Outer Membrane Of Gram-negative Bacteria Research Articles

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.

Molecular mechanism of action of imidazolium carbosilane dendrimers on the outer bacterial membrane – From membrane damage to permeability to antimicrobial endolysin

The outer bacterial membrane of drug-resistant bacteria is a significant barrier to many antimicrobials. Therefore, the development of new antibacterials primarily focuses on damaging the outer bacterial membrane of Gram-negative bacteria. Among many membrane-disrupting substances, the most promising are cationic dendritic systems. However, the mode of action may vary among different strains due to variations in the lipid compositions of the membrane. Here, we investigated the interaction of two types of cationic imidazolium carbosilane dendrimers: one with a single cationic group (methyl imidazolium) and the other with the same cationic group but attached to a functional group (a pendant pyridyl moiety), capable of establishing interactions with membranes through H-bonding or ion–dipole electrostatic interactions. We used different models of the outer membrane of Gram-negative bacteria – Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumannii. Additionally, we assessed the combined effect of the dendrimers and the antibacterial endolysin on P. aeruginosa. Our results show that the mechanism of action depends on the type of dendrimer and the lipid composition of the membrane. We also demonstrate that the alteration of membrane fluidity and permeability to endolysin by the methyl imidazolium and pyridyl imidazolium dendrimers may play a more significant role in antimicrobial activity compared to membrane damage caused by positively charged dendrimers.

Активность липополисахарида типового штамма Azospirillum palustre B2 в отношении проростков пшеницы (Triticum aestivum L.)

Lipopolysaccharide is the main structural component of the outer membrane of Gram-negative bacteria, which can also be a part of extracellular polymeric substances. Lipopolysaccharides of bacteria that stimulate the growth and development of plants belong to the group of molecules that form a microbe-associated molecular pattern (MAMP). These glycoconjugates of both symbiotic and phytopathogenic bacteria induce the activation of immune responses in plants. However, the level of plant response under the influence of symbiont lipopolysaccharides differs significantly, also due to their structural features, which make it possible to bypass or weaken the reactions of innate autoimmunity. In this paper, we present the results of the analysis of the reactions of wheat seedlings Triticum aestivum L. after incubation with lipopolysaccharide of associative bacteria Azospirillum palustre B2(T). Incubation of wheat seedlings in the presence of A. palustre B2 lipopolysaccharide for three days led to the activation of plant growth processes, namely an increase in the length of shoots, roots, the area of the first leaf, and a change in the content of pigments in the leaves.

Monitoring Drug-Protein Interactions in the Bacterial Periplasm by Solution Nuclear Magnetic Resonance Spectroscopy.

The rapid spread of antimicrobial resistance across bacterial pathogens poses a serious risk to the efficacy and sustainability of available treatments. This puts pressure on research concerning the development of new drugs. Here, we present an in-cell NMR-based research strategy to monitor the activity of the enzymes located in the periplasmic space delineated by the inner and outer membranes of Gram-negative bacteria. We demonstrate its unprecedented analytical power in monitoring in situ and in real time (i) the hydrolysis of β-lactams by β-lactamases, (ii) the interaction of drugs belonging to the β-lactam family with their essential targets, and (iii) the binding of inhibitors to these enzymes. We show that in-cell NMR provides a powerful analytical tool for investigating new drugs targeting the molecular components of the bacterial periplasm.

Intestinal epithelial cells of Japanese flounder (Paralichthys olivaceus) as an in vitro model for studying intestine immune function based on transcriptome analysis

