The microbiome shapes immunity in a sex-specific manner in mouse models of Alzheimer's disease.

INTRODUCTION: Preclinical studies reveal that the microbiome broadly affects immune responses and the deposition and/or clearance of amyloid-beta (Aß) in mouse models of Alzheimer's disease (AD). Whether the microbiome shapes central and peripheral immune profiles in AD models remains unknown. METHODS: We examined adaptive immune responses in two mouse models containing AD-related genetic predispositions (3xTg and 5xFAD) in the presence or absence of the microbiome. RESULTS: T and B cells were altered in brain-associated and systemic immune tissues between genetic models and wildtype mice, with earlier signs if inflammation in female mice. Systemic immune responses were modulated by the microbiome and differed by sex. Further, the absence of a microbiome in germ-free mice resulted in reduced cognitive deficits, primarily in female mice. DISCUSSION: These data reveal sexual dimorphism in early signs of inflammation and the effects of the microbiome, and highlight a previously unrecognized interaction between sex and the microbiome in mouse models of AD. Research in Context: Systemic review: We reviewed the literature related to Alzheimer's disease (AD), inflammation, and the microbiome using PubMed. We cite several studies that demonstrate the influence of the microbiome on inflammation and cognitive performance in both animal models and humans. However, the mechanisms linking immunity to AD are not well understood. Interpretation: Using two well-established mouse models of AD, we found that the microbiome does not strongly influence the onset of inflammation in brain-draining lymph nodes; rather, it largely modulates systemic immune responses, local cytokine production, and cognitive performance. Notably, the inflammatory state in mice was affected by sex, and this sex effect differed between local and systemic tissues and mice with or without a microbiome. Future directions: Our work identified a sex- and microbiome-mediated effect on inflammation and cognitive performance. Future studies may focus on microbiome-dependent mechanisms that intersect with sex hormone and immune responses to determine peripheral effects on AD outcomes. Highlights: Adaptive immunity is activated at early ages and differentially by sex in mouse models of AD.Inflammation in 5xFAD mice is characterized by increased IL-17A-producing T cells.Inflammation in 3xTg mice is characterized by increased cytokine responses in males, but attenuated cytokine responses in female mice.Longitudinal immune responses differ between 3xTg mice and 5xFAD mice.Both 3xTg and 5xFAD female mice show improved learning and cognition in the absence of a microbiome.

Microbe-derived uremic solutes enhance thrombosis potential in the host.

p-Cresol sulfate (pCS) and indoxyl sulfate (IS), gut microbiome-derived metabolites, are traditionally associated with cardiovascular disease (CVD) risks in the setting of impaired kidney function. While pharmacologic provision of pCS or IS can promote pro-thrombotic phenotypes, neither the microbial enzymes involved nor direct gut microbial production have been linked to CVD. Untargeted metabolomics was performed on a discovery cohort (n = 1,149) with relatively preserved kidney function, followed by stable isotope-dilution mass spectrometry quantification of pCS and IS in an independent validation cohort (n = 3,954). Genetic engineering of human commensals to produce p-cresol and indole gain-of-function and loss-of-function mutants, followed by colonization of germ-free mice, and studies on host thrombosis were performed. Systemic pCS and IS levels were independently associated with all-cause mortality. Both in vitro and within colonized germ-free mice p-cresol productions were recapitulated by collaboration of two organisms: a Bacteroides strain that converts tyrosine to 4-hydroxyphenylacetate, and a Clostridium strain that decarboxylates 4-hydroxyphenylacetate to p-cresol. We then engineered a single organism, Bacteroides thetaiotaomicron, to produce p-cresol, indole, or both metabolites. Colonizing germ-free mice with engineered strains, we show the gut microbial genes for p-cresol (hpdBCA) and indole (tryptophanase) are sufficient to confer a pro-thrombotic phenotype in vivo. Moreover, human fecal metagenomics analyses show that abundances of hpdBCA and tryptophanase are associated with CVD. These studies show that pCS and IS, two abundant microbiome-derived metabolites, play a broader potential role in CVD than was previously known. They also suggest that therapeutic targeting of gut microbial p-cresol- and indole-producing pathways represent rational targets for CVD.IMPORTANCEAlterations in gut microbial composition and function have been linked to numerous diseases. Identifying microbial pathways responsible for producing molecules that adversely impact the host is an important first step in the development of therapeutic interventions. Here, we first use large-scale clinical observations to link blood levels of defined microbial products to cardiovascular disease risks. Notably, the previously identified uremic toxins p-cresol sulfate and indoxyl sulfate were shown to predict 5-year mortality risks. After identifying the microbes and microbial enzymes involved in the generation of these uremic toxins, we used bioengineering technologies coupled with colonization of germ-free mice to show that the gut microbial genes that generate p-cresol and indole are sufficient to confer p-cresol sulfate and indoxyl sulfate formation, and a pro-thrombotic phenotype in vivo. The findings and tools developed serve as a critical step in both the study and targeting of these gut microbial pathways in vivo.

