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Staphylococcus aureus is a prevalent commensal bacterium which represents one of the leading causes in health care-associated bacterial infections worldwide and can cause a variety of different diseases ranging from simple abscesses to severe and life threatening infections including pneumonia, osteomyelitis and sepsis.
In recent times multi-resistant strains have emerged, causing severe problems in nosocomial as well as community-acquired (CA) infection settings, especially in the United States (USA). Therefore S. aureus has been termed as a superbug by the WHO, underlining the severe health risk originating from it. Today, infections in the USA are dominated by S. aureus genotypes which are classified as USA300 and USA400, respectively. Strains of genotype USA300 are responsible for about 70% of the CA infections.
The molecular mechanisms which render S. aureus such an effective pathogen are still not understood in its entirety. For decades S. aureus was thought to be a strictly extracellular pathogen relying on pore-forming toxins like α-hemolysin to damage human cells and tissue. Only recently it has been shown that S. aureus can enter non-professional phagocytes, using adhesins like the fibronectin-binding proteins which mediate an endocytotic uptake into the host cells. The bacteria are consequently localized to endosomes, where the degradation of enclosed bacterial cells through phagosome maturation would eventually occur.
S. aureus can avoid degradation, and translocate to the cellular cytoplasm, where it can replicate. The ability to cause this so-called phagosomal escape has mainly been attributed to a family of amphiphilic peptides called phenol soluble modulins (PSMs), but as studies have shown, they are not sufficient.
In this work I used a transposon mutant library in combination with automated fluorescence microscopy to screen for genes involved in the phagosomal escape process and intracellular survival of S. aureus. I thereby identified a number of genes, including a non-ribosomal peptide synthetase (NRPS). The NRPS, encoded by the genes ausA and ausB, produces two types of small peptides, phevalin and tyrvalin. Mutations in the ausAB genes lead to a drastic decrease in phagosomal escape rates in epithelial cells, which were readily restored by genetic complementation in trans as well as by supplementation of synthetic phevalin. In leukocytes, phevalin interferes with calcium fluxes and activation of neutrophils and promotes cytotoxicity of intracellular bacteria in both, macrophages and neutrophils. Further ausAB is involved in survival and virulence of the bacterium during mouse lung pneumoniae.
The here presented data demonstrates the contribution of the bacterial cyclic dipeptide phevalin to S. aureus virulence and suggests, that phevalin directly acts on a host cell target to promote cytotoxicity of intracellular bacteria.
Motoneuron diseases form a heterogeneous group of pathologies characterized by the progressive degeneration of motoneurons. More and more genetic factors associated with motoneuron diseases encode proteins that have a function in RNA metabolism, suggesting that disturbed RNA metabolism could be a common underlying problem in several, perhaps all, forms of motoneuron diseases. Recent results suggest that SMN interacts with hnRNP R and TDP-43 in neuronal processes, which are not part of the classical SMN complex. This point to an additional function of SMN, which could contribute to the high vulnerability of spinal motoneurons in spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). The current study elucidates functional links between SMN, the causative factor of SMA (spinal muscular atrophy), hnRNP R, and TDP-43, a genetic factor in ALS (amyotrophic lateral sclerosis). In order to characterize the functional interaction of SMN with hnRNP R and TDP-43, we produced recombinant proteins and investigated their interaction by co-immunoprecipitation. These proteins bind directly to each other, indicating that no other co-factors are needed for this interaction. SMN potentiates the ability of hnRNP R and TDP-43 to bind to ß-actin mRNA. Depletion of SMN alters the subcellular distribution of hnRNP R in motoneurons both in SMN-knockdown motoneurons and SMA mutant mouse (delta7 SMA). These data point to functions of SMN beyond snRNP assembly which could be crucial for recruitment and transport of RNA particles into axons and axon terminals, a mechanism which may contribute to SMA pathogenesis and ALS.
