TY  - JOUR
A1  - Koch, Rebecca-Diana
A1  - Hörner, Eva-Maria
A1  - Münch, Nadine
A1  - Maier, Elke
A1  - Kozjak-Pavlovic, Vera
T1  - Modulation of Host Cell Death and Lysis Are Required for the Release of Simkania negevensis
JF  - Frontiers in Cellular and Infection Microbiology
N2  - Simkania negevensis is a Chlamydia-like bacterium and emerging pathogen of the respiratory tract. It is an obligate intracellular bacterium with a biphasic developmental cycle, which replicates in a wide range of host cells. The life cycle of S. negevensis has been shown to proceed for more than 12 days, but little is known about the mechanisms that mediate the cellular release of these bacteria. This study focuses on the investigation of host cell exit by S. negevensis and its connection to host cell death modulation. We show that Simkania-infected epithelial HeLa as well as macrophage-like THP-1 cells reduce in number during the course of infection. At the same time, the infectivity of the cell culture supernatant increases, starting at the day 3 for HeLa and day 4 for THP-1 cells and reaching maximum at day 5 post infection. This correlates with the ability of S. negevensis to block TNFα-, but not staurosporin-induced cell death up to 3 days post infection, after which cell death is boosted by the presence of bacteria. Mitochondrial permeabilization through Bax and Bak is not essential for host cell lysis and release of S. negevensis. The inhibition of caspases by Z-VAD-FMK, caspase 1 by Ac-YVAD-CMK, and proteases significantly reduces the number of released infectious particles. In addition, the inhibition of myosin II by blebbistatin also strongly affects Simkania release, pointing to a possible double mechanism of exit through host cell lysis and potentially extrusion.
KW  - exit
KW  - release
KW  - cell death
KW  - caspases
Y1  - 2020
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-215158
SN  - 2235-2988
VL  - 10
ER  - 
TY  - THES
A1  - Stelzner, Kathrin
T1  - Identification of factors involved in Staphylococcus aureus- induced host cell death
T1  - Identifizierung von Faktoren, die am Staphylococcus aureus-induzierten Wirtszelltod beteiligt sind
N2  - Staphylococcus aureus is a Gram-positive commensal bacterium, that asymptomatically colonizes human skin and mucosal surfaces. Upon opportune conditions, such as immunodeficiency or breached barriers of the host, it can cause a plethora of infections ranging from local, superficial infections to life-threatening diseases. Despite being regarded as an extracellular pathogen, S. aureus can invade and survive within non-phagocytic and phagocytic cells. Eventually, the pathogen escapes from the host cell resulting in killing of the host cell, which is associated with tissue destruction and spread of infection. However, the exact molecular mechanisms underlying S. aureus-induced host cell death remain to be elucidated. 
In the present work, a genome-wide haploid genetic screen was performed to identify host cell genes crucial for S. aureus intracellular cytotoxicity. A mutant library of the haploid cell line HAP1 was infected with the pathogen and cells surviving the infection were selected. Twelve genes were identified, which were significantly enriched when compared to an infection with a non-cytotoxic S. aureus strain.
Additionally, characteristics of regulated cell death pathways and the role of Ca2+ signaling in S. aureus-infected cells were investigated. Live cell imaging of Ca2+ reporter cell lines was used to analyze single cells. S. aureus-induced host cell death exhibited morphological features of apoptosis and activation of caspases was detected. Cellular H2O2 levels were elevated during S. aureus intracellular infection. Further, intracellular S. aureus provoked cytosolic Ca2+ overload in epithelial cells. This resulted from Ca2+ release from endoplasmic reticulum and Ca2+ influx via the plasma membrane and led to mitochondrial Ca2+ overload. The final step of S. aureus-induced cell death was plasma membrane permeabilization, a typical feature of necrotic cell death.
In order to identify bacterial virulence factors implicated in S. aureus-induced host cell killing, the cytotoxicity of selected mutants was investigated. Intracellular S. aureus employs the bacterial cysteine protease staphopain A to activate an apoptosis-like cell death characterized by cell contraction and membrane bleb formation. Phagosomal escape represents a prerequisite staphopain A-induced cell death, whereas bacterial intracellular replication is dispensable. Moreover, staphopain A contributed to efficient colonization of the lung in a murine pneumonia model. 
In conclusion, this work identified at least two independent cell death pathways activated by intracellular S. aureus. While initially staphopain A mediates S. aureus-induced host cell killing, cytosolic Ca2+-overload follows later and leads to the final demise of the host cell.
N2  - Staphylococcus aureus ist ein Gram-positives, kommensales Bakterium, welches menschliche Haut- und Schleimhautoberflächen asymptomatisch kolonisiert. Unter günstigen Bedingungen, wie z. B. Immunschwäche oder verletzten Barrieren des Wirtes, kann es eine Vielzahl von Infektionen verursachen, die von lokalen, oberflächlichen Infektionen bis hin zu lebensbedrohlichen Krankheiten reichen. Obwohl S. aureus als extrazellulärer Erreger angesehen wird, kann das Bakterium von nicht-phagozytischen und phagozytischen Zellen aufgenommen werden und dort überleben. Schließlich bricht das Pathogen aus der Wirtszelle aus und die damit einhergehende Tötung der Wirtszelle wird mit Gewebezerstörung und Ausbreitung der Infektion in Verbindung gebracht. Die genauen molekularen Mechanismen, die dem S. aureus induzierten Wirtszelltod zugrunde liegen, müssen jedoch noch geklärt werden. 
