Martin von Bergen

Sex Differences in the Gut Microbiome Drive Hormone-Dependent Regulation of Autoimmunity

Mighty Male Microbes Both genetic and environmental factors contribute to an individuals susceptibility to autoimmune disease, but the specific environmental influences are not well characterized. Markle et al. (p. 1084, published online 17 January; see the Perspective by Flak et al.) explored how microbial factors, in particular the gut microbiota, influence susceptibility to type 1 diabetes in mice. In the non-obese diabetic (NOD) mouse model of type 1 diabetes, female mice are significantly more susceptible to disease than males; however, this difference was not apparent under germ-free conditions. Transfer of cecal contents from male NOD mice to female NOD mice prior to disease onset protected against pancreatic islet inflammation, autoantibody production, and the development of diabetes and was associated with increased testosterone in female mice. Blocking androgen receptor activity abrogated protection. Thus, the microbiota may be able to regulate sex hormones and influence an individuals susceptibility to autoimmunity. In mice, the gut microbiota influences levels of sex hormones and the development of autoimmune disease. [Also see Perspective by Flak et al.] Microbial exposures and sex hormones exert potent effects on autoimmune diseases, many of which are more prevalent in women. We demonstrate that early-life microbial exposures determine sex hormone levels and modify progression to autoimmunity in the nonobese diabetic (NOD) mouse model of type 1 diabetes (T1D). Colonization by commensal microbes elevated serum testosterone and protected NOD males from T1D. Transfer of gut microbiota from adult males to immature females altered the recipients microbiota, resulting in elevated testosterone and metabolomic changes, reduced islet inflammation and autoantibody production, and robust T1D protection. These effects were dependent on androgen receptor activity. Thus, the commensal microbial community alters sex hormone levels and regulates autoimmune disease fate in individuals with high genetic risk.

Complete nitrification by Nitrospira bacteria

Nitrification, the oxidation of ammonia via nitrite to nitrate, has always been considered to be a two-step process catalysed by chemolithoautotrophic microorganisms oxidizing either ammonia or nitrite. No known nitrifier carries out both steps, although complete nitrification should be energetically advantageous. This functional separation has puzzled microbiologists for a century. Here we report on the discovery and cultivation of a completely nitrifying bacterium from the genus Nitrospira, a globally distributed group of nitrite oxidizers. The genome of this chemolithoautotrophic organism encodes the pathways both for ammonia and nitrite oxidation, which are concomitantly activated during growth by ammonia oxidation to nitrate. Genes affiliated with the phylogenetically distinct ammonia monooxygenase and hydroxylamine dehydrogenase genes of Nitrospira are present in many environments and were retrieved on Nitrospira-contigs in new metagenomes from engineered systems. These findings fundamentally change our picture of nitrification and point to completely nitrifying Nitrospira as key components of nitrogen-cycling microbial communities.

Mutations of tau protein in frontotemporal dementia promote aggregation of paired helical filaments by enhancing local beta-structure

The microtubule-associated protein tau is a natively unfolded protein in solution, yet it is able to polymerize into the ordered paired helical filaments (PHF) of Alzheimers disease. In the splice isoforms lacking exon 10, this process is facilitated by the formation of β-structure around the hexapeptide motif PHF6 (306VQIVYK311) encoded by exon 11. We have investigated the structural requirements for PHF polymerization in the context of adult tau isoforms containing four repeats (including exon 10). In addition to the PHF6 motif there exists a related PHF6* motif (275VQIINK280) in the repeat encoded by the alternatively spliced exon 10. We show that this PHF6* motif also promotes aggregation by the formation of β-structure and that there is a cross-talk between the two hexapeptide motifs during PHF aggregation. We also show that two of the tau mutations found in hereditary frontotemporal dementias, ΔK280 and P301L, have a much stronger tendency for PHF aggregation which correlates with their high propensity for β-structure around the hexapeptide motifs.