ALS and FTLD (frontotemporal lobar degeneration) are linked by several lines of evidence with respect to clinical and pathological characteristics. Both sporadic and familial forms are a feature of the ALS-FTLD spectrum, with numerous genes having been associated with these pathological conditions. Both diseases are characterized by the pathological cellular aggregation of proteins. Interestingly, some of these proteins such as TDP-43 and FUS have also common relations not only with ALS-FTLD but also with SMA. Intronic hexanucleotide expansions in C9ORF72 are common in ALS and FTLD but it is unknown whether loss of function, toxicity by the expanded RNA or dipeptides from non ATG-initiated translation is responsible for the pathophysiology. This study tries to characterize the cellular function of C9ORF72 protein. To address this, lentiviral based knockdown and overexpression of C9ORF72 was used in isolated mouse motoneurons. The results clearly show that survival of these motoneurons was not affected by altered C9ORF72 levels, whereas adverse effects on axon growth and growth cone size became apparent after C9ORF72 suppression. Determining the protein interactome revealed several proteins in complexes with C9ORF72. Interestingly, C9ORF72 is present in a complex with cofilin and other actin binding proteins that modulate actin dynamics. These interactions were confirmed both by co-precipitation analyses and in particular by functional studies showing altered actin dynamics in motoneurons with reduced levels of C9ORF72. Importantly, the phosphorylation of cofilin is enhanced in C9ORF72 depleted motoneurons and patient derived lymphoblastoid cells with reduced C9ORF72 levels. These findings indicate that C9ORF72 regulates axonal actin dynamics and the loss of this function could contribute to disease pathomechanisms in ALS and FTLD.
The enteric nervous system (ENS) innervates the gastrointestinal (GI) tract and controls central aspects of GI physiology including contractility of the intestinal musculature, glandular secretion and intestinal blood flow. The ENS is composed of neurons that conduct electrical signals and of enteric glial cells (EGCs). EGCs resemble central nervous system (CNS) astrocytes in their morphology and in the expression of shared markers such as the intermediate filament protein glial fibrillary acidic protein (GFAP). They are strategically located at the interface of ENS neurons and their effector cells to modulate intestinal motility, epithelial barrier stability and inflammatory processes. The specific contributions of EGCs to the maintenance of intestinal homeostasis are subject of current research.
From a clinical point of view EGC involvement in pathophysiological processes such as intestinal inflammation is highly relevant. Like CNS astrocytes ECGs can acquire a reactive, tissue-protective phenotype in response to intestinal injury. In patients with chronic inflammatory bowel diseases (IBD) such as Crohn's disease and ulcerative colitis, alterations in the EGC network are well known, particularly a differential expression of GFAP, which is a hallmark of reactive gliosis in the CNS.
With increasing recognition of the role of EGCs in intestinal health and disease comes the need to study the glial population in its complexity. The overall aim of this thesis was to comprehensively study EGCs with focus on the reactive GFAP-expressing subpopulation under inflammatory conditions in vivo and in vitro. In a first step, a novel in vivo rat model of acute systemic inflammation mimicking sepsis was employed to investigate rapidly occuring responses of EGCs to inflammation. This study revealed that within a short time frame of a few hours, EGCs responded to the inflammation with an upregulation of Gfap gene expression. This inflammation-induced upregulation was confined to the myenteric plexus and varied in intensity along the intestinal rostro-caudal axis. This highly responsive myenteric GFAP-expressing EGC population was further characterized in vivo andin vitro using a transgenic mouse model (hGFAP-eGFP mice). Primary purified murine GFAP-EGC cultures in vitro were established and it was assessed how the transcriptomic and proteomic profiles of these cells change upon inflammatory stimulation. Here, myenteric GFAP-EGCs were found to undergo a shift in gene expression profile that predominantly affects expression of genes associated with inflammatory responses. Further, a secretion of inflammatory mediators was validated on protein level. The GFAP+ subpopulation is hence an active participant in inflammatory pathophysiology. In an acute murine IBD model in vivo, GFAP-EGCs were found to express components of the major histocompatibility complex (MHC) class II in inflamed tissue, which also indicates a crosstalk of EGCs with the innate and the adaptive lamina propria immune system in acute inflammation.
