Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/92261
標題: Characterization of the Acinetobacter baumannii antigens NcsP and TonB-R
鮑氏不動桿菌抗原 NcsP 與 TonB-R 之特性分析
作者: 莊斯凱
Sih-Kai Jhuang
關鍵字: 鮑氏不動桿菌
鐵離子
疫苗
Acinetobacter baumannii
iron
vaccine
引用: Antunes, L., Imperi, F., Towner, K. J. & Visca, P. (2011). Genome-assisted identification of putative iron-utilization genes in Acinetobacter baumannii and their distribution among a genotypically diverse collection of clinical isolates. Research in microbiology 162, 279-284. Bateman, A. & Bycroft, M. (2000). The structure of a LysM domain from E. coli membrane-bound lytic murein transglycosylase D (MltD). Journal of molecular biology 299, 1113-1119. Boyer, H. W., & Roulland-dussoix, D. (1969). A complementation analysis of the restriction and modification of DNA in Escherichia coli. Journal of molecular biology 41, 459-472. Choi, C. H., Lee, E. Y., Lee, Y. C., Park, T. I., Kim, H. J., Hyun, S. H., Kim, S., Lee, S. K. & Lee, J. C. (2005). Outer membrane protein 38 of Acinetobacter baumannii localizes to the mitochondria and induces apoptosis of epithelial cells. Cellular microbiology 7, 1127-1138. Choi, C. H., Lee, J. S., Lee, Y. C., Park, T. I. & Lee, J. C. (2008). Acinetobacter baumannii invades epithelial cells and outer membrane protein A mediates interactions with epithelial cells. BMC microbiology 8, 216. Conlon, J. M., Ahmed, E. & Condamine, E. (2009). Antimicrobial Properties of Brevinin‐2‐Related Peptide and its Analogs: Efficacy Against Multidrug‐Resistant Acinetobacter baumannii. Chemical biology & drug design 74, 488-493. Davis, K. A., Moran, K. A., McAllister, C. K. & Gray, P. J. (2005). Multidrug-resistant Acinetobacter extremity infections in soldiers. Emerging infectious diseases 11, 1218-1224. Dorsey, C. W. (2003). Genetic organization of an Acinetobacter baumannii chromosomal region harbouring genes related to siderophore biosynthesis and transport. Microbiology 149, 1227-1238. Dorsey, C. W., Beglin, M. S. & Actis, L. A. (2003). Detection and analysis of iron uptake components expressed by Acinetobacter baumannii clinical isolates. Journal of clinical microbiology 41, 4188-4193. Dorsey, C. W., Tomaras, A. P., Connerly, P. L., Tolmasky, M. E., Crosa, J. H. & Actis, L. A. (2004). The siderophore-mediated iron acquisition systems of Acinetobacter baumannii ATCC 19606 and Vibrio anguillarum 775 are structurally and functionally related. Microbiology 150, 3657-3667. Ellis, T. N. & Kuehn, M. J. (2010). Virulence and immunomodulatory roles of bacterial outer membrane vesicles. Microbiology and Molecular Biology Reviews 74, 81-94. Gaddy, J. A., Tomaras, A. P. & Actis, L. 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. Infection and immunity 77, 3150-3160. Gaddy, J. A., Arivett, B. A., McConnell, M. J., Lopez-Rojas, R., Pachon, J. & Actis, L. A. (2012). Role of acinetobactin-mediated iron acquisition functions in the interaction of Acinetobacter baumannii strain ATCC 19606T with human lung epithelial cells, Galleria mellonella caterpillars, and mice. Infection and immunity 80, 1015-1024. Giacometti, A., Cirioni, O., Kamysz, W., D'Amato, G., Silvestri, C., Del Prete, M. S., Lukasiak, J. & Scalise, G. (2003). Comparative activities of cecropin A, melittin, and cecropin A–melittin peptide CA (1–7) M (2–9) NH2 against multidrug-resistant nosocomial isolates of Acinetobacter baumannii. Peptides 24, 1315-1318. Goel, V. K. & Kapil, A. (2001). Monoclonal antibodies against the iron regulated outer membrane proteins of Acinetobacter baumannii are bactericidal. BMC microbiology 1, 16. Gordon, N. C. & Wareham, D. W. (2010). Multidrug-resistant Acinetobacter baumannii: mechanisms of virulence and resistance. International journal of antimicrobial agents 35, 