Analysis of Human TAAR8 and Murine Taar8b Mediated Signaling Pathways and Expression Profile

The thyroid hormone derivative 3-iodothyronamine (3-T1AM) exerts metabolic effects in vivo that contradict known effects of thyroid hormones. 3-T1AM acts as a trace amine-associated receptor 1 (TAAR1) agonist and activates Gs signaling in vitro. Interestingly, 3-T1AM-meditated in vivo effects persist in Taar1 knockout-mice indicating that further targets of 3-T1AM might exist. Here, we investigated another member of the TAAR family, the only scarcely studied mouse and human trace-amine-associated receptor 8 (Taar8b, TAAR8). By RT-qPCR and locked-nucleic-acid (LNA) in situ hybridization, Taar8b expression in different mouse tissues was analyzed. Functionally, we characterized TAAR8 and Taar8b with regard to cell surface expression and signaling via different G-protein-mediated pathways. Cell surface expression was verified by ELISA, and cAMP accumulation was quantified by AlphaScreen for detection of Gs and/or Gi/o signaling. Activation of G-proteins Gq/11 and G12/13 was analyzed by reporter gene assays. Expression analyses revealed at most marginal Taar8b expression and no gender differences for almost all analyzed tissues. In heart, LNA-in situ hybridization demonstrated the absence of Taar8b expression. We could not identify 3-T1AM as a ligand for TAAR8 and Taar8b, but both receptors were characterized by a basal Gi/o signaling activity, a so far unknown signaling pathway for TAARs

In vivo proteomics identifies the competence regulon and AliB oligopeptide transporter as pathogenic factors in pneumococcal meningitis

Streptococcus pneumoniae (pneumococci) is a leading cause of severe bacterial meningitis in many countries worldwide. To characterize the repertoire of fitness and virulence factors predominantly expressed during meningitis we performed niche-specific analysis of the in vivo proteome in a mouse meningitis model, in which bacteria are directly inoculated into the cerebrospinal fluid (CSF) cisterna magna. We generated a comprehensive mass spectrometry (MS) spectra library enabling bacterial proteome analysis even in the presence of eukaryotic proteins. We recovered 200,000 pneumococci from CSF obtained from meningitis mice and by MS we identified 685 pneumococci proteins in samples from in vitro filter controls and 249 in CSF isolates. Strikingly, the regulatory two-component system ComDE and substrate-binding protein AliB of the oligopeptide transporter system were exclusively detected in pneumococci recovered from the CSF. In the mouse meningitis model, AliB-, ComDE-, or AliB-ComDE-deficiency resulted in attenuated meningeal inflammation and disease severity when compared to wild-type pneumococci indicating the crucial role of ComDE and AliB in pneumococcal meningitis. In conclusion, we show here mechanisms of pneumococcal adaptation to a defined host compartment by a proteome-based approach. Further, this study provides the basis of a promising strategy for the identification of protein antigens critical for invasive disease caused by pneumococci and other meningeal pathogens

<i>Staphylococcus aureus </i>Transcriptome Architecture:From Laboratory to Infection-Mimicking Conditions

Staphylococcus aureus is a major pathogen that colonizes about 20% of the human population. Intriguingly, this Gram-positive bacterium can survive and thrive under a wide range of different conditions, both inside and outside the human body. Here, we investigated the transcriptional adaptation of S. aureus HG001, a derivative of strain NCTC 8325, across experimental conditions ranging from optimal growth in vitro to intracellular growth in host cells. These data establish an extensive repertoire of transcription units and non-coding RNAs, a classification of 1412 promoters according to their dependence on the RNA polymerase sigma factors SigA or SigB, and allow identification of new potential targets for several known transcription factors. In particular, this study revealed a relatively low abundance of antisense RNAs in S. aureus, where they overlap only 6% of the coding genes, and only 19 antisense RNAs not co-transcribed with other genes were found. Promoter analysis and comparison with Bacillus subtilis links the small number of antisense RNAs to a less profound impact of alternative sigma factors in S. aureus. Furthermore, we revealed that Rho-dependent transcription termination suppresses pervasive antisense transcription, presumably originating from abundant spurious transcription initiation in this A+T-rich genome, which would otherwise affect expression of the overlapped genes. In summary, our study provides genome-wide information on transcriptional regulation and non-coding RNAs in S. aureus as well as new insights into the biological function of Rho and the implications of spurious transcription in bacteria

A global Staphylococcus aureus proteome resource applied to the in vivo characterization of host-pathogen interactions.