Taken together, this work advances our knowledge on EGC (patho-)physiology by identifying and characterizing an EGC subpopulation rapidly responsive to inflammation. This study further provides the transcriptomic profile of this population in vivo and in vitro, which can be used to identify targets for therapeutic intervention. Due to the modulating influence of EGCs on the intestinal microenvironment, the study further underlines the importance of integrating EGCs into in vitro test systems that aim to model intestinal tissues in vitro and presents an outlook on a potential strategy.
G-quadruplex structures are highly stable alternative DNA structures that can, when not properly regulated, impede replication fork progression and cause genome instability (Castillo Bosch et al, 2014; Crabbe et al, 2004; Koole et al, 2014; Kruisselbrink et al, 2008; London et al, 2008; Lopes et al, 2011; Paeschke et al, 2013; Paeschke et al, 2011; Piazza et al, 2015; Piazza et al, 2010; Piazza et al, 2012; Ribeyre et al, 2009; Sabouri et al, 2014; Sarkies et al, 2012; Sarkies et al, 2010; Schiavone et al, 2014; Wu & Spies, 2016; Zimmer et al, 2016). The aim of this thesis was to identify novel G-quadruplex interacting proteins in Saccharomyces cerevisiae and to unravel their regulatory function at these structures to maintain genome integrity. Mms1 and Rtt101 were identified as G-quadruplex binding proteins in vitro via a pull-down experiment with subsequent mass spectrometry analysis. Rtt101, Mms1 and Mms22, which are all components of an ubiquitin ligase (Rtt101Mms1/Mms22), are important for the progression of the replication fork following fork stalling (Luke et al, 2006; Vaisica et al, 2011; Zaidi et al, 2008). The in vivo binding of endogenously tagged Mms1 to its target regions was analyzed genome-wide using chromatin-immunoprecipitation followed by deep-sequencing. Interestingly, Mms1 bound independently of Mms22 and Rtt101 to G-rich regions that have the potential to form G-quadruplex structures. In vitro, formation of G-quadruplex structures could be shown for the G-rich regions Mms1 bound to. This binding was observed throughout the cell cycle. Furthermore, the deletion of MMS1 caused replication fork stalling as evidenced by increased association of DNA Polymerase 2 at Mms1 dependent sites. A gross chromosomal rearrangement assay revealed that deletion of MMS1 results in a significantly increased genome instability at G-quadruplex motifs compared to G-rich or non-G-rich regions. Additionally, binding of the helicase Pif1, which unwinds G4 structures in vitro (Paeschke et al, 2013; Ribeyre et al, 2009; Sanders, 2010; Wallgren et al, 2016), to Mms1 binding sites was reduced in mms1 cells. The data presented in this thesis, together with published data, suggests a novel mechanistic model in which Mms1 binds to G-quadruplex structures and enables Pif1 association. This allows for replication fork progression and genome integrity.
The rotation of the earth around its own axis determines periodically changing environmental conditions, like alterations in light and temperature. For the purpose of adapting all organisms’ behavior, physiology and metabolism to recurring changes, endogenous clocks have evolved, which allow the organisms to anticipate environmental changes. In chronobiology, the scientific field dealing with the investigation of the underlying mechanisms of the endogenous clock, the fruit fly Drosophila melanogaster serves as a beneficial model organism. The fruit fly’s circadian clock exhibits a rather simple anatomical organization, but nevertheless constitutes homologies to the mammalian system. Thus also in this PhD-thesis the fruit fly was used to decipher general features of the circadian clock’s interneuronal communication.
