Potential roles of gut microbiome and metabolites in modulating ALS in mice.

Amyotrophic lateral sclerosis (ALS) is a complex neurodegenerative disorder, in which the clinical manifestations may be influenced by genetic and unknown environmental factors. Here we show that ALS-prone Sod1 transgenic (Sod1-Tg) mice have a pre-symptomatic, vivarium-dependent dysbiosis and altered metabolite configuration, coupled with an exacerbated disease under germ-free conditions or after treatment with broad-spectrum antibiotics. We correlate eleven distinct commensal bacteria at our vivarium with the severity of ALS in mice, and by their individual supplementation into antibiotic-treated Sod1-Tg mice we demonstrate that Akkermansia muciniphila (AM) ameliorates whereas Ruminococcus torques and Parabacteroides distasonis exacerbate the symptoms of ALS. Furthermore, Sod1-Tg mice that are administered AM are found to accumulate AM-associated nicotinamide in the central nervous system, and systemic supplementation of nicotinamide improves motor symptoms and gene expression patterns in the spinal cord of Sod1-Tg mice. In humans, we identify distinct microbiome and metabolite configurations-including reduced levels of nicotinamide systemically and in the cerebrospinal fluid-in a small preliminary study that compares patients with ALS with household controls. We suggest that environmentally driven microbiome-brain interactions may modulate ALS in mice, and we call for similar investigations in the human form of the disease.

Calcium-activated chloride channel regulator 1 (CLCA1) forms non-covalent oligomers in colonic mucus and has mucin 2-processing properties.

Calcium-activated chloride channel regulator 1 (CLCA1) is one of the major nonmucin proteins found in intestinal mucus. It is part of a larger family of CLCA proteins that share highly conserved features and domain architectures. The CLCA domain arrangement is similar to proteins belonging to the ADAM (a disintegrin and metalloproteinase) family, known to process extracellular matrix proteins. Therefore, CLCA1 is an interesting candidate in the search for proteases that process intestinal mucus. Here, we investigated CLCA1's biochemical properties both in vitro and in mucus from mouse and human colon biopsy samples. Using immunoblotting with CLCA1-specific antibodies and recombinant proteins, we observed that the CLCA1 C-terminal self-cleavage product forms a disulfide-linked dimer that noncovalently interacts with the N-terminal part of CLCA1, which further interacts to form oligomers. We also characterized a second, more catalytically active, N-terminal product of CLCA1, encompassing the catalytic domain together with its von Willebrand domain type A (VWA). This fragment was unstable but could be identified in freshly prepared mucus. Furthermore, we found that CLCA1 can cleave the N-terminal part of the mucus structural component MUC2. We propose that CLCA1 regulates the structural arrangement of the mucus and thereby takes part in the regulation of mucus processing.

Identifying transglutaminase reaction products via mass spectrometry as exemplified by the MUC2 mucin - Pitfalls and traps.

In order to demonstrate transglutaminase activity in biological samples immunological as well as glutamine- and amine-donor based assays are commonly used. However, the identification of the transglutaminase reaction product, i. e. the isopeptide cross-linked peptides/proteins or the deamidated protein/peptide are often neglected. This article describes a workflow for the detection of the products of transglutaminase-catalyzed reactions. In particular, possible pitfalls and traps that can arise during the mass spectrometry-based identification of isopeptide cross-links are addressed and characterised on actual samples.

The human transmembrane mucin MUC17 responds to TNFα by increased presentation at the plasma membrane.

