Outer Membrane Research Articles

Genetic Insights on Meropenem Resistance Concerning Klebsiella pneumoniae Clinical Isolates

The transferable genetic elements are associated with the dissemination of virulence determinants amongst Klebsiella pneumoniae. Thus, we assessed the correlated antimicrobial resistance in carbapenem-resistant Klebsiella pneumoniae clinical isolates. Each isolate’s ability to biosynthesize biofilm, carbapenemase, and extended-spectrum β-lactamase were examined. Genotypically, the biofilm-, outer membrane porin-, and some plasmid-correlated antimicrobial resistance genes were screened. About 50% of the isolates were multidrug-resistant while 98.4% were extended-spectrum β-lactamase producers and 89.3% were carbapenem-resistant. Unfortunately, 93.1% of the multidrug-resistant isolates produced different biofilm levels. Additionally, fimD and mrkD genes encoding adhesins were detected in 100% and 55.2% of the tested isolates, respectively. Also, the blaKPC, blaOXA-48-like, and blaNDM-encoding carbapenemases were observed in 16.1%, 53.6%, and 55.4% of the tested isolates, respectively. Moreover, the blaSHV and blaCTX-M extended-spectrum β-lactamase-associated genes were detected at 95.2% and 61.3%, respectively. Furthermore, aac(3)IIa, qnrB, and tetB resistance-correlated genes were observed in 38.1%, 46%, and 7.9% of the tested isolates, respectively. Certainly, the tested antimicrobial resistance-encoding genes were concurrently observed in 3.2% of the tested isolates. These findings confirmed the elevated prevalence of various antimicrobial resistance-associated genes in Klebsiella pneumoniae. The concurrent transferring of plasmid-encoding antimicrobial resistance-related genes could be associated with the possible acquisition of multidrug-resistant Klebsiella pneumoniae phenotypes.

Role and mechanism of the outer membrane porin LamB in T-2 mycotoxin-mediated extensive drug resistance in Escherichia coli

The influence of mycotoxins in ecological niches shared with antibiotic-resistant bacteria (ARB) remains underexplored. This study examined the impact of T-2 mycotoxin on the evolution of antibiotic resistance in Escherichia coli, highlighting the role of specific porins. Our findings revealed that exposure to 10ng/mL of T-2 toxin induced multi-drug resistant (MDR) phenotypes in three E. coli. At 10-5ng/mL, T-2 toxin caused E. coli ATCC 25922 to develop stable resistance to 13 critical antibiotics, with minimum inhibitory concentrations (MICs) increasing 16- to several thousand-fold. This resistance was linked to the downregulation of the mal gene cluster. Notably, T-2 toxin reduced membrane permeability by downregulating lamB, facilitating its own entry and reducing the intracellular accumulation of both the toxin and antibiotics, thereby enhancing resistance development. LamB mediated the XDR phenotypes in E. coli, particularly by blocking last-resort antibiotics such as cephalosporins, carbapenems, tigecycline, and colistin, complicating treatment strategies. LamB demonstrated high binding affinities for T-2 toxin and various antibiotics, with specific binding sites identified for meropenem (Arg134), imipenem (Ser148, Arg170, Lys129), ceftazidime (Phe106, Lys129), and cefepime (Tyr66, Gln267, Lys269), exhibiting binding energies of -2.93, -2.58, -2.53, and -4.3, respectively. These findings suggest that even low levels of T-2 mycotoxin pose a substantial public health risk. They underscore the urgent need to address these contaminants and open new avenues for antibiotic resistance research.

The effect of clinically relevant antibiotics on bacterial extracellular vesicle release from E. coli.

Sepsis, a leading cause of death in hospitals, can be defined as a dysregulated host inflammatory response to infection, which can lead to tissue damage, organ failure, and cardiovascular complications. Although there is no cure for sepsis, the condition is typically managed with broad-spectrum antibiotics to eliminate any potential bacterial source of infection. However, a potential side-effect of antibiotic treatment is the enhanced release of bacterial extracellular vesicles (BEVs), membrane-bound nanoparticles containing proteins and other biological molecules from their parent bacterium. Some of the Gram-negative EV cargo, including Peptidoglycan associated lipoprotein (Pal) and Outer membrane protein A (OmpA), have been shown to induce both acute and chronic inflammation in host tissue. We hypothesize that antibiotic concentration and its mechanism of action can have an effect on the amount of released BEVs, which could potentially exacerbate the host inflammatory response. In this study, we evaluated nine clinically relevant antibiotics for their effect on EV release from Escherichia coli. Several beta-lactam antibiotics caused significantly more EV release, while quinolone and aminoglycosides caused relatively less vesiculation. Further study is warranted to corroborate the correlation between an antibiotic's mechanism of action and its effect on EV release, but these results underline the importance of antibiotic choice when treating sepsis patients.