Gut microbiota disturbance during antibiotic therapy: a multi-omic approach

Objective Antibiotic (AB) usage strongly affects microbial intestinal metabolism and thereby impacts human health. Understanding this process and the underlying mechanisms remains a major research goal. Accordingly, we conducted the first comparative omic investigation of gut microbial communities in faecal samples taken at multiple time points from an individual subjected to β-lactam therapy. Methods The total (16S rDNA) and active (16S rRNA) microbiota, metagenome, metatranscriptome (mRNAs), metametabolome (high-performance liquid chromatography coupled to electrospray ionisation and quadrupole time-of-flight mass spectrometry) and metaproteome (ultra high performing liquid chromatography coupled to an Orbitrap MS2 instrument [UPLC-LTQ Orbitrap-MS/MS]) of a patient undergoing AB therapy for 14 days were evaluated. Results Apparently oscillatory population dynamics were observed, with an early reduction in Gram-negative organisms (day 6) and an overall collapse in diversity and possible further colonisation by ‘presumptive’ naturally resistant bacteria (day 11), followed by the re-growth of Gram-positive species (day 14). During this process, the maximum imbalance in the active microbial fraction occurred later (day 14) than the greatest change in the total microbial fraction, which reached a minimum biodiversity and richness on day 11; additionally, major metabolic changes occurred at day 6. Gut bacteria respond to ABs early by activating systems to avoid the antimicrobial effects of the drugs, while ‘presumptively’ attenuating their overall energetic metabolic status and the capacity to transport and metabolise bile acid, cholesterol, hormones and vitamins; host–microbial interactions significantly improved after treatment cessation. Conclusions This proof-of-concept study provides an extensive description of gut microbiota responses to follow-up β-lactam therapy. The results demonstrate that ABs targeting specific pathogenic infections and diseases may alter gut microbial ecology and interactions with host metabolism at a much higher level than previously assumed.

Inducible Expression of Tau Repeat Domain in Cell Models of Tauopathy AGGREGATION IS TOXIC TO CELLS BUT CAN BE REVERSED BY INHIBITOR DRUGS

We generated several cell models of tauopathy in order to study the mechanisms of neurodegeneration in diseases involving abnormal changes of tau protein. N2a neuroblastoma cell lines were created that inducibly express different variants of the repeat domain of tau (tauRD) when exposed to doxycycline (Tet-On system). The following three constructs were chosen: (i) the repeat domain of tau that coincides with the core of Alzheimer paired helical filaments; (ii) the repeat domain with the deletion mutation ΔK280 known from frontotemporal dementia and highly prone to spontaneous aggregation; and (iii) the repeat domain with ΔK280 and two proline point mutations that inhibit aggregation. The comparison of wild-type, pro-aggregation, and anti-aggregation mutants shows the following. (a) Aggregation of tauRD is toxic to cells. (b) The degree of aggregation and toxicity depends on the propensity for β-structure. (c) Soluble mutants of tauRD that cannot aggregate are not toxic. (d) Aggregation is preceded by fragmentation. (e) Fragmentation of tauRD in cells is initially due to a thrombin-like protease activity. (f) Phosphorylation of tauRD (at KXGS motifs) precedes aggregation but is not correlated with the degree of aggregation. (g) Aggregates of tauRD disappear when the expression is silenced, showing that aggregation is reversible. (h) Aggregation can be prevented by drugs and even pre-formed aggregates can be dissolved again by drugs. Thus, the cell models open up new insights into the relationship between the structure, expression, phosphorylation, aggregation, and toxicity of tauRD that can be used to test current hypotheses on tauopathy and to develop drugs that prevent the aggregation and degeneration of cells.

Functional metaproteome analysis of protein extracts from contaminated soil and groundwater

