利用蛋白質體學分析胃幽門桿菌之生物膜組成與嗜熱菌Meiothermus taiwanensis之可溶性蛋白

Abstract

胃幽門桿菌可以引起十二指腸潰瘍、胃潰瘍跟胃癌。臨床治療上通常會長期使用多種抗生素治療胃幽門桿菌感染，但是要完全清除胃幽門桿菌卻很困難，即使使用抗生素治療後，大約還有10~20 %的人會持續感染胃幽門桿菌。常利用的抗生素治療不能根除胃幽門桿菌的原因被推測為生物膜的形成。生物膜的主要成分為biofilm matrix，它可以保護菌體逃避寄主的免疫系統攻擊，也可以抵抗外界壓力，包含抗生素使用。我們的研究目的為鑑定biofilm matrix的組成，biofilm matrix主要成分為胞外醣類跟少許的蛋白質。胃幽門桿菌產生的胞外醣類成分別為mannose、galactose跟glucose。分析培養四天的生物膜胞外物質(biofilm matrix)發現mannose為主要部分(80%)，galactose跟glucose所占的比例較少只有7%跟13%，然而在第七天的生物膜胞外物質中，galactose跟glucose所占的比例增加為15.5跟16.5%。之後利用SDS-PAGE跟MALDI-Q-TOF之MS/MS分析生物膜胞外物質的蛋白質成分，這些蛋白質分別為urease subunit beta、60 kDa chaperonin、catalase、citrate synthase、urease subunit alpha、probable peroxiredoxin、ferritin跟neutrophil-activating protein A。在urease活性測試實驗中發現，生物膜細胞的urease活性高於流動細胞(planktonic cell)。之後利用抑制urease activity的AHA作用於胃幽門桿菌，觀察其生物膜生成情況，結果顯示加入AHA作用的細菌生物膜生成量有明顯下降。另外也觀察napA 突變株的生物膜生成情況，發現napA突變株仍然可以形成生物膜，但是在顯微鏡下觀察生物膜在早期的菌落型態，發現napA突變株不容易往上推疊成較大的菌落，菌落型態呈零散狀，可以推測NapA的功能為影響細菌之間的作用力，進而影響細菌的聚集堆疊，但是不影響生物膜的生成。 Meiothermus taiwanensis WR-220-R 跟 Meiothermus taiwanensis WR-220-W的16 rRNA呈現99.8 %的相似度，它們也都會形成絲狀型態，但是唯一不同在於M. taiwanensis WR-220-R會產生紅色色素，而M. taiwanensis WR-220-W不會。從生長曲線觀察可以發現，這兩株菌都可以在55℃跟65 ℃生長，唯一不同在於M. taiwanensis WR-220-W的細菌生成量有明顯下降。在細胞膜的脂肪酸組成方面，兩者都可以觀察到iso- 跟anteiso 分支的脂肪酸為主要脂肪酸的成分，然而當溫度升高培養時，iso/anteiso的比例也提升，至於直鏈狀的脂肪酸所占的比例較少，其中含量最多的脂肪酸為iso-C15:0、iso-C17:0、 anteiso-C15:0跟anteiso-C17:0，然而兩者之間比較不同的地方在於，M. taiwanensis WR-220-W有較高的normal-C16:0、normal-C18:1、normal-C18:0含量、iso-anteiso的比例跟average acyl chain。另外他們的生化活性也接近。最後利用二維蛋白質電泳跟MALTI-Q-TOF之MS/MS分析兩者之間蛋白質差異性，在M. taiwanensis WR-220-R過度表現的蛋白為chaperonin GroEL、elongation factor Tu、trigger factor、enolase、FeS assembly ATPase SufC、DNA-directed RNA polymerase subunit beta、proabable transaldolase、30S ribosomal protein S、inorganic pyrophosphatase跟DNA-directed RNA polymerase subunit omega。在M. taiwanensis WR-220-W過度表現的蛋白為chaperonin GroEL、elongation factor Tu、Candida agglutinin-like protein (ALS)跟deoxyhypusine synthase。

H. pylori can induce duodenal ulcers, gastric ulcers, chronic gastritis, and gastric carcinoma. The eradication of H. pylori can be difficult, usually requiring a multi-drug regimen and a lengthy treatment period. Despite antimicrobial therapy, 10–20% of infections manage to persist. The persistency of H. pylori infection may due to be associated with the biofilm formation. The biofilm protects the bacteria from host immunological defense as well as antibiotic killing. In this study, our purpose was to identify the composition of the biofilm, especially in polysaccharides and proteins. The biofilm matrix mainly included exopolysaccharides and proteins. The exopolysaccharide produced by H. pylori was found to be composed of mannose, glucose and galactose. For four-day liquid culture, mannose was the major component, and galactose and glucose as minor components (80:7:13%). For seven day liquid culture, the ratios of galactose and glucose increased. (15.5%:16.5%). The protein components of biofilm matrix for four and seven day cultures were analyzed and identified by SDS-PAGE and MALDI-Q-TOF-MS/MS, repectively. Those proteins included urease subunit beta, 60 kDa chaperonin, catalase, citrate synthase, urease subunit alpha, probable peroxiredoxin, ferritin, neutrophil-activating protein A. The urease activity experiment showed that the biofilm cell has stronger urease activity than planktonic cell. In addition, H pylori treated with urease inhibitor AHA attenuated the biofilm formation. Nevertheless, the napA mutant strain can still develop biofilm, but not easy to form microcolony in early stage of biofilm life cycle. Its role in biofilm formation was predicted to be associated with cell-cell interaction.

The 16 rRNA of Meiothermus taiwanensis WR-220-R and Meiothermus taiwanensis WR-220-W showed 99.8 % similarity. They both form filamentous morphology. The difference of cell morphology was that M. taiwanensis WR-220-R produce red pigment, but M. taiwanensis WR-220-W dose not. From the growth curve, M. taiwanensis WR-220-R and M. taiwanensis WR-220-W both could grow in 55℃ and 65 ℃. The major difference between them was decreased the growth intensity in M. taiwanensis WR-220-W. In the analysis of lipid composition the results showed that iso- and anteiso-branched fatty acids were predominant in the composition of fatty acids. Moreover, the ratios of iso- to anteiso-branched fatty acