Yersinia V–Antigen Exploits Toll-like Receptor 2 and CD14 for Interleukin 10–mediated Immunosuppression

A characteristic of the three human-pathogenic Yersinia spp. (the plague agent Yersinia pestis and the enteropathogenic Yersinia pseudotuberculosis and Yersinia enterocolitica) is the expression of the virulence (V)-antigen (LcrV). LcrV is a released protein which is involved in contact-induced secretion of yersinia antihost proteins and in evasion of the host's innate immune response. Here we report that recombinant LcrV signals in a CD14- and toll-like receptor 2 (TLR2)-dependent fashion leading to immunosuppression by interleukin 10 induction. The impact of this immunosuppressive effect for yersinia pathogenesis is underlined by the observation that TLR2-deficient mice are less susceptible to oral Y. enterocolitica infection than isogenic wild-type animals. In summary, these data demonstrate a new ligand specificity of TLR2, as LcrV is the first known secreted and nonlipidated virulence-associated protein of a Gram-negative bacterium using TLR2 for cell activation. We conclude that yersiniae might exploit host innate pattern recognition molecules and defense mechanisms to evade the host immune response

Novel genetically encoded fluorescent probes enable real-time detection of potassium in vitro and in vivo

Changes in intra-and extracellular potassium ion (K+) concentrations control many important cellular processes and related biological functions. However, our current understanding of the spatiotemporal patterns of physiological and pathological K+ changes is severely limited by the lack of practicable detection methods. We developed K+-sensitive genetically encoded, Forster resonance energy transfer-(FRET) based probes, called GEPIIs, which enable quantitative real-time imaging of K+ dynamics. GEPIIs as purified biosensors are suitable to directly and precisely quantify K+ levels in different body fluids and cell growth media. GEPIIs expressed in cells enable time-lapse and real-time recordings of global and local intracellular K+ signals. Hitherto unknown Ca2+-triggered, organelle-specific K+ changes were detected in pancreatic beta cells. Recombinant GEPIIs also enabled visualization of extracellular K+ fluctuations in vivo with 2-photon microscopy. Therefore, GEPIIs are relevant for diverse K+ assays and open new avenues for live-cell K+ imaging

PHIP-associated Chung-Jansen syndrome: Report of 23 new individuals

In 2016 and 2018, Chung, Jansen and others described a new syndrome caused by haploinsufficiency of PHIP (pleckstrin homology domain interacting protein, OMIM *612,870) and mainly characterized by developmental delay (DD), learning difficulties/intellectual disability (ID), behavioral abnormalities, facial dysmorphism and obesity (CHUJANS, OMIM #617991). So far, PHIP alterations appear to be a rare cause of DD/ID. “Omics” technologies such as exome sequencing or array analyses have led to the identification of distinct types of alterations of PHIP, including, truncating variants, missense substitutions, splice variants and large deletions encompassing portions of the gene or the entire gene as well as adjacent genomic regions. We collected clinical and genetic data of 23 individuals with PHIP-associated Chung-Jansen syndrome (CHUJANS) from all over Europe. Follow-up investigations (e.g. Sanger sequencing, qPCR or Fluorescence-in-situ-Hybridization) and segregation analysis showed either de novo occurrence or inheritance from an also (mildly) affected parent. In accordance with previously described patients, almost all individuals reported here show developmental delay (22/23), learning disability or ID (22/23), behavioral abnormalities (20/23), weight problems (13/23) and characteristic craniofacial features (i.e. large ears/earlobes, prominent eyebrows, anteverted nares and long philtrum (23/23)). To further investigate the facial gestalt of individuals with CHUJANS, we performed facial analysis using the GestaltMatcher approach. By this, we could establish that PHIP patients are indistinguishable based on the type of PHIP alteration (e.g. missense, loss-of-function, splice site) but show a significant difference to the average face of healthy individuals as well as to individuals with Prader-Willi syndrome (PWS, OMIM #176270) or with a CUL4B-alteration (Intellectual developmental disorder, X-linked, syndromic, Cabezas type, OMIM #300354). Our findings expand the mutational and clinical spectrum of CHUJANS. We discuss the molecular and clinical features in comparison to the published individuals. The fact that some variants were inherited from a mildly affected parent further illustrates the variability of the associated phenotype and outlines the importance of a thorough clinical evaluation combined with genetic analyses for accurate diagnosis and counselling

