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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 -
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 -
Frontiers in Chemistry 2024Andrographolide is one of the main biologically active molecules isolated from , which is a traditional Chinese herb used extensively throughout Eastern Asia, India, and...
Andrographolide is one of the main biologically active molecules isolated from , which is a traditional Chinese herb used extensively throughout Eastern Asia, India, and China. often known as is a common clinical opportunistic pathogen with remarkable adaptability to harsh settings and resistance to antibiotics. possesses a wide array of virulence traits, one of which is biofilm formation, which contributes to its pathogenicity. One of the main modulators of the -controlled intramembrane proteolysis pathway is AlgW, a membrane-bound periplasmic serine protease. In this work, we have used a set of density functional theory (DFT) calculations to understand the variety of chemical parameters in detail between andrographolide and levofloxacin, which show strong bactericidal activity against . Additionally, the stability and interaction of andrographolide and levofloxacin with the protein AlgW have been investigated by molecular docking and molecular dynamics (MD) simulations . Moreover, the growth and inhibition of biofilm production by experiments were also investigated, providing insight that andrographolide could be a potential natural product to inhibit
PubMed: 38680458
DOI: 10.3389/fchem.2024.1388545 -
ACS Synthetic Biology May 2024is often used as a factory to produce recombinant proteins. In many cases, the recombinant protein needs disulfide bonds to fold and function correctly. These proteins...
is often used as a factory to produce recombinant proteins. In many cases, the recombinant protein needs disulfide bonds to fold and function correctly. These proteins are genetically fused to a signal peptide so that they are secreted to the oxidizing environment of the periplasm (where the enzymes required for disulfide bond formation exist). Currently, it is difficult to determine whether a recombinant protein is efficiently secreted from the cytoplasm and folded in the periplasm or if there is a bottleneck in one of these steps because cellular capacity has been exceeded. To address this problem, we have developed a biosensor that detects cellular stress caused by (1) inefficient secretion of proteins from the cytoplasm and (2) aggregation of proteins in the periplasm. We demonstrate how the fluorescence fingerprint obtained from the biosensor can be used to identify induction conditions that do not exceed the capacity of the cell and therefore do not cause cellular stress. These induction conditions result in more effective biomass and in some cases higher titers of soluble recombinant proteins.
Topics: Biosensing Techniques; Escherichia coli; Periplasmic Proteins; Recombinant Proteins; Periplasm; Stress, Physiological; Escherichia coli Proteins
PubMed: 38676700
DOI: 10.1021/acssynbio.3c00720 -
Viruses Mar 2024The molecular mechanism of how the infecting DNA of bacteriophage T4 passes from the capsid through the bacterial cell wall and enters the cytoplasm is essentially...
The molecular mechanism of how the infecting DNA of bacteriophage T4 passes from the capsid through the bacterial cell wall and enters the cytoplasm is essentially unknown. After adsorption, the short tail fibers of the infecting phage extend from the baseplate and trigger the contraction of the tail sheath, leading to a puncturing of the outer membrane by the tail tip needle composed of the proteins gp5.4, gp5 and gp27. To explore the events that occur in the periplasm and at the inner membrane, we constructed T4 phages that have a modified gp27 in their tail tip with a His-tag. Shortly after infection with these phages, cells were chemically cross-linked and solubilized. The cross-linked products were affinity-purified on a nickel column and the co-purified proteins were identified by mass spectrometry, and we found that predominantly the inner membrane proteins DamX, SdhA and PpiD were cross-linked. The same partner proteins were identified when purified gp27 was added to spheroplasts, suggesting a direct protein-protein interaction.
Topics: Bacteriophage T4; Cell Division; Escherichia coli; Escherichia coli Proteins; Viral Proteins
PubMed: 38675830
DOI: 10.3390/v16040487 -
Molecules (Basel, Switzerland) Apr 2024Antibiotic resistance in Gram-negative bacteria remains one of the most pressing challenges to global public health. Blocking the transportation of lipopolysaccharides...
