Outer Membrane Of Gram-negative Bacteria Research Articles

Faster but Not Sweeter: A Model of Escherichia coli Re-level Lipopolysaccharide for Martini 3 and a Martini 2 Version with Accelerated Kinetics.

Lipopolysaccharide (LPS) is a complex glycolipid molecule that is the main lipidic component of the outer leaflet of the outer membrane of Gram-negative bacteria. It has very limited lateral motion compared to phospholipids, which are more ubiquitous in biological membranes, including in the inner leaflet of the outer membrane of Gram-negative bacteria. The slow-moving nature of LPS can present a hurdle for molecular dynamics simulations, given that the (pragmatically) accessible timescales to simulations are currently limited to microseconds, during which LPS displays some conformational dynamics but hardly any lateral diffusion. Thus, it is not feasible to observe phenomena such as insertion of molecules, including antibiotics/antimicrobials, directly into the outer membrane from the extracellular side nor to observe LPS dissociating from proteins via molecular dynamics using currently available models at the atomistic and more coarse-grained levels of granularity. Here, we present a model of deep rough LPS compatible with the Martini 2 coarse-grained force field with scaled down nonbonded interactions to enable faster diffusion. We show that the faster-diffusing LPS model is able to reproduce the salient biophysical properties of the standard models, but due to its faster lateral motion, molecules are able to penetrate deeper into membranes containing the faster model. We show that the fast ReLPS model is able to reproduce experimentally determined patterns of interaction with outer membrane proteins while also allowing for LPS to associate and dissociate with proteins within microsecond timescales. We also complete the Martini 3 LPS toolkit for Escherichia coli by presenting a (standard) model of deep rough LPS for this force field.

Bacteriophage Removal from Infected Salmonella Cultures.

Bacteriophages, or simply phages, play a vital role in microbial environments, impacting bacterial populations and shaping their evolution and interactions. These organisms are viruses that infect and replicate within bacterial hosts. Phages are ubiquitous on Earth, highly diverse, and very abundant. While bacteriophages have valuable roles in different environments and are a key area of research in microbiology and ecology, their presence can be undesirable in certain industrial processes or products. Considering the abundance and ubiquity of bacteriophages on Earth, the design of procedures for the removal of bacteriophages from bacterial cultures is crucial in diverse laboratory and industrial applications to preserve the integrity of the cultures and ensure accurate experimental results or product quality. Here, we have fine-tuned a protocol to eliminate the bacteriophages from infected Salmonella enterica cultures, using a strategy based on the use of lipopolysaccharides (LPS) located in the outer membrane of Gram-negative bacteria. Bacterial LPS plays an important role in host recognition by phages, and we make use of this property to design an effective procedure for the removal of phages, which use LPS as a receptor, in Salmonella bacterial cultures.

Genetic evidence for functional diversification of gram-negative intermembrane phospholipid transporters.

The outer membrane of gram-negative bacteria is a barrier to chemical and physical stress. Phospholipid transport between the inner and outer membranes has been an area of intense investigation and, in E. coli K-12, it has recently been shown to be mediated by YhdP, TamB, and YdbH, which are suggested to provide hydrophobic channels for phospholipid diffusion, with YhdP and TamB playing the major roles. However, YhdP and TamB have different phenotypes suggesting distinct functions. It remains unclear whether these functions are related to phospholipid metabolism. We investigated a synthetic cold sensitivity caused by deletion of fadR, a transcriptional regulator controlling fatty acid degradation and unsaturated fatty acid production, and yhdP, but not by ΔtamB ΔfadR or ΔydbH ΔfadR. Deletion of tamB recuses the ΔyhdP ΔfadR cold sensitivity further demonstrating the phenotype is related to functional diversification between these genes. The ΔyhdP ΔfadR strain shows a greater increase in cardiolipin upon transfer to the non-permissive temperature and genetically lowering cardiolipin levels can suppress cold sensitivity. These data also reveal a qualitative difference between cardiolipin synthases in E. coli, as deletion of clsA and clsC suppresses cold sensitivity but deletion of clsB does not. Moreover, increased fatty acid saturation is necessary for cold sensitivity and lowering this level genetically or through supplementation of oleic acid suppresses the cold sensitivity of the ΔyhdP ΔfadR strain. Together, our data clearly demonstrate that the diversification of function between YhdP and TamB is related to phospholipid metabolism. Although indirect regulatory effects are possible, we favor the parsimonious hypothesis that YhdP and TamB have differential phospholipid-substrate transport preferences. Thus, our data provide a potential mechanism for independent control of the phospholipid composition of the inner and outer membranes in response to changing conditions based on regulation of abundance or activity of YhdP and TamB.

