Outer Membrane Biosynthesis Research Articles

Thanatin: A Promising Antimicrobial Peptide Targeting the Achilles' Heel of Multidrug-Resistant Bacteria.

Antimicrobial resistance poses an escalating threat to human health, necessitating the development of novel antimicrobial agents capable of addressing challenges posed by antibiotic-resistant bacteria. Thanatin, a 21-amino acid β-hairpin insect antimicrobial peptide featuring a single disulfide bond, exhibits broad-spectrum antibacterial activity, particularly effective against multidrug-resistant strains. The outer membrane biosynthesis system is recognized as a critical vulnerability in antibiotic-resistant bacteria, which thanatin targets to exert its antimicrobial effects. This peptide holds significant promise for diverse applications. This review begins with an examination of the structure-activity relationship and synthesis methods of thanatin. Subsequently, it explores thanatin's antimicrobial activity, detailing its various mechanisms of action. Finally, it discusses prospective clinical, environmental, food, and agricultural applications of thanatin, offering valuable insights for future research endeavors.

Toward understanding the antibacterial mechanism of chitosan: Experimental approach and in silico analysis

This study investigated the antibacterial mechanisms of chitosan (Cs), alkylated form (AlkCs), and chitosan nanoparticles (CsNPs) against Escherichia coli MG1655, a gram-negative model microorganism. CsNPs displayed a hemispherical shape with an average diameter of 169 nm using scanning electron microscopy (SEM) and a hydrodynamic diameter of 240 nm with a polydispersity index (PDI) of 0.154 based on dynamic light scattering (DSL) analysis. Energy Dispersive X-Ray (EDX), Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), and UV–Vis spectroscopy (UV) techniques were employed to verify the presence of unique elements and characteristics in Cs, AlkCs, and CsNPs. AlkCs demonstrated the highest growth inhibition zone against E. coli MG1655 compared to Cs and CsNPs. Furthermore, AlkCs and CsNPs exhibited lower minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values compared to Cs. Flow cytometry showed Cs caused significant membrane permeability at 200 μg/mL, while AlkCs and CsNPs induced greater permeability at 20, 50, and 750 μg/mL. Exposure to Cs, AlkCs, and CsNPs caused significant changes in cell size, number, and morphology, as observed through light and atomic force microscopy (AFM) images, compared to control cells. Microarray analysis, gene ontology (GO), and VENN diagram analysis were employed to analyze the differential expression of target genes (DEGs) and predict the mechanism of action of chitosan-based compounds. The findings indicated that CsNPs and AlkCs have the potential to act as antibacterial agents by inhibiting outer membrane biosynthesis, metabolic activities, and membrane-related signaling pathways. This study provides insights into the antibacterial mechanism of chitosan and its derivatives.

Coordination of bacterial cell wall and outer membrane biosynthesis

Gram-negative bacteria surround their cytoplasmic membrane with a peptidoglycan (PG) cell wall and an outer membrane (OM) with an outer leaflet composed of lipopolysaccharide (LPS)1. This complex envelope presents a formidable barrier to drug entry and is a major determinant of the intrinsic antibiotic resistance of these organisms2. The biogenesis pathways that build the surface are also targets of many of our most effective antibacterial therapies3. Understanding the molecular mechanisms underlying the assembly of the Gram-negative envelope therefore promises to aid the development of new treatments effective against the growing problem of drug-resistant infections. Although the individual pathways for PG and OM synthesis and assembly are well characterized, almost nothing is known about how the biogenesis of these essential surface layers is coordinated. Here we report the discovery of a regulatory interaction between the committed enzymes for the PG and LPS synthesis pathways in the Gram-negative pathogen Pseudomonas aeruginosa. We show that the PG synthesis enzyme MurA interacts directly and specifically with the LPS synthesis enzyme LpxC. Moreover, MurA was shown to stimulate LpxC activity in cells and in a purified system. Our results support a model in which the assembly of the PG and OM layers in many proteobacterial species is coordinated by linking the activities of the committed enzymes in their respective synthesis pathways.

(p)ppGpp and moonlighting RNases influence the first step of lipopolysaccharide biosynthesis in Escherichia coli.