Drosophila melanogaster’s circadian clock consists of about 150 clock neurons, which are located in the central nervous system of the fly. These clock neurons can be subdivided regarding to their anatomical position in the brain into the dorsal neurons (DN1s, DN2s, DN3s), as well as into the lateral neurons (LPNs, LNds, s-LNvs, l-LNvs). Functionally these clock neuron clusters can be classified as Morning- and Evening oscillators (M- and E- oscillators), driving different parts of the fly’s locomotor activity in light-dark conditions (LD). The Morning-oscillators are represented by the s-LNvs and are known to be the main pacemakers, driving the pace of the clock in constant conditions (constant darkness; DD). The group of Evening-oscillators consists of the LNds, the DN1s and the 5th s-LNv and is important for the proper timing of the evening activity in LD. All of these clock neurons are not functionally independent, but form complex neuronal connections, which are highly plastic in their response to different environmental stimuli (Zeitgebers), like light or temperature.
Even though a lot is known about the function and the importance of some clock neuron clusters, the exact interplay between the neurons is not fully known yet. To investigate the mechanisms, which are involved in communication processes among different clock neurons, we depolarized specific clock cells in a temporally and cell-type restricted manner using dTrpA1, a thermosensitive cation channel, which allows the depolarization of neurons by application of temperature pulses (TP) above 29°C to the intact and freely moving fly. Using different clock specific GAL4-driver lines and applying TPs at different time points within the circadian cycle in DD enabled us with the help of phase shift experiments to draw conclusions on the properties of the endogenous clock. The obtained phase shifts in locomotor behavior elicited by specific clock neuronal activation were plotted as phase response curves (PRCs).
The depolarization of all clock neurons shifted the phase of activity the strongest, especially in the delay zone of the PRC. The exclusive depolarization of the M oscillators together with the l-LNvs (PDF+ neurons: s-LNvs & l-LNvs) caused shifts in the delay and in the advance zone as well, however the advances were severely enhanced in their temporal occurrence ranging into the subjective day. We concluded that light might have inhibitory effects on the PDF+ cells in that particular part of the PRC, as typical light PRCs do not exhibit that kind of distinctive advances. By completely excluding light in the PRC-experiments of this PhD-thesis, this photic inhibitory input to the PDF+ neurons is missing, probably causing the broadened advance zone. These findings suggest the existence of an inhibitory light-input pathway to the PDF+ cells from the photoreceptive organs (Hofbauer-Buchner eyelet, photoreceptor cells of compound eyes, ocelli) or from other clock neurons, which might inhibit phase advances during the subjective day.
To get an impression of the molecular state of the clock in the delay and advance zone, staining experiments against Period (PER), one of the most important core clock components, and against the neuropeptide Pigment Dispersing Factor (PDF) were performed. The cycling of PER levels mirrored the behavioral phase shifts in experimental flies, whereas the controls were widely unaffected. As just those neurons, which had been depolarized, exhibited immediate shifted PER oscillations, this effect has to be rapidly regulated in a cell-autonomous manner.
However, the molecular link between clock neuron depolarization and shifts in the molecular clock’s cycling is still missing. This issue was addressed by CREB (cAMP responsive element binding protein) quantification in the large ventrolateral neurons (l-LNvs), as these neurons responded unexpectedly and strongest to the artificial depolarization exhibiting a huge increase in PER levels. It had been previously suggested that CREB is involved in circadian rhythms by binding to regulatory sequences of the period gene (Belvin et al., 1999), thus activating its transcription. We were able to show, that CREB levels in the l-LNvs are under circadian regulation, as they exhibit higher CREB levels at the end of the subjective night relative to the end of the subjective day. That effect was further reinforced by artificial depolarization, independently of the time point of depolarization. Furthermore the data indicate that rises in CREB levels are coinciding with the time point of increases of PER levels in the l-LNvs, suggesting CREB being the molecular link between the neuronal electrical state and the molecular clock.
Taking together, the results indicate that a temporal depolarization using dTrpA1 is able to significantly phase shift the clock on the behavioral and protein level. An artificial depolarization at the beginning of the subjective night caused phase delays, whereas a depolarization at the end of the subjective night resulted in advances. The activation of all clock neurons caused a PRC that roughly resembled a light-PRC. However, the depolarization of the PDF+ neurons led to a PRC exhibiting a shape that did not resemble that of a light-mediated PRC, indicating the complex processing ability of excitatory and inhibitory input by the circadian clock. Even though this experimental approach is highly artificial, just the exclusion of light-inputs enabled us to draw novel conclusions on the network communication and its light input pathways.