Mg2+-dependent mechanism of environmental versatility in a multidrug efflux pump.

Tripartite resistance nodulation and cell division multidrug efflux pumps span the periplasm and are a major driver of multidrug resistance among Gram-negative bacteria. The periplasm provides a distinct environment between the inner and outer membranes of Gram-negative bacteria. Cations, such as Mg2+, become concentrated within the periplasm and, in contrast to the cytoplasm, its pH is sensitive to conditions outside the cell. Here, we reveal an interplay between Mg2+ and pH in modulating the dynamics of the periplasmic adaptor protein, AcrA, and its function within the prototypical AcrAB-TolC multidrug efflux pump from Escherichia coli. In the absence of Mg2+, AcrA becomes increasingly plastic within acidic conditions, but when Mg2+ is bound this is ameliorated, resulting in domain specific organisation in neutral to weakly acidic regimes. We establish a unique histidine residue directs these structural dynamics and is essential for sustaining pump efflux activity across acidic, neutral, and alkaline conditions. Overall, we propose Mg2+ conserves the structural mobility of AcrA to ensure optimal AcrAB-TolC function within rapid changing environments commonly faced by the periplasm during bacterial infection and colonization. This work highlights that Mg2+ is an important mechanistic component in this pump class and possibly across other periplasmic lipoproteins.

Genetic evidence for functional diversification of gram-negative intermembrane phospholipid transporters.

The outer membrane of Gram-negative bacteria is a barrier to chemical and physical stress. Phospholipid transport between the inner and outer membranes has been an area of intense investigation and, in E. coli K-12, it has recently been shown to be mediated by YhdP, TamB, and YdbH, which are suggested to provide hydrophobic channels for phospholipid diffusion, with YhdP and TamB playing the major roles. However, YhdP and TamB have different phenotypes suggesting distinct functions. We investigated these functions using synthetic cold sensitivity (at 30 °C) caused by deletion of yhdP and fadR, a transcriptional regulator controlling fatty acid degradation and unsaturated fatty acid production, but not by ΔtamB ΔfadR or ΔydbH ΔfadR,. Deletion of tamB suppresses the ΔyhdP ΔfadR cold sensitivity suggesting this phenotype is related to phospholipid transport. The ΔyhdP ΔfadR strain shows a greater increase in cardiolipin upon transfer to the non-permissive temperature and genetically lowering cardiolipin levels can suppress cold sensitivity. These data also reveal a qualitative difference between cardiolipin synthases in E. coli, as deletion of clsA and clsC suppresses cold sensitivity but deletion of clsB does not despite lower cardiolipin levels. In addition to increased cardiolipin, increased fatty acid saturation is necessary for cold sensitivity and lowering this level genetically or through supplementation of oleic acid suppresses the cold sensitivity of the ΔyhdP ΔfadR strain. Although indirect effects are possible, we favor the parsimonious hypothesis that YhdP and TamB have differential substrate transport preferences, most likely with YhdP preferentially transporting more saturated phospholipids and TamB preferentially transporting more unsaturated phospholipids. We envision cardiolipin contributing to this transport preference by sterically clogging TamB-mediated transport of saturated phospholipids. Thus, our data provide a potential mechanism for independent control of the phospholipid composition of the inner and outer membranes in response to changing conditions.

Top-Down Characterization of Bacterial Lipopolysaccharides and Lipooligosaccharides Using Activated-Electron Photodetachment Mass Spectrometry.

Lipopolysaccharides (LPS) and lipooligosaccharides (LOS) are located in the outer membrane of Gram-negative bacteria and are comprised of three distinctive parts: lipid A, core oligosaccharide (OS), and O-antigen. The structure of each region influences bacterial stability, toxicity, and pathogenesis. Here, we highlight the use of targeted activated-electron photodetachment (a-EPD) tandem mass spectrometry to characterize LPS and LOS from two crucial players in the human gut microbiota, Escherichia coli Nissle and Bacteroides fragilis. a-EPD is a hybrid activation method that uses ultraviolet photoirradiation to generate charge-reduced radical ions followed by collisional activation to produce informative fragmentation patterns. We benchmark the a-EPD method for top-down characterization of triacyl LOS from E. coli R2, then focus on characterization of LPS from E. coli Nissle and B. fragilis. Notably, a-EPD affords extensive fragmentation throughout the backbone of the core OS and O-antigen regions of LPS from E. coli Nissle. This hybrid approach facilitated the elucidation of structural details for LPS from B. fragilis, revealing a putative hexuronic acid (HexA) conjugated to lipid A.