The outer membrane (OM) protects Gram-negative bacteria from harsh environmental conditions and provides intrinsic resistance to many antimicrobial compounds. The asymmetric OM is characterized by phospholipids in the inner leaflet and lipopolysaccharides (LPS) in the outer leaflet. Previous reports suggested an involvement of the signaling nucleotide ppGpp in cell envelope homeostasis in Escherichia coli. Here, we investigated the effect of ppGpp on OM biosynthesis. We found that ppGpp inhibits the activity of LpxA, the first enzyme of LPS biosynthesis, in a fluorometric in vitro assay. Moreover, overproduction of LpxA resulted in elongated cells and shedding of outer membrane vesicles (OMVs) with altered LPS content. These effects were markedly stronger in a ppGpp-deficient background. We further show that RnhB, an RNase H isoenzyme, binds ppGpp, interacts with LpxA, and modulates its activity. Overall, our study uncovered new regulatory players in the early steps of LPS biosynthesis, an essential process with many implications in the physiology and susceptibility to antibiotics of Gram-negative commensals and pathogens.

Stress response genes to membrane fluidizers in mycobacteria

Plasma membrane is a heterogeneous mixture of lipids and proteins that includes laterally discrete domains. Membrane domains have been extensively described in eukaryotes, but little is known in prokaryotes. Mycobacterium, a genus that includes several notorious human pathogens, organizes its plasma membrane by creating the intracellular membrane domain (IMD), which is spatially and biochemically separable from the conventional plasma membrane (PM-CW). This membrane architecture is proposed to optimize the synthesis of cell envelope precursors such as peptide glycan (PG) and arabinogalactan (AG) (Fig. 1). However, the mechanisms that govern the formation and maintenance of the IMD is completely unknown. We recently found that membrane fluidizers such as benzyl alcohol and dibucaine can reversibly disrupt the formation of the IMD. To reveal the mechanism of this stress response, in this study, we carried out transposon sequencing (Tn-seq), comparing pools of mutants that were treated or mock-treated with either benzyl alcohol or dibucaine. We found genes involved in peptidoglycan biosynthesis and outer membrane biosynthesis important for withstanding membrane fluidizer treatments, suggesting that the physical pinning by the loadbearing exoskeleton structures is critical for the IMD maintenance and recovery. We also found genes involved in modulating fatty acid structures, indicating the role of membrane lipid composition in the membrane stress response. We are currently verifying the roles of these genes by making gene deletion mutants, and testing the sensitivities of these mutants to the membrane fluidizers.

Disrupting Gram-Negative Bacterial Outer Membrane Biosynthesis through Inhibition of the Lipopolysaccharide Transporter MsbA.

There is a critical need for new antibacterial strategies to counter the growing problem of antibiotic resistance. In Gram-negative bacteria, the outer membrane (OM) provides a protective barrier against antibiotics and other environmental insults. The outer leaflet of the outer membrane is primarily composed of lipopolysaccharide (LPS). Outer membrane biogenesis presents many potentially compelling drug targets as this pathway is absent in higher eukaryotes. Most proteins involved in LPS biosynthesis and transport are essential; however, few compounds have been identified that inhibit these proteins. The inner membrane ABC transporter MsbA carries out the first essential step in the trafficking of LPS to the outer membrane. We conducted a biochemical screen for inhibitors of MsbA and identified a series of quinoline compounds that kill Escherichia coli through inhibition of its ATPase and transport activity, with no loss of activity against clinical multidrug-resistant strains. Identification of these selective inhibitors indicates that MsbA is a viable target for new antibiotics, and the compounds we identified serve as useful tools to further probe the LPS transport pathway in Gram-negative bacteria.

Cold Stress Makes Escherichia coli Susceptible to Glycopeptide Antibiotics by Altering Outer Membrane Integrity

A poor understanding of the mechanisms by which antibiotics traverse the outer membrane remains a considerable obstacle to the development of novel Gram-negative antibiotics. Herein, we demonstrate that the Gram-negative bacterium Escherichia coli becomes susceptible to the narrow-spectrum antibiotic vancomycin during growth at low temperatures. Heterologous expression of an Enterococcus vanHBX vancomycin resistance cluster in E. coli confirmed that the mechanism of action was through inhibition of peptidoglycan biosynthesis. To understand the nature of vancomycin permeability, we screened for strains of E. coli that displayed resistance to vancomycin at low temperature. Surprisingly, we observed that mutations in outer membrane biosynthesis suppressed vancomycin activity. Subsequent chemical analysis of lipopolysaccharide from vancomycin-sensitive and -resistant strains confirmed that suppression was correlated with truncations in the core oligosaccharide of lipopolysaccharide. These unexpected observations challenge the current understanding of outer membrane permeability, and provide new chemical insights into the susceptibility of E. coli to glycopeptide antibiotics.