A unique case of Sylvian arachnoid cyst complicated by chronic subdural hematoma

Background: Arachnoid cysts (ACs) complicated by chronic subdural hematoma (CSDH) are a rare but distinct entity. Case Description: A 27-year-old man previously diagnosed with Sylvian AC presented to the hospital with a persistent headache. He was not aware of any preceding head trauma. However, he frequently performed bench presses at the gymnasium, especially 4 weeks prior. The patient did not exhibit any neurological deficits at presentation. Computed tomography revealed slightly low-density areas in the right cerebral convexity. Magnetic resonance imaging revealed compressive masses in the right middle fossa and cerebral convexity. The patient underwent a craniotomy. Reflection of the dura mater exposed thickened arachnoid membrane. Making an incision resulted in the egress of fluid hematoma. The membrane separating the subdural hematoma and inner AC possessed fine vasculature and adjacent holes. Furthermore, there were fragile clots adhered to the inner wall of the cyst. Microscopic findings of the separating membrane were consistent with inflammatory granulation tissue, similar to those of the outer membrane of CSDH. Conclusion: Exertional hypertension associated with the bench press may result in the rupture of fine arteries distributed over the AC wall. Under certain circumstances, the AC wall may transform into the outer membrane of CSDH.

Caspase-2 kills cells with extra centrosomes.

Centrosomes are membrane-less organelles that orchestrate a wide array of biological functions by acting as microtubule organizing centers. Here, we report that caspase-2-driven apoptosis is elicited in blood cells failing cytokinesis and that extra centrosomes are necessary to trigger this cell death. Activation of caspase-2 depends on the PIDDosome multi-protein complex, and priming of PIDD1 at extra centrosomes is necessary for pathway activation. Accordingly, loss of its centrosomal adapter, ANKRD26, allows for cell survival and unrestricted polyploidization in response to cytokinesis failure. Mechanistically, cell death is initiated upstream of mitochondria via caspase-2-mediated processing of the BCL2 family protein BID, driving BAX/BAK-dependent mitochondrial outer membrane permeabilization (MOMP). Remarkably, BID-deficient cells enforce apoptosis by engaging p53-dependent proapoptotic transcriptional responses initiated by caspase-2. Consistently, BID and MDM2 act as shared caspase-2 substrates, with BID being kinetically favored. Our findings document that the centrosome limits its own unscheduled duplication by the induction of PIDDosome-driven mitochondrial apoptosis to avoid potentially pathogenic polyploidization events.

Increased antimicrobial resistance of acid-adapted pathogenic Escherichia coli, and transcriptomic analysis of polymyxin-resistant strain

This study investigated the acid adaptation and antimicrobial resistance of seven pathogenic Escherichia coli strains and one commensal strain under nutrient-rich acidic conditions. After acid adaptation, three pathogenic E. coli survived during 100 h incubation in tryptic soy broth at pH 3.25. Acid-adapted (AA) strains showed increased resistance to antimicrobials including ampicillin, ciprofloxacin and especially polymyxins (colistin and polymyxin B), the last resort antimicrobial for multidrug-resistant Gram-negative bacteria. Enterotoxigenic E. coli strain (NCCP 13717) showed significantly increased resistance to acids and polymyxins. Transcriptome analysis of the AA NCCP 13717 revealed upregulation of genes related to the acid fitness island and the arn operon, which reduces lipopolysaccharide binding affinity at the polymyxin site of action. Genes such as eptA, tolC, and ompCF were also upregulated to alter the structure of the cell membrane, reducing the outer membrane permeability compared to the control, which is likely to be another mechanism for polymyxin resistance. This study highlights the emergence of antimicrobial resistance in AA pathogenic E. coli strains, particularly polymyxin resistance, and the mechanisms behind the increased antimicrobial resistance, providing important insights for the development of risk management strategies to effectively control the antimicrobial resistant foodborne pathogens.