Multi-level analysis of the gut-brain axis shows autism spectrum disorder-associated molecular and microbial profiles.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by heterogeneous cognitive, behavioral and communication impairments. Disruption of the gut-brain axis (GBA) has been implicated in ASD although with limited reproducibility across studies. In this study, we developed a Bayesian differential ranking algorithm to identify ASD-associated molecular and taxa profiles across 10 cross-sectional microbiome datasets and 15 other datasets, including dietary patterns, metabolomics, cytokine profiles and human brain gene expression profiles. We found a functional architecture along the GBA that correlates with heterogeneity of ASD phenotypes, and it is characterized by ASD-associated amino acid, carbohydrate and lipid profiles predominantly encoded by microbial species in the genera Prevotella, Bifidobacterium, Desulfovibrio and Bacteroides and correlates with brain gene expression changes, restrictive dietary patterns and pro-inflammatory cytokine profiles. The functional architecture revealed in age-matched and sex-matched cohorts is not present in sibling-matched cohorts. We also show a strong association between temporal changes in microbiome composition and ASD phenotypes. In summary, we propose a framework to leverage multi-omic datasets from well-defined cohorts and investigate how the GBA influences ASD.

Global metabolic profiles in a non-human primate model of maternal immune activation: implications for neurodevelopmental disorders.

Epidemiological evidence implicates severe maternal infections as risk factors for neurodevelopmental disorders, such as ASD and schizophrenia. Accordingly, animal models mimicking infection during pregnancy, including the maternal immune activation (MIA) model, result in offspring with neurobiological, behavioral, and metabolic phenotypes relevant to human neurodevelopmental disorders. Most of these studies have been performed in rodents. We sought to better understand the molecular signatures characterizing the MIA model in an organism more closely related to humans, rhesus monkeys (Macaca mulatta), by evaluating changes in global metabolic profiles in MIA-exposed offspring. Herein, we present the global metabolome in six peripheral tissues (plasma, cerebrospinal fluid, three regions of intestinal mucosa scrapings, and feces) from 13 MIA and 10 control offspring that were confirmed to display atypical neurodevelopment, elevated immune profiles, and neuropathology. Differences in lipid, amino acid, and nucleotide metabolism discriminated these MIA and control samples, with correlations of specific metabolites to behavior scores as well as to cytokine levels in plasma, intestinal, and brain tissues. We also observed modest changes in fecal and intestinal microbial profiles, and identify differential metabolomic profiles within males and females. These findings support a connection between maternal immune activation and the metabolism, microbiota, and behavioral traits of offspring, and may further the translational applications of the MIA model and the advancement of biomarkers for neurodevelopmental disorders such as ASD or schizophrenia.

A gut-derived metabolite alters brain activity and anxiety behaviour in mice.

Integration of sensory and molecular inputs from the environment shapes animal behaviour. A major site of exposure to environmental molecules is the gastrointestinal tract, in which dietary components are chemically transformed by the microbiota1 and gut-derived metabolites are disseminated to all organs, including the brain2. In mice, the gut microbiota impacts behaviour3, modulates neurotransmitter production in the gut and brain4,5, and influences brain development and myelination patterns6,7. The mechanisms that mediate the gut-brain interactions remain poorly defined, although they broadly involve humoral or neuronal connections. We previously reported that the levels of the microbial metabolite 4-ethylphenyl sulfate (4EPS) were increased in a mouse model of atypical neurodevelopment8. Here we identified biosynthetic genes from the gut microbiome that mediate the conversion of dietary tyrosine to 4-ethylphenol (4EP), and bioengineered gut bacteria to selectively produce 4EPS in mice. 4EPS entered the brain and was associated with changes in region-specific activity and functional connectivity. Gene expression signatures revealed altered oligodendrocyte function in the brain, and 4EPS impaired oligodendrocyte maturation in mice and decreased oligodendrocyte-neuron interactions in ex vivo brain cultures. Mice colonized with 4EP-producing bacteria exhibited reduced myelination of neuronal axons. Altered myelination dynamics in the brain have been associated with behavioural outcomes7,9-14. Accordingly, we observed that mice exposed to 4EPS displayed anxiety-like behaviours, and pharmacological treatments that promote oligodendrocyte differentiation prevented the behavioural effects of 4EPS. These findings reveal that a gut-derived molecule influences complex behaviours in mice through effects on oligodendrocyte function and myelin patterning in the brain.