Delivery of endolysin across outer membrane of Gram-negative bacteria using translocation domain of botulinum neurotoxin

The emergence of multidrug-resistant pathogens has outpaced the development of new antibiotics, leading to renewed interest in endolysins. Endolysins have been investigated as novel biocontrol agents for Gram-positive bacteria. However, their efficacy against Gram-negative species is limited by the barrier presented by their outer membrane, which prevents endolysin access to the peptidoglycan substrate. Here, we used the translocation domain of botulinum neurotoxin to deliver endolysin across the outer membrane of Gram-negative bacteria. The translocation domain selectively interacts with and penetrates membranes composed of anionic lipids, which have been used in nature to deliver various proteins into animal cells. In addition to the botulinum neurotoxin translocation domain, we have fused bacteriophage-derived receptor binding protein to endolysins. This allows the attached protein to efficiently bind to a broad spectrum of Gram-negative bacteria. By attaching these target-binding and translocation machineries to endolysins, we aimed to develop an engineered endolysin with broad-spectrum targeting and enhanced antibacterial activity against Gram-negative species. To validate our strategy, we designed engineered endolysins using two well-known endolysins, T5 and LysPA26, and tested them against 23 strains from six species of Gram-negative bacteria, confirming that our machinery can act broadly. In particular, we observed a 2.32 log reduction in 30 min with only 0.5 µM against an Acinetobacter baumannii isolate. We also used the SpyTag/SpyCatcher system to easily attach target-binding proteins, thereby improving its target-binding ability. Overall, our newly developed endolysin engineering strategy may be a promising approach to control multidrug-resistant Gram-negative bacterial strains.

Effect of the bacterial lipopolysaccharide (LPS) on vitamin B1 (thiamin) uptake along the intestinal tract: inhibition mediated via reduction in level of expression of thiamin transporter-1 & -2 proteins at the cell membrane

Background: LPS is a potent bacterial virulence factor and is a source of considerable clinical morbidity and mortality. This endotoxin is a predominant component of the outer membrane of Gram-negative bacteria. The gut is exposed to high levels of LPS in conditions like Inflammatory Bowel Diseases, necrotizing enterocolitis, sepsis, and following infection with enteric pathogens. Studies have shown that exposure of gut epithelial/other cells to LPS exerts profound and differential effects on membrane transport processes. Nothing, however, is known about the effect of LPS on uptake of free thiamin in the small intestine (where absorption of dietary thiamin occurs), and the large intestine (where absorption of the microbiota-generated thiamin occurs). Aim: To examine the effect of LPS on the small and large intestinal uptake of free thiamin and to determine the mechanism(s) involved. Methods: We used in vitro (human duodenum-derived HuTu 80 cells and colon-derived non-transformed NCM460 cells), ex vivo (human differentiated polarized enteroid and colonoid monolayers), and in vivo (mouse) models in our studies. Uptake studies were performed using 3H-thiamin as substrate. Results: Treating intestinal HuTu 80 and colonic NCM460 epithelial cells with LPS led to a significant inhibition in carrier-mediated 3H-thiamin uptake. Similar results were obtained when human differentiated enteroid and colonoid monolayers ex vivo, and mouse in vivo were treated with LPS. No changes in total cellular levels of thiamin transporter-1 & -2 (THTR-1 and THTR-2, respectively) proteins and mRNAs were observed upon LPS treatments; however, a significant decrease in the fraction of the THTR-1 & THTR-2 proteins that are expressed at the cell membrane were observed. These findings suggest that the LPS inhibitory effect on thiamin uptake in the small and large intestine is likely mediated via a decrease in the fraction of the thiamin transporters that are expressed at the cell membrane. Conclusion: This study shows that bacterial LPS inhibits thiamin uptake along the intestinal tract, and that this inhibition is likely mediated via reduction in level of the THTR-1 & THTR-2 proteins at the cell membrane. Supported by NIH grants DK-56061 and AA-018071, and VA grants 101BX001142 & IK6BX006189. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Development of novel indole-quinoline hybrid molecules targeting bacterial proton motive force.