Data-independent acquisition mass spectrometry promises higher performance in terms of quantification and reproducibility compared to data-dependent acquisition mass spectrometry methods. To enable high-accuracy quantification of Staphylococcus aureus proteins, we have developed a global ion library for data-independent acquisition approaches employing high-resolution time of flight or Orbitrap instruments for this human pathogen. We applied this ion library resource to investigate the time-resolved adaptation of S. aureus to the intracellular niche in human bronchial epithelial cells and in a murine pneumonia model. In epithelial cells, abundance changes for more than 400 S. aureus proteins were quantified, revealing, e.g., the precise temporal regulation of the SigB-dependent stress response and differential regulation of translation, fermentation, and amino acid biosynthesis. Using an in vivo murine pneumonia model, our data-independent acquisition quantification analysis revealed for the first time the in vivo proteome adaptation of S. aureus. From approximately 2.15 × 1

Increased Expression of Bcl11b Leads to Chemoresistance Accompanied by G1 Accumulation

BACKGROUND: The expression of BCL11B was reported in T-cells, neurons and keratinocytes. Aberrations of BCL11B locus leading to abnormal gene transcription were identified in human hematological disorders and corresponding animal models. Recently, the elevated levels of Bcl11b protein have been described in a subset of squameous cell carcinoma cases. Despite the rapidly accumulating knowledge concerning Bcl11b biology, the contribution of this protein to normal or transformed cell homeostasis remains open. METHODOLOGY/PRINCIPAL FINDINGS: Here, by employing an overexpression strategy we revealed formerly unidentified features of Bcl11b. Two different T-cell lines were forced to express BCL11B at levels similar to those observed in primary T-cell leukemias. This resulted in markedly increased resistance to radiomimetic drugs while no influence on death-receptor apoptotic pathway was observed. Apoptosis resistance triggered by BCL11B overexpression was accompanied by a cell cycle delay caused by accumulation of cells at G1. This cell cycle restriction was associated with upregulation of CDKN1C (p57) and CDKN2C (p18) cyclin dependent kinase inhibitors. Moreover, p27 and p130 proteins accumulated and the SKP2 gene encoding a protein of the ubiquitin-binding complex responsible for their degradation was repressed. Furthermore, the expression of the MYCN oncogene was silenced which resulted in significant depletion of the protein in cells expressing high BCL11B levels. Both cell cycle restriction and resistance to DNA-damage-induced apoptosis coincided and required the histone deacetylase binding N-terminal domain of Bcl11b. The sensitivity to genotoxic stress could be restored by the histone deacetylase inhibitor trichostatine A. CONCLUSIONS: The data presented here suggest a potential role of BCL11B in tumor survival and encourage developing Bcl11b-inhibitory approaches as a potential tool to specifically target chemoresistant tumor cells

Genomewide Characterization of Host-Pathogen Interactions by Transcriptomic Approaches