Transmembrane mucin MUC17 is an integral part of the glycocalyx as it covers the brush border membrane of small intestinal enterocytes and presents an extended O-glycosylated mucin domain to the intestinal lumen. Here, we identified two unknown phosphorylated serine residues, S4428 and S4492, in the cytoplasmic tail of human MUC17. We have previously demonstrated that MUC17 is anchored to the apical membrane domain via an interaction with the scaffolding protein PDZK1. S4492, localized in the C-terminal PDZ binding motif of MUC17, was mutated to generate phosphomimetic and phosphodeficient variants of MUC17. Using Caco-2 cells as a model system, we found that induction of an inflammatory state by long-term stimulation with the proinflammatory cytokine TNFα resulted in an increase of MUC17 protein levels and enhanced insertion of MUC17 and its two phospho-variants into apical membranes. Up-regulation and apical insertion of MUC17 was followed by shedding of MUC17-containing vesicles. Transmembrane mucins have previously been shown to play a role in the prevention of bacterial colonization by acting as sheddable decoys for encroaching bacteria. Overexpression and increased presentation at the plasma membrane of wild-type MUC17 and its phosphodeficient variant MUC17 S-4492A protected Caco-2 cells against adhesion of enteropathogenic Escherichia coli, indicating that C-terminal phosphorylation of MUC17 may play a functional role in epithelial cell protection. We propose a new function for MUC17 in inflammation, where MUC17 acts as a second line of defense by preventing attachment of bacteria to the epithelial cell glycocalyx in the small intestine.

Structural weakening of the colonic mucus barrier is an early event in ulcerative colitis pathogenesis.

OBJECTIVE: The colonic inner mucus layer protects us from pathogens and commensal-induced inflammation, and has been shown to be defective in active UC. The aim of this study was to determine the underlying compositional alterations, their molecular background and potential contribution to UC pathogenesis. DESIGN: In this single-centre case-control study, sigmoid colon biopsies were obtained from patients with UC with ongoing inflammation (n=36) or in remission (n=28), and from 47 patients without colonic disease. Mucus samples were collected from biopsies ex vivo, and their protein composition analysed by nanoliquid chromatography-tandem mass spectrometry. Mucus penetrability and goblet cell responses to microbial stimulus were assessed in a subset of patients. RESULTS: The core mucus proteome was found to consist of a small set of 29 secreted/transmembrane proteins. In active UC, major structural mucus components including the mucin MUC2 (p<0.0001) were reduced, also in non-inflamed segments. Active UC was associated with decreased numbers of sentinel goblet cells and attenuation of the goblet cell secretory response to microbial challenge. Abnormal penetrability of the inner mucus layer was observed in a subset of patients with UC (12/40; 30%). Proteomic alterations in penetrable mucus samples included a reduction of the SLC26A3 apical membrane anion exchanger, which supplies bicarbonate required for colonic mucin barrier formation. CONCLUSION: Core mucus structural components were reduced in active UC. These alterations were associated with attenuation of the goblet cell secretory response to microbial challenge, but occurred independent of local inflammation. Thus, mucus abnormalities are likely to contribute to UC pathogenesis.

Intestinal Muc2 mucin O-glycosylation is affected by microbiota and regulated by differential expression of glycosyltranferases.

Intestinal cells are covered by mucus. In the small intestine, a single unattached mucus is present whereas the colon has both an inner attached mucus layer and an outer loose mucus. The attached mucus of the colon is impenetrable to bacteria while the loose mucus acts as a habitat for commensal bacteria. In germ-free (GF) mice, small intestinal mucus is attached to the epithelium and the inner colon mucus is penetrable. O-glycosylation plays an important role in the host-microbiota interactions as the commensal bacteria use glycans as nutrient sources and attachment sites. While mucus protein composition is relatively homogenous along the intestine, its main component the Muc2 mucin shows regiospecific O-glycan patterns. We have now analyzed the glycosyltransferase relative concentrations in the epithelial cells along the intestine in GF and conventionally raised mice and compared this with the O-glycans formed. As Muc2 is the main O-glycosylated product in mucus, we made the simplified assumption that most of the glycosyltransferases found in the epithelial cells are involved in Muc2 O-glycan biosynthesis. The O-glycosyltransferase abundances along the intestine correlated well with the Muc2 O-glycan patterns. Some of the glycosyltransferases involved in the O-glycan elongation were decreased in GF mice, something that is in concordance with the observed shorter Muc2 O-glycans.

Genome-wide investigation of mRNA lifetime determinants in Escherichia coli cells cultured at different growth rates.