Eclosion is the emergence of an adult insect from the pupal case at the end of development. In the fruit fly Drosophila melanogaster, eclosion is a circadian clock-gated event and is regulated by various peptides. When studied on the population level, eclosion reveals a clear rhythmicity with a peak at the beginning of the light-phase that persists also under constant conditions. It is a long standing hypothesis that eclosion gating to the morning hours with more humid conditions is an adaption to reduce water loss and increase the survival. Eclosion behavior, including the motor pattern required for the fly to hatch out of the puparium, is orchestrated by a well-characterized cascade of peptides. The main components are ecdysis-triggering hormone (ETH), eclosion hormone (EH) and crustacean cardioactive peptide (CCAP). The molt is initiated by a peak level and pupal ecdysis by a subsequent decline of the ecdysteroid ecdysone. Ecdysteroids are produced by the prothoracic gland (PG), an endocrine tissue that contains a peripheral clock and degenerates shortly after eclosion. Production and release of ecdysteroids are regulated by the prothoracicotropic hormone (PTTH).
Although many aspects of the circadian clock and the peptidergic control of the eclosion behavior are known, it still remains unclear how both systems are interconnected. The aim of this dissertation research was to dissect this connection and evaluate the importance of different Zeitgebers on eclosion rhythmicity under natural conditions.
Potential interactions between the central clock and the peptides regulating ecdysis motor behavior were evaluated by analyzing the influence of CCAP on eclosion rhythmicity. Ablation and silencing of CCAP neurons, as well as CCAP null-mutation did not affect eclosion rhythmicity under either light or temperature entrainment nor under natural conditions.
To dissect the connection between the central and the peripheral clock, PTTH neurons were ablated. Monitoring eclosion under light and temperature entrainment revealed that eclosion became arrhythmic under constant conditions. However, qPCR expression analysis revealed no evidence for cycling of Ptth mRNA in pharate flies. To test for a connection with pigment-dispersing factor (PDF)-expressing neurons, the PDF receptor (PDFR) and short neuropeptide F receptor (sNPFR) were knocked down in the PTTH neurons. Knockdown of sNPFR, but not PDFR, resulted in arrhythmic eclosion under constant darkness conditions. PCR analysis of the PTTH receptor, Torso, revealed its expression in the PG and the gonads, but not in the brain or eyes, of pharate flies. Knockdown of torso in the PG lead to arrhythmicity under constant conditions, which provides strong evidence for the specific effect of PTTH on the PG. These results suggest connections from the PDF positive lateral neurons to the PTTH neurons via sNPF signaling, and to the PG via PTTH and Torso. This interaction presumably couples the period of the peripheral clock in the PG to that of the central clock in the brain.
To identify a starting signal for eclosion and possible further candidates in the regulation of eclosion behavior, chemically defined peptidergic and aminergic neurons were optogenetically activated in pharate pupae via ChR2-XXL. This screen approach revealed two candidates for the regulation of eclosion behavior: Dromyosuppressin (DMS) and myo-inhibitory peptides (MIP). However, ablation of DMS neurons did not affect eclosion rhythmicity or success and the exact function of MIP must be evaluated in future studies.