This study aimed to develop an editable structural scaffold for improving drug development, including pharmacokinetics and pharmacodynamics of antibiotics by using synthetic compounds derived from a (hetero)aryl-quinoline hybrid scaffold. In this study, 18 CF3-substituted (hetero)aryl-quinoline hybrid molecules were examined for their potential antibacterial activity against Staphylococcus aureus by determining minimal inhibitory concentrations. These 18 synthetic compounds represent modifications to key regions of the quinoline N-oxide scaffold, enabling us to conduct a structure-activity relationship analysis for antibacterial potency. Among the compounds, 3m exhibited potency against with both methicillin resistant S. aureus strains, as well as other Gram-positive bacteria, including Enterococcus faecalis and Bacillus subtilis. We demonstrated that 3m disrupted the bacterial proton motive force (PMF) through monitoring the PMF and conducting the molecular dynamics simulations. Furthermore, we show that this mechanism of action, disrupting PMF, is challenging for S. aureus to overcome. We also validated this PMF inhibition mechanism of 3m in an Acinetobacter baumannii strain with weaken lipopolysaccharides. Additionally, in Gram-negative bacteria, we demonstrated that 3m exhibited a synergistic effect with colistin that disrupts the outer membrane of Gram-negative bacteria. Our approach to developing editable synthetic novel antibacterials underscores the utility of CF3-substituted (hetero)aryl-quinoline scaffold for designing compounds targeting the bacterial proton motive force, and for further drug development, including pharmacokinetics and pharmacodynamics.

Pseudomonas aeruginosa Lipid A Structural Variants Induce Altered Immune Responses.

Pseudomonas aeruginosa causes chronic lung infection in cystic fibrosis (CF), resulting in structural lung damage and progressive pulmonary decline. P. aeruginosa in the CF lung undergoes numerous changes, adapting to host-specific airway pressures while establishing chronic infection. P. aeruginosa undergoes lipid A structural modification during CF chronic infection, not seen in any other disease state. Lipid A, the membrane anchor of lipopolysaccharide (i.e., endotoxin), comprises the majority of the outer membrane of Gram-negative bacteria and is a potent toll-like receptor (TLR)4 agonist. The structure of P. aeruginosa lipid A is intimately linked with its recognition by TLR4, and subsequent immune response. Prior work has identified P. aeruginosa strains with altered lipid A structures that arise during chronic CF lung infection; however, the impact of P. aeruginosa lipid A structure on airway disease has not been investigated. Here, we show that P. aeruginosa lipid A lacks PagL-mediated deacylation during human airway infection using a direct-from-sample mass spectrometry approach on human bronchoalveolar lavage fluid. This structure triggers increased pro-inflammatory cytokine production by primary human macrophages. Furthermore, alterations in lipid A 2-hydroxylation impact cytokine response in a site-specific manner, independent of CFTR function. Interestingly, there is a CF-specific reduction in IL-8 secretion within the epithelial-cell compartment that only occurs in CF bronchial epithelial cells when infected with CF-adapted P. aeruginosa that lack PagL-mediated lipid A deacylation. Taken together, we show that P. aeruginosa alters its lipid A structure during acute lung infection and that this lipid A structure induces stronger signaling through TLR4.

Cerastecins inhibit membrane lipooligosaccharide transport in drug-resistant Acinetobacter baumannii.

Carbapenem-resistant Acinetobacter baumannii infections have limited treatment options. Synthesis, transport and placement of lipopolysaccharide or lipooligosaccharide (LOS) in the outer membrane of Gram-negative bacteria are important for bacterial virulence and survival. Here we describe the cerastecins, inhibitors of the A. baumannii transporter MsbA, an LOS flippase. These molecules are potent and bactericidal against A. baumannii, including clinical carbapenem-resistant Acinetobacter baumannii isolates. Using cryo-electron microscopy and biochemical analysis, we show that the cerastecins adopt a serpentine configuration in the central vault of the MsbA dimer, stalling the enzyme and uncoupling ATP hydrolysis from substrate flipping. A derivative with optimized potency and pharmacokinetic properties showed efficacy in murine models of bloodstream or pulmonary A. baumannii infection. While resistance development is inevitable, targeting a clinically unexploited mechanism avoids existing antibiotic resistance mechanisms. Although clinical validation of LOS transport remains undetermined, the cerastecins may open a path to narrow-spectrum treatment modalities for important nosocomial infections.

