Abstract
Stenotrophomonas maltophilia (S. maltophilia) is a common opportunistic pathogen in intensive care units and causes infections most often after surgeries in immune-compromised patients such as those undergoing chemotherapy. Outer membrane protein A (OmpA) is the most abundant of the outer membrane proteins in S. maltophilia. Previous studies on OmpA usually focus on its interaction with the host cells and its role in vaccine development. However, the impact of OmpA on the virulence of S. maltophilia to host cells and the effects on apoptosis remain unclear. In this study, we exposed purified recombinant S. maltophilia OmpA (rOmpA) to HEp-2 cells and investigated the effects of OmpA on epithelial cell apoptosis. Morphologic and flow cytometric analyses revealed that HEp-2 cells stimulated with rOmpA multiple apoptosis features, including nuclear roundness and pyknosis, chromatin aggregation, and phosphatidylserine eversion. We found that rOmpA regulated the protein levels of Bax and Bcl-xL in HEp-2 cells, leading to changes in mitochondria permeability and the release of cytochrome c and apoptosis-inducing factors into the cytoplasm. These subsequently activate the caspase-9/caspase-3 pathway that promote apoptosis. We also observed that rOmpA enhanced the generation of reactive oxygen species and increased intracellular Ca2+ levels in HEp-2 cells. Collectively, our data suggested that rOmpA induced epithelial cells apoptosis via mi-tochondrial pathways.
Article PDF
Similar content being viewed by others
References
An, Z., Huang, X., Zheng, C., and Ding, W. 2019. Acinetobacter baumannii outer membrane protein A induces HeLa cell autophagy via MAPK/JNK signaling pathway. Int. J. Med. Microbiol. 309, 97–107.
Atale, N., Gupta, S., Yadav, U.C.S., and Rani, V. 2014. Cell-death assessment by fluorescent and nonfluorescent cytosolic and nuclear staining techniques. J. Microsc. 225, 7–19.
Birkinshaw, R.W. and Czabotar, P.E. 2017. The Bcl-2 family of proteins and mitochondrial outer membrane permeabilisation. Semin. Cell Dev. Biol. 72, 152–162.
Chang, Y.T., Lin, C.Y., Lu, P.L., Lai, C.C., Chen, T.C., Chen, C.Y., Wu, D.C., Wang, T.P., Lin, C.M., Wang, T.P., et al. 2012. Stenotrophomonas maltophilia blood stream infection: comparison between community onset and hospital-acquired infections. J. Microbiol. Immunol. Infect. 47, 28–35.
Choi, C.C., Hyun, S.H., Lee, J.Y., Lee, J.S., Lee, Y.S., Kim, S.A., Chae, J.P., Yoo, S.M., and Lee, J.C. 2008. Acinetobacter baumannii outer membrane protein A targets the nucleus and induces cytotoxicity. Cell. Microbiol. 10, 309–319.
Choi, C.C., Lee, E.Y., Lee, Y.C., Park, T.I., Kim, H.J., Hyun, S.H., Kim, S.A., Lee, S.K., and Lee, J.C. 2005. Outer membrane protein 38 of Acinetobacter baumannii localizes to the mitochondria and induces apoptosis of epithelial cells. Cell. Microbiol. 7, 1127–1138.
Confer, A.W. and Ayalew, S. 2013. The OmpA family of proteins: roles in bacterial pathogenesis and immunity. Vet. Microbiol. 163, 207–222.
Cregan, S.P., Dawson, V.L., and Slack, R.S. 2004. Role of AIF in caspase-dependent and caspase-independent cell death. Oncogene 23, 2785–2796.
Elmore, S. 2007. Apoptosis:a review of programmed cell death. Toxicol. Pathol. 35, 495–516.
Gaddy, J.A., Tomaras, A.P., and Luis, A.A. 2009. The Acinetobacter baumannii 19606 OmpA protein plays a role in biofilm formation on abiotic surfaces and in the interaction of this pathogen with eukaryotic cells. Infect. Immun. 77, 3150–3160.
Galia, A., Calogero, A.E., Condorelli, R.A., Fraggetta, F., La Corte, C., Ridolfo, F., Bosco, P., Castiglione, R., and Salemi, M. 2012. PARP-1 protein expression in glioblastoma multiforme. Eur. J. Histochem. 56, e9.
Gnopo, Y.M.D., Mirsa, A., Hsu, H.L., DeLisa, M.P., Daniel, S., and Putnam, D. 2020. Induced fusion and aggregation of bacterial outer membrane vesicles: experimental and theoretical analysis. J. Colloid Interf. Sci. 578, 522–532.
Gyulkhandanyan, A.V., Mutlu, A., Freedman, J., and Leytin, V. 2015. Mitochondrial permeability transition pore (MPTP)-dependent and -independent pathways of mitochondrial membrane depolarization, cell shrinkage and microparticle formation during platelet apoptosis. Br. J. Haematol. 169, 142–145.