BACKGROUND: Changes to mRNA lifetime adjust mRNA concentration, facilitating the adaptation of growth rate to changes in growth conditions. However, the mechanisms regulating mRNA lifetime are poorly understood at the genome-wide scale and have not been investigated in bacteria growing at different rates. RESULTS: We used linear covariance models and the best model selected according to the Akaike information criterion to identify and rank intrinsic and extrinsic general transcript parameters correlated with mRNA lifetime, using mRNA half-life datasets for E. coli, obtained at four growth rates. The principal parameter correlated with mRNA stability was mRNA concentration, the mRNAs most concentrated in the cells being the least stable. However, sequence-related features (codon adaptation index (CAI), ORF length, GC content, polycistronic mRNA), gene function and essentiality also affected mRNA lifetime at all growth rates. We also identified sequence motifs within the 5'UTRs potentially related to mRNA stability. Growth rate-dependent effects were confined to particular functional categories (e.g. carbohydrate and nucleotide metabolism). Finally, mRNA stability was less strongly correlated with the amount of protein produced than mRNA concentration and CAI. CONCLUSIONS: This study provides the most complete genome-wide analysis to date of the general factors correlated with mRNA lifetime in E. coli. We have generalized for the entire population of transcripts or excluded determinants previously defined as regulators of stability for some particular mRNAs and identified new, unexpected general indicators. These results will pave the way for discussions of the underlying mechanisms and their interaction with the growth physiology of bacteria.

Protein turnover forms one of the highest maintenance costs in Lactococcus lactis.

Protein turnover plays an important role in cell metabolism by regulating metabolic fluxes. Furthermore, the energy costs for protein turnover have been estimated to account for up to a third of the total energy production during cell replication and hence may represent a major limiting factor in achieving either higher biomass or production yields. This work aimed to measure the specific growth rate (µ)-dependent abundance and turnover rate of individual proteins, estimate the ATP cost for protein production and turnover, and compare this with the total energy balance and other maintenance costs. The lactic acid bacteria model organism Lactococcus lactis was used to measure protein turnover rates at µ = 0.1 and 0.5 h(-1) in chemostat experiments. Individual turnover rates were measured for ~75% of the total proteome. On average, protein turnover increased by sevenfold with a fivefold increase in growth rate, whilst biomass yield increased by 35%. The median turnover rates found were higher than the specific growth rate of the bacterium, which suggests relatively high energy consumption for protein turnover. We found that protein turnover costs alone account for 38 and 47% of the total energy produced at µ = 0.1 and 0.5 h(-1), respectively, and gene ontology groups Energy metabolism and Translation dominated synthesis costs at both growth rates studied. These results reflect the complexity of metabolic changes that occur in response to changes in environmental conditions, and signify the trade-off between biomass yield and the need to produce ATP for maintenance processes.

Magnetic fractionation and proteomic dissection of cellular organelles occupied by the late replication complexes of Semliki Forest virus.

Alphavirus replicase complexes are initially formed at the plasma membrane and are subsequently internalized by endocytosis. During the late stages of infection, viral replication organelles are represented by large cytopathic vacuoles, where replicase complexes bind to membranes of endolysosomal origin. In addition to viral components, these organelles harbor an unknown number of host proteins. In this study, a fraction of modified lysosomes carrying functionally intact replicase complexes was obtained by feeding Semliki Forest virus (SFV)-infected HeLa cells with dextran-covered magnetic nanoparticles and later magnetically isolating the nanoparticle-containing lysosomes. Stable isotope labeling with amino acids in cell culture combined with quantitative proteomics was used to reveal 78 distinct cellular proteins that were at least 2.5-fold more abundant in replicase complex-carrying vesicles than in vesicles obtained from noninfected cells. These host components included the RNA-binding proteins PCBP1, hnRNP M, hnRNP C, and hnRNP K, which were shown to colocalize with the viral replicase. Silencing of hnRNP M and hnRNP C expression enhanced the replication of SFV, Chikungunya virus (CHIKV), and Sindbis virus (SINV). PCBP1 silencing decreased SFV-mediated protein synthesis, whereas hnRNP K silencing increased this synthesis. Notably, the effect of hnRNP K silencing on CHIKV- and SINV-mediated protein synthesis was opposite to that observed for SFV. This study provides a new approach for analyzing the proteome of the virus replication organelle of positive-strand RNA viruses and helps to elucidate how host RNA-binding proteins exert important but diverse functions during positive-strand RNA viral infection.