The Prediction of LptA and LptC Protein-Protein Interactions and Virtual Screening for Potential Inhibitors.

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.

On the Microstructure and Dynamics of Membranes Formed by Lipid as From Stenotrophomonas maltophilia, a Member of Gut Microbiome: An EPR Study

AbstractLipid As are the main components of the external leaflet of the outer membrane of Gram-negative bacteria. Their molecular structure has evolved to allow the bacteria survival in specific environments. In the present work, we investigate how and to what extent lipid membranes that include in their composition lipid A molecules of a bacterium of the gut microbiota, Stenotrophomonas maltophilia, differ from those formed by the lipid A of the common Gram-negative bacterium Salmonella enterica, which is not specific to the gut and is here used as a reference. Electron Paramagnetic Resonance (EPR) spectroscopy, using spin-labelled lipids as molecular probes, allows the segmental order of the acyl chain and the polarity across the bilayer to be analyzed in detail. Both considered lipid As cause a stiffening of the outermost segments of the acyl chains. This effect increases with increasing the lipid A content and is stronger for the lipid A extracted from Stenotrophomonas maltophilia than for that extracted from Salmonella enterica. At the same time, the local polarity of the bilayer region just below the interface increases. As the inner core of the bilayer is considered, it is found that the lipid A from Salmonella enterica causes a local disorder and a significant reduction of the local polarity, an effect not found for the lipid A from Stenotrophomonas maltophilia. These results are interpreted in terms of the different lengths and distributions of the acyl tails in the two lipid As. It can be concluded that the symmetrically distributed short tails of the lipid A from Stenotrophomonas maltophilia favors a regular packing within the bilayer.

Electrochemical and Infrared Studies of a Model Bilayer of the Outer Membrane of Gram-Negative Bacteria and its Interaction with polymyxin─the Last-Resort Antibiotic.

A model bilayer of the outer membrane (OM) of Gram-negative bacteria, composed of lipid A and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), was assembled on the β-Tg modified gold (111) single crystal surface using a combination of Langmuir-Blodgett and Langmuir-Schaefer transfer. Electrochemical and spectroscopic methods were employed to study the properties of the model bilayer and its interaction with polymyxin. The model bilayer is stable on the gold surface in the transmembrane potential region between 0.0 and -0.7 V. The presence of Mg2+ coordinates with the phosphate and carboxylate groups in the leaflet of lipid A and stabilizes the structure of the model bilayer. Polymyxin causes the model bilayer leakage and damage in the transmembrane potential region between 0.2 and -0.4 V. At transmembrane potentials lower than -0.5 V, polymyxin does not affect the membrane integrity. Polymyxin binds to the phosphate and carboxylate groups in lipid A molecules and causes the increase of the tilt angle of acyl chains and the decrease of the tilt of the C═O bond. The results in this paper indicate that the antimicrobial activity of polymyxin depends on the transmembrane potential at the model bilayer and provides useful information for the development of new antibiotics.

Lessons learned from contamination with endotoxin originated from the supplement in the cell culture medium

IntroductionEndotoxin is a typical pyrogen derived from the outer membrane of Gram-negative bacteria. In fabricating cell-based medicinal products, it is necessary to control endotoxin in the process and the products. In the quality control tests of our clinical study, endotoxin concentration in the culture supernatant of autologous oral mucosal epithelial cell sheets exceeded the criterion value. Therefore, endotoxin measurements were conducted to clarify the cause of the endotoxin contamination. MethodsThe reagents used to prepare the culture medium, the unused culture medium, and the culture supernatants were diluted with pure water. Endotoxin concentrations in the diluted samples were measured. ResultsEndotoxin was detected in both the unused culture medium and the culture supernatant of the epithelial cell sheets at higher concentrations than the criterion value. Therefore, endotoxin concentrations in the reagents used to prepare the culture medium were measured and were found to be below the criterion value, except for cholera toxin. On the other hand, three lots of cholera toxin products were used for the measurement, and the endotoxin concentrations were higher than the criterion value. The results indicate that the endotoxin contamination is caused by the cholera toxin product. ConclusionsTo prevent endotoxin contamination in cell-based medicinal products, endotoxin concentrations in reagents used for the fabrication should be measured in the facility conducting clinical research or confirmed by an adequate certificate of analysis from the manufacturers of the reagents.