Antibiotic resistance in Gram-negative bacteria remains one of the most pressing challenges to global public health. Blocking the transportation of lipopolysaccharides (LPS), a crucial component of the outer membrane of Gram-negative bacteria, is considered a promising strategy for drug discovery. In the transportation process of LPS, two components of the LPS transport (Lpt) complex, LptA and LptC, are responsible for shuttling LPS across the periplasm to the outer membrane, highlighting their potential as targets for antibacterial drug development. In the current study, a protein-protein interaction (PPI) model of LptA and LptC was constructed, and a molecular screening strategy was employed to search a protein-protein interaction compound library. The screening results indicated that compound 18593 exhibits favorable binding free energy with LptA and LptC. In comparison with the molecular dynamics (MD) simulations on currently known inhibitors, compound 18593 shows more stable target binding ability at the same level. The current study suggests that compound 18593 may exhibit an inhibitory effect on the LPS transport process, making it a promising hit compound for further research.
Topics: Anti-Bacterial Agents; Bacterial Proteins; Drug Discovery; Gram-Negative Bacteria; Lipopolysaccharides; Molecular Docking Simulation; Molecular Dynamics Simulation; Protein Binding; Carrier Proteins
PubMed: 38675646
DOI: 10.3390/molecules29081827 -
Microorganisms Mar 2024Peptidoglycan hydrolases are enzymes responsible for breaking the peptidoglycan present in the bacterial cell wall, facilitating cell growth, cell division and...
Peptidoglycan hydrolases are enzymes responsible for breaking the peptidoglycan present in the bacterial cell wall, facilitating cell growth, cell division and peptidoglycan turnover. subsp. (), the causal agent of citrus canker, encodes an M23 peptidase EnvC homolog. EnvC is a LytM factor essential for cleaving the septal peptidoglycan, thereby facilitating the separation of daughter cells. In this study, the investigation focused on EnvC contribution to the virulence and cell separation of . It was observed that disruption of the gene (Δ) led to a reduction in virulence. Upon inoculation into leaves of Rangpur lime ( Osbeck), the Δ exhibited a delayed onset of citrus canker symptoms compared with the wild-type . Mutant complementation restored the wild-type phenotype. Sub-cellular localization confirmed that EnvC is a periplasmic protein. Moreover, the Δ mutant exhibited elongated cells, indicating a defect in cell division. These findings support the role of EnvC in the regulation of cell wall organization, cell division, and they clarify the role of this peptidase in virulence.
PubMed: 38674634
DOI: 10.3390/microorganisms12040691 -
Pathogens (Basel, Switzerland) Apr 2024() utilizes FimA fimbriae to colonize the gingival sulcus and evade the host immune system. The biogenesis of all FimA-related components is positively regulated by the...
() utilizes FimA fimbriae to colonize the gingival sulcus and evade the host immune system. The biogenesis of all FimA-related components is positively regulated by the FimS-FimR two-component system, making the FimS sensory protein an attractive target for preventing infection. However, the specific environmental signal received by FimS remains unknown. We constructed random mutant libraries to identify critical amino acid residues for signal sensing by FimS. Optimized error-prone polymerase chain reaction (PCR) was used to introduce a limited number of random mutations in the periplasmic-domain-coding sequence of , and expression vectors carrying various mutants were generated by inverse PCR. More than 500 transformants were obtained from the -knockout strain using the - conjugal transfer system, whereas only ~100 transformants were obtained using electroporation. Four and six transformant strains showed increased and decreased expression, respectively. Six strains had single amino acid substitutions in the periplasmic domain, indicating critical residues for signal sensing by FimS. This newly developed strategy should be generally applicable and contribute to molecular genetics studies of , including the elucidation of structure-function relationships of proteins of interest.
PubMed: 38668264
DOI: 10.3390/pathogens13040309