This thesis contains results from transcriptome studies on different aspects of host-pathogen interactions. First, liver gene expression profiles from a murine chronic stress model served to elucidate aspects of the influence of stress on metabolism and immune response state. Chronic stress in female BALB/c mice was shown to lead to a hypermetabolic syndrome including induction of gluconeogenesis, hypercholesteremia, and loss of essential amino acids, to the induction of the acute phase response, but also of immune suppressive pathways and to the repression of hepatic antigen presentation. Increased leukocyte trafficking, increased oxidative stress together with counter-regulatory gene expression changes, and an induction of apoptosis were detected. The influence of intra-venous infection on the host kidney gene expression was analyzed in another murine model using the wild type strain Staphylococcus aureus RN1HG and its isogenic sigB mutant. Gene expression profiling indicated a highly reproducible host kidney response to infection. The comparison of infected with non-infected samples revealed a strong inflammatory reaction of kidney tissue, e. g. Toll-like receptor signaling, complement system, antigen presentation, interferon and IL-6 signaling. However, the results of this study did not provide any hints for differences in the pathomechanism of the S. aureus strains RN1HG and &Delta;sigB, since the host response did not differ between infections with the two strains analyzed. Effects of SigB might be transient, only apparent at earlier time points, or might also be compensated for in the in vivo infection by the interlaced pattern of other regulators. SigB might possess only to a lesser extent characteristics attributed to virulence factors and might act in vivo more like a virulence modulator and fine tune bacterial reactions. In addition to the analysis of tissue samples, different in vitro models were furthermore studied. The third part of this thesis focuses on bone-marrow derived macrophages (BMM) of the two mouse strains BALB/c and C57BL/6, which are described in literature to exhibit genetically determined differences in their reaction to infection. Expression profiling was performed on control and IFN-&gamma; treated samples from a serum-free cultivation system and revealed mainly induction of gene expression after treatment of BMM with IFN-&gamma;. Gene expression changes confirmed known IFN-&gamma; effects like induction of immunoproteasome, antigen presentation, interferon signaling related genes, GTPase/GBPs, and inducible NO synthase. IFN-&gamma; dependent gene expression changes were highly similar in BALB/c and C57BL/6 BMM. Considering gene expression differences between BMM of both strains, a similar expression trend was visible on the level of untreated controls as well as after IFN-&gamma; treatment. Differentially expressed genes between BMM of both strains included immune-relevant genes as well as genes linked to cell death, but the coverage of functional groups was limited. The bronchial epithelial cell line S9 was used as an in vitro model system for the infection with S. aureus RN1HG. The fourth chapter in this thesis includes S9 cell gene expression signatures 2.5 h and 6.5 h after start of infection. At the early time point, only 40 genes were differentially expressed, which nevertheless indicated a beginning pro-inflammatory response, e. g. induction of cytokines (IL-6, IFN-&beta;, LIF) or prostaglandin-endoperoxide synthase 2 (PTGS2), but also counter-regulatory processes, e. g. induction of CD274. The host cell response was dramatically aggravated at the later 6.5 h time point. Differential expression was detected for 1196 genes. These included induced cytokines, pattern recognition receptor signaling, antigen presentation, and genes involved in immune defense (e. g. GBPs, MX, APOL). Negative effects on growth and proliferation were even more enhanced in comparison to the early time point, and signs for apoptotic processes were revealed. Finally, the last chapter addresses amongst others the pathogen’s expression profile in the S9 cell in vitro infection model at the two time points 2.5 h and 6.5 h after start of infection by tiling array gene expression analysis. The pathogen expression profiling revealed the activity of the SaeRS two-component system in internalized staphylococci. Partly dependent on SaeRS, the induction of adhesins (e. g. fnbAB, clfAB), toxins (hlgBC, lukDE, hla), and immune evasion genes (e. g. chp, eap) was observed. Furthermore, expression changes of metabolic genes were recorded (gene induction of amino acid biosynthesis, TCA cycle, gluconeogenesis; gene repression of glycolysis, purine biosynthesis, tRNA synthetases). Expression analysis recorded a distinct bacterial expression program, which supported literature results of a specific, bacterial strain and host cell line dependent transcriptional adaptation of the pathogen.Diese