Engineering strategies and challenges of endolysin as an antibacterial agent against Gram-negative bacteria.

Bacteriophage endolysin is a novel antibacterial agent that has attracted much attention in the prevention and control of drug-resistant bacteria due to its unique mechanism of hydrolysing peptidoglycans. Although endolysin exhibits excellent bactericidal effects on Gram-positive bacteria, the presence of the outer membrane of Gram-negative bacteria makes it difficult to lyse them extracellularly, thus limiting their application field. To enhance the extracellular activity of endolysin and facilitate its crossing through the outer membrane of Gram-negative bacteria, researchers have adopted physical, chemical, and molecular methods. This review summarizes the characterization of endolysin targeting Gram-negative bacteria, strategies for endolysin modification, and the challenges and future of engineering endolysin against Gram-negative bacteria in clinical applications, to promote the application of endolysin in the prevention and control of Gram-negative bacteria.

Diversity, Complexity, and Specificity of Bacterial Lipopolysaccharide (LPS) Structures Impacting Their Detection and Quantification.

Endotoxins are toxic lipopolysaccharides (LPSs), extending from the outer membrane of Gram-negative bacteria and notorious for their toxicity and deleterious effects. The comparison of different LPSs, isolated from various Gram-negative bacteria, shows a global similar architecture corresponding to a glycolipid lipid A moiety, a core oligosaccharide, and outermost long O-chain polysaccharides with molecular weights from 2 to 20 kDa. LPSs display high diversity and specificity among genera and species, and each bacterium contains a unique set of LPS structures, constituting its protective external barrier. Some LPSs are not toxic due to their particular structures. Different, well-characterized, and highly purified LPSs were used in this work to determine endotoxin detection rules and identify their impact on the host. Endotoxin detection is a major task to ensure the safety of human health, especially in the pharma and food sectors. Here, we describe the impact of different LPS structures obtained under different bacterial growth conditions on selective LPS detection methods such as LAL, HEK-blue TLR-4, LC-MS2, and MALDI-MS. In these various assays, LPSs were shown to respond differently, mainly attributable to their lipid A structures, their fatty acid numbers and chain lengths, the presence of phosphate groups, and their possible substitutions.

β-lactam-induced OMV release promotes polymyxin tolerance in Salmonella enterica sv. Typhi.

The rise of multidrug-resistant bacteria is a global concern, leading to a renewed reliance on older antibiotics like polymyxins as a last resort. Polymyxins, cationic cyclic peptides synthesized nonribosomally, feature a hydrophobic acyl tail and positively charged residues. Their antimicrobial mechanism involves initial interaction with Gram-negative bacterial outer-membrane components through polar and hydrophobic interactions. Outer membrane vesicles (OMVs), nano-sized proteoliposomes secreted from the outer membrane of Gram-negative bacteria, play a crucial role in tolerating harmful molecules, including cationic peptides such as polymyxins. Existing literature has documented environmental changes' impact on modulating OMV properties in Salmonella Typhimurium. However, less information exists regarding OMV production and characteristics in Salmonella Typhi. A previous study in our laboratory showed that S. Typhi ΔmrcB, a mutant associated with penicillin-binding protein (PBP, a β-lactam antibiotic target), exhibited hypervesiculation. Consequently, this study investigated the potential impact of β-lactam antibiotics on promoting polymyxin tolerance via OMVs in S. Typhi. Our results demonstrated that sub-lethal doses of β-lactams increased bacterial survival against polymyxin B in S. Typhi. This phenomenon stems from β-lactam antibiotics inducing hypervesiculation of OMVs with higher affinity for polymyxin B, capturing and diminishing its biologically effective concentration. These findings suggest that β-lactam antibiotic use may inadvertently contribute to decreased polymyxin effectivity against S. Typhi or other Gram-negative bacteria, complicating the effective treatment of infections caused by these pathogens. This study emphasizes the importance of evaluating the influence of β-lactam antibiotics on the interaction between OMVs and other antimicrobial agents.