Immunmodulation durch Interaktion des Yersinia enterocolitica V-Antigens mit Rezeptoren des angeborenen Immunsystems

In der vorliegenden Dissertation konnte der immunsuppressive Effekt von LcrV, über IL-10- Induktion eine TNF-alpha- Suppression auszulösen, auf TLR2 und Nod1/2 zurückgeführt werden. Damit konnte gezeigt werden, dass ein bakterieller Virulenzfaktor den für die Vermeidung einer überschiessenden Immunreaktion wichtigen Mechanismus einer TLR- Toleranz im Rahmen einer TLR- Homo- bzw. Heterotoleranzinduktion für seine Zwecke ausnutzt. Weiter konnte die LcrV- induzierte Signaltransduktion analysiert werden. Hierbei wurde herausgefunden, dass das LcrV- Signaling CD14- und TLR2- abhängig ist. Die in der Arbeit dargestellten in vitro und in vivo- Ergebnisse zeigen, dass Yersinien TLR2 ausnutzen, um über diesen Rezeptor der angeborenen Immunität die erste Immunantwort des Wirtes zu unterwandern. Als nicht- lipidiertes Protein eines Gram- negativen Bakteriums begründet LcrV nicht nur eine neue Klasse von bakteriellen TLR2- Agonisten, sondern als virulenz- assoziiertes Protein auch eine neue bakterielle Strategie, Rezeptoren (TLR2) und auch Effektoren (IL-10, TNFalpha) des angeborenen Immunsystems zur Umgehung der Wirtsimmunantwort zu nutzen. Für das LcrV-homologe Protein PcrV des opportunistischen Erregers P. aeruginosa, das weder eine IL-10- Induktion noch eine TNF-alpha- Suppression zeigte, konnte eine CD14/TLR2- Aktivität ausgeschlossen werden. Der Vergleich dieser Proteine zeigt auf, dass die Ausnutzung wirtseigener Effektoren eine pathogenetische Strategie ist, die obligat pathogene Bakterien von opportunistischen Erregern unterscheidet. Aufgrund der Diskrepanz in der immunmodulatorischen Wirkung von LcrV und PcrV wurde eine N-terminal lokalisierte LcrV- Domäne, die bei PcrV fehlt, als mögliche CD14/TLR2- aktive Domäne gefunden und näher untersucht. Unter Verwendung synthetischer Oligopeptide gelang es, die aktive Region von LcrV für das immunmodulierende CD14/TLR2- Signalling auf die Aminosäureregion aa31-aa57 einzugrenzen und durch punktmutierte Peptide und die zugehörigen Proteine mit voller LcrV- Länge schliesslich an der Existenz bestimmter Aminosäuren festzumachen. Ausserdem wurde hiermit der erste Nachweis einer TLR- Stimulierbarkeit durch nicht- lipidierte Peptide erbracht. Der endgültige Beweis für eine immunsupprimierende Wirkung der aktiven Region von LcrV über TLR2 wurde durch Konstruktion einer Yersinien- Mutante mit einem Aminosäureaustausch an Position 42 von LcrV erbracht, die keine LcrV- abhängige Immunmodulation über CD14/TLR2 mehr zeigte. Diese konnte in Mausinfektionsversuchen auch die Relevanz der LcrV- Wirkung für den Ausgang einer Y. enterocolitica Infektion beweisen. So konnte mit einer Yersinie mit TLR2- inaktivem LcrV, das alle übrigen Funktionen jedoch noch erfüllte, der nicht unbeträchtliche Beitrag von LcrV zur Gesamtpathogenität einer Yersinie verdeutlicht werden. Durch Verwendung von TLR2-/- und IL-10-/- Mäusen konnte auch die entscheidende Rolle von TLR2 und des endogenen Effektors IL-10 für die LcrV- induzierte Immunsuppression im Wirtsorganismus bewiesen werden. Auf der Suche nach möglichen intrazellulären Rezeptoren für LcrV wurden Nod1 und Nod2 identifiziert. Die Nod- aktive Region konnte auf die N- terminalen 130 Aminosäuren von LcrV eingegrenzt werden. Y. enterocolitica scheint die angeborene Immunantwort mit Hilfe des N- Terminus von LcrV doppelt- über den Transmembranrezeptor TLR2 und über die intrazellulären, löslichen Rezeptoren Nod1 und Nod2 zur Induktion einer Immunmodulation über IL-10 zu nutzen. Bei der Untersuchung der Frage, wie LcrV in die Zelle gelangt, wurde eine neuartige Shuttle- Funktion von TLR2 entdeckt, welches LcrV der Nod- Signalkaskade zuführt. Damit wurde erstmals gezeigt, dass ein TLR für die intrazelluläre Aufnahme eines bakteriellen Liganden benötigt wird. Darüber hinaus stellt LcrV das erste bekannte Protein und den dritten mikrobiellen Faktor dar, der von Nod1/2 erkannt wird. Insgesamt gesehen wurde in dieser Arbeit die elaborierte Strategie eines pathogenen Erregers genauer beleuchtet, der Effektoren (IL-10 im Rahmen einer TLR-Toleranz- Induktion) und extra- sowie intrazelluläre Rezeptoren des angeborenen Immunsystems ausnutzt, um so durch Vortäuschung des Endes einer Bedrohung durch ein Pathogen das Immunsystem zu überlisten und sich im Wirt auszubreiten. Aus der Arbeit können neue Einsichten in die Pathogen- Wirts- Interaktionen und in Struktur- Wirkungsbeziehungen von bakteriellen Proteinen in ihren Rezeptorinteraktionen mit LRR- haltigen Rezeptoren gewonnen werden. Es werden Ansatzpunkte geliefert, um durch Manipulation des TLR- bzw. Nod- Systems neue therapeutische Strategien zu entwickeln. Im Hinblick auf die Mutation im NOD2- Gen, die mit Morbus Crohn in Verbindung gebracht wird, kann die vorliegende Arbeit neue Ideen für die Ursachenforschung entzündlicher Erkrankungen liefern