“Dandelion” magnetic bead-driven CDs@CeO2 based-multifunctional nanoplatform for tri-modal ultrasensitive detection and efficient eradication of pathogenic bacteria

To address the challenge of foodborne pathogens, we have developed an ultra-sensitive tri-modal detection and effective pathogen eradication strategy utilizing a “dandelion” magnetic bead-driven cerium dioxide doped with carbon dots (CDs@CeO2) multifunctional nanoplatform. Aptamer (Apt) modification CDs@CeO2 specifically recognizes and binds to the outer membrane proteins of Salmonella typhimurium (S. typhimurium). Concurrently, mannose-functionalized dandelion-like magnetic beads selectively recognize adhesins of S. typhimurium, avoiding competition with Apt for binding sites and leveraging the strong bacterial capture capability for high-sensitivity detection. The prepared CDs@CeO2 has been demonstrated to exhibit favourable fluorescence characteristics. In the presence of H2O2, CDs@CeO2 is capable of oxidising colourless 3,3',5,5'-tetramethylbenzidine (TMB) into blue oxide, displaying exceptional photothermal properties. CDs@CeO2 have the potential to generate •OH through a Fenton-like reaction, which could directly damage cells. Notably, coupling the photosensitizer Ce6 to CDs@CeO2 and subsequent illumination generates 1O2, which synergistically eradicates bacteria with •OH. Detection limits of the fluorescence, colorimetry, and photothermal methods were 3.85 cfu/mL, 9.44 cfu/mL, and 5.00 cfu/mL, respectively. The platform successfully achieves precise detection of S. typhimurium in actual pre-prepared food samples through fluorescence, colorimetry, and photothermal tri-modal signals, and effectively kills bacteria, achieving trace detection and source control.

Antimicrobial and antibiofilm potential of α-MSH derived cationic and hydrophobic peptides against Escherichia coli: Mechanistic insight through peptide-lipopolysaccharide interactions

The prevalence of infections caused by various Gram-negative pathogens specifically Escherichia coli continuously poses a significant challenge in health care as well as community settings owing to their ability to form biofilm and escalating tolerance towards available antibiotics. While most treatment regimes are targeted at eliminating the E. coli cells, the pathogenicity factors called endotoxin (lipopolysaccharides), associated with the sepsis initiation and the leading cause of death in intensive care units globally, are often ignored. In this study, the potency of alpha-Melanocyte Stimulating Hormone based-peptides, particularly Ana-9 and Ana-10 against E. coli was investigated through microbiological, biophysical, and microscopic assays. Both Ana-9 and Ana-10 demonstrated enhanced activity against planktonic E. coli cells, and retained their activity against biofilm, which was supported by confocal microscopy. From the mechanistic perspective, spectroscopic studies indicated that the binding of peptides with LPS led to structural alteration of peptides due to their insertion into the hydrophobic environment of LPS. The electrostatic interaction of the peptide with LPS leads to outer membrane disorganization, allowing the peptide to access the inner membrane, depolarize it and ultimately inhibit the bacterial cells within the biofilm. These observations were further confirmed by atomic force and scanning electron microscopy. Thus, this study deepens our understanding of the structural characteristics of peptides attached to LPS, which could lead to the gradual improvement in developing more potent, broad-spectrum endotoxin neutralizers.

Probing marine macroalgal phlorotannins as an antibacterial candidate against Salmonella typhi: Molecular docking and dynamics simulation approach

The increasing prevalence of drug-resistant bacterial strains, including multidrug-resistant Salmonella typhi, has raised significant concerns about the effectiveness of traditional antimicrobial treatments. To address this issue, the study employed computational techniques to evaluate the potential of natural phlorotannins derived from marine algae as alternative antibacterial agents against S. typhi. A total of 104 phlorotannins were retrieved from PubChem and subjected to molecular docking with three specific target proteins of S. typhi. Among the compounds tested, Compound-10 a derivative of dieckol exhibited the highest affinity for the Omptin family outer membrane protease [pgtE] protein and for the cell invasion protein sipB protein. The docking results revealed strong interactions between the phlorotannins and the target proteins, indicating their potential as antimicrobial agents. Additionally, pharmacokinetic studies using the QikProp module demonstrated that the top-ranked compounds showed favourable drug-like properties with good druggable efficiency, moderate gut-blood barrier transport, and high human oral absorption. Most compounds passed the rule of three and five drug-likeness criteria, indicating their potential as drug candidates. Furthermore, MM-GBSA analysis provided relative binding affinity estimations, ranking the compounds based on their calculated binding energies. These results align reasonably well with experimental binding affinities, reinforcing the potential of the identified compounds as potent binders to their target proteins. This study highlights the promising antibacterial potential of marine phlorotannins against S. typhi. Further in vitro studies are warranted to validate these compounds' efficacy and anti-microbial activity, ultimately paving the way for developing new therapeutic strategies to combat drug-resistant Salmonella infections.

