Functional studies of the virulence-associated proteins (vapD) from Helicobacter pylori

胃幽門螺旋桿菌毒性相關蛋白(vapD)的功能分析

梁剛豪

指導教授 ： 黃海美

國立清華大學/生命科學院/生物科技研究所/碩士(2008年)

摘要

毒性相關蛋白基因被廣泛地發現在其他細菌的染色體或質體上，而且在其他細菌上的功能已有一些相關的研究。被預測為毒性相關蛋白的HP0315在胃幽門螺旋桿菌裡還沒有被通盤研究，只有應用微陣列方式有證明：胃幽門螺旋桿菌在酸性環境下（pH≦5.5）30分鐘此基因的mRNA會增加表現量。在本實驗室中，以基因轉殖方式已將重組的HP0315蛋白表現在大腸桿菌SG13009中，並利用Ni-NTA管柱純化此蛋白，作為抗原以製備多株抗體（Lin, 2006）。利用RT-PCR和西方墨點轉漬法，HP0315的mRNA和蛋白質被發現在酸處理1小時和H2O2處理的胃幽門螺旋桿菌中有較多的表現（Wang, 2007）。然而其致病相關的角色還沒有被了解，所以此HP0315蛋白的特性及其生物上角色在本實驗中被進一步研究。
以RT-PCR和西方墨點轉漬法證明：HP0315的mRNA和蛋白質在胃幽門螺旋桿菌中有表現。細菌樣本在酸性環境下（pH 4.5 or 5.5）處理30分鐘和1小時其HP0315 mRNA會比中性環境下（pH7.2）所得多表現1.6-2倍。胃幽門螺旋桿菌處在pH 4.5的酸性環境下30分鐘會有比正常培養液所得細菌樣本較多的HP0315蛋白（1.58倍）會被誘導。然而在pH 7.2, 5.5和4.5的環境下，細菌處理1小時，其HP0315蛋白並沒有明顯的改變（1.2~1.3倍）。
胃幽門螺旋桿菌處理H2O2後，HP0315的mRNA有明顯的增加。在20和40 mM H2O2處理40分鐘後，所得的HP0315的mRNA 會比未處理組增加1.5-2倍。當處理細菌濃度0-75 mM H2O2, 40分鐘，HP0315蛋白的最大表現量（2.43倍）會出現在45 mM H2O2處理組。細菌的HP0315蛋白會隨著其所處理的H2O2劑量增加（0-45 mM, 40 分鐘）而增加表現量（1~2.43倍）。
HP0315的mRNA在胃幽門螺旋桿菌處理SNP（一氧化氮提供者）後會增加。在400和800 uM SNP 1小時處理組，HP0315的mRNA 會比未處理組增加1.1-1.6倍。當細菌處理SNP (0-1000 uM), 1小時，HP0315蛋白的最大表現量（1.64倍）會出現在800 uM SNP細菌處理組。細菌的HP0315蛋白會隨著處理之SNP濃度增加（0-800 uM, 1小時）而增加表現量（1~1.64倍）。SNP對胃幽門螺旋桿菌及大腸桿菌的半數致死劑量大約為1000 uM SNP。在正常、酸、H2O2及SNP環境所得之細菌樣品，其HP0315蛋白表現也用流式細胞儀證實。
經由（a）酸處理（pH 3，在37℃ 15分鐘）過後的重組HP0315蛋白（66 ug/ml）比（b）未處理組（pH 8）較容易進入共同培養的AGS細胞核中。和AGS細胞共同培養1小時後，前者（a）大約有48%會出現在AGS細胞核，在之後2-6小時和AGS的共同培養，大約有50-60%會進入AGS細胞核中。但是在只有利用重組的HP0315蛋白不管有無酸的前處理都不能對AGS有細胞毒性，或對AGS的細胞週期產生影響。
利用HP0315抗體將細菌extract進行免疫沈澱，再用VacA（細胞液泡毒素，胃幽門螺旋桿菌中的一種重要毒性因子，HP0887）作西方墨點轉漬法確認HP0315和VacA之交互作用，然後交換抗體順序再確認兩者也有交互作用。利用共同免疫沈澱法和西方墨點轉漬法確認HP0315和VacA在胃幽門螺旋桿菌中的交互作用。由此結果推測：HP0315蛋白可能有助於VacA毒性的表現。
另一方面，HP0315蛋白被證明存在於胃幽門螺旋桿菌中。利用免疫沈澱法從胃幽門螺旋桿菌中獲得HP0315蛋白轉印到PVDF膜上，並以Coomassie Blue染色。染色後，所穫之一條假設存在的HP0315蛋白（11.5 kD）加以蛋白質定序，證實其存在於胃幽門螺旋桿菌中，而且占log時期胃幽門螺旋桿菌全部蛋白的1/500.