Japanese flounder (Paralichthys olivaceus) is an economically crucial marine species, but diseases like hemorrhagic septicemia caused by Edwardsiella tarda have resulted in significant economic losses. E. tarda infects various hosts, and its pathogenicity in fish is not fully understood. Lipopolysaccharides (LPS) are components of the outer membrane of Gram-negative bacteria and are representative of typical PAMP molecules that cause activation of the immune system. The PoIEC cell line is a newly established intestinal epithelial cell line from P. olivaceus. In order to investigate whether it can be used as an in vitro model for studying the pathogenesis of E. tarda and LPS stimulation, we conducted RNA-seq experiments for the PoIECs model of E. tarda infection and LPS stimulation. In this study, transcriptome sequencing was carried out in the PoIEC cell line after treatment with LPS and E. tarda. A total of 62.52G of high-quality data from transcriptome sequencing results were obtained in nine libraries, of which an average of 87.96% data could be aligned to the P. olivaceus genome. Data analysis showed that 283 and 414 differentially expressed genes (DEGs) in the LPS versus Control (LPS-vs-Con) and E. tarda versus Control groups (Et-vs-Con), respectively, of which 60 DEGs were shared in two comparation groups. The GO terms were predominantly enriched in the extracellular space, inflammatory response, and cytokine activity in the LPS-vs-Con group, whereas GO terms were predominantly enriched in nucleus and positive regulation of transcription by RNA polymerase II in the Et-vs-Con group. KEGG analysis revealed that three immune-related pathways were co-enriched in both comparison groups, including the Toll-like receptor signaling pathway, C-type lectin receptor signaling pathway, and Cytokine-cytokine receptor interaction. Five genes were randomly screened to confirm the validity and accuracy of the transcriptome data. These results suggest that PoIEC cell line can be an ideal in vitro model for studies of marine fish gut immunity and pathogenesis of Edwardsiellosis.

Characterization and immunological effect of outer membrane vesicles from Pasteurella multocida on macrophages

Pasteurella multocida is an important bacterial pathogen that can cause diseases in both animals and humans. Its elevated morbidity and mortality rates in animals result in substantial economic repercussions within the livestock industry. The prevention of diseases caused by P. multocida through immunization is impeded by the absence of a safe and effective vaccine. Outer membrane vesicles (OMVs) secreted from the outer membrane of Gram-negative bacteria are spherical vesicular structures that encompass an array of periplasmic components in conjunction with a diverse assortment of lipids and proteins. These vesicles can induce antibacterial immune responses within the host. P. multocida has been shown to produce OMVs. Nonetheless, the precise characteristics and immunomodulatory functions of P. multocida OMVs have not been fully elucidated. In this study, OMVs were isolated from P. multocida using an ultrafiltration concentration technique, and their morphology, protein constitution, and immunomodulatory properties in RAW264.7 cells were studied. Transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) revealed that the OMVs exhibited typical spherical and bilayered lipid vesicular architecture, exhibiting an average diameter of approximately 147.5 nm. The yield of OMVs was 2.6 × 1011 particles/mL. Proteomic analysis revealed a high abundance of membrane-associated proteins within P. multocida OMVs, with the capability to instigate the host’s immune response. Furthermore, OMVs stimulated the proliferation and cellular uptake of macrophages and triggered the secretion of cytokines, such as TNF-ɑ, IL-1β, IL-6, IL-10, and TGF-β1. Consequently, our results indicated that OMVs from P. multocida could directly interact with macrophages and regulate their immune function in vitro. These results supported the prospective applicability of P. multocida OMVs as a platform in the context of vaccine development.Key points• Preparation and characterization of P. multocida OMVs.• P. multocida OMVs possess a range of antigens and lipoproteins associated with the activation of the immune system.• P. multocida OMVs can activate the proliferation, internalization, and cytokine secretion of macrophages in vitro.

The C-terminus is essential for the stability of the mycobacterial channel protein MspA.