Metaproteomics reveals parallel utilization of colonic mucin glycans and dietary fibers by the human gut microbiota.

A diet lacking dietary fibers promotes the expansion of gut microbiota members that can degrade host glycans, such as those on mucins. The microbial foraging on mucin has been associated with disruptions of the gut-protective mucus layer and colonic inflammation. Yet, it remains unclear how the co-utilization of mucin and dietary fibers affects the microbiota composition and metabolic activity. Here, we used 14 dietary fibers and porcine colonic and gastric mucins to study the dynamics of mucin and dietary fiber utilization by the human fecal microbiota in vitro. Combining metaproteome and metabolites analyses revealed the central role of the Bacteroides genus in the utilization of complex fibers together with mucin while Akkermansia muciniphila was the main utilizer of sole porcine colonic mucin but not gastric mucin. This study gives a broad overview of the colonic environment in response to dietary and host glycan availability.

MUC17 is an essential small intestinal glycocalyx component that is disrupted in Crohn's disease.

Crohn's disease (CD) is the chronic inflammation of the ileum and colon triggered by bacteria, but insights into molecular perturbations at the bacteria-epithelium interface are limited. We report that membrane mucin MUC17 protects small intestinal enterocytes against commensal and pathogenic bacteria. In non-inflamed CD ileum, reduced MUC17 levels correlated with a compromised glycocalyx, allowing bacterial contact with enterocytes. Muc17 deletion in mice rendered the small intestine prone to atypical infection while maintaining resistance to colitis. The loss of Muc17 resulted in spontaneous deterioration of epithelial homeostasis and extra-intestinal translocation of bacteria. Finally, Muc17-deficient mice harbored specific small intestinal bacterial taxa observed in CD. Our findings highlight MUC17 as an essential line of defense in the small intestine with relevance for early epithelial defects in CD.

BPP43_05035 is a Brachyspira pilosicoli cell surface adhesin that weakens the integrity of the epithelial barrier during infection.

The anaerobic spirochete Brachyspira causes intestinal spirochetosis, characterized by the intimate attachment of bacterial cells to the colonic mucosa, potentially leading to symptoms such as diarrhea, abdominal pain, and weight loss. Despite the clinical significance of Brachyspira infections, the mechanism of the interaction between Brachyspira and the colon epithelium is not known. We characterized the molecular mechanism of the B. pilosicoli-epithelium interaction and its impact on the epithelial barrier during infection. Through a proteomics approach, we identified BPP43_05035 as a candidate B. pilosicoli surface protein that mediates bacterial attachment to cultured human colonic epithelial cells. The crystal structure of BPP43_05035 revealed a globular lipoprotein with a six-bladed beta-propeller domain. Blocking the native BPP43_05035 on B. pilosicoli, either with a specific antibody or via competitive inhibition, abrogated its binding to epithelial cells, which required cell surface-exposed N-glycans. Proximity labeling and interaction assays revealed that BPP43_05035 bound to tight junctions, thereby increasing the permeability of the epithelial monolayer. Extending our investigation to humans, we discovered a downregulation of tight junction and brush border genes in B. pilosicoli-infected patients carrying detectable levels of epithelium-bound BPP43_05035. Collectively, our findings identify BPP43_05035 as a B. pilosicoli adhesin that weakens the colonic epithelial barrier during infection.

Neonatal microbiota colonization drives maturation of primary and secondary goblet cell mediated protection in the pre-weaning colon.

In the distal colon, mucus secreting goblet cells primarily confer protection from luminal microorganisms via generation of a sterile inner mucus layer barrier structure. Bacteria-sensing sentinel goblet cells provide a secondary defensive mechanism that orchestrates mucus secretion in response to microbes that breach the mucus barrier. Previous reports have identified mucus barrier deficiencies in adult germ-free mice, thus implicating a fundamental role for the microbiota in programming mucus barrier generation. In this study, we have investigated the natural neonatal development of the mucus barrier and sentinel goblet cell-dependent secretory responses upon postnatal colonization. Combined in vivo and ex vivo analyses of pre- and post-weaning colonic mucus barrier and sentinel goblet cell maturation demonstrated a sequential microbiota-dependent development of these primary and secondary goblet cell-intrinsic protective functions, with dynamic changes in mucus processing dependent on innate immune signalling via MyD88, and development of functional sentinel goblet cells dependent on the NADPH/Dual oxidase family member Duox2. Our findings therefore identify new mechanisms of microbiota-goblet cell regulatory interaction and highlight the critical importance of the pre-weaning period for the normal development of colonic barrier function.