Cajaninstilbene acid derivatives conjugated with siderophores of 3-hydroxypyridin-4(1H)-ones as novel antibacterial agents against Gram-negative bacteria based on the Trojan horse strategy

The low permeability of the outer membrane of Gram-negative bacteria is a serious obstacle to the development of new antibiotics against them. Conjugation of antibiotic with siderophore based on the “Trojan horse strategy” is a promising strategy to overcome the outer membrane obstacle. In this study, series of antibacterial agents were designed and synthesized by conjugating the 3-hydroxypyridin-4(1H)-one based siderophores with cajaninstilbene acid (CSA) derivative 4 which shows good activity against Gram-positive bacteria by targeting their cell membranes but is ineffective against Gram-negative bacteria. Compared to the inactive parent compound 4, the conjugates 45c or 45d exhibits significant improvement in activity against Gram-negative bacteria, including Escherichia coli, Klebsiella pneumoniae and especially P. aeruginosa (minimum inhibitory concentrations, MICs = 7.8–31.25 μM). The antibacterial activity of the conjugates is attributed to the CSA derivative moiety, and the action mechanism is by disruption of bacterial cell membranes. Further studies on the uptake mechanisms showed that the bacterial siderophore-dependent iron transport system was involved in the uptake of the conjugates. In addition, the conjugates 45c and 45d showed a lower cytotoxic effects in vivo and in vitro and a positive therapeutic effect in the treatment of C. elegans infected by P. aeruginosa. Overall, our work describes a new class and a promising 3-hydroxypyridin-4(1H)-one-CSA derivative conjugates for further development as antibacterial agents against Gram-negative bacteria.

Abstract 2179: Microbiome-derived metabolites mediate carcinogenic alterations of breast tissue in the context of obesity

Abstract Obesity increases the relative risk for breast cancer incidence. Multiple molecular mechanisms linked to obesity drive breast cancer progression. Obesity shifts the gut microbiome in ways that may increase breast cancer risk. Microbial-associated molecular pattern (MAMP)-proteins and metabolites could directly affect breast epithelial cell signaling. In this study, we focused on lipopolysaccharide (LPS), a structural component of the outer membranes of gram-negative bacteria and a toll-like receptor 4 (TLR4)-agonist. Systemic LPS levels are known to increase in obesity. We hypothesize that chronic low-grade inflammation caused by LPS contributes to breast cancer initiation. To test this hypothesis, we first quantified levels of LPS, along with a panel of adipokines/cytokines, in serum samples from women (n= 33; BMI range= 18.3-45.3 Kg/m2; Age range= 18-51 years). Women with elevated serum LPS had significantly higher BMI, leptin, and leptin/adiponectin ratio, confirming a link between systemic LPS and metabolic markers of obesity. Other biomarkers of breast cancer risk include DNA damage. Analyses of non-cancerous breast tissue sections from matched donors revealed higher densities of DNA double-strand breaks (estimated based on 53BP1 foci counts) in women with high serum LPS. Experiments with HMT-3522 S1 breast acini showed that LPS increases DNA break frequencies and oxidative stress levels in 3D structures of mammary epithelium. Moreover, LPS increases oxidative lesions in the DNA (estimated based on 8-oxoguanine fluorescence in MCF-10A cells) that may lead to increased LPS-mediated DNA damage. Patient obesity status was associated with alterations in oxidative modifications in the breast proteome. We also found that LPS disrupts epithelial polarity, a hallmark of breast tissue homeostasis. In mammary epithelium, LPS activates the nuclear factor-kappa B (NFκB) pathway by binding to the TLR4 receptor, leading to an increased expression of inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α). Interestingly, TLR4 blockade prevented the loss of apical polarity and DNA damage induced by LPS. Last, but not least, we show an association between LPS immunogenicity (anti-LPS IgA concentrations) and participant BMI in non-cancerous breast tissue samples. These novel findings show that LPS is a key systemic and local mediator of the obesity-modulated microbiome that increases breast cancer risk. The outcomes of our study underscore the importance of considering the microbiome in the prevention of breast cancer. Citation Format: Mohamed Gaber, Kayla Neary, Julia Holmes, Adam S. Wilson, Adam J. Katz, Pierre-Alexandre Vidi, Katherine L. Cook. Microbiome-derived metabolites mediate carcinogenic alterations of breast tissue in the context of obesity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2179.