Outer membrane proteins perform essential functions in uptake and secretion processes in bacteria. MspA is an octameric channel protein in the outer membrane of Mycobacterium smegmatis and is structurally distinct from any other known outer membrane protein. MspA is the founding member of a family with more than 3000 homologs and is one of the most widely used proteins in nanotechnological applications due to its advantageous pore structure and extraordinary stability. While a conserved C-terminal signal sequence is essential for folding and protein assembly in the outer membrane of Gram-negative bacteria, the molecular determinants of these processes are unknown for MspA. In this study, we show that mutation and deletion of methionine 183 in the highly conserved C-terminus of MspA and mutation of the conserved tryptophan 40 lead to a complete loss of protein in heat extracts of M. smegmatis. Swapping these residues partially restores the heat stability of MspA indicating that methionine 183 and tryptophan 40 form a conserved sulfur-π electron interaction, which stabilizes the MspA monomer. Flow cytometry showed that all MspA mutants are surface-accessible demonstrating that oligomerization and membrane integration in M. smegmatis are not affected. Thus, the conserved C-terminus of MspA is essential for its thermal stability, but it is not required for protein assembly in its native membrane, indicating that this process is mediated by a mechanism distinct from that in Gram-negative bacteria. These findings will benefit the rational design of MspA-like pores to tailor their properties in current and future applications.

Generation of Endotoxin-Specific Monoclonal Antibodies by Phage and Yeast Display for Capturing Endotoxin.

Endotoxin, a synonym for lipopolysaccharide (LPS), is anchored in the outer membranes of Gram-negative bacteria. Even minute amounts of LPS entering the circulatory system can have a lethal immunoactivating effect. Since LPS is omnipresent in the environment, it poses a great risk of contaminating any surface or solution, including research products and pharmaceuticals. Therefore, monitoring LPS contamination and taking preventive or decontamination measures to ensure human safety is of the utmost importance. Nevertheless, molecules used for endotoxin detection or inhibition often suffer from interferences, low specificity, and low affinity. For this reason, the selection of new binders that are biocompatible, easy to produce, and that can be used for biopharmaceutical applications, such as endotoxin removal, is of high interest. Powerful techniques for selecting LPS-binding molecules in vitro are display technologies. In this study, we established and compared the selection and production of LPS-specific, monoclonal, human single-chain variable fragments (scFvs) through two display methods: yeast and phage display. After selection, scFvs were fused to a human constant fragment crystallizable (Fc). To evaluate the applicability of the constructs, they were conjugated to polystyrene microbeads. Here, we focused on comparing the functionalized beads and their LPS removal capacity to a polyclonal anti-lipid A bead. Summarized, five different scFvs were selected through phage and yeast display, with binding properties comparable to a commercial polyclonal antibody. Two of the conjugated scFv-Fcs outperformed the polyclonal antibody in terms of the removal of LPS in aqueous solution, resulting in 265 times less residual LPS in solution, demonstrating the potential of display methods to generate LPS-specific binding molecules.

Antibacterial synergy between a phage endolysin and citric acid against the Gram-negative kiwifruit pathogen Pseudomonas syringae pv. actinidiae.

The phytopathogen Pseudomonas syringae pv. actinidiae (Psa) causes major impacts to kiwifruit horticulture, and the current control strategies are heavily reliant on copper and antibiotics. The environmental impact and increasing resistance to these agrichemicals are driving interest in alternative antimicrobials including bacteriophage-derived therapies. In this study, we characterize the endolysin from the Otagovirus Psa374 which infects Psa. When combined with citric acid, this endolysin displays an impressive antibacterial synergy to reduce viable Psa below the limit of detection. The use of citric acid as a synergistic agent with endolysins has not been extensively studied and has never been evaluated against a plant pathogen. We determined that the synergy involved a combination of the chelation activity of citric acid, acidic pH, and the specific activity of the ΦPsa374 endolysin. Our study highlights an exciting opportunity for alternative antimicrobials in agriculture.

Structure, Physicochemical Properties and Biological Activity of Lipopolysaccharide from the Rhizospheric Bacterium Ochrobactrum quorumnocens T1Kr02, Containing d-Fucose Residues.