Dissertation beschreibt Ergebnisse aus Transkriptomstudien zu verschiedenen Aspekten von Wirt-Erreger-Wechselwirkungen. Zunächst wurde das Lebergenexpressionsprofil aus einem Mausmodell für chronischen, psychischen Streß verwendet, um den Einfluß von Streß auf Metabolismus und Immunantwort zu verdeutlichen. Chronischer Streß führte in weiblichen BALB/c-Mäusen zu einem hypermetabolischen Syndrom einschließlich Auslösung von Gluconeogenese und Hypercholesterinämie und dem Verlust von essentiellen Aminosäuren, zur Auslösung einer Akute-Phase-Antwort, aber auch zu immunsupprimierenden Abläufen und zur Unterdrückung von hepatischer Antigenpräsentation. Gesteigerte Leukozyteneinwanderung, verstärkter oxidativer Streß einhergehend mit gegenregulatorischen Expressionsänderungen sowie Apoptose wurden detektiert. Der Einfluß einer i. v. Infektion auf die Nierengenexpression des Wirts wurde in einem Mausmodell vergleichend zwischen Staphylococcus aureus RN1HG und seiner isogenen sigB-Mutante analysiert. Das Expressionsprofil infizierter Nieren war sehr gut reproduzierbar. Der Vergleich mit nichtinfizierten Kontrollen zeigte eine starke, proinflammatorische Reaktion der Niere, z. B. „Toll-like“-Rezeptor-Signalweitergabe, Komplementsystem, Antigenpräsentation, IFN- und IL-6-Signalwege. Die Studie konnte keine Unterschiede im Mechanismus der Pathogenese von S. aureus RN1HG und seiner isogenen sigB-Mutante belegen, da sich die Wirtsantwort in beiden Fällen nicht unterschied. Effekte von SigB sind womöglich transienter Natur, nur zu früheren Zeitpunkten auffällig oder könnten in der in vivo Infektion auch von Einflüssen anderer Regulatoren überlagert sein. Möglicherweise besitzt SigB weniger Eigenschaften eines Virulenzfaktors als vielmehr eines Virulenzmodulators, der in vivo die Feinabstimmung der bakteriellen Reaktion übernimmt. Zusätzlich zu Expressionsanalysen an Gewebeproben wurden in vitro Modelle untersucht. Im dritten Teil dieser Dissertation wurde das Genexpressionsprofil von „bone-marrow derived macrophages“ (BMM) der Mausstämme BALB/c und C57BL/6, die nach Literaturangaben genetisch bedingte Unterschiede in ihrer Reaktion auf Infektion aufweisen, nach IFN-&gamma;-Behandlung in serumfreien Kulturbedingungen analysiert. IFN-&gamma;-Behandlung bewirkte in BMM beider Stämme hauptsächlich Geninduktion. Bekannte IFN-&gamma;-Effekte wie die Induktion von Immunproteasom, Antigenpräsentation, Genen der Interferonsignalwege und von GTPasen/GBPs, sowie der induzierbaren NO-Synthase wurden bestätigt. IFN-&gamma;-abhängige Expressionsänderungen waren zwischen BALB/c- und C57BL/6-BMM in hohem Maße ähnlich. Genexpressionsunterschiede zwischen BMM beider Mausstämme wiesen in Kontroll-BMM und nach IFN-&gamma;-Behandlung einen sehr ähnlichen Trend auf. Die stammabhängig unterschiedlich exprimierten Gene schlossen immunrelevante und zelltodassoziierte Gene ein, aber die Abdeckung der funktionellen Gruppen war begrenzt. Die Bronchialepithelzellinie S9 wurde für die in vitro Infektion mit S. aureus RN1HG verwendet. Das vierte Kapitel beschreibt S9-Expressionssignaturen für die zwei Zeitpunkte 2,5 h und 6,5 h nach Beginn der Infektion. Zum frühen 2,5 h-Zeitpunkt wurde nur für 40 Gene differentielle Expression gemessen, die dennoch eine beginnende pro-inflammatorische Antwort zeigte, z. B. verstärkte Expression von Cytokinen (IL-6, IFN-&beta;, LIF) oder Prostaglandinendoperoxidsynthase 2 (PTGS2), aber auch gegenregulatorische Prozesse wie die verstärkte Expression von CD274. Die Wirtsantwort verstärkte sich zum späteren 6,5 h-Zeitpunkt dramatisch, an dem differentielle Expression für 1196 Gene detektiert wurde. Darin waren Cytokine, Signalwege der Rezeptoren für bakterielle Strukturen, Antigenpräsentation und an der Immunantwort beteiligte Gene (z. B. GBP, MX, APOL) enthalten. Negative Auswirkungen auf Wachstum und Proliferation traten noch stärker auf als schon beim früheren Zeitpunkt beobachtet, und Zeichen apoptotischer Prozesse wurden sichtbar. Das letzte Kapitel befaßt sich schließlich mit Tiling-Array Genexpressionsanalysen von S. aureus RN1HG u. a. 2,5 h und 6,5 h nach Beginn der Infektion in dem in vitro Infektionsmodell der S9-Zellen. Das bakterielle Expressionsprofil zeigte die Aktivität des SaeRS Zwei-Komponenten-Systems in internalisierten Staphylokokken an. Zum Teil in Abhängigkeit von SaeRS wurde die stärkere Expression von Adhesinen (z. B. fnbAB, clfAB), Toxinen (hlgBC, lukDE, hla) und immunausweichenden Genen (z. B. chp, eap) beobachtet. Außerdem wurden Expressionsveränderungen metabolischer Gene deutlich (verstärkte Expression von Aminosäurebiosynthese, TCA-Zyklus , Gluconeogenese; verringerte Expression von Glycolyse, Purinbiosynthese, tRNA-Synthetasen). Die Expressionsanalyse erfaßte ein bakterielles Genexpressionsprogramm, welches Literaturangaben einer spezifischen, von der Bakterienstamm-Wirtszellinien-Kombination abhängigen, transkriptionellen Adaptation des Erregers bestätigte