Identification of Additional Players in the Alternative Biosynthesis Pathway to Isovaleryl‐CoA in the Myxobacterium Myxococcus xanthus

Isovaleryl-CoA (IV-CoA) is usually derived from the degradation of leucine by using the Bkd (branched-chain keto acid dehydrogenase) complex. We have previously identified an alternative pathway for IV-CoA formation in myxobacteria that branches from the well-known mevalonate-dependent isoprenoid biosynthesis pathway. We identified 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase (MvaS) to be involved in this pathway in Myxococcus xanthus, which is induced in mutants with impaired leucine degradation (e.g., bkd(-)) or during myxobacterial fruiting-body formation. Here, we show that the proteins required for leucine degradation are also involved in the alternative IV-CoA biosynthesis pathway through the efficient catalysis of the reverse reactions. Moreover, we conducted a global gene-expression experiment and compared vegetative wild-type cells with bkd mutants, and identified a five-gene operon that is highly up-regulated in bkd mutants and contains mvaS and other genes that are directly involved in the alternative pathway. Based on our experiments, we assigned roles to the genes required for the formation of IV-CoA from HMG-CoA. Additionally, several genes involved in outer-membrane biosynthesis and a plethora of genes encoding regulatory proteins were decreased in expression levels in the bkd(-) mutant; this explains the complex phenotype of bkd mutants including a lack of adhesion in developmental submerse culture.

Probing outer membrane biogenesis

The outer membrane (OM) of Gram-negative bacteria is a complex organelle composed of phospholipids, glycolipids and proteins that acts as a barrier to harmful compounds such as antibiotics. Studies of OM biosynthesis using OM-deficient mutants have provided unclear results, as OM-deficient mutants generally have nonspecific permeability defects. In the 22 April issue of Cell, Ruiz et al. describe 'chemical conditionality' screens to identify mutant E. coli strains that confer enhanced resistance to antibiotics. A paper in the same issue by Wu et al. describes the identification of a multiprotein complex that is required for biosynthesis of OM -barrel proteins in E. coli.

Moving folded proteins across the bacterial cell membrane.

The Tat protein export system is located in the bacterial cytoplasmic membrane and operates in parallel to the well-known Sec pathway. While the Sec system only transports unstructured substrates, the function of the Tat pathway is to translocate folded proteins. The Tat translocase thus faces the formidable challenge of moving structured macromolecular substrates across the bacterial cytoplasmic membrane without rendering the membrane freely permeable to protons and other ions. The substrates of the Tat pathway are often proteins that bind cofactor molecules in the cytoplasm, and are thus folded, prior to export. Such periplasmic cofactor-containing proteins are essential for most types of bacterial respiratory and photosynthetic energy metabolism. In addition, the Tat pathway is involved in outer membrane biosynthesis and in bacterial pathogenesis. Substrates are targeted to the Tat pathway by amino-terminal signal sequences harbouring consecutive, essentially invariant, arginine residues, and movement of proteins through the Tat system is energized by the transmembrane proton electrochemical gradient. The TatA protein probably forms the transport channel while the TatBC proteins act as a receptor complex that recognizes the signal peptide of the substrate protein.

Crystallization and preliminary X-ray diffraction studies of the lipopolysaccharide core biosynthetic enzyme ADP-L-glycero-D-mannoheptose 6-epimerase from Escherichia coli K-12.

ADP-L-glycero-D-mannoheptose 6-epimerase is a 240 kDa NAD-dependent nucleotide diphosphosugar epimerase from Escherichia coli K12 which catalyzes the interconversion of ADP-D-glycero-D-mannoheptose and ADP-L-glycero-D-mannoheptose. ADP-L-glycero-D-mannoheptose is a required intermediate for lipopolysaccharide inner-core and outer-membrane biosynthesis in several genera of pathogenic and non-pathogenic Gram-negative bacteria. ADP-L-glycero-D-mannoheptose 6-epimerase was overexpressed in E. coli and purified to apparent homogeneity by chromatographic methods. Three crystal forms of the epimerase were obtained by a hanging-drop vapor-diffusion method. A native data set for crystal form III was collected in-house on a Rigaku R-AXIS-IIC image plate at 3.0 A resolution. The form III crystals belong to the monoclinic space group P21. The unit-cell parameters are a = 98.94, b = 110.53, c = 180.68 A and beta = 90.94 degrees. Our recent results show that these crystals diffract to 2.0 A resolution at the Cornell High Energy Synchrotron Source. The crystal probably contains six 40 kDa monomers per asymmetric unit, with a corresponding volume per protein mass (Vm) of 4.11 A3 Da-1 and a solvent fraction of 70%.