Safety and target engagement of an oral small-molecule sequestrant in adolescents with autism spectrum disorder: an open-label phase 1b/2a trial.

Autism spectrum disorder (ASD) is defined by hallmark behaviors involving reduced communication and social interaction as well as repetitive activities and restricted interests. ASD represents a broad spectrum, from minimally affected individuals to those requiring intense support, with additional manifestations often including anxiety, irritability/aggression and altered sensory processing. Gastrointestinal (GI) issues are also common in ASD, and studies have identified changes in the gut microbiome of individuals with ASD compared to control populations, complementing recent findings of differences in gut-derived metabolites in feces and circulation. However, a role for the GI tract or microbiome in ASD remains controversial. Here we report that an oral GI-restricted adsorbent (AB-2004) that has affinity for small aromatic or phenolic molecules relieves anxiety-like behaviors that are driven by a gut microbial metabolite in mice. Accordingly, a pilot human study was designed and completed to evaluate the safety of AB-2004 in an open-label, single-cohort, multiple-ascending-dose clinical trial that enrolled 30 adolescents with ASD and GI symptoms in New Zealand and Australia. AB-2004 was shown to have good safety and tolerability across all dose levels, and no drug-related serious adverse events were identified. Significant reductions in specific urinary and plasma levels of gut bacterial metabolites were observed between baseline and end of AB-2004 treatment, demonstrating likely target engagement. Furthermore, we observed improvements in multiple exploratory behavioral endpoints, most significantly in post hoc analysis of anxiety and irritability, as well as GI health, after 8 weeks of treatment. These results from an open-label study (trial registration no. ACTRN12618001956291) suggest that targeting gut-derived metabolites with an oral adsorbent is a safe and well-tolerated approach to improving symptoms associated with ASD, thereby emboldening larger placebo-controlled trials.

Microbiota regulate social behaviour via stress response neurons in the brain.

Social interactions among animals mediate essential behaviours, including mating, nurturing, and defence1,2. The gut microbiota contribute to social activity in mice3,4, but the gut-brain connections that regulate this complex behaviour and its underlying neural basis are unclear5,6. Here we show that the microbiome modulates neuronal activity in specific brain regions of male mice to regulate canonical stress responses and social behaviours. Social deviation in germ-free and antibiotic-treated mice is associated with elevated levels of the stress hormone corticosterone, which is primarily produced by activation of the hypothalamus-pituitary-adrenal (HPA) axis. Adrenalectomy, antagonism of glucocorticoid receptors, or pharmacological inhibition of corticosterone synthesis effectively corrects social deficits following microbiome depletion. Genetic ablation of glucocorticoid receptors in specific brain regions or chemogenetic inactivation of neurons in the paraventricular nucleus of the hypothalamus that produce corticotrophin-releasing hormone (CRH) reverse social impairments in antibiotic-treated mice. Conversely, specific activation of CRH-expressing neurons in the paraventricular nucleus induces social deficits in mice with a normal microbiome. Via microbiome profiling and in vivo selection, we identify a bacterial species, Enterococcus faecalis, that promotes social activity and reduces corticosterone levels in mice following social stress. These studies suggest that specific gut bacteria can restrain the activation of the HPA axis, and show that the microbiome can affect social behaviours through discrete neuronal circuits that mediate stress responses in the brain.

Plasma and Fecal Metabolite Profiles in Autism Spectrum Disorder.

BACKGROUND: Autism spectrum disorder (ASD) is a neurodevelopmental condition with hallmark behavioral manifestations including impaired social communication and restricted repetitive behavior. In addition, many affected individuals display metabolic imbalances, immune dysregulation, gastrointestinal dysfunction, and altered gut microbiome compositions. METHODS: We sought to better understand nonbehavioral features of ASD by determining molecular signatures in peripheral tissues through mass spectrometry methods (ultrahigh performance liquid chromatography-tandem mass spectrometry) with broad panels of identified metabolites. Herein, we compared the global metabolome of 231 plasma and 97 fecal samples from a large cohort of children with ASD and typically developing control children. RESULTS: Differences in amino acid, lipid, and xenobiotic metabolism distinguished ASD and typically developing samples. Our results implicated oxidative stress and mitochondrial dysfunction, hormone level elevations, lipid profile changes, and altered levels of phenolic microbial metabolites. We also revealed correlations between specific metabolite profiles and clinical behavior scores. Furthermore, a summary of metabolites modestly associated with gastrointestinal dysfunction in ASD is provided, and a pilot study of metabolites that can be transferred via fecal microbial transplant into mice is identified. CONCLUSIONS: These findings support a connection between metabolism, gastrointestinal physiology, and complex behavioral traits and may advance discovery and development of molecular biomarkers for ASD.