Lipopolysaccharides (LPSs) are major components of the outer membranes of Gram-negative bacteria. In this work, the structure of the O-polysaccharide of Ochrobactrum quorumnocens T1Kr02 was identified by nuclear magnetic resonance (NMR), and the physical-chemical properties and biological activity of LPS were also investigated. The NMR analysis showed that the O-polysaccharide has the following structure: →2)-β-d-Fucf-(1→3)-β-d-Fucp-(1→. The structure of the periplasmic glucan coextracted with LPS was established by NMR spectroscopy and chemical methods: →2)-β-d-Glcp-(1→. Non-stoichiometric modifications were identified in both polysaccharides: 50% of d-fucofuranose residues at position 3 were O-acetylated, and 15% of d-Glcp residues at position 6 were linked with succinate. This is the first report of a polysaccharide containing both d-fucopyranose and d-fucofuranose residues. The fatty acid analysis of the LPS showed the prevalence of 3-hydroxytetradecanoic, hexadecenoic, octadecenoic, lactobacillic, and 27-hydroxyoctacosanoic acids. The dynamic light scattering demonstrated that LPS (in an aqueous solution) formed supramolecular particles with a size of 72.2 nm and a zeta-potential of -21.5 mV. The LPS solution (10 mkg/mL) promoted the growth of potato microplants under in vitro conditions. Thus, LPS of O. quorumnocens T1Kr02 can be recommended as a promoter for plants and as a source of biotechnological production of d-fucose.

MSphere of Influence: The discovery of a missing link in bacterial cell envelope biogenesis.

Gregory Harrison is a bacteriologist researching essential pathways in bacteria as potential therapeutic targets. In this mSphere of Influence article, he reflects on a series of studies that employ complementary genetic approaches to define the crucial role of AsmA-family proteins in transporting phospholipids between the inner and outer membranes of Gram-negative bacteria. The authors of these three studies identify this family of lipid transporters through the means of bacterial genetics, answering a long-standing question in bacterial physiology, and serving as a reminder that a well-designed genetic strategy can go a long way in uncovering new biology.

Regulation of virulence in Chromobacterium violaceum and strategies to combat it.

Chromobacterium is a rod-shaped, Gram-negative, facultatively anaerobic bacteria with a cosmopolitan distribution. Just about 160 Chromobacterium violaceum incidents have been reported globally, but then once infected, it has the ability to cause deadly septicemia, and infections in the lungs, liver, brain, spleen, and lymphatic systems that might lead to death. C. violaceum produces and utilizes violacein to kill bacteria that compete with it in an ecological niche. Violacein is a hydrophobic bisindole that is delivered through an efficient transport route termed outer membrane vesicles (OMVs) through the aqueous environment. OMVs are small, spherical segments detached from the outer membrane of Gram-negative bacteria. C. violaceum OMV secretions are controlled by a mechanism called the quorum sensing system CviI/CviR, which enables cell-to-cell communication between them and regulation of various virulence factors such as biofilm formation, and violacein biosynthesis. Another virulence factor bacterial type 3 secretion system (T3SS) is divided into two types: Cpi-1 and Cpi-2. Cpi-1's needle and rod effector proteins are perhaps recognized by NAIP receptors in humans and mice, activating the NLRC4 inflammasome cascade, effectively clearing spleen infections via pyroptosis, and cytotoxicity mediated by IL-18-driven Natural killer (NK) cells in the liver. In this paper, we attempt to interrelate quorum-controlled biofilm formation, violacein production, violacein delivery by OMVs and T3SS effector protein production and host mediated immunological effects against the Cpi1 of T3SS. We suggest a research path with natural bioactive molecule like palmitic acid that can act as an anti-quorum agent by reducing the expression of virulence factors as well as an immunomodulatory agent that can augment innate immune defense by hyperactivation of NLRC4 inflammasome hence dramatically purge C. violaceum infections.

How do Antimicrobial Peptides Interact with the Outer Membrane of Gram-Negative Bacteria? Role of Lipopolysaccharides in Peptide Binding, Anchoring, and Penetration.