Muc2-dependent microbial colonization of the jejunal mucus layer is diet sensitive and confers local resistance to enteric pathogen infection.

Intestinal mucus barriers normally prevent microbial infections but are sensitive to diet-dependent changes in the luminal environment. Here we demonstrate that mice fed a Western-style diet (WSD) suffer regiospecific failure of the mucus barrier in the small intestinal jejunum caused by diet-induced mucus aggregation. Mucus barrier disruption due to either WSD exposure or chromosomal Muc2 deletion results in collapse of the commensal jejunal microbiota, which in turn sensitizes mice to atypical jejunal colonization by the enteric pathogen Citrobacter rodentium. We illustrate the jejunal mucus layer as a microbial habitat, and link the regiospecific mucus dependency of the microbiota to distinctive properties of the jejunal niche. Together, our data demonstrate a symbiotic mucus-microbiota relationship that normally prevents jejunal pathogen colonization, but is highly sensitive to disruption by exposure to a WSD.

Trans-Golgi protein TVP23B regulates host-microbe interactions via Paneth cell homeostasis and Goblet cell glycosylation.

A key feature in intestinal immunity is the dynamic intestinal barrier, which separates the host from resident and pathogenic microbiota through a mucus gel impregnated with antimicrobial peptides. Using a forward genetic screen, we have found a mutation in Tvp23b, which conferred susceptibility to chemically induced and infectious colitis. Trans-Golgi apparatus membrane protein TVP23 homolog B (TVP23B) is a transmembrane protein conserved from yeast to humans. We found that TVP23B controls the homeostasis of Paneth cells and function of goblet cells, leading to a decrease in antimicrobial peptides and more penetrable mucus layer. TVP23B binds with another Golgi protein, YIPF6, which is similarly critical for intestinal homeostasis. The Golgi proteomes of YIPF6 and TVP23B-deficient colonocytes have a common deficiency of several critical glycosylation enzymes. TVP23B is necessary for the formation of the sterile mucin layer of the intestine and its absence disturbs the balance of host and microbe in vivo.

Transglutaminase 3 crosslinks the secreted gel-forming mucus component Mucin-2 and stabilizes the colonic mucus layer.

The colonic mucus layer is organized as a two-layered system providing a physical barrier against pathogens and simultaneously harboring the commensal flora. The factors contributing to the organization of this gel network are not well understood. In this study, the impact of transglutaminase activity on this architecture was analyzed. Here, we show that transglutaminase TGM3 is the major transglutaminase-isoform expressed and synthesized in the colon. Furthermore, intrinsic extracellular transglutaminase activity in the secreted mucus was demonstrated in vitro and ex vivo. Absence of this acyl-transferase activity resulted in faster degradation of the major mucus component the MUC2 mucin and changed the biochemical properties of mucus. Finally, TGM3-deficient mice showed an early increased susceptibility to Dextran Sodium Sulfate-induced colitis. Here, we report that natural isopeptide cross-linking by TGM3 is important for mucus homeostasis and protection of the colon from inflammation, reducing the risk of colitis.

Multi-omics approach to study the growth efficiency and amino acid metabolism in Lactococcus lactis at various specific growth rates.