Gut microbial molecules in behavioural and neurodegenerative conditions.

Mounting evidence suggests that the gut microbiome impacts brain development and function. Gut-brain connections may be mediated by an assortment of microbial molecules that are produced in the gastrointestinal tract, which can subsequently permeate many organs, including sometimes the brain. Studies in animal models have identified molecular cues propagated from intestinal bacteria to the brain that can affect neurological function and/or neurodevelopmental and neurodegenerative conditions. Herein, we describe bacterial metabolites with known or suspected neuromodulatory activity, define mechanisms of signalling pathways from the gut microbiota to the brain and discuss direct effects that gut bacterial molecules are likely exerting on specific brain cells. Many discoveries are recent, and the findings described in this Perspective are largely novel and yet to be extensively validated. However, expanding research into the dynamic molecular communications between gut microorganisms and the CNS continues to uncover critical and previously unappreciated clues in understanding the pathophysiology of behavioural, psychiatric and neurodegenerative diseases.

A gut bacterial amyloid promotes α-synuclein aggregation and motor impairment in mice.

Amyloids are a class of protein with unique self-aggregation properties, and their aberrant accumulation can lead to cellular dysfunctions associated with neurodegenerative diseases. While genetic and environmental factors can influence amyloid formation, molecular triggers and/or facilitators are not well defined. Growing evidence suggests that non-identical amyloid proteins may accelerate reciprocal amyloid aggregation in a prion-like fashion. While humans encode ~30 amyloidogenic proteins, the gut microbiome also produces functional amyloids. For example, curli are cell surface amyloid proteins abundantly expressed by certain gut bacteria. In mice overexpressing the human amyloid α-synuclein (αSyn), we reveal that colonization with curli-producing Escherichia coli promotes αSyn pathology in the gut and the brain. Curli expression is required for E. coli to exacerbate αSyn-induced behavioral deficits, including intestinal and motor impairments. Purified curli subunits accelerate αSyn aggregation in biochemical assays, while oral treatment of mice with a gut-restricted amyloid inhibitor prevents curli-mediated acceleration of pathology and behavioral abnormalities. We propose that exposure to microbial amyloids in the gastrointestinal tract can accelerate αSyn aggregation and disease in the gut and the brain.

Searching for the gut microbial contributing factors to social behavior in rodent models of autism spectrum disorder.

Social impairment is one of the major symptoms in multiple psychiatric disorders, including autism spectrum disorder (ASD). Accumulated studies indicate a crucial role for the gut microbiota in social development, but these mechanisms remain unclear. This review focuses on two strategies adopted to elucidate the complicated relationship between gut bacteria and host social behavior. In a top-down approach, researchers have attempted to correlate behavioral abnormalities with altered gut microbial profiles in rodent models of ASD, including BTBR mice, maternal immune activation (MIA), maternal valproic acid (VPA) and maternal high-fat diet (MHFD) offspring. In a bottom-up approach, researchers use germ-free (GF) animals, antibiotics, probiotics or pathogens to manipulate the intestinal environment and ascertain effects on social behavior. The combination of both approaches will hopefully pinpoint specific bacterial communities that control host social behavior. Further discussion of how brain development and circuitry is impacted by depletion of gut microbiota is also included. The converging evidence strongly suggests that gut microbes affect host social behavior through the alteration of brain neural circuits. Investigation of intestinal microbiota and host social behavior will unveil any bidirectional communication between the gut and brain and provide alternative therapeutic targets for ASD. © 2018 Wiley Periodicals, Inc. Develop Neurobiol 78: 474-499, 2018.

Controlled Measurement and Comparative Analysis of Cellular Components in E. coli Reveals Broad Regulatory Changes in Response to Glucose Starvation.