Gram-negative bacteria possess a complex structural cell envelope that constitutes a barrier for antimicrobial peptides that neutralize the microbes by disrupting their cell membranes. Computational and experimental approaches were used to study a model outer membrane interaction with an antimicrobial peptide, melittin. The investigated membrane included di[3-deoxy-d-manno-octulosonyl]-lipid A (KLA) in the outer leaflet and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) in the inner leaflet. Molecular dynamics simulations revealed that the positively charged helical C-terminus of melittin anchors rapidly into the hydrophilic headgroup region of KLA, while the flexible N-terminus makes contacts with the phosphate groups of KLA, supporting melittin penetration into the boundary between the hydrophilic and hydrophobic regions of the lipids. Electrochemical techniques confirmed the binding of melittin to the model membrane. To probe the peptide conformation and orientation during interaction with the membrane, polarization modulation infrared reflection absorption spectroscopy was used. The measurements revealed conformational changes in the peptide, accompanied by reorientation and translocation of the peptide at the membrane surface. The study suggests that melittin insertion into the outer membrane affects its permeability and capacitance but does not disturb the membrane's bilayer structure, indicating a distinct mechanism of the peptide action on the outer membrane of Gram-negative bacteria.

Interaction of designed cationic antimicrobial peptides with the outer membrane of gram-negative bacteria

The outer membrane (OM) is a hallmark feature of gram-negative bacteria that provides the species with heightened resistance against antibiotic threats while cationic antimicrobial peptides (CAPs) are natural antibiotics broadly recognized for their ability to disrupt bacterial membranes. It has been well-established that lipopolysaccharides present on the OM are among major targets of CAP activity against gram-negative species. Here we investigate how the relative distribution of charged residues along the primary peptide sequence, in conjunction with its overall hydrophobicity, affects such peptide-OM interactions in the natural CAP Ponericin W1. Using a designed peptide library derived from Ponericin W1, we determined that the consecutive placement of Lys residues at the peptide N- or C-terminus (ex. “PonN”: KKKKKKWLGSALIGALLPSVVGLFQ) enhances peptide binding affinity to OM lipopolysaccharides compared to constructs where Lys residues are interspersed throughout the primary sequence (ex. “PonAmp”: WLKKALKIGAKLLPSVVKLFKGSGQ). Antimicrobial activity against multidrug resistant strains of Pseudomonas aeruginosa was similarly found to be highest among Lys-clustered sequences. Our findings suggest that while native Ponericin W1 exerts its initial activity at the OM, Lys-clustering may be a promising means to enhance potency towards this interface, thereby augmenting peptide entry and activity at the IM, with apparent advantage against multidrug-resistant species.

Dual recognition of multiple signals in bacterial outer membrane proteins enhances assembly and maintains membrane integrity

Outer membrane proteins (OMPs) are essential components of the outer membrane of Gram-negative bacteria. In terms of protein targeting and assembly, the current dogma holds that a ‘β-signal’ imprinted in the final β-strand of the OMP engages the β-barrel assembly machinery (BAM) complex to initiate membrane insertion and assembly of the OMP into the outer membrane. Here, we revealed an additional rule that signals equivalent to the β-signal are repeated in other, internal β-strands within bacterial OMPs, by peptidomimetic and mutational analysis. The internal signal is needed to promote the efficiency of the assembly reaction of these OMPs. BamD, an essential subunit of the BAM complex, recognizes the internal signal and the β-signal, arranging several β-strands and partial folding for rapid OMP assembly. The internal signal-BamD ordering system is not essential for bacterial viability but is necessary to retain the integrity of the outer membrane against antibiotics and other environmental insults.

Dual recognition of multiple signals in bacterial outer membrane proteins enhances assembly and maintains membrane integrity.

Outer membrane proteins (OMPs) are essential components of the outer membrane of Gram-negative bacteria. In terms of protein targeting and assembly, the current dogma holds that a 'β-signal' imprinted in the final β-strand of the OMP engages the β-barrel assembly machinery (BAM) complex to initiate membrane insertion and assembly of the OMP into the outer membrane. Here, we revealed an additional rule that signals equivalent to the β-signal are repeated in other, internal β-strands within bacterial OMPs, by peptidomimetic and mutational analysis. The internal signal is needed to promote the efficiency of the assembly reaction of these OMPs. BamD, an essential subunit of the BAM complex, recognizes the internal signal and the β-signal, arranging several β-strands and partial folding for rapid OMP assembly. The internal signal-BamD ordering system is not essential for bacterial viability but is necessary to retain the integrity of the outer membrane against antibiotics and other environmental insults.