Microsatellite Markers: A Tool to Assess the Genetic Diversity of Yellow Mustard (<i>Sinapis alba</i> L.)

Microsatellite markers were used for the assessment of genetic diversity and genetic structure in a germplasm collection of yellow mustard, Sinapis alba L. The comprehensive collection of genetic resources represented 187 registered varieties, landraces, and breeding materials. Microsatellites generated 44 polymorphic alleles in 15 loci. Eleven of them were medium to highly polymorphic, and the high levels of observed heterozygosity (0.12–0.83) and Nei’s gene diversity index (0.11–0.68) indicated a high level of polymorphism. Based on PCoA and neighbor joining analyses, the genetic resources were divided into two groups. The range of genetic dissimilarity in the analysed collection was in the range of 0.00–1.00. The high level of dissimilarity between the accessions was documented by the high WAM value (33.82%). Bayesian clustering algorithms were performed in the STRUCTURE 2.3.4 software. The number of clusters was estimated at K = 2. The accessions were classified according to Q1/Q2 values. The low average values of the parameters Fst_1 (0.3482), Fst_2 (0.1916), and parameter alpha (0.0602) indicated substantial mating barriers between varieties and reproductive isolation due to the limited exchange of genetic resources between breeders. These results demonstrated the importance of extensive collections of genetic resources for the maintenance of genetic diversity and indicated considerable genetic differentiation among accessions

Mechanisms of Yersinia enterocolitica evasion of the host innate immune response by V antigen.