Preparation of a thermosensitive and antibacterial in situ gel using poloxamer-quaternized chitosan for sustained ocular delivery of Levofloxacin hydrochloride

In this study, a thermosensitive in situ gel with porous structure was developed using poloxamer (Po) and N-(2-hydroxy-3-trimethyl ammonium) propyl chitosan chloride (HTCC). The poloxamer-quaternized chitosan (Po-HTCC) in situ gel exhibited superior rheological property, water absorption capacity and antibacterial activity against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Streptococcus pyogenes, making it well-suited for ocular applications. Scanning electron microscope revealed a macroporous architecture with pore sizes ranging from 1 to 2 μm, suggesting that the gel has desirable breathability, corneal adhesion capability, and overall conformability. In vitro drug release assay was conducted with levofloxacin hydrochloride, demonstrating that sustained release over 48 h could be achieved at 34 °C, with approximately 80 % of the drug released within this timeframe. Computational simulations revealed substantial binding affinity between the material and the Escherichia coli outer membrane lipopolysaccharide-associated protein and corneal mucin. The protein showing the strongest binding energy to N-(2-hydroxy-3-trimethyl ammonium) propyl chitosan chloride (HTCC), as calculated by the Molecular Mechanics Generalized Born Surface Area Method (MM-GBSA), was LptD-LptE, with a binding energy of −61.14 ± 4.72 kcal/mol. These results underscore the potential of this system for effective and convenient ocular delivery with sustained drug release.

Elucidating Antibiotic Permeation through the Escherichia coli Outer Membrane: Insights from Molecular Dynamics.

Antibiotic resistance represents a critical public health threat, with an increasing number of Gram-negative pathogens demonstrating resistance to a broad range of clinical drugs. A primary challenge in enhancing antibiotic efficacy is overcoming the robust barrier presented by the bacterial outer membrane. Our research addresses a longstanding question: What is the rate of antibiotic permeation across the outer membrane (OM) of Gram-negative bacteria? Utilizing molecular dynamics (MD) simulations, we assess the passive permeability profiles of four commercially available antibiotics─gentamicin, novobiocin, rifampicin, and tetracycline across an asymmetric atomistic model of the Escherichia coli (E. coli) OM, employing the inhomogeneous solubility-diffusion model. Our examination of the interactions between these drugs and their environmental context during OM permeation reveals that extended hydrogen bond formation and drug-cation interactions significantly hinder the energetics of passive permeation, notably affecting novobiocin. Our MD simulations corroborate well with experimental data and reveal new implications of solvation on drug permeability, overall advancing the possible use of computational prediction of membrane permeability in future antibiotic discovery.

Bioorthogonal Monomycolate of Trehalose Disclosed the O-Mycoloylation of Mycoloyltransferases and Other Cell Envelope Proteins in C. glutamicum.

Protein mycoloylation is a recently identified unusual post-translational modification (PTM) exclusively observed in Mycobacteriales, an order of bacteria that includes several human pathogens. These bacteria possess a distinctive outer membrane, known as the mycomembrane, composed of very long-chain fatty acids called mycolic acids. It has been demonstrated that a few mycomembrane proteins undergo covalent modification with mycolic acids in the model organism Corynebacterium glutamicum through the action of mycoloyltransferase MytC. This PTM represents the first example of protein O-acylation in prokaryotes and also the first example of protein modification by mycolic acid. Many questions about the specificity of protein O-mycoloylation remain crucial for understanding its evolutionary significance in Mycobacteriales and its role in cell physiology. We have developed the first bioorthogonal mycolate donor featuring the natural mycolic acid pattern, enabling direct, unambiguous transfer of the lipid moiety to its acceptors and efficient metabolic labeling and enrichment of MytC protein substrates. Mass spectrometry analysis of the labeled proteins and comparative proteomic analysis of the cell envelope proteome between wild-type and ΔmytC strains identified an unbiased list of 21 proteins likely mycoloylated in the cell. The robustness of our approach is demonstrated by the successful biological validation of mycoloylation in 6 candidate proteins within wild-type cells, revealing the characteristic profile of proteins modified with natural mycolates. These findings provide interesting insights into the significance of this new lipidation pathway and pave the way for understanding their function, especially concerning the mycoloyltransferase family that includes the essential Antigen85 enzymes in Mycobacteria.