To assess the importance of the clock and of possible Zeitgebers in nature, eclosion of the wildtype Canton S and the clock mutant per01 and the PDF signaling mutants pdf01 and han5304 was monitored under natural conditions. For this purpose, the Würzburg eclosion monitor (WEclMon) was developed, which is a new open monitoring system that allows direct exposure of pupae to the environment. A general decline of rhythmicity under natural conditions compared to laboratory conditions was observed in all tested strains. While the wildtype and the pdf01 and han5304 mutants stayed weakly rhythmic, the per01 mutant flies eclosed mostly arrhythmic. PDF and its receptor (PDFR encoded by han) are required for the synchronization of the clock network and functional loss can obviously be compensated by a persisting synchronization to external Zeitgebers. The loss of the central clock protein PER, however, lead to a non-functional clock and revealed the absolute importance of the clock for eclosion rhythmicity. To quantitatively analyze the effect of the clock and abiotic factors on eclosion rhythmicity, a statistical model was developed in cooperation with Oliver Mitesser and Thomas Hovestadt. The modelling results confirmed the clock as the most important factor for eclosion rhythmicity. Moreover, temperature was found to have the strongest effect on the actual shape of the daily emergence pattern, while light has only minor effects. Relative humidity could be excluded as Zeitgeber for eclosion and therefore was not further analyzed.
Taken together, the present dissertation identified the so far unknown connection between the central and peripheral clock regulating eclosion. Furthermore, a new method for the analysis of eclosion rhythms under natural conditions was established and the necessity of a functional clock for rhythmic eclosion even in the presence of multiple Zeitgebers was shown.
A large fraction of human tumors exhibits aberrant expression of the oncoprotein MYC. As a transcription factor regulating various cellular processes, MYC is also crucially involved in normal development. Direct targeting of MYC has been a major challenge for molecular cancer drug discovery. The proof of principle that its inhibition is nevertheless feasible came from in vivo studies using a dominant-negative allele of MYC termed OmoMYC. Systemic expression of OmoMYC triggered long-term tumor regression with mild and fully reversible side effects on normal tissues.
In this study, OmoMYC’s mode of action was investigated combining methods of structural biology and functional genomics to elucidate how it is able to preferentially affect oncogenic functions of MYC.
The crystal structure of the OmoMYC homodimer, both in the free and the E-box-bound state, was determined, which revealed that OmoMYC forms a stable homodimer, and as such, recognizes DNA via the same base-specific DNA contacts as the MYC/MAX heterodimer. OmoMYC binds DNA with an equally high affinity as MYC/MAX complexes. RNA-sequencing showed that OmoMYC blunts both MYC-dependent transcriptional activation and repression. Genome-wide DNA-binding studies using chromatin immunoprecipitation followed by high-throughput sequencing revealed that OmoMYC competes with MYC/MAX complexes on chromatin, thereby reducing their occupancy at consensus DNA binding sites. The most prominent decrease in MYC binding was seen at low-affinity promoters, which were invaded by MYC at oncogenic levels. Strikingly, gene set enrichment analyses using OmoMYC-regulated genes enabled the identification of tumor subgroups with high MYC levels in multiple tumor entities. Together with a targeted shRNA screen, this identified novel targets for the eradication of MYC-driven tumors, such as ATAD3A, BOP1, and ADRM1.
In summary, the findings suggest that OmoMYC specifically inhibits tumor cell growth by attenuating the expression of rate-limiting proteins in cellular processes that respond to elevated levels of MYC protein using a DNA-competitive mechanism. This opens up novel strategies to target oncogenic MYC functions for tumor therapy.
Amyotrophic lateral sclerosis and spinal muscular atrophy are the two most common motoneuron diseases. Both are characterized by destabilization of axon terminals, axon degeneration and alterations in neuronal cytoskeleton. Accumulation of neurofilaments has been observed in several neurodegenerative diseases but the mechanisms how elevated neurofilament levels destabilize axons are unknown so far. Here, I show that increased neurofilament expression in motor nerves of pmn mutant mice causes disturbed microtubule dynamics. Depletion of neurofilament by Nefl knockout increases the number and regrowth of microtubules in pmn mutant motoneurons and restores axon elongation. This effect is mediated by interaction of neurofilament with the stathmin complex. Depletion of neurofilament increases stathmin-Stat3 interaction and stabilizes the microtubules. Consequently, the axonal maintenance is improved and the pmn mutant mice survive longer. We propose that this mechanism could also be relevant for other neurodegenerative diseases in which neurofilament accumulation is a prominent feature.
