TY - JOUR A1 - Sunkavalli, Ushasree A1 - Aguilar, Carmen A1 - Silva, Ricardo Jorge A1 - Sharan, Malvika A1 - Cruz, Ana Rita A1 - Tawk, Caroline A1 - Maudet, Claire A1 - Mano, Miguel A1 - Eulalio, Ana T1 - Analysis of host microRNA function uncovers a role for miR-29b-2-5p in Shigella capture by filopodia JF - PLoS Pathogens N2 - MicroRNAs play an important role in the interplay between bacterial pathogens and host cells, participating as host defense mechanisms, as well as exploited by bacteria to subvert host cellular functions. Here, we show that microRNAs modulate infection by Shigella flexneri, a major causative agent of bacillary dysentery in humans. Specifically, we characterize the dual regulatory role of miR-29b-2-5p during infection, showing that this microRNA strongly favors Shigella infection by promoting both bacterial binding to host cells and intracellular replication. Using a combination of transcriptome analysis and targeted high-content RNAi screening, we identify UNC5C as a direct target of miR-29b-2-5p and show its pivotal role in the modulation of Shigella binding to host cells. MiR-29b-2-5p, through repression of UNC5C, strongly enhances filopodia formation thus increasing Shigella capture and promoting bacterial invasion. The increase of filopodia formation mediated by miR-29b-2-5p is dependent on RhoF and Cdc42 Rho-GTPases. Interestingly, the levels of miR-29b-2-5p, but not of other mature microRNAs from the same precursor, are decreased upon Shigella replication at late times post-infection, through degradation of the mature microRNA by the exonuclease PNPT1. While the relatively high basal levels of miR-29b-2-5p at the start of infection ensure efficient Shigella capture by host cell filopodia, dampening of miR-29b-2-5p levels later during infection may constitute a bacterial strategy to favor a balanced intracellular replication to avoid premature cell death and favor dissemination to neighboring cells, or alternatively, part of the host response to counteract Shigella infection. Overall, these findings reveal a previously unappreciated role of microRNAs, and in particular miR-29b-2-5p, in the interaction of Shigella with host cells. KW - hos tcells KW - Salmonellosis KW - Shigellosis KW - microRNAs KW - Shigella KW - small interfering RNAs KW - HeLa cells KW - Cell binding Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-158204 VL - 13 IS - 4 ER - TY - THES A1 - Konradt, Christoph T1 - Cross-talk between Shigella and cells of the adaptive immunity: The TTS effector IpgD inhibits T cell migration T1 - Cross-talk zwischen Shigella und Zellen des Adaptiven Immunsystems: Der TTS Effektor IpgD inhibiert die T Zell Migration N2 - Shigellosis, or bacillary dysentery, is a rectocolitis caused by the gram-negative, enteroinvasive bacteria of the genus Shigella. Shigellosis still remains a major public health burden with an estimated 80 million cases of bloody diarrhoea and 700.000 deaths per year, primarily in children under the age of 5. Shigella disrupts, invades, and causes inflammatory destruction of the colonic epithelium in humans through virulence effectors secreted by the type III secretion apparatus (TTSA). In contrast to the Shigella-induced manipulation of the host innate immune response, the impact of Shigella on the adaptive immunity has been poorly studied thus far. In order to understand why the naturally induced protective humoral response requires several infections to be primed and is of short duration, the work presented here investigates if Shigella is able to directly interact with T cells. Indeed, it has been shown that Shigella was able to invade and proliferate inside T cells. Furthermore, Shigella was able to inhibit T cell migration through a TTSA effector. Moreover, the Shigella effector IpgD, a phosphoinositide 4-phosphatase that specifically dephosphorylates phosphatidylinositol-(4,5)-bisphosphate (PIP2) into phosphatidylinositol-(5)-monophosphate (PI(5)P), was identified as the effector responsible for the observed inhibition. It could be demonstrated that IpgD was responsible for a reduction of intracellular PIP2 levels in T cells. Further experiments showed a reduced level of phosphorylated ezrin, radixin and moesin (ERM) proteins in infected, as well as with IpgD transfected, T cells. The ERM protein family plays an imported role in signal transduction and motility and their activity is closely related to the binding of PIP2. Therefore, the low level of PIP2 leads to a dephosphorylation of the ERM proteins which inhibits T cells response to chemokine stimulation. Indeed, IpgD transfected T cells show a reduced ability to re-localise the ERM proteins upon chemokine stimulation. Targeting T cell motility, via TTSA effectors, could explain the low level of specific T cell priming during Shigella infection. This is the first report of Shigella induced manipulation of T cell function and on the inhibition of T cell migration by a bacterial effector. N2 - Shigellose oder Bakterieruhr ist eine von Bakterien der Gattung Shigella ausgelöste Dysenterie Erkrankung des Dickdarms. Mit jährlich über 80 Millionen Fällen von blutigen Durchfällen und 700000 Todesfällen, hauptsächlich bei Kindern unter 5 Jahren, stellt Shigella immer noch ein ernsthaftes Gesundheitsproblem dar. Shigella destabilisiert das menschliche Dickdarmgewebe und dringt in dieses ein, wo es eine akute Entzündung auslöst, die das Gewebe weiterhin zerstört. Verursacht wird dies durch bakterielle Effektoren, die durch ein Type III Sekretionssytem (TTSA) sekretiert werden. Verglichen mit der Anzahl an Studien über die Manipulation der angeborenen Immunabwehr gibt es nur wenige Studien über die Interaktionen von Shigella mit dem adaptiven Immunsystem. Um zu verstehen, warum für die Entwicklung einer humoralen Immunantwort mehrere Infektionen erforderlich sind, wurde im Rahmen dieser Arbeit untersucht, ob Shigella in der Lage ist, direkt mit TZellen zu interagieren. Es konnte gezeigt werden, dass Shigella in T-Zellen eindringen und sich vermehren kann. Darüber hinaus zeigt sich, dass Shigella in der Lage ist, durch TTSA-Effektoren die T-Zell-Migration zu hemmen. Der Shigella Effektor IpgD konnte als der für die Hemmung verantwortliche Effektor identifiziert werden. Bei IpgD handelt es sich um eine 4-Phosphoinositid-Phosphatase, die Phosphatidylinositol-(4,5)-bisphosphat (PIP2) zu Phosphatidyl-inositol-(5)- monophosphat (PI(5)P) dephosphoryliert. Es wurde deutlich, dass der Effektor IpgD, neben der Menge an PIP2, auch die Menge an phosphorylierten Ezrin, Radixin und Moesin (ERM) Proteinen in T-Zellen reduziert. Die ERM-Protein-Familie spielt in der Signaltransduktion und bei der Motilität von T-Zellen eine wichtige Rolle und ihre Phosphorylierung ist eng an die Bindung von PIP2 gekoppelt. Daher führt eine geringe Menge an PIP2 zu einer Dephosphorylierung der ERM-Proteine, was eine Stimulierung der T-Zellen durch Chemokine hemmt. In der Tat zeigten IpgDtransfizierte T-Zellen eine verminderte Fähigkeit zur Relokalisierung der ERM-Proteine nach einer Chemokine-Stimulation. In dieser Arbeit konnte erstmals die Manipulation von T-Zell-Funktionen durch Shigella und die Hemmung der T-Zell-Migration, ausgelöst durch einen bakteriellen TTSA-Effektor, gezeigt werden. KW - Medizinische Mikrobiologie KW - Shigella KW - Immunsystem KW - Shigella KW - immune sytem KW - host-pathogen-interaction Y1 - 2010 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-55397 ER -