Characterizing the interplay between Acinetobacter baumannii, A549 cells, and anti-Omp34 antibodies: implications for adherence, internalization, and cytotoxicity.

Acinetobacter baumannii thrives within eukaryotic cells, influencing persistence, treatment approaches, and progression of disease. We probed epithelial cell invasion by A. baumannii and the influence of antibodies raised to outer membrane protein 34 (Omp34) on epithelial interactions. We expressed and purified recombinant Omp34 and induced anti-Omp34 antibodies in Bagg albino or BALB/c mice. Omp34 was evaluated for acute toxicity in mice through histological analysis of six organs. The host cell line, A549, was exposed to both A. baumannii 19606 and a clinical isolate. The study also investigated serum resistance, adherence, internalization, and proliferation of A. baumannii in A549 cells, with and without anti-Omp34 sera, utilizing cell culture techniques and light microscopy. A549 cell viability was evaluated by A. baumannii challenge and exposure to anti-Omp34 sera. Actin disruption experiments using cytochalasin D probed microfilament and microtubule roles in A. baumannii invasion. Omp34 prompted antibody production without toxicity in mice. The serum showed bactericidal effects on both strains. Additionally, both A. baumannii strains were found to form biofilms. Omp34 serum was observed to decrease biofilm formation, bacterial adherence, internalization, and proliferation in A549 cells. Furthermore, the use of anti-Omp34 serum enhanced the post-infection survival of the host cell. Pre-exposure of A549 cells to cytochalasin D reduced bacterial internalization, highlighting the role of actin polymerization in the invasion process. Microscopic analysis revealed various interactions, such as adherence, membrane alterations, vacuolization, apoptosis, and cellular damage. Anti-Omp34 serum-exposed A549 cells were protected and showed reduced damage. The findings reveal that A. baumannii can significantly multiply intracellularly within host cells. This suggests the bacterium's ability to establish an environment conducive to its replication by preventing fusion with degradative lysosomes and inhibiting acidification. This finding contributes to the understanding of A. baumannii's intracellular persistence and highlights the role of Omp34 in influencing apoptosis, autophagy, and bacterial adherence, which may impact the development of effective treatments against A. baumannii infections.

Self‐Oxygenated Biomimetic Nanozyme for Tumor Catalytic Immunotherapy

AbstractThe treatment of solid tumors remains a significant challenge due to the complex, immunosuppressive tumor microenvironment (TME). This manuscript introduces the self‐oxygenated biomimetic nanozyme system, SS‐MSN@Au‐BOM, which innovatively combines catalytic therapy with immunotherapy to modulate the TME for enhanced therapeutic efficacy. Employing mesoporous silicon nanoparticles doped with disulfide bonds, the system integrates gold nanozymes that exhibit glucose oxidase (GOx)‐like and peroxidase (POD)‐like activities, along with a catalase (CAT) function derived from bacterial outer membrane (BOM) coatings. SS‐MSN@Au‐BOM significantly disrupted tumor growth by catalyzing endogenous hydrogen peroxide to generate cytotoxic ROS and essential oxygen, enhancing intratumoral ROS levels while alleviating hypoxia. This catalytic action facilitates immunogenic cell death, enhances dendritic cell maturation, and T‐cell activation. Notably, the system shows excellent biocompatibility, effective tumor targeting, and prolonged systemic circulation. SS‐MSN@Au‐BOM offers a dual‐functional approach that effectively disrupts the TME and promotes robust antitumor immunity. This study paves the way for further clinical investigation and the development of enzyme‐based therapeutics, potentially transforming the landscape of cancer treatment.

BlaGES-producing ST654 comprises a quarter of all carbapenem-resistant Pseudomonas aeruginosa in blood isolates from 15 hospitals.