Henry, C.M., Hollville, E., and Martin, S.J. 2013. Measuring apoptosis by microscopy and flow cytometry. Methods 61, 90–97.
Kang, Y., Sun, Y., Zhang, Y., and Wang, Z. 2018. Cytochrome c is important in apoptosis of labial glands in primary Sjogren’s syndrome. Mol. Med. Rep. 17, 1993–1997.
Kim, R. 2005. Unknotting the roles of Bcl-2 and Bcl-xL. Biochem. Biophys. Res. Commun. 333, 336–343.
Lee, J.S., Choi, C.H., Kim, J.W., and Lee, J.C. 2010. Acinetobacter baumannii outer membrane protein A induces dendritic cell death through mitochondrial targeting. J. Microbiol. 48, 387–392.
Lee, M.R., Wang, H.C., Yang, C.Y., Lin, C.K., Kuo, H.Y., Ko, J.C., Sheng, W.H., Lee, L.N., Yu, C.J., and Hsueh, P.R. 2014. Clinical characteristics and outcomes of patients with pleural infections due to Stenotrophomonas maltophilia at a medical center in Taiwan, 2004–2012. Eur. J. Clin. Microbiol. Infect. Dis. 33, 1143–1148.
Li, Y., Tang, X., Zhao, Z., Wang, H., Wang, X., Shang, X., Liu, P., Kou, Z., Jiang, Y., and Li, Y. 2019. Intranasal immunization with recombinant outer membrane protein A induces protective immune response against S. maltophilia infection. PLoS ONE 14, e0214596.
Li, Y., Zhao, Z.Q., Liu, P., Shang, X.Y., Tang, X.P., and Li, Y. 2017a. Bioinformatics analysis of Stenotrophomonas maltophilia outer membrane protein OmpA. Mil. Med. Sci. 41, 987–990.
Li, P., Zhou, L., Zhao, T., Liu, X., Zhang, P., Liu, Y., Zheng, X., and Li, Q. 2017b. Caspase-9: structure, mechanisms and clinical application. Oncotarget. 8, 23996–24008.
Liu, J., Yao, Y., Ding, H., and Chen, R. 2014. Oxymatrine triggers apoptosis by regulating Bcl-2 family proteins and activating cas-pase-3/caspase-9 pathway in human leukemia HL-60 cells. Tumour Biol. 35, 5409–5415.
Lopes, B.R.P., Ribeiro, A.G., Silva, T.F., Barbosa, L.V., Jesus, T.I., Matsuda, B.K., Costa, M.F., and Toledo, K.A. 2020. Diagnosis and treatment of HEp-2 cells contaminated with mycoplasma. Braz. J. Biol. Epub Apr. 22.
Mancini, F., Rossi, O., Necchi, F., and Micoli, F. 2020. OMV vaccines and the role of TLR agonists in immune response. Int. J. Mol. Sci. 21, 4416.
March, C., Moranta, D., Regueiro, V., Llobet, E., Tomás, A., Garmendia, J., and Bengoechea, J.A. 2011. Klebsiella pneumoniae outer membrane protein A is required to prevent the activation of airway epithelial cells. J. Biol. Chem. 286, 9956–9967.
Meng, G., Pan, L., Li, C., Hu, F., Shi, X., Lee, I., Drevensek-Olenik, I., Zhang, X., and Xua, J. 2014. Temperature-induced labelling of Fluo-3 AM selectively yields brighter nucleus in adherent cells. Biochem. Biophys. Res. Commun. 443, 888–893.
Morris, G., Walker, A.J., Berk, M., Maes, M., and Puri, B.K. 2018. Cell death pathways: a novel therapeutic approach for neuroscientists. Mol. Neurobiol. 55, 5767–5786.
Namba, A., Mano, N., Takano, H., Beppu, T., Ueda, K., and Hirose, H. 2008. OmpA is an adhesion factor of Aeromonas veronii, an optimistic pathogen that habituates in carp intestinal tract. J. Appl. Microbiol. 105, 1441–1451.
Negara, K.S., Suwiyoga, K., Pemayun, T.G.A., Sudewi, A.A.R., Astawa, N.M., Arijana, I.G.N.K., and Tunas, K. 2018. The role of caspase-3, apoptosis-inducing factor, and B-cell lymphoma-2 expressions in term premature rupture of membrane. Rev. Bras. Ginecol. Obstet. 40, 733–739.
Pistritto, G., Trisciuoglio, D., Ceci, C., Garufi, A., and D’Orazi, G. 2016. Apoptosis as anticancer mechanism: function and dysfunction of its modulators and targeted therapeutic strategies. Aging 8, 603–619.
Renault, T.T., Dejean, L.M., and Manon, S. 2016. A brewing understanding of the regulation of Bax function by Bcl-xL and Bcl-2. Mech. Ageing Dev. 161, 201–210.
Rumbo, C., Tomás, M., Moreira, E.F., Soares, N.C., Carvajal, M., Santillana, E., Beceiro, A., Romero, A., and Bou, G. 2014. The Acinetobacter baumannii Omp33-36 porin is a virulence factor that induces apoptosis and modulates autophagy in human cells. Infect. Immun. 82, 4666–4680.