關鍵字

胃幽門螺旋桿菌 ； 毒性相關蛋白

並列摘要

Virulence-associated protein gene has been extensively discovered in the chromosome or plasmid from many species. Its function has been studied in several species recently. HP0315, annotated as virulence-associated protein D, was not yet completely studied in H. pylori. Only microarray data indicated that HP0315 up regulated in H. pylori after 30 min culture at pH≦5.5 (Wen et al., 2003). The recombinant HP0315 protein was cloned, expressed in E. coli strain SG13009, purified by Ni-NTA beads column, and used to prepare antibodies for its functional studies previously in my laboratory (Lin, 2006). Increasing HP0315 mRNA and protein expression was found in bacteria cultured in acid condition for 1 hr and H2O2 exposure by RT-PCR and western blotting recently (Wang, 2007). However, its virulence-associated roles were not yet understood. Further characterization of this protein and its biological roles are currently investigated in this study.
RT-PCR and western blotting results showed that H. pylori have HP0315 mRNA and protein expression (Table 3). Furthermore, more HP0315 mRNA expression showed in samples from 0.5-1 h medium at pH 5.5 (2-fold) or 4.5 (1.6-fold) than that at pH 7.2 (Fig. 6A and Fig. 6B). More HP0315 protein expression was found in bacteria 0.5-h cultured in 4.5 (1.58-fold) medium than in pH 7.2 (Fig. 7A). However, no significant changes (1.2-~1.3-fold) on HP0315 protein expression was observed in bacteria after 1 h cultured in pH 4.5, 5.5, and 7.2 medium (Fig. 8A).
Significant HP0315 mRNA expression was observed in H. pylori after H2O2 exposure. At 20 and 40 mM treatment dose for 40 min, HP0315 mRNA increased 1.5- and 2.0-fold of untreated control (Fig. 9). Exposed bacteria at 0-75 mM H2O2 for 40 min, most HP0315 protein expression (2.43-fold) showed in samples after 45 mM H2O2 exposure (Fig. 10). Dose-response increase (1-~2.43-fold) on HP0315 expression was observed in samples exposed at dose 0-45 mM H2O2 for 40 min (Fig. 10A).
HP0315 mRNA expression was observed in H. pylori after SNP (NO donor) exposure for 1 h. At 400 and 800 uM exposure dose, HP0315 mRNA increased 1.1- and 1.6-fold of untreated control (Fig. 11). Exposed bacteria at 0-1000 uM SNP for 1 hr, most HP0315 protein expression (1.64-fold) showed in samples after 800 uM exposure (Fig. 10). Dose-response increase (1-~1.64-fold) on HP0315 expression was observed in samples exposed at dose 0-800 uM SNP for 1 hr (Fig. 10A). The LD50 dose was approximate 1000 uM SNP for either H. pylori or E. coli (Fig. 13). HP0315 protein expression from all samples treated in acid condition, medium having H2O2 or SNP was also confirmed by means of flow cytometry (Fig. 8B, 10B, 12 B).
Acid pretreated rec-HP0315 protein (37 ℃ for 15 min at pH 3.0; 66 ug/ml) would get into nucleus more quickly than that at pH 8 when co-cultured with AGS cells. About 48% acid pretreated protein reached AGS nuclei after 1 h co-cultured and remained 50-60% of them in nuclei after co-cultured with AGS cells for 2-6 h (Fig. 17). However, rec-HP0315 protein either having pH 3 acid-pretreatment or in pH 8 did not show cytotoxic effects and did not affect cell cycle progression on AGS cells (Fig. 18 and 19).

After first applied co-immunoprecipitation (co-IP) with antibodies against HP0315 and blotted with antibodies against VacA in H. pylori extract, then vice versa, then western blotting results indicated there has interaction between HP0315 and VacA (vacuolating cytotoxin, an important virulent factor in H. pylori, HP0887) within H. pylori lysate (Fig. 16). This suggested that HP0315 might play a VacA-association role in bacteria.
On the other hand, HP0315 protein does actually present in H. pylori bacteria. It was detected from 1/500 of total amount log phase untreated bacterial extract after IP, transferred to PVDF membrane, and coomassie blue staining. The protein sequence of a presumed HP0315 protein band (PDVF/staining) from this conduct was confirmed (Fig. 14 and Fig. 15).

參考文獻

Alpuche Aranda, C. M., J. A. Swanson, et al. (1992). "Salmonella typhimurium activates virulence gene transcription within acidified macrophage phagosomes." Proc Natl Acad Sci U S A 89(21): 10079-83.

Atherton, J. C., P. Cao, et al. (1995). "Mosaicism in vacuolating cytotoxin alleles of Helicobacter pylori. Association of specific vacA types with cytotoxin production and peptic ulceration." J Biol Chem 270(30): 17771-7.

Benoit, S., A. Benachour, et al. (2001). "Induction of vap genes encoded by the virulence plasmid of Rhodococcus equi during acid tolerance response." Res Microbiol 152(5): 439-49.

Benoit, S., A. Benachour, et al. (2002). "H(2)O(2), which causes macrophage-related stress, triggers induction of expression of virulence-associated plasmid determinants in Rhodococcus equi." Infect Immun 70(7): 3768-76.

Billington, S. J., J. L. Johnston, et al. (1996). "Virulence regions and virulence factors of the ovine footrot pathogen, Dichelobacter nodosus." FEMS Microbiol Lett 145(2): 147-56.