Next, using Smn-/-;SMN2 mouse as a model, the molecular mechanism behind synapse loss in SMA is studied. SMA is characterized by degeneration of lower α-motoneurons in spinal cord; however, how reduction of ubiquitously expressed SMN leads to MN-specific degeneration remains unclear. SMN is involved in pre-mRNA splicing (Pellizzoni, Kataoka et al. 1998) and its deficiency in SMA affects the splicing machinery. Neuromuscular junction denervation precedes neurodegeneration in SMA. However, there is no evidence of a link between aberrant splicing of transcripts downstream of Smn and reduced presynaptic axon excitability observed in SMA. In this study, we observed that expression and splicing of Nrxn2, that encodes a presynaptic protein is affected in the SMA mouse and that Nrxn2 could be a candidate that relates aberrant splicing to synaptic motoneuron defects in SMA.
The microbial communities that live inside the human gastrointestinal tract -the human gut
microbiome- are important for host health and wellbeing. Characterizing this new “organ”,
made up of as many cells as the human body itself, has recently become possible through
technological advances. Metagenomics, the high-throughput sequencing of DNA directly from
microbial communities, enables us to take genomic snapshots of thousands of microbes living
together in this complex ecosystem, without the need for isolating and growing them.
Quantifying the composition of the human gut microbiome allows us to investigate its
properties and connect it to host physiology and disease. The wealth of such connections was
unexpected and is probably still underestimated. Due to the fact that most of our dietary as well
as medicinal intake affects the microbiome and that the microbiome itself interacts with our
immune system through a multitude of pathways, many mechanisms have been proposed to
explain the observed correlations, though most have yet to be understood in depth.
An obvious prerequisite to characterizing the microbiome and its interactions with the host is
the accurate quantification of its composition, i.e. determining which microbes are present and
in what numbers they occur. Historically, standard practices have existed for sample handling,
DNA extraction and data analysis for many years. However, these were generally developed for
single microbe cultures and it is not always feasible to implement them in large scale
metagenomic studies. Partly because of this and partly because of the excitement that new
technology brings about, the first metagenomic studies each took the liberty to define their own
approach and protocols. From early meta-analysis of these studies it became clear that the
differences in sample handling, as well as differences in computational approaches, made
comparisons across studies very difficult. This restricts our ability to cross-validate findings of
individual studies and to pool samples from larger cohorts. To address the pressing need for
standardization, we undertook an extensive comparison of 21 different DNA extraction methods
as well as a series of other sample manipulations that affect quantification. We developed a
number of criteria for determining the measurement quality in the absence of a mock
community and used these to propose best practices for sampling, DNA extraction and library
preparation. If these were to be accepted as standards in the field, it would greatly improve
comparability across studies, which would dramatically increase the power of our inferences
and our ability to draw general conclusions about the microbiome.
Most metagenomics studies involve comparisons between microbial communities, for example
between fecal samples from cases and controls. A multitude of approaches have been proposed
to calculate community dissimilarities (beta diversity) and they are often combined with
various preprocessing techniques. Direct metagenomics quantification usually counts
sequencing reads mapped to specific taxonomic units, which can be species, genera, etc. Due to
technology-inherent differences in sampling depth, normalizing counts is necessary, for
instance by dividing each count by the sum of all counts in a sample (i.e. total sum scaling), or by
subsampling. To derive a single value for community (dis-)similarity, multiple distance
measures have been proposed. Although it is theoretically difficult to benchmark these
approaches, we developed a biologically motivated framework in which distance measures can
be evaluated. This highlights the importance of data transformations and their impact on the
measured distances.