International audienc

Monoglyceride Lipase Deficiency Is Associated with Altered Thrombogenesis in Mice

Monoglyceride lipase (MGL) hydrolyzes monoacylglycerols (MG) to glycerol and one fatty acid. Among the various MG species, MGL also degrades 2-arachidonoylglycerol, the most abundant endocannabinoid and potent activator of the cannabinoid receptors 1 and 2. We investigated the consequences of MGL deficiency on platelet function using systemic (Mgl−/−) and platelet-specific Mgl-deficient (platMgl−/−) mice. Despite comparable platelet morphology, loss of MGL was associated with decreased platelet aggregation and reduced response to collagen activation. This was reflected by reduced thrombus formation in vitro, accompanied by a longer bleeding time and a higher blood volume loss. Occlusion time after FeCl3-induced injury was markedly reduced in Mgl−/− mice, which is consistent with contraction of large aggregates and fewer small aggregates in vitro. The absence of any functional changes in platelets from platMgl−/− mice is in accordance with lipid degradation products or other molecules in the circulation, rather than platelet-specific effects, being responsible for the observed alterations in Mgl−/− mice. We conclude that genetic deletion of MGL is associated with altered thrombogenesis

Monoglyceride Lipase Deficiency Is Associated with Altered Thrombogenesis in Mice

Monoglyceride lipase (MGL) hydrolyzes monoacylglycerols (MG) to glycerol and one fatty acid. Among the various MG species, MGL also degrades 2-arachidonoylglycerol, the most abundant endocannabinoid and potent activator of the cannabinoid receptors 1 and 2. We investigated the consequences of MGL deficiency on platelet function using systemic (Mgl&minus;/&minus;) and platelet-specific Mgl-deficient (platMgl&minus;/&minus;) mice. Despite comparable platelet morphology, loss of MGL was associated with decreased platelet aggregation and reduced response to collagen activation. This was reflected by reduced thrombus formation in vitro, accompanied by a longer bleeding time and a higher blood volume loss. Occlusion time after FeCl3-induced injury was markedly reduced in Mgl&minus;/&minus; mice, which is consistent with contraction of large aggregates and fewer small aggregates in vitro. The absence of any functional changes in platelets from platMgl&minus;/&minus; mice is in accordance with lipid degradation products or other molecules in the circulation, rather than platelet-specific effects, being responsible for the observed alterations in Mgl&minus;/&minus; mice. We conclude that genetic deletion of MGL is associated with altered thrombogenesis

Presenilin-1 Established ER-Ca2+ Leak: a Follow Up on Its Importance for the Initial Insulin Secretion in Pancreatic Islets and β-Cells upon Elevated Glucose

BACKGROUND/AIMS: In our recent work, the importance of GSK3β-mediated phosphorylation of presenilin-1 as crucial process to establish a Ca2+ leak in the endoplasmic reticulum and, subsequently, the pre-activation of resting mitochondrial activity in β-cells was demonstrated. The present work is a follow-up and reveals the importance of GSK3β-phosphorylated presenilin-1 for responsiveness of pancreatic islets and β-cells to elevated glucose in terms of cytosolic Ca2+ spiking and insulin secretion. METHODS: Freshly isolated pancreatic islets and the two pancreatic β-cell lines INS-1 and MIN-6 were used. Cytosolic Ca2+ was fluorometrically monitored using Fura-2/AM and cellular insulin content and secretion were measured by ELISA. RESULTS: Our data strengthened our previous findings of the existence of a presenilin-1-mediated ER-Ca2+ leak in β-cells, since a reduction of presenilin-1 expression strongly counteracted the ER Ca2+ leak. Furthermore, our data revealed that cytosolic Ca2+ spiking upon administration of high D-glucose was delayed in onset time and strongly reduced in amplitude and frequency upon siRNA-mediated knock-down of presenilin-1 or the inhibition of GSK3β in the pancreatic β-cells. Moreover, glucose-triggered initial insulin secretion disappeared by depletion from presenilin-1 and inhibition of GSK3β in the pancreatic β-cells and isolated pancreatic islets, respectively. CONCLUSION: These data complement our previous work and demonstrate that the sensitivity of pancreatic islets and β-cells to glucose illustrated as glucose-triggered cytosolic Ca2+ spiking and initial but not long-lasting insulin secretion crucially depends on a strong ER Ca2+ leak that is due to the phosphorylation of presenilin-1 by GSK3β, a phenomenon that might be involved in the development of type 2 diabetes