Shin, S., Lu, G., Cai, M., and Kim K.S. 2005. Escherichia coli outer membrane protein A adheres to human brain microvascular endothelial cells. Biochem. Biophys. Res. Commun. 330, 1199–1204.
Siegel, S.J. and Weiser, J.N. 2015. Mechanisms of bacterial colonization of the respiratory tract. Annu. Rev. Microbiol. 69, 425–444.
Sinha, K., Das, J., Pal, P.P., and Sil, P.C. 2013. Oxidative stress: the mitochondria-dependent and mitochondria-independent pathways of apoptosis. Arch. Toxicol. 87, 1157–1180.
Smith, S.G.J., Mahon, M., Lambert, M.A., and Fagan, R.P. 2007. A molecular Swiss army knife: OmpA structure, function and expression. FEMS Microbiol. Lett. 273, 1–11.
Sukumaran, S.K., Selvaraj, S.K., and Prasadarao, N.V. 2004. Inhibition of apoptosis by Escherichia coli K1 is accompanied by increased expression of Bcl-xL and blockade of mitochondrial cytochrome c release in macrophages. Infect. Immun. 72, 6012–6022.
Wallberg, F., Tenev, T., and Meier, P. 2016. Analysis of apoptosis and necroptosis by fluorescence-activated cell sorting. Cold Spring Harb. Protoc. 2016, 347–352.
Wang, Y., Kim, N.S., Haince, J.F., Kang, H.C., David, K.K., Andrabi, S.A., Poirier, G.G., Dawson, V.L., and Dawson, T.M. 2011. Poly (ADP-ribose) (PAR) binding to apoptosis-inducing factor is critical for PAR Polymerase-1-dependent cell death (parthanatos). Sci. Signal. 4, ra20.
Wang, C., Liu, C., Niu, L., Wang, L., Hou, L., and Cao, X. 2013. Surfactin-induced apoptosis through ROS–ERS–Ca2+-ERK pathways in HepG2 cells. Cell Biochem. Biophys. 67, 1433–1439.
Wu, B.T, Bao, L., Sun, Z., Li, D.K., and Zhang, Y. 2011. The OmpA-like protein Loa22 from Leptospira interrogans serovar lai induces apoptosis in A549 via Ca2+ signal pathway. J. Sichuan Univ. Med. Sci. Edn. 42, 298–302.
Xu, W., Guo, G., Li, J., Ding, Z., Sheng, J., Li, J., and Tan, W. 2016. Activation of Bcl-2-caspase-9 apoptosis pathway in the testis of asthmatic mice. PLoS ONE 11, e0149353.
Xu, G., Tang, X., Shang, X., Li, Y., Wang, J., Yue, J., and Li, Y. 2018. Identification of immunogenic outer membrane proteins and evaluation of their protective efficacy against Stenotrophomonas maltophilia. BMC Infect. Dis. 18, 347.
Zhang, Y., Bao, L., Zhu, H., Huang, B., and Zhang, H. 2010. OmpA-like protein Loa 22 from Leptospira interogans serovar Lai is cytotoxic to cultured rat renal cells and promotes inflammatory responses. Acta Biochim. Biophys. Sin. 42, 70–79.
Zhang, J., Wang, X., Vikash, V., Ye, Q., Wu, D., Liu, Y., and Dong, W. 2016. ROS and ROS-mediated cellular signaling. Oxid. Med. Cell. Longev. 2016, 4350965.
Zhang, M., Zheng, J., Nussinov, R., and Ma, B. 2017. Release of cytochrome c from Bax pores at the mitochondrial membrane. Sci. Rep. 7, 2635.
Zhao, X., Tao, X., Xu, L., Yin, L., Qi, Y., Xu, Y., Han, X., and Peng, J. 2016. Dioscin induces apoptosis in human cervical carcinoma HeLa and SiHa cells through ROS-mediated DNA damage and the mitochondrial signaling pathway. Molecules 21, 730.
Zhou, F., Yang, Y., and Xing, D. 2011. Bcl-2 and Bcl-xL play important roles in the crosstalk between autophagy and apoptosis. FEBS J. 278, 403–413.
Acknowledgments
This work was supported by the National Natural Science Foundation of China (81571959, 81071399). The funder had no role in study design, data collectionand analysis, decision to publish, or preparation of the manuscript.
Author information
Authors and Affiliations
Corresponding author
Additional information
Conflict of Interest
The authors declare that they have no competing interests.
Supplemental material for this article may be found at http://www.springerlink.com/content/120956.
Electronic Supplementary Material
Rights and permissions
About this article
Cite this article
Wang, X., Li, Y., Tang, X. et al. Stenotrophomonas maltophilia outer membrane protein A induces epithelial cell apoptosis via mitochondrial pathways. J Microbiol. 58, 868–877 (2020). https://doi.org/10.1007/s12275-020-0235-9
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12275-020-0235-9