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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 -
Trends in Biochemical Sciences Apr 2024The survival and virulence of Gram-negative bacteria require proper biogenesis and maintenance of the outer membrane (OM), which is densely packed with β-barrel OM... (Review)
Review
The survival and virulence of Gram-negative bacteria require proper biogenesis and maintenance of the outer membrane (OM), which is densely packed with β-barrel OM proteins (OMPs). Before reaching the OM, precursor unfolded OMPs (uOMPs) must cross the whole cell envelope. A network of periplasmic chaperones and proteases maintains unfolded but folding-competent conformations of these membrane proteins in the aqueous periplasm while simultaneously preventing off-pathway aggregation. These periplasmic proteins utilize different strategies, including conformational heterogeneity, oligomerization, multivalency, and kinetic partitioning, to perform and regulate their functions. Redundant and unique characteristics of the individual periplasmic players synergize to create a protein quality control team capable responding to changing environmental stresses.
PubMed: 38677921
DOI: 10.1016/j.tibs.2024.03.015 -
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 -
Memorias Do Instituto Oswaldo Cruz 2024The availability of genes and protein sequences for parasites has provided valuable information for drug target identification and vaccine development. One such parasite...
BACKGROUND
The availability of genes and protein sequences for parasites has provided valuable information for drug target identification and vaccine development. One such parasite is Bartonella quintana, a Gram-negative, intracellular pathogen that causes bartonellosis in mammalian hosts.
OBJECTIVE
Despite progress in understanding its pathogenesis, limited knowledge exists about the virulence factors and regulatory mechanisms specific to B. quintana.
METHODS AND FINDINGS
To explore these aspects, we have adopted a subtractive proteomics approach to analyse the proteome of B. quintana. By subtractive proteins between the host and parasite proteome, a set of proteins that are likely unique to the parasite but absent in the host were identified. This analysis revealed that out of the 1197 protein sequences of the parasite, 660 proteins are non-homologous to the human host. Further analysis using the Database of Essential Genes predicted 159 essential proteins, with 28 of these being unique to the pathogen and predicted as potential putative targets. Subcellular localisation of the predicted targets revealed 13 cytoplasmic, eight membranes, one periplasmic, and multiple location proteins. The three-dimensional structure and B cell epitopes of the six membrane antigenic protein were predicted. Four B cell epitopes in KdtA and mraY proteins, three in lpxB and BQ09550, whereas the ftsl and yidC proteins were located with eleven and six B cell epitopes, respectively.
MAINS CONCLUSIONS
This insight prioritises such proteins as novel putative targets for further investigations on their potential as drug and vaccine candidates.
Topics: Proteomics; Bartonella quintana; Bacterial Vaccines; Bacterial Proteins; Humans; Computer Simulation; Virulence Factors; Proteome
PubMed: 38655925
DOI: 10.1590/0074-02760230040 -
Applied Magnetic Resonance Mar 2024The majority of pathogenic Gram-negative bacteria benefit from intrinsic antibiotic resistance, attributed primarily to the lipopolysaccharide (LPS) coating of the...
The majority of pathogenic Gram-negative bacteria benefit from intrinsic antibiotic resistance, attributed primarily to the lipopolysaccharide (LPS) coating of the bacterial envelope. To effectively coat the bacterial cell envelope, LPS is transported from the inner membrane by the LPS transport (Lpt) system, which comprises seven distinct Lpt proteins, LptA-G, that form a stable protein bridge spanning the periplasm to connect the inner and outer membranes. The driving force of this process, LptBFG, is an asymmetric ATP binding cassette (ABC) transporter with a novel architecture and function that ejects LPS from the inner membrane and facilitates transfer to the periplasmic bridge. Here, we utilize site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy to probe conformational differences between the periplasmic domains of LptF and LptG. We show that LptC solely interacts with the edge β-strand of LptF and does not directly interact with LptG. We also quantify the interaction of periplasmic LptC with LptF. Additionally, we show that LPS cannot enter the protein complex externally, supporting the unidirectional LPS transport model. Furthermore, we present our findings that the presence of LPS within the LptBFGC binding cavity and the membrane reconstitution environment affect the structural orientation of the periplasmic domains of LptF and LptG, but overall are relatively fixed with respect to one another. This study will provide insight into the structural asymmetry associated with the newly defined type VI ABC transporter class.
PubMed: 38645307
DOI: 10.1007/s00723-023-01590-3 -
BioRxiv : the Preprint Server For... Apr 2024Phosphoethanolamine (pEtN) cellulose is a naturally occurring modified cellulose produced by several Enterobacteriaceae. The minimal components of the cellulose...
Phosphoethanolamine (pEtN) cellulose is a naturally occurring modified cellulose produced by several Enterobacteriaceae. The minimal components of the cellulose synthase complex include the catalytically active BcsA enzyme, an associated periplasmic semicircle of hexameric BcsB, as well as the outer membrane (OM)-integrated BcsC subunit containing periplasmic tetratricopeptide repeats (TPR). Additional subunits include BcsG, a membrane-anchored periplasmic pEtN transferase associated with BcsA, and BcsZ, a conserved periplasmic cellulase of unknown biological function. While events underlying the synthesis and translocation of cellulose by BcsA are well described, little is known about its pEtN modification and translocation across the cell envelope. We show that the N-terminal cytosolic domain of BcsA positions three copies of BcsG near the nascent cellulose polymer. Further, the terminal subunit of the BcsB semicircle tethers the N-terminus of a single BcsC protein to establish a trans-envelope secretion system. BcsC's TPR motifs bind a putative cello-oligosaccharide near the entrance to its OM pore. Additionally, we show that only the hydrolytic activity of BcsZ but not the subunit itself is necessary for cellulose secretion, suggesting a secretion mechanism based on enzymatic removal of mislocalized cellulose. Lastly, we introduce pEtN modification of cellulose in orthogonal cellulose biosynthetic systems by protein engineering.
PubMed: 38645035
DOI: 10.1101/2024.04.04.588173