219-226. Hood, M. I., Mortensen, B. L., Moore, J. L., Zhang, Y., Kehl-Fie, T. E., Sugitani, N., Chazin, W. J., Caprioli, R. M. & Skaar, E. P. (2012). Identification of an Acinetobacter baumannii zinc acquisition system that facilitates resistance to calprotectin-mediated zinc sequestration. PLoS pathogens 8, e1003068. Hsueh, P. R., Teng, L. J., Chen, C. Y., Chen, W. H., Yu, C. J., Ho, S. W. & Luh, K. T. (2002). Pandrug-resistant Acinetobacter baumannii causing nosocomial infections in a university hospital, Taiwan. Emerging infectious diseases 8, 827-832. Jawad, A., Seifert, H., Snelling, A., Heritage, J. & Hawkey, P. (1998). Survival of Acinetobacter baumannii on dry surfaces: comparison of outbreak and sporadic isolates. Journal of clinical microbiology 36, 1938-1941. Jin, J. S., Kwon, S.-O., Moon, D. C., Gurung, M., Lee, J. H., Kim, S. I. & Lee, J. C. (2011). Acinetobacter baumannii secretes cytotoxic outer membrane protein A via outer membrane vesicles. PloS one 6, e17027. Jun, S. H., Lee, J. H., Kim, B. R., Kim, S. I., Park, T. I., Lee, J. C. & Lee, Y. C. (2013). Acinetobacter baumannii Outer Membrane Vesicles Elicit a Potent Innate Immune Response via Membrane Proteins. PloS one 8, e71751. Jyothisri, K., Deepak, V. & Rajeswari, M. R. (1999). Purification and characterization of a major 40 kDa outer membrane protein of Acinetobacter baumannii. FEBS letters 443, 57-60. Kim, S. W., Choi, C. H., Moon, D. C. & other authors (2009). Serum resistance of Acinetobacter baumannii through the binding of factor H to outer membrane proteins. FEMS microbiology letters 301, 224-231. Kuehn, M. J. & Kesty, N. C. (2005). Bacterial outer membrane vesicles and the host–pathogen interaction. Genes & development 19, 2645-2655. Kuo, S. C., Chang, S. C., Wang, H. Y., Lai, J. F., Chen, P. C., Shiau, Y. R., Huang, I. W. & Lauderdale, T. L. (2012). Emergence of extensively drug-resistant Acinetobacter baumannii complex over 10 years: nationwide data from the Taiwan Surveillance of Antimicrobial Resistance (TSAR) program. BMC infectious diseases 12, 200. Lee, H. W., Koh, Y., Kim, J., Lee, J. C., Lee, Y. C., Seol, S. Y. & Cho, D. T. (2008). Capacity of multidrug‐resistant clinical isolates of Acinetobacter baumannii to form biofilm and adhere to epithelial cell surfaces. Clinical Microbiology and Infection 14, 49-54. McConnell, M. J., Actis, L. & Pachon, J. (2013). Acinetobacter baumannii: human infections, factors contributing to pathogenesis and animal models. FEMS microbiology reviews 37, 130-155. Melillo, A., Sledjeski, D. D., Lipski, S., Wooten, R. M., Basrur, V. & Lafontaine, E. R. (2006). Identification of a Francisella tularensis LVS outer membrane protein that confers adherence to A549 human lung cells. FEMS microbiology letters 263, 102-108. Mihara, K., Tanabe, T., Yamakawa, Y., Funahashi, T., Nakao, H., Narimatsu, S. & Yamamoto, S. (2004). Identification and transcriptional organization of a gene cluster involved in biosynthesis and transport of acinetobactin, a siderophore produced by Acinetobacter baumannii ATCC 19606T. Microbiology 150, 2587-2597. Neely, A. N. (2000). A survey of gram-negative bacteria survival on hospital fabrics and plastics. Journal of Burn Care & Research 21, 523&hyhen. Nemec, A., Dolzani, L., Brisse, S., van den Broek, P. & Dijkshoorn, L. (2004). Diversity of aminoglycoside-resistance genes and their association with class 1 integrons among strains of pan-European Acinetobacter baumannii clones. J Med Microbiol 53, 1233-1240. Nucleo, E., Steffanoni, L., Fugazza, G., Migliavacca, R., Giacobone, E., Navarra, A., Pagani, L. & Landini, P. (2009). Growth in glucose-based medium and exposure to subinhibitory concentrations of imipenem induce biofilm formation in a multidrug-resistant clinical isolate of