TY - JOUR A1 - Stelzner, Kathrin A1 - Boyny, Aziza A1 - Hertlein, Tobias A1 - Sroka, Aneta A1 - Moldovan, Adriana A1 - Paprotka, Kerstin A1 - Kessie, David A1 - Mehling, Helene A1 - Potempa, Jan A1 - Ohlsen, Knut A1 - Fraunholz, Martin J. A1 - Rudel, Thomas T1 - Intracellular Staphylococcus aureus employs the cysteine protease staphopain A to induce host cell death in epithelial cells JF - PLoS Pathogens N2 - Staphylococcus aureus is a major human pathogen, which can invade and survive in non-professional and professional phagocytes. Uptake by host cells is thought to contribute to pathogenicity and persistence of the bacterium. Upon internalization by epithelial cells, cytotoxic S. aureus strains can escape from the phagosome, replicate in the cytosol and induce host cell death. Here, we identified a staphylococcal cysteine protease to induce cell death after translocation of intracellular S. aureus into the host cell cytoplasm. We demonstrated that loss of staphopain A function leads to delayed onset of host cell death and prolonged intracellular replication of S. aureus in epithelial cells. Overexpression of staphopain A in a non-cytotoxic strain facilitated intracellular killing of the host cell even in the absence of detectable intracellular replication. Moreover, staphopain A contributed to efficient colonization of the lung in a mouse pneumonia model. In phagocytic cells, where intracellular S. aureus is exclusively localized in the phagosome, staphopain A did not contribute to cytotoxicity. Our study suggests that staphopain A is utilized by S. aureus to exit the epithelial host cell and thus contributes to tissue destruction and dissemination of infection. Author summary Staphylococcus aureus is an antibiotic-resistant pathogen that emerges in hospital and community settings and can cause a variety of diseases ranging from skin abscesses to lung inflammation and blood poisoning. The bacterium can asymptomatically colonize the upper respiratory tract and skin of humans and take advantage of opportune conditions, like immunodeficiency or breached barriers, to cause infection. Although S. aureus was not regarded as intracellular bacterium, it can be internalized by human cells and subsequently exit the host cells by induction of cell death, which is considered to cause tissue destruction and spread of infection. The bacterial virulence factors and underlying molecular mechanisms involved in the intracellular lifestyle of S. aureus remain largely unknown. We identified a bacterial cysteine protease to contribute to host cell death of epithelial cells mediated by intracellular S. aureus. Staphopain A induced killing of the host cell after translocation of the pathogen into the cell cytosol, while bacterial proliferation was not required. Further, the protease enhanced survival of the pathogen during lung infection. These findings reveal a novel, intracellular role for the bacterial protease staphopain A. KW - Staphylococcus aureus KW - Staphylococcal infection KW - host cells KW - HeLa cells KW - cytotoxicity KW - intracellular pathogens KW - apoptosis KW - epithelial cells Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-263908 VL - 17 IS - 9 ER - TY - JOUR A1 - Wagner, Fabienne A1 - Kunz, Tobias C. A1 - Chowdhury, Suvagata R. A1 - Thiede, Bernd A1 - Fraunholz, Martin A1 - Eger, Debora A1 - Kozjak-Pavlovic, Vera T1 - Armadillo repeat-containing protein 1 is a dual localization protein associated with mitochondrial intermembrane space bridging complex JF - PLoS ONE N2 - Cristae architecture is important for the function of mitochondria, the organelles that play the central role in many cellular processes. The mitochondrial contact site and cristae organizing system (MICOS) together with the sorting and assembly machinery (SAM) forms the mitochondrial intermembrane space bridging complex (MIB), a large protein complex present in mammalian mitochondria that partakes in the formation and maintenance of cristae. We report here a new subunit of the mammalian MICOS/MIB complex, an armadillo repeat-containing protein 1 (ArmC1). ArmC1 localizes both to cytosol and mitochondria, where it associates with the outer mitochondrial membrane through its carboxy-terminus. ArmC1 interacts with other constituents of the MICOS/MIB complex and its amounts are reduced upon MICOS/MIB complex depletion. Mitochondria lacking ArmC1 do not show defects in cristae structure, respiration or protein content, but appear fragmented and with reduced motility. ArmC1 represents therefore a peripheral MICOS/MIB component that appears to play a role in mitochondrial distribution in the cell. KW - Mitochondria KW - Outer membrane proteins KW - HeLa cells KW - Immunoprecipitation KW - Cytosol KW - Small interfering RNAs KW - Confocal microscopy KW - Cell stainin Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-202670 VL - 14 IS - 10 ER - TY - JOUR A1 - Fathy, Moustafa A1 - Fawzy, Michael Atef A1 - Hintzsche, Henning A1 - Nikaido, Toshio A1 - Dandekar, Thomas A1 - Othman, Eman M. T1 - Eugenol exerts apoptotic effect and modulates the sensitivity of HeLa cells to cisplatin and radiation JF - Molecules N2 - Eugenol is a phytochemical present in different plant products, e.g., clove oil. Traditionally, it is used against a number of different disorders and it was suggested to have anticancer activity. In this study, the activity of eugenol was evaluated in a human cervical cancer (HeLa) cell line and cell proliferation was examined after treatment