Carbapenem-resistant Pseudomonas aeruginosa (CRPA) are of major clinical concern. We analyzed 85 P. aeruginosa blood isolates non-susceptible to carbapenems collected during 2021-2023 from 15 medical centers in Israel. We aimed to determine the prevalence of high-risk clones, examine clonality, test antibiotic susceptibility, and assess the presence of acquired resistance genes, including carbapenemases. Whole-genome sequencing was performed using Illumina sequencing technology. Susceptibly was determined using the broth microdilution method. In the entire sample, 43.5% were high-risk clones. A main clade (27.1% of isolates) found in multiple hospitals comprised 19 isolates belonging to the high-risk ST654 clone and four closely related isolates. The isolates in this main clade harbored a broad set of resistance genes, including GES-type genes, and 91% had a mutated outer membrane protein (OprD). Isolates in the main clade were uniformly tobramycin (TOB) resistant and 83% were ceftolozane/tazobactam resistant. In the entire sample, we found high resistance to most antipseudomonal agents, including new beta-lactam/beta-lactamase inhibitor combinations. No uniform susceptibility to an antipseudomonal agent was found. Carbapenemases were carried by 9.4% of isolates (5.9% blaGES-5 and 3.5% blaNDM-1) and oprD was mutated in 67% of isolates. Thus, the epidemiology of CRPA is explained by a combination of clonal expansion of a dominant high-risk clade and sporadic occurrence of mutated strains. Our findings highlight the importance of susceptibility testing using a wide panel of antibiotics when CRPA is detected. Prevention measures tracking and controlling emerging high-risk clades and clones are crucial to limit the spread of CRPA.

Guanethidine Enhances the Antibacterial Activity of Rifampicin Against Multidrug-Resistant Bacteria

The escalating global threat of antibiotic resistance necessitates innovative strategies, such as the combination of antibiotics with adjuvants. Monotherapy with rifampicin is more likely to induce resistance in pathogens compared to other antibiotics. Herein, we found that the antihypertensive drug guanethidine enhanced the activity of rifampicin against certain clinically resistant Gram-negative bacteria, resulting in a reduction of up to 128-fold in the minimum inhibitory concentration. In infected animal models, this combination has achieved treatment benefits, including increased survival and decreased bacterial burden. The antimicrobial mechanism of guanethidine in synergy with rifampicin involves the disruption of the outer membrane of Gram-negative bacteria, leading to dissipation of the proton motive force. This results in an increase in reactive oxygen species and a reduction in ATP synthesis, severely disturbing energy metabolism and ultimately increasing bacterial mortality. In summary, guanethidine has the potential to become a novel adjuvant for rifampicin, offering a new option for the treatment of clinical Gram-negative bacterial infections.

PlsX and PlsY: Additional roles beyond glycerophospholipid synthesis in Gram-negative bacteria.

The unique asymmetry of the Gram-negative outer membrane, with glycerophospholipids (GPLs) in the inner leaflet and lipopolysaccharide (LPS) in the outer leaflet, works to resist external stressors and prevent the entry of toxic compounds. Thus, GPL and LPS synthesis must be tightly controlled to maintain the integrity of this essential structure. We sought to decipher why organisms like Escherichia coli possess two redundant pathways-PlsB and PlsX/Y-for synthesis of the GPL precursor lysophosphatidic acid (LPA). LPA is then converted by PlsC to the universal precursor for GPL synthesis, phosphatidic acid (PA). PlsB and PlsC are essential in E. coli, indicating they serve as the major pathway for PA synthesis. While loss of PlsX or PlsY individually has little consequence on the cell, the absence of both was lethal. To understand the synthetic lethality of this seemingly redundant PlsX/Y pathway, we performed a suppressor screen. Suppressor analysis indicated that ∆plsXY requires increased levels of glycerol-3-phosphate (G3P), a GPL precursor. In agreement, ∆plsXY required supplementation with G3P for survival. Furthermore, loss of PlsX dysregulated fatty acid synthesis, resulting in increased long-chain fatty acids. We show that although PlsX/Y together contribute to PA synthesis, they also contribute to the regulation of overall membrane biogenesis. Thus, synthetic lethality of ∆plsXY is multifactorial, suggesting that PlsX/Y has been maintained as a redundant system to fine-tune the synthesis of major lipids and promote cell envelope homeostasis.IMPORTANCEGram-negative bacteria must maintain optimal ratios of glycerophospholipids and lipopolysaccharide within the cell envelope for viability. Maintenance of proper outer membrane asymmetry allows for resistance to toxins and antibiotics. Here, we describe additional roles of PlsX and PlsY in Escherichia coli beyond lysophosphatidic acid synthesis, a key precursor of all glycerophospholipids. These findings suggest that PlsX and PlsY also play a larger role in impacting homeostasis of lipid synthesis.