Acinetobacter baumannii. BMC microbiology 9, 270. Oldfield, N. J., Bland, S. J., Taraktsoglou, M., Ramos, F. J. D., Robinson, K., Wooldridge, K. G. & Ala'Aldeen, D. A. (2007). T‐cell stimulating protein A (TspA) of Neisseria meningitidis is required for optimal adhesion to human cells. Cellular microbiology 9, 463-478. Ostorhazi, E., Rozgonyi, F., Sztodola, A. & other authors (2010). Preclinical advantages of intramuscularly administered peptide A3-APO over existing therapies in Acinetobacter baumannii wound infections. Journal of antimicrobial chemotherapy 65, 2416-2422. Penwell, W. F., Arivett, B. A. & Actis, L. A. (2012). The Acinetobacter baumannii entA gene located outside the acinetobactin cluster is critical for siderophore production, iron acquisition and virulence. PloS one 7, e36493. Russo, T. A., MacDonald, U., Beanan, J. M., Olson, R., MacDonald, I. J., Sauberan, S. L., Luke, L. W. & Umland, T. C. (2009). Penicillin-binding protein 7/8 contributes to the survival of Acinetobacter baumannii in vitro and in vivo. Journal of Infectious Diseases 199, 513-521. Russo, T. A., Luke, N. R., Beanan, J. M., Olson, R., Sauberan, S. L., MacDonald, U., Schultz, L. W., Umland, T. C. & Campagnari, A. A. (2010). The K1 capsular polysaccharide of Acinetobacter baumannii strain 307-0294 is a major virulence factor. Infection and immunity 78, 3993-4000. Smani, Y., Docobo-Perez, F., Lopez-Rojas, R., Dominguez-Herrera, J., Ibanez-Martinez, J. & Pachon, J. (2012a). Platelet-activating factor receptor initiates contact of Acinetobacter baumannii expressing phosphorylcholine with host cells. Journal of Biological Chemistry 287, 26901-26910. Smani, Y., McConnell, M. J. & Pachon, J. (2012b). Role of fibronectin in the adhesion of Acinetobacter baumannii to host cells. PloS one 7, e33073. Smani, Y., Dominguez-Herrera, J. & Pachon, J. (2013). Association of the Outer Membrane Protein Omp33 With Fitness and Virulence of Acinetobacter baumannii. Journal of Infectious Diseases, jit386. Taiwan CDC,2013,101 年傳染病統計暨監視年報。 Tien, H. C., Battad, A., Bryce, E. A. & other authors (2007). Multi-drug resistant Acinetobacter infections in critically injured Canadian forces soldiers. BMC infectious diseases 7, 95. Vidal, R., Dominguez, M., Urrutia, H., Bello, H., Gonzalez, G., Garcia, A. & Zemelman, R. (1995). Biofilm formation by Acinetobacter baumannii. Microbios 86, 49-58. Wang, W., Reitzer, L., Rasko, D. A., Pearson, M. M., Blick, R. J., Laurence, C. & Hansen, E. J. (2007). Metabolic analysis of Moraxella catarrhalis and the effect of selected in vitro growth conditions on global gene expression. Infection and immunity 75, 4959-4971. Wendt, C., Dietze, B., Dietz, E. & Ruden, H. (1997). Survival of Acinetobacter baumannii on dry surfaces. Journal of clinical microbiology 35, 1394-1397. Yamamoto, S., Okujo, N., Kataoka, H. & Narimatsu, S. (1999). Siderophore-mediated utilization of transferrin- and lactoferrin-bound iron by Acinetobacter baumannii. Journal of Health Science 45, 297-302. Zimbler, D. L., Penwell, W. F., Gaddy, J. A., Menke, S. M., Tomaras, A. P., Connerly, P. L. & Actis, L. A. (2009). Iron acquisition functions expressed by the human pathogen Acinetobacter baumannii. Biometals : an international journal on the role of metal ions in biology, biochemistry, and medicine 22, 23-32. 潘健群,2014,Moraxella catarrhalis 保守抗原 NcsP 與 Mp16 的免疫特性分析。國立中興大學分子生物學研究所,碩士論文。