How do bacteria regulate their cellular physiology in response to starvation? Here, we present a detailed characterization of Escherichia coli growth and starvation over a time-course lasting two weeks. We have measured multiple cellular components, including RNA and proteins at deep genomic coverage, as well as lipid modifications and flux through central metabolism. Our study focuses on the physiological response of E. coli in stationary phase as a result of being starved for glucose, not on the genetic adaptation of E. coli to utilize alternative nutrients. In our analysis, we have taken advantage of the temporal correlations within and among RNA and protein abundances to identify systematic trends in gene regulation. Specifically, we have developed a general computational strategy for classifying expression-profile time courses into distinct categories in an unbiased manner. We have also developed, from dynamic models of gene expression, a framework to characterize protein degradation patterns based on the observed temporal relationships between mRNA and protein abundances. By comparing and contrasting our transcriptomic and proteomic data, we have identified several broad physiological trends in the E. coli starvation response. Strikingly, mRNAs are widely down-regulated in response to glucose starvation, presumably as a strategy for reducing new protein synthesis. By contrast, protein abundances display more varied responses. The abundances of many proteins involved in energy-intensive processes mirror the corresponding mRNA profiles while proteins involved in nutrient metabolism remain abundant even though their corresponding mRNAs are down-regulated.

Top-Down Strategies for the Structural Elucidation of Intact Gram-negative Bacterial Endotoxins.

Re-modelling of lipopolysaccharides, which are the primary constituent of the outer cell membrane of Gram-negative bacteria, modulates pathogenesis and resistance to microbials. Reported herein is the characterization of intact Gram-negative bacterial lipooligosaccharides (LOS) via a new strategy utilizing online liquid chromatography (LC) coupled with ultraviolet photodissociation (UVPD) mass spectrometry. Compared to collision-based MS/MS methods, UVPD and UVPD/HCD promoted a greater array of cleavages within both the glycan and lipid moieties, including C-C, C-N, C-O cleavages in the acyl chains as well as glycosidic and cross-ring cleavages, thus providing the most far-reaching structural characterization of LOS. This LC-MS/MS strategy affords a robust analytical method to structurally characterize complex mixtures of bacterial endotoxins that maintains the integrity of the core oligosaccharide and lipid A domains of LOS, providing direct feedback about the cell envelope architectures and LOS modification strategies involved in resistance host innate immune defense.

The Vps/VacJ ABC transporter is required for intercellular spread of Shigella flexneri.

The Vps/VacJ ABC transporter system is proposed to function in maintaining the lipid asymmetry of the outer membrane. Mutations in vps or vacJ in Shigella flexneri resulted in increased sensitivity to lysis by the detergent sodium dodecyl sulfate (SDS), and the vpsC mutant showed minor differences in its phospholipid profile compared to the wild type. vpsC mutants were unable to form plaques in cultured epithelial cells, but this was not due to a failure to invade, to replicate intracellularly, or to polymerize actin via IcsA for movement within epithelial cells. The addition of the outer membrane phospholipase gene pldA on a multicopy plasmid in a vpsC or vacJ mutant restored its resistance to SDS, suggesting a restoration of lipid asymmetry to the outer membrane. However, the pldA plasmid did not restore the mutant's ability to form plaques in tissue culture cells. Increased PldA levels also failed to restore the mutant's phospholipid profile to that of the wild type. We propose a dual function of the Vps/VacJ ABC transporter system in S. flexneri in both the maintenance of lipid asymmetry in the outer membrane and the intercellular spread of the bacteria between adjacent epithelial cells.

193 nm ultraviolet photodissociation mass spectrometry for the structural elucidation of lipid A compounds in complex mixtures.

Here we implement ultraviolet photodissociation (UVPD) in an online liquid chromatographic tandem mass spectrometry (MS/MS) strategy to support analysis of complex mixtures of lipid A combinatorially modified during development of vaccine adjuvants. UVPD mass spectrometry at 193 nm was utilized to characterize the structures and fragment ion types of lipid A from Escherichia coli, Vibrio cholerae, and Pseudomonas aeruginosa using an Orbitrap mass spectrometer. The fragment ions generated by UVPD were compared to those from collision induced dissociation (CID) and higher energy collision dissociation (HCD) with respect to the precursor charge state. UVPD afforded the widest array of fragment ion types including acyl chain C-O, C-N, and C-C bond cleavages and glycosidic C-O and cross ring cleavages, thus providing the most comprehensive structural analysis of the lipid A. UVPD exhibited virtually no dependence on precursor ion charge state and was best at determining lipid A structure including acyl chain length and composition, giving it an advantage over collision based methods. UVPD was incorporated into an LC-MS/MS methodology for the analysis of a number of structural variants in a complex mixture of combinatorially engineered Escherichia coli lipid A.