PilY1 regulates the dynamic architecture of the type IV pilus machine in Pseudomonas aeruginosa

Type IV pili (T4P) produced by the pathogen Pseudomonas aeruginosa play a pivotal role in adhesion, surface motility, biofilm formation, and infection in humans. Despite the significance of T4P as a potential therapeutic target, key details of their dynamic assembly and underlying molecular mechanisms of pilus extension and retraction remain elusive, primarily due to challenges in isolating intact T4P machines from the bacterial cell envelope. Here, we combine cryo-electron tomography with subtomogram averaging and integrative modelling to resolve in-situ architectural details of the dynamic T4P machine in P. aeruginosa cells. The T4P machine forms 7-fold symmetric cage-like structures anchored in the cell envelope, providing a molecular framework for the rapid exchange of major pilin subunits during pilus extension and retraction. Our data suggest that the T4P adhesin PilY1 forms a champagne-cork-shaped structure, effectively blocking the secretin channel in the outer membrane whereas the minor-pilin complex in the periplasm appears to contact PilY1 via the central pore of the secretin gate. These findings point to a hypothetical model where the interplay between the secretin protein PilQ and the PilY1-minor-pilin priming complex is important for optimizing conformations of the T4P machine in P. aeruginosa, suggesting a gate-keeping mechanism that regulates pilus dynamics.

Antibacterial, Antibiofilm, and Anti-inflammatory Effects of a Novel Thrombin-Derived Peptide in Sepsis Models: Insights into Underlying Mechanisms.

We developed two short helical antimicrobial peptides, HVF18-a3 and its d-enantiomer, HVF18-a3-d, derived from the thrombin C-terminal peptide HVF18. These peptides exhibit potent antimicrobial activity against various bacteria by compromising both the outer and inner membranes, with low hemolytic activity. They are stable in the presence of physiological salts and human serum, exhibiting a low potential for developing drug resistance and excellent antibiofilm activity against Gram-negative bacteria. HVF18-a3-d also neutralized lipopolysaccharide (LPS) through direct binding interactions and suppressed the production of inflammatory cytokines through the inflammatory signaling pathway mediated by Toll-like receptor 4 in RAW264.7 cells stimulated with LPS. Both pre- and post-treatment with HVF18-a3-d significantly protected mice against fatal septic shock induced by carbapenem resistant Acinetobacter baumannii. These findings suggest HVF18-a3 and HVF18-a3-d are promising candidates for developing antibiotics against Gram-negative sepsis.

Helicobacter pylori biofilm interference by N-acyl homoserine lactonases: in vitro and in silico approaches.

Qurom quenching enzyme have an impact on treatment efficacy and prevent the recurrence of Helicobacter pylori biofilm-related infections, although it has not been thoroughly investigated in vitro and in silico. The current study aims to characterize the N-acyl homoserine lactonase, the quorum quenching AiiA protein of Bacillus licheniformis against H. pylori biofilm. In this study, AiiA protein were screened for their anti-biofilm activity, was found to effectively control biofilm formation of H. pylori with concentrations ranging from 2 to 10µg/mL. According to CLSM and COMSTAT analysis, the untreated substratum had the robust biofilm biomass of 25-18 µM and biovolume of 3-4 mm3 /mm2. The total biofilm biovolume and average biofilm thickness were considerably reduced by 40% with a single application of 10µg/mL of AiiA protein. The biofilm treated with AiiA exhibited a lower urease and polysaccharides than to the untreated biofilm. Further, in silico analysis, exhibited a greater interaction of AiiA against the outer membrane proteins of H. pylori compared to virulence factors. The conserved domains in the binding pockets of AiiA proteins showed a highest binding affinity proving the catalytic activity of the protein. In this study, the H. pylori biofilm architecture, exopolysaccharide and urease were significantly controlled by our purified N-acyl homoserine lactonase from B. licheniformis. Furthermore, the molecular docking showed the significant interaction between AiiA and key biofilm forming and virulence proteins proved an excellent antibiofilm activity controlling the infections of H. pylori human pathogen.