Altered hepatic mRNA expression of immune response and apoptosis-associated genes after acute and chronic psychological stress in mice

Using a combination of transcriptional profiling and Ingenuity Pathway Analysis (IPA, www.ingenuity.com) we investigated acute and chronic psychological stress induced alterations of hepatic gene expression of BALB/c mice. Already after a 2-h single stress session, up-regulation of several LPS and glucocorticoid-sensitive immune response genes and markers related to oxidative stress and apoptotic processes were observed. Support for the existence of oxidative stress was gained by measuring increased protein carbonylation, but no alterations of immune responsiveness or cell death were measured in mice after acute stress compared to the control group. When animals were repeatedly stressed during 4.5-days, we found reduced transcription of antigen presentation molecules, altered mRNA levels of immune cell signaling mediators and persisting high expression of apoptosis-related genes. These alterations were associated with a measurable immune suppression characterized by a reduced ability to clear experimental Salmonella typhimurium infection from the liver and a heightened hepatocyte apoptosis. Moreover, genes associated with anti-oxidative functions and regenerative processes were induced in the hepatic tissue of chronically stressed mice. These findings indicate that modulation of the immune response and of apoptosis-related genes is initiated already during a single acute stress exposure. However, immune suppression will only manifest in repeatedly stressed mice which additionally show induction of protective and liver regenerative genes to prevent further hepatocyte damage

Hypermetabolic syndrome as a consequence of repeated psychological stress in mice.

Stress is a powerful modulator of neuroendocrine, behavioral, and immunological functions. After 4.5-d repeated combined acoustic and restraint stress as a murine model of chronic psychological stress, severe metabolic dysregulations became detectable in female BALB/c mice. Stress-induced alterations of metabolic processes that were found in a hepatic mRNA expression profiling were verified by in vivo analyses. Repeatedly stressed mice developed a hypermetabolic syndrome with the severe loss of lean body mass, hyperglycemia, dyslipidemia, increased amino acid turnover, and acidosis. This was associated with hypercortisolism, hyperleptinemia, insulin resistance, and hypothyroidism. In contrast, after a single acute stress exposure, changes in expression of metabolic genes were much less pronounced and predominantly confined to gluconeogenesis, probably indicating that metabolic disturbances might be initiated already early but will only manifest in repeatedly stressed mice. Thus, in our murine model, repeated stress caused severe metabolic dysregulations, leading to a drastic reduction of the individual's energy reserves. Under such circumstances stress may further reduce the ability to cope with new stressors such as infection or cancer