Fortifying the barrier: the impact of lipid A remodelling on bacterial pathogenesis.

Gram-negative bacteria decorate their outermost surface structure, lipopolysaccharide, with elaborate chemical moieties, which effectively disguises them from immune surveillance and protects them from the onslaught of host defences. Many of these changes occur on the lipid A moiety of lipopolysaccharide, a component that is crucial for host recognition of Gram-negative infection. In this Review, we describe the regulatory mechanisms controlling lipid A modification and discuss the impact of modifications on pathogenesis, bacterial physiology and bacterial interactions with the host immune system.

Virulence and stress-related periplasmic protein (VisP) in bacterial/host associations.

Gram-negative bacteria have an outer membrane containing LPS. LPS is constituted of an oligosaccharide portion and a lipid-A moiety that embeds this molecule within the outer membrane. LPS is a pathogen-associated molecular pattern, and several pathogens modify their lipid-A as a stealth strategy to avoid recognition by the innate immune system and gain resistance to host factors that disrupt the bacterial cell envelope. An essential feature of Salmonella enterica Typhimurium pathogenesis is its ability to replicate within vacuoles in professional macrophages. S. Typhimurium modifies its lipid-A by hydroxylation by the Fe2+/α-ketoglutarate-dependent dioxygenase enzyme (LpxO). Here, we show that a periplasmic protein of the bacterial oligonucleotide/oligosaccharide-binding fold family, herein named virulence and stress-related periplasmic protein (VisP), on binding to the sugar moiety of peptidoglycan interacts with LpxO. This interaction inhibits LpxO function, leading to decreased LpxO-dependent lipid-A modifications and increasing resistance to stressors within the vacuole environment during intramacrophage replication promoting systemic disease. Consequently, ΔvisP is avirulent in systemic murine infections, where VisP acts through LpxO. Several Gram-negative pathogens harbor both VisP and LpxO, suggesting that this VisP-LpxO mechanism of lipid-A modifications has broader implications in bacterial pathogenesis. Bacterial species devoid of LpxO (e.g., Escherichia coli) have no lipid-A phenotypes associated with the lack of VisP; however, VisP also controls LpxO-independent phenotypes. VisP and LpxO act independently in the S. Typhimurium murine colitis model, with both mutants being attenuated for diverging reasons; ΔvisP is less resistant to cationic antimicrobial peptides, whereas ΔlpxO is deficient for epithelial cell invasion. VisP converges bacterial cell wall homeostasis, stress responses, and pathogenicity.

Modulating the innate immune response by combinatorial engineering of endotoxin.

Despite its highly inflammatory nature, LPS is a molecule with remarkable therapeutic potential. Lipid A is a glycolipid that serves as the hydrophobic anchor of LPS and constitutes a potent ligand of the Toll-like receptor (TLR)4/myeloid differentiation factor 2 receptor of the innate immune system. A less toxic mixture of monophosphorylated lipid A species (MPL) recently became the first new Food and Drug Administration-approved adjuvant in over 70 y. Whereas wild-type Escherichia coli LPS provokes strong inflammatory MyD88 (myeloid differentiation primary response gene 88)-mediated TLR4 signaling, MPL preferentially induces less inflammatory TRIF (TIR-domain-containing adaptor-inducing IFN-ß)-mediated responses. Here, we developed a system for combinatorial structural diversification of E. coli lipid A, yielding a spectrum of bioactive variants that display distinct TLR4 agonist activities and cytokine induction. Mice immunized with engineered lipid A/antigen emulsions exhibited robust IgG titers, indicating the efficacy of these molecules as adjuvants. This approach demonstrates how combinatorial engineering of lipid A can be exploited to generate a spectrum of immunostimulatory molecules for vaccine and therapeutics development.

The outer membrane of Gram-negative bacteria: lipid A isolation and characterization.

The isolation and characterization of the lipid A domain of lipopolysaccharide (LPS) are important methodologies utilized to gain understanding of the Gram-negative cell envelope. Here, we describe protocols often employed by our laboratory for small- and large-scale isolation of lipid A from bacterial cells. Additionally, we describe various methodologies including isolation of radiolabeled lipid A, thin layer chromatography, and various mass spectrometry methods. Tandem mass spectrometry is an integral tool for the structural characterization of lipid A molecules, and both coventional collision induced dissociation (CID) and new ultraviolet photodissociation (UVPD) methods are described.