Investigation of an AIDA-I based expression system for display of various affinity proteins on Escherichia coli

Autotransporters constitute a large family of natural proteins that are essential for delivering many types of proteins and peptides across the outer membrane in Gram-negative bacteria. In biotechnology, autotransporters have been explored for display of recombinant proteins and peptides on the surface of Escherichia coli and have potential as tools for directed evolution of affinity proteins. Here, we investigate conditions for AIDA-I autotransporter-mediated display of recombinant proteins. A new expression vector was designed and engineered for this purpose, and a panel of proteins from different affinity-protein classes were subcloned to the vector, followed by evaluation of expression, surface display and functionality. Surface expression was explored in ten different E. coli strains together with assessment of transformation efficiencies. Furthermore, the most promising strain and expression vector combination was used in mock library selections for evaluation of magnetic-assisted cell sortings (MACS). The results demonstrated dramatically different performances depending on the type of affinity protein and choice of E. coli strain. The optimized MACS protocol showed efficient enrichment, and thus potential for the new AIDA-I display system to be used in methods for directed evolution of affinity proteins.

A Lipopolysaccharide-Enriched Cow's Milk Allergy Microbiome Promotes a TLR4-Dependent Proinflammatory Intestinal Immune Response.

We have previously reported that the gut microbiota of healthy infants harbors allergy-protective bacteria taxa that are depleted in infants with cow's milk allergy (CMA). Few reports have investigated the role of the gut microbiota in promoting allergic responses. In this study we selected a CMA-associated microbiota with increased abundance of Gram-negative bacteria for analysis of its proinflammatory potential. LPS is the major component of the outer membrane of Gram-negative bacteria. Colonization of mice with a global or conditional mutation of the LPS receptor TLR4 with this CMA microbiota induced expression of serum amyloid A1 (Saa1) and other Th17-, B cell-, and Th2-associated genes in the ileal epithelium in a TLR4-dependent manner. In agreement with the gene expression data, mice colonized with the CMA microbiota have expanded populations of Th17 and regulatory T cells and elevated concentrations of fecal IgA. Importantly, we used both antibiotic-treated specific pathogen-free and germ-free rederived mice with a conditional mutation of TLR4 in the CD11c+ compartment to demonstrate that the induction of proinflammatory genes, fecal IgA, and Th17 cells is dependent on TLR4 signaling. Furthermore, metagenomic sequencing revealed that the CMA microbiota has an increased abundance of LPS biosynthesis genes. Taken together, our results show that a microbiota displaying a higher abundance of LPS genes is associated with TLR4-dependent proinflammatory gene expression and a mixed type 2/type 3 response in mice, which may be characteristic of a subset of infants with CMA.

Recombinant Expression and Overproduction of Transmembrane β-Barrel Proteins.

Transmembrane β-barrel proteins reside in the outer membrane of Gram-negative bacteria and are thus in direct contact with the environment. Because of that, they are involved in many key processes stretching from cellular survival to virulence. Hence, they are an attractive target for the development of novel antimicrobials, in addition to being of fundamental biological interest. To study this class of proteins, they are often required to be expressed in Escherichia coli. Recombinant expression of β-barrel proteins can be achieved using two fundamentally different strategies. The first alternative uses a complete coding sequence that includes a signal peptide for targeting the protein to its native cellular location, the bacterial outer membrane. The second alternative omits the signal peptide in the gene, leading to mislocalization and aggregation of the protein in the bacterial cytoplasm. These aggregates, called inclusion bodies, can be solubilized and the protein can be folded into its native form in vitro. In this chapter, we present example protocols for both strategies and discuss their advantages and disadvantages.