Building on our experience with accurate abundance estimation and data preprocessing
techniques, we can now try and understand some of the basic properties of microbial
communities. In 2011, it was proposed that the space of genus level variation of the human gut
microbial community is structured into three basic types, termed enterotypes. These were
described in a multi-country cohort, so as to be independent of geography, age and other host
properties. Operationally defined through a clustering approach, they are “densely populated
areas in a multidimensional space of community composition”(source) and were proposed as a
general stratifier for the human population. Later studies that applied this concept to other
datasets raised concerns about the optimum number of clusters and robustness of the
clustering approach. This heralded a long standing debate about the existence of structure and
the best ways to determine and capture it. Here, we reconsider the concept of enterotypes, in
the context of the vastly increased amounts of available data. We propose a refined framework
in which the different types should be thought of as weak attractors in compositional space and
we try to implement an approach to determining which attractor a sample is closest to. To this
end, we train a classifier on a reference dataset to assign membership to new samples. This way,
enterotypes assignment is no longer dataset dependent and effects due to biased sampling are
minimized. Using a model in which we assume the existence of three enterotypes characterized
by the same driver genera, as originally postulated, we show the relevance of this stratification
and propose it to be used in a clinical setting as a potential marker for disease development.
Moreover, we believe that these attractors underline different rules of community assembly and
we recommend they be accounted for when analyzing gut microbiome samples.
While enterotypes describe structure in the community at genus level, metagenomic sequencing
can in principle achieve single-nucleotide resolution, allowing us to identify single nucleotide
polymorphisms (SNPs) and other genomic variants in the gut microbiome. Analysis
methodology for this level of resolution has only recently been developed and little exploration
has been done to date. Assessing SNPs in a large, multinational cohort, we discovered that the
landscape of genomic variation seems highly structured even beyond species resolution,
indicating that clearly distinguishable subspecies are prevalent among gut microbes. In several
cases, these subspecies exhibit geo-stratification, with some subspecies only found in the
Chinese population. Generally however, they present only minor dispersion limitations and are
seen across most of our study populations. Within one individual, one subspecies is commonly
found to dominate and only rarely are several subspecies observed to co-occur in the same
ecosystem. Analysis of longitudinal data indicates that the dominant subspecies remains stable
over periods of more than three years. When interrogating their functional properties we find
many differences, with specific ones appearing relevant to the host. For example, we identify a
subspecies of E. rectale that is lacking the flagellum operon and find its presence to be
significantly associated with lower body mass index and lower insulin resistance of their hosts;
it also correlates with higher microbial community diversity. These associations could not be
seen at the species level (where multiple subspecies are convoluted), which illustrates the
importance of this increased resolution for a more comprehensive understanding of microbial
interactions within the microbiome and with the host.
Taken together, our results provide a rigorous basis for performing comparative metagenomics
of the human gut, encompassing recommendations for both experimental sample processing
and computational analysis. We furthermore refine the concept of community stratification into
enterotypes, develop a reference-based approach for enterotype assignment and provide
compelling evidence for their relevance. Lastly, by harnessing the full resolution of
metagenomics, we discover a highly structured genomic variation landscape below the
microbial species level and identify common subspecies of the human gut microbiome. By
developing these high-precision metagenomics analysis tools, we thus hope to contribute to a
greatly improved understanding of the properties and dynamics of the human gut microbiome.
MYC is a transcription factor, whose expression is elevated or deregulated in many human cancers (up to 70%) and is often associated with aggressive and poorly differentiated tumors. Although MYC is extensively studied, discrepancies have emerged about how this transcription factor works. In primary lymphocytes, MYC promotes transcriptional amplification of virtually all genes with an open promoter, whereas in tumor cells MYC regulates specific sets of genes that have significant prognostic value. Furthermore, the set of target genes that distinguish MYC’s physiological function from the pathological/oncogenic one, whether it exists or not, has not been fully understood yet.
In this study, it could be shown that MYC protein levels within a cell and promoter affinity (determined by E-box presence or interaction with other proteins) of target genes toward MYC are important factors that influence MYC activity. At low levels, MYC can amplify a certain transcriptional program, which includes high affinity binding sites, whereas at high levels MYC leads to the specific up- and down regulation of genes with low affinity. Moreover, the promoter affinity characterizes different sets of target genes which can be distinguished in the physiological or oncogenic MYC signatures.
MYC-mediated repression requires higher MYC levels than activation and formation of a complex with MIZ1 is necessary for inhibiting expression of a subset of MYC target genes.