Using proteins from soil or groundwater as functional biomarkers requires efficient extraction. We developed an extraction method in which the separation of proteins from the inorganic and organic constituents of the soil matrix was achieved by a combination of 0.1 M NaOH treatment and phenol extraction. Incubation with NaOH released humic acids and proteins from soil minerals, and simultaneously, disrupted microorganisms. The subsequent phenol extraction separated the proteins from the humic organic matter. Protein extracts were applied to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and 2D-electrophoresis (2-DE). Spots and bands were excised and individual proteins identified by liquid chromatography online linked to mass spectrometry (MS) via electrospray ionization source (LC-ESI-MS). To assess the suitability of the method for the functional analysis of environmental metaproteomes, it was applied to soil that had been enriched in chlorophenoxy acid-degrading bacteria by incubation with 2,4-dichlorophenoxy acetic acid (2,4-D) for 22 days. The method was also used to analyze groundwater from the aquifer of a chlorobenzene-contaminated site. The identification of enzymes such as chlorocatechol dioxygenases was consistent with bacterial metabolic pathways expected to be expressed in these samples. The protocol enabled thus the analysis of the metaproteome of soil and groundwater samples. It thereby provides a means to study the diversity of environmental microbial communities while addressing functional aspects more directly than metagenome or even metatranscriptome analysis.

Structural Principles of Tau and the Paired Helical Filaments of Alzheimer’s Disease

Tau, a major microtubule‐associated protein in brain, forms abnormal fibers in Alzheimer’s disease and several other neurodegenerative diseases. Tau is highly soluble and adopts a natively unfolded structure in solution. In the paired helical filaments of Alzheimer’s disease, small segments of tau adopt a β‐conformation and interact with other tau molecules. In the filament core, the microtubule‐binding repeat region of tau has a cross‐β structure, while the rest of the protein retains its largely unfolded structure and gives rise to the fuzzy coat of the filaments.

Maternal and newborn vitamin D status and its impact on food allergy development in the German LINA cohort study

Vitamin D levels are known to be associated with atopic disease development; however, existing data are controversial. The aim of this study was to investigate whether corresponding maternal and cord blood vitamin D levels are associated with atopic outcomes in early infancy.

Combined Proteomic and Metabolomic Profiling of Serum Reveals Association of the Complement System with Obesity and Identifies Novel Markers of Body Fat Mass Changes

Obesity is associated with multiple adverse health effects and a high risk of developing metabolic and cardiovascular diseases. Therefore, there is a great need to identify circulating parameters that link changes in body fat mass with obesity. This study combines proteomic and metabolomic approaches to identify circulating molecules that discriminate healthy lean from healthy obese individuals in an exploratory study design. To correct for variations in physical activity, study participants performed a one hour exercise bout to exhaustion. Subsequently, circulating factors differing between lean and obese individuals, independent of physical activity, were identified. The DIGE approach yielded 126 differentially abundant spots representing 39 unique proteins. Differential abundance of proteins was confirmed by ELISA for antithrombin-III, clusterin, complement C3 and complement C3b, pigment epithelium-derived factor (PEDF), retinol binding protein 4 (RBP4), serum amyloid P (SAP), and vitamin-D binding protein (VDBP). Targeted serum metabolomics of 163 metabolites identified 12 metabolites significantly related to obesity. Among those, glycine (GLY), glutamine (GLN), and glycero-phosphatidylcholine 42:0 (PCaa 42:0) serum concentrations were higher, whereas PCaa 32:0, PCaa 32:1, and PCaa 40:5 were decreased in obese compared to lean individuals. The integrated bioinformatic evaluation of proteome and metabolome data yielded an improved group separation score of 2.65 in contrast to 2.02 and 2.16 for the single-type use of proteomic or metabolomics data, respectively. The identified circulating parameters were further investigated in an extended set of 30 volunteers and in the context of two intervention studies. Those included 14 obese patients who had undergone sleeve gastrectomy and 12 patients on a hypocaloric diet. For determining the long-term adaptation process the samples were taken six months after the treatment. In multivariate regression analyses, SAP, CLU, RBP4, PEDF, GLN, and C18:2 showed the strongest correlation to changes in body fat mass. The combined serum proteomic and metabolomic profiling reveals a link between the complement system and obesity and identifies both novel (C3b, CLU, VDBP, and all metabolites) and confirms previously discovered markers (PEDF, RBP4, C3, ATIII, and SAP) of body fat mass changes.

Structure of tau protein and assembly into paired helical filaments

Over the past few years the systematic investigation of paired helical filament assembly from tau protein in vitro has become feasible. We review our current understanding of the structure and conformations of tau protein and how this affects taus assembly into the pathological paired helical filaments in Alzheimers disease.