifying enzyme genes (narG, nirK, napA, norB and nosZ) in Liaohe Estuary sediments were examined, and the community structure of nirK denitrifying bacteria was also analyzed. The results demonstrate a significant positive correlation between heavy metal content (Cu, Zn, and Cr) and the denitrification rate, and the abundance of napA/norB (periplasmic nitrate reductase and nitric-oxide reductase) in sediments. The dominant narG denitrifiers were Pseudomonas, Hydrogenophaga, and Serratia known to be tolerant to heavy metal pollution. Sediment particle size, NO, NO, Zn, and Cd were the key factors influencing the denitrifying community structure. These findings suggest that heavy metals may enhance the aerobic denitrification process in sediments and mitigate the adverse effects of high dissolved oxygen levels.
Topics: Denitrification; Geologic Sediments; Metals, Heavy; Estuaries; Water Pollutants, Chemical; Bacteria; China; Nitrate Reductase
PubMed: 38696947
DOI: 10.1016/j.marpolbul.2024.116408 -
Biochemistry May 2024Periplasmic solute-binding proteins (SBPs) are key ligand recognition components of bacterial ATP-binding cassette (ABC) transporters that allow bacteria to import...
Periplasmic solute-binding proteins (SBPs) are key ligand recognition components of bacterial ATP-binding cassette (ABC) transporters that allow bacteria to import nutrients and metabolic precursors from the environment. Periplasmic SBPs comprise a large and diverse family of proteins, of which only a small number have been empirically characterized. In this work, we identify a set of 610 unique uncharacterized proteins within the SBP_bac_5 family that are found in conserved operons comprising genes encoding (i) ABC transport systems and (ii) putative amidases from the FmdA_AmdA family. From these uncharacterized SBP_bac_5 proteins, we characterize a representative periplasmic SBP from sp. A09 (Ami_SBP) and show that Ami_SBP binds l-amino acid amides but not the corresponding l-amino acids. An X-ray crystal structure of Ami_SBP bound to l-serinamide highlights the residues that impart distinct specificity for l-amino acid amides and reveals a structural Ca binding site within one of the lobes of the protein. We show that the residues involved in ligand and Ca binding are conserved among the 610 SBPs from experimentally uncharacterized FmdA_AmdA amidase-associated ABC transporter systems, suggesting these homologous systems are also likely to be involved in the sensing, uptake, and metabolism of l-amino acid amides across many Gram-negative nitrogen-fixing soil bacteria. We propose that Ami_SBP is involved in the uptake of such solutes to supplement pathways such as the citric acid cycle and the glutamine synthetase-glutamate synthase pathway. This work expands our currently limited understanding of microbial interactions with l-amino acid amides and bacterial nitrogen utilization.
Topics: Amides; Crystallography, X-Ray; Periplasmic Binding Proteins; ATP-Binding Cassette Transporters; Amino Acids; Mesorhizobium; Bacterial Proteins; Binding Sites; Models, Molecular; Amidohydrolases; Calcium; Protein Binding
PubMed: 38696389
DOI: 10.1021/acs.biochem.4c00096 -
Protein Expression and Purification Aug 2024Phytate (inositol hexaphosphate) is the major storage form of phosphorus (P) in nature, and phytases catalyze the hydrolysis of P from phytate and the formation of...
Phytate (inositol hexaphosphate) is the major storage form of phosphorus (P) in nature, and phytases catalyze the hydrolysis of P from phytate and the formation of inositol phosphate isomers. In this study, a bacterium that produces phytase was isolated in a phytase screening medium. The bacterium was identified as Klebsiella sp. using phenotypic and molecular techniques. The PhyK phytase gene was successfully amplified from the genome, inserted into the pET-21a (+) vector, and expressed as a recombinant protein in E. Coli BL21. The efficiency of a laboratory phytase (Lab-Ph, PhyK phytase) was determined and compared with a commercial phytase (Com-Ph, Quantum Blue 40P phytase, AB Vista) under an in vitro digestion assay. The native signal peptide effectively facilitated the translocation of the protein to the periplasmic space of E. Coli BL21, resulting in the proper folding of the protein and the manifestation of desirable enzyme activity. The Lab-Ph displayed the temperature and pH optima at 50 °C and 5 respectively. In addition, the Lab-Ph was inactivated at 80 °C. Under an in vitro digestion assay condition, Lab-Ph improved the P solubility coefficient in broiler diets. In comparison, the Com-Ph significantly increased the P solubility coefficient even when compared with the Lab-Ph. In summary, this study has shown that Lab-Ph possesses the necessary biochemical properties to be used in various industrial applications. However, Lab-Ph is extremely sensitive to heat treatment. The Lab-Ph and Com-Ph under an in vitro digestion assay improved the solubility coefficient of P in the broiler diet.
Topics: Animals; Chickens; Recombinant Proteins; 6-Phytase; Solubility; Klebsiella; Escherichia coli; Animal Feed; Bacterial Proteins; Hydrogen-Ion Concentration; Minerals; Phytic Acid
PubMed: 38685535
DOI: 10.1016/j.pep.2024.106489