with various concentrations of eugenol and different treatment durations. Cytotoxicity was tested using lactate dehydrogenase (LDH) enzyme leakage. In order to assess eugenol’s potential to act synergistically with chemotherapy and radiotherapy, cell survival was calculated after eugenol treatment in combination with cisplatin and X-rays. To elucidate its mechanism of action, caspase-3 activity was analyzed and the expression of various genes and proteins was checked by RT-PCR and western blot analyses. Eugenol clearly decreased the proliferation rate and increased LDH release in a concentration- and time-dependent manner. It showed synergistic effects with cisplatin and X-rays. Eugenol increased caspase-3 activity and the expression of Bax, cytochrome c (Cyt-c), caspase-3, and caspase-9 and decreased the expression of B-cell lymphoma (Bcl)-2, cyclooxygenase-2 (Cox-2), and interleukin-1 beta (IL-1β) indicating that eugenol mainly induced cell death by apoptosis. In conclusion, eugenol showed antiproliferative and cytotoxic effects via apoptosis and also synergism with cisplatin and ionizing radiation in the human cervical cancer cell line. KW - eugenol KW - HeLa cells KW - cisplatin KW - radiation KW - apoptosis Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-193227 SN - 1420-3049 VL - 24 IS - 21 ER - TY - JOUR A1 - Subbarayal, Prema A1 - Karunakaran, Karthika A1 - Winkler, Ann-Cathrin A1 - Rother, Marion A1 - Gonzalez, Erik A1 - Meyer, Thomas F. A1 - Rudel, Thomas T1 - EphrinA2 Receptor (EphA2) Is an Invasion and Intracellular Signaling Receptor for Chlamydia trachomatis JF - PLoS Pathogens N2 - The obligate intracellular bacterium Chlamydia trachomatis invades into host cells to replicate inside a membrane-bound vacuole called inclusion. Multiple different host proteins are recruited to the inclusion and are functionally modulated to support chlamydial development. Invaded and replicating Chlamydia induces a long-lasting activation of the PI3 kinase signaling pathway that is required for efficient replication. We identified the cell surface tyrosine kinase EphrinA2 receptor (EphA2) as a chlamydial adherence and invasion receptor that induces PI3 kinase (PI3K) activation, promoting chlamydial replication. Interfering with binding of C. trachomatis serovar L2 (Ctr) to EphA2, downregulation of EphA2 expression or inhibition of EphA2 activity significantly reduced Ctr infection. Ctr interacts with and activates EphA2 on the cell surface resulting in Ctr and receptor internalization. During chlamydial replication, EphA2 remains active accumulating around the inclusion and interacts with the p85 regulatory subunit of PI3K to support the activation of the PI3K/Akt signaling pathway that is required for normal chlamydial development. Overexpression of full length EphA2, but not the mutant form lacking the intracellular cytoplasmic domain, enhanced PI3K activation and Ctr infection. Despite the depletion of EphA2 from the cell surface, Ctr infection induces upregulation of EphA2 through the activation of the ERK pathway, which keeps the infected cell in an apoptosis-resistant state. The significance of EphA2 as an entry and intracellular signaling receptor was also observed with the urogenital C. trachomatis-serovar D. Our findings provide the first evidence for a host cell surface receptor that is exploited for invasion as well as for receptor-mediated intracellular signaling to facilitate chlamydial replication. In addition, the engagement of a cell surface receptor at the inclusion membrane is a new mechanism by which Chlamydia subverts the host cell and induces apoptosis resistance. KW - membrane proteins KW - chlamydia infection KW - chlamydia trachomatis KW - chlamydia KW - HeLa cells KW - apoptosis KW - host cells KW - membrane receptor signaling Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-125566 VL - 11 IS - 4 ER - TY - JOUR A1 - Rudel, Thomas A1 - Prusty, Bhupesh K. A1 - Siegl, Christine A1 - Gulve, Nitish A1 - Mori, Yasuko T1 - GP96 Interacts with HHV-6 during Viral Entry and Directs It for Cellular Degradation N2 - CD46 and CD134 mediate attachment of Human Herpesvirus 6A (HHV-6A) and HHV-6B to host cell, respectively. But many cell types interfere with viral infection through rapid degradation of viral DNA. Hence, not all cells expressing these receptors are permissive to HHV-6 DNA replication and production of infective virions suggesting the involvement of additional factors that influence HHV-6 propagation. Here, we used a proteomics approach to identify other host cell proteins necessary for HHV-6 binding and entry. We found host cell chaperone protein GP96 to interact with HHV-6A and HHV-6B and to interfere with virus propagation within the host cell. In human peripheral blood mononuclear cells (PBMCs), GP96 is transported to the cell surface upon infection with HHV-6 and interacts with HHV-6A and -6B through its C-terminal end. Suppression of GP96 expression decreased initial viral binding but increased viral DNA replication. Transient expression of human GP96 allowed HHV-6 entry into CHO-K1 cells even in the absence of CD46. Thus, our results suggest an important role for GP96 during HHV-6 infection, which possibly supports the cellular degradation of the virus. KW - host cells KW - immunoprecipitation KW - HeLa cells KW - antibodies KW - cell binding KW - viral transmission and infection KW - viral entry KW - flow cytometry Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-111068 ER -