BACKGROUND: Lactococcus lactis is recognised as a safe (GRAS) microorganism and has hence gained interest in numerous biotechnological approaches. As it is fastidious for several amino acids, optimization of processes which involve this organism requires a thorough understanding of its metabolic regulations during multisubstrate growth. RESULTS: Using glucose limited continuous cultivations, specific growth rate dependent metabolism of L. lactis including utilization of amino acids was studied based on extracellular metabolome, global transcriptome and proteome analysis. A new growth medium was designed with reduced amino acid concentrations to increase precision of measurements of consumption of amino acids. Consumption patterns were calculated for all 20 amino acids and measured carbon balance showed good fit of the data at all growth rates studied. It was observed that metabolism of L. lactis became more efficient with rising specific growth rate in the range 0.10-0.60 h(-1), indicated by 30% increase in biomass yield based on glucose consumption, 50% increase in efficiency of nitrogen use for biomass synthesis, and 40% reduction in energy spilling. The latter was realized by decrease in the overall product formation and higher efficiency of incorporation of amino acids into biomass. L. lactis global transcriptome and proteome profiles showed good correlation supporting the general idea of transcription level control of bacterial metabolism, but the data indicated that substrate transport systems together with lower part of glycolysis in L. lactis were presumably under allosteric control. CONCLUSIONS: The current study demonstrates advantages of the usage of strictly controlled continuous cultivation methods combined with multi-omics approach for quantitative understanding of amino acid and energy metabolism of L. lactis which is a valuable new knowledge for development of balanced growth media, gene manipulations for desired product formation etc. Moreover, collected dataset is an excellent input for developing metabolic models.

Metabolic changes underlying the higher accumulation of glutathione in Saccharomyces cerevisiae mutants.

Molecular mechanisms leading to glutathione (GSH) over-accumulation in a Saccharomyces cerevisiae strain produced by UV irradiation-induced random mutagenesis were studied. The mutant accumulated GSH but also cysteine and γ-glutamylcysteine in concentrations that were several fold higher than in its wild-type parent strain under all studied cultivation conditions (chemostat, fed-batch, and turbidostat). Transcript analyses along with shotgun proteome quantification indicated a difference in the expression of a number of genes and proteins, the most pronounced of which were several fold higher expression of CYS3, but also that of GSH1 and its transcriptional activator YAP1. This together with the higher intracellular cysteine concentration is most likely the primary factor underlying GSH over-accumulation in the mutant. Comparative sequencing of GSH1 and the fed-batch experiments with continuous cysteine addition demonstrated that the feedback inhibition of Gsh1p by GSH was still operational in the mutant.

Metaproteomics Analysis of Host-Microbiota Interfaces.

Metaproteomics of host-microbiome interfaces comprises the analysis of complex mixtures of bacteria, archaea, fungi, and viruses in combination with its host cells. Microbial niches can be found all over the host including the skin, oral cavity, and the intestine and are considered to be essential for the homeostasis. The complex interactions between the host and diverse commensal microbiota are poorly characterized while of great interest as dysbiosis is associated with the development of various inflammatory and metabolic diseases. The metaproteomics workflows to study these interfaces are currently being established, and many challenges remain. The major challenge is the large diversity in species composition that make up the microbiota, which results in complex samples that require extended mass spectrometry analysis time. In addition, current database search strategies are not developed to the size of the search space required for unbiased microbial protein identification.Here, we describe a workflow for the proteomics analysis of microbial niches with a focus on intestinal mucus layer. We will cover step-by-step the sample collection, sample preparation, liquid chromatography-mass spectrometry, and data analysis.

IL-22 promotes the formation of a MUC17 glycocalyx barrier in the postnatal small intestine during weaning.

The intestine is under constant exposure to chemicals, antigens, and microorganisms from the external environment. Apical aspects of transporting epithelial cells (enterocytes) form a brush-border membrane (BBM), shaped by packed microvilli coated with a dense glycocalyx. We present evidence showing that the glycocalyx forms an epithelial barrier that prevents exogenous molecules and live bacteria from gaining access to BBM. We use a multi-omics approach to investigate the function and regulation of membrane mucins exposed on the BBM during postnatal development of the mouse small intestine. Muc17 is identified as a major membrane mucin in the glycocalyx that is specifically upregulated by IL-22 as part of an epithelial defense repertoire during weaning. High levels of IL-22 at time of weaning reprogram neonatal postmitotic progenitor enterocytes to differentiate into Muc17-expressing enterocytes, as found in the adult intestine during homeostasis. Our findings propose a role for Muc17 in epithelial barrier function in the small intestine.