In dieser Arbeit wurde ein genomweiter haploid genetischer Screen durchgeführt, um Wirtszellgene zu identifizieren, die für die intrazelluläre Zytotoxizität von S. aureus entscheidend sind. Eine Mutantenbibliothek der haploiden Zelllinie HAP1 wurde mit dem Erreger infiziert und die Zellen, die die Infektion überlebten, wurden selektiert. Dabei wurden zwölf Gene identifiziert, die signifikant angereichert waren gegenüber einer Infektion mit einem nicht-zytotoxischen S. aureus Stamm.
Des Weiteren wurden Eigenschaften regulierter Zelltod-Signalwege und die Rolle der Ca2+-Signalübertragung in S. aureus infizierten Zellen untersucht. Lebendzellbildgebung von Ca2+-Reporterzelllinien wurde zur Analyse von einzelnen Zellen eingesetzt. Der S. aureus induzierte Wirtszelltod wies morphologische Merkmale von Apoptose auf und die Aktivierung von Caspasen wurde nachgewiesen. Der zelluläre H2O2-Spiegel wurde durch die intrazelluläre Infektion mit S. aureus erhöht. Zusätzlich rief der intrazelluläre S. aureus eine zytosolische Ca2+-Überbelastung in Epithelzellen hervor. Dies resultierte aus der Ca2+-Freisetzung vom endoplasmatischen Retikulum und dem Einstrom von Ca2+ über die Plasmamembran und führte zu einer mitochondrialen Ca2+-Überbelastung. Der finale Schritt des durch S. aureus induzierten Zelltods war die Permeabilisierung der Plasmamembran, ein typisches Merkmal des nekrotischen Zelltods.
Um bakterielle Virulenzfaktoren zu identifizieren, die am S. aureus-induzierten Wirtszelltod beteiligt sind, wurde die Zytotoxizität von ausgewählten Mutanten untersucht. Der intrazelluläre S. aureus nutzt die bakterielle Cysteinprotease Staphopain A, um einen Apoptose-artigen Zelltod zu aktivieren, der durch Zellkontraktion und Blasenbildung der Membran gekennzeichnet ist. Der phagosomale Ausbruch stellt eine Voraussetzung für den Staphopain A-induzierten Zelltod da, während die intrazelluläre Replikation der Bakterien nicht notwendig ist. Darüber hinaus trug Staphopain A zu einer effizienten Kolonisation der Lunge in einem murinen Pneumonie-Modell bei.
Zusammenfassend lässt sich sagen, dass diese Arbeit mindestens zwei unabhängige Zelltod-Signalwege identifiziert hat, die durch den intrazellulären S. aureus aktiviert werden. Während zunächst Staphopain A den Tod der Wirtszelle einleitet, folgt später die zytosolische Ca2+-Überlastung und führt zum endgültigen Untergang der Wirtszelle.
KW  - Staphylococcus aureus
KW  - Zelltod
KW  - Wirtszelle
KW  - cell death
KW  - host cell
Y1  - 2020
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-188991
N1  - Zusatzmaterial (Videos) befinden sich auch auf einer CD in der gedruckten Ausgabe
ER  - 
TY  - JOUR
A1  - Stelzner, Kathrin
A1  - Winkler, Ann-Cathrin
A1  - Liang, Chunguang
A1  - Boyny, Aziza
A1  - Ade, Carsten P.
A1  - Dandekar, Thomas
A1  - Fraunholz, Martin J.
A1  - Rudel, Thomas
T1  - Intracellular Staphylococcus aureus Perturbs the Host Cell Ca\(^{2+}\) Homeostasis To Promote Cell Death
JF  - mBio
N2  - The opportunistic human pathogen Staphylococcus aureus causes serious infectious diseases that range from superficial skin and soft tissue infections to necrotizing pneumonia and sepsis. While classically regarded as an extracellular pathogen, S. aureus is able to invade and survive within human cells. Host cell exit is associated with cell death, tissue destruction, and the spread of infection. The exact molecular mechanism employed by S. aureus to escape the host cell is still unclear. In this study, we performed a genome-wide small hairpin RNA (shRNA) screen and identified the calcium signaling pathway as being involved in intracellular infection. S. aureus induced a massive cytosolic Ca\(^{2+}\) increase in epithelial host cells after invasion and intracellular replication of the pathogen. This was paralleled by a decrease in endoplasmic reticulum Ca\(^{2+}\) concentration. Additionally, calcium ions from the extracellular space contributed to the cytosolic Ca2+ increase. As a consequence, we observed that the cytoplasmic Ca\(^{2+}\) rise led to an increase in mitochondrial Ca\(^{2+}\) concentration, the activation of calpains and caspases, and eventually to cell lysis of S. aureus-infected cells. Our study therefore suggests that intracellular S. aureus disturbs the host cell Ca\(^{2+}\) homeostasis and induces cytoplasmic Ca\(^{2+}\) overload, which results in both apoptotic and necrotic cell death in parallel or succession.

IMPORTANCE Despite being regarded as an extracellular bacterium, the pathogen Staphylococcus aureus can invade and survive within human cells. The intracellular niche is considered a hideout from the host immune system and antibiotic treatment and allows bacterial proliferation. Subsequently, the intracellular bacterium induces host cell death, which may facilitate the spread of infection and tissue destruction. So far, host cell factors exploited by intracellular S. aureus to promote cell death are only poorly characterized. We performed a genome-wide screen and found the calcium signaling pathway to play a role in S. aureus invasion and cytotoxicity. The intracellular bacterium induces a cytoplasmic and mitochondrial Ca\(^{2+}\) overload, which results in host cell death. Thus, this study first showed how an intracellular bacterium perturbs the host cell Ca\(^{2+}\) homeostasis."
KW  - Staphylococcus aureus
KW  - calcium signaling pathway
KW  - cell death
KW  - facultatively intracellular pathogens
Y1  - 2020
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-231448
VL  - 11
ER  -