摘要: Acinetobacter baumannii (AB), which is a Gram-negative opportunistic respiratory pathogen, often causes nosocomial infections. In recent years, many multidrug-resistant AB (MRAB) strains have been found because of antibiotic overuse. Besides searching of new antibiotics for control of the MRAB infections, one alternative strategy is to develop effective vaccines. TonB-R and NcsP are two human serum-reactive antigens previously identified in our laboratory. This study aims to investigate the characteristic of these two proteins and to evaluate their feasibility to be used as anti-AB vaccines. At first, mice were immunized with purified recombinant TonB-R (rTonB-R) and rNcsP, respectively, to prepare antigen-specific antisera. Western blot analysis revealed that TonB-R and NcsP were detected in 89 % (65/73) and 100 % (73/73) of the tested AB isolates suggesting that these two antigens are highly conserved in AB. The results of bacterial fractionation showed that both proteins were presented in the outer membrane. ELISA performed with the heat-killed AB and immunostaining the viable AB followed by flow cytometry and immunomicroscopy demonstrated that both antigens are surface exposed. Next, the levels of TonB-R and NcsP at different growth phases and under iron-limiting conditions were monitored. The results showed that in the presence of 2,2'- bipyridyl (BIP), an iron chelator, the levels of TonB-R were higher than those bacteria grown in LB and LB supplemented with FeCl3 (LB/FeCl3); particularly in the late log phase. In contrast, higher NcsP levels were observed only at the early log phase in the bacteria grown in LB/BIP compared to those in LB and LB/FeCl3. Nevertheless, the results indicate that the expression of TonB-R and NcsP may be iron-regulated. To evaluate whether these two proteins can be used as AB vaccines, mice immunized with rTonB-R and rNcsP in the presence of Freund,s adjuvant (FA) and the control group (immunized with FA only) were challenged via the respiratory tract with a clinical isolated AB. AB was recoverd from the lung four hours post challenged. The median bacterial burdens were 4176, 3605, and 8513 CFU/g for TonB-R, NcsP, and control mice, respectively. The differences between the immunized and the control group were significant (TonB-R, P < 0.05; NcsP, P < 0.01) indicating that both TonB-R and NcsP are able to elicit protective immune responses. The bactericidal/bacteriostatic activies of anti-TonB-R and anti-NcsP antisera were further confirmed in vitro. Finally, MTT assay was performed to evaluate whether rTonB-R and rNcsP are cytotoxic to human cells. The results showed that both proteins were not cytotoxic to human A549 cells. Collectively, these results indicate that TonB-R and NcsP are good vaccine candidates against AB infection.
鮑氏不動桿菌 ( Acinetobacter baumannii,AB ) 是革蘭氏陰性菌,為伺機性呼吸道病原菌,常造成院內感染。近年來抗生素的濫用,使得多重抗藥性 AB (multidrug-resistant AB,MRAB) 不斷地被發現。在 MRAB 的防疫中,除了尋找更強效的抗生素,研發有效的疫苗為另一可行之策略。先前本實驗室鑑定到兩個可被人類血清辨認的蛋白質 TonB-R (putative TonB-dependent siderophore receptor) 及 NcsP (non-classical secretory protein)。本研究主要探討 TonB-R 及 NcsP 之生理特性及評估作為 AB 菌疫苗之可行性。首先將重組蛋白質 TonB-R (rTonB-R) 及 NcsP (rNcsP) 純化並免疫小鼠製備具抗原專一性之抗血清,西方墨點法分析各分離菌株,其中 TonB-R 及 NcsP 於 AB 菌分別具有 89 % (65/73) 及 100 % (73/73) 之高度保守性。菌體分層分析結果顯示 TonB-R 及 NcsP皆存在於外膜。經 ELISA 分析經熱失活處理之 AB 菌菌體表面,以及利用免疫螢光染色經流式細胞儀及螢光顯微鏡分析活菌表面,結果皆顯示兩目標抗原外露於菌體表面。接著,本研究分析 AB 菌於缺鐵生長環境下的生長狀況及目標抗原表現情形,結果顯示提供鐵離子螯合劑 2, 2'- bipyridyl (BIP) 之 LB 於晚期 log phase 發現 AB 菌 TonB-R 表現量高於 LB 及添加 FeCl3 之 LB (LB/FeCl3)。相反的,相較於 LB 及 LB/FeCl3,NcsP 於 LB/BIP 具較高表現量於早期 log phase。這些結果指出 TonB-R 及 NcsP 的表現可能受鐵離子調控。為了評估目標抗原作為 AB 菌疫苗之可行性,將小鼠個別免疫含費氏佐劑 (Freund,s adjuvant,FA ) 的 rTonB-R、rNcsP 重組蛋白質及控制組 (僅施打 FA ) 後,以臨床分離之 AB 進行呼吸道感染。感染四小時後,取其肺葉計算菌數,中位數 (median) 計算結果 TonB-R、NcsP、控制組依序為 4176、3605、8513 CFU/g,這些具顯著差異 (TonB-R,P < 0.05;NcsP,P < 0.01) 之結果證明 TonB-R 及 NcsP 所引起的免疫反應可提供有效的保護力。進一步於in vitro 數據證實 anti-TonB-R 及 anti-NcsP 抗血清對 AB 菌具殺菌/抑菌效力。最後,利用 MTT assay 分析 rTonB-R 及 rNcsP 對人類細胞是否具細胞毒性,結果顯示對 A549 不具毒性。綜合上述結果,面對 AB 菌的感染,TonB-R 及 NcsP 適合作為疫苗研發之目標。
URI: http://hdl.handle.net/11455/92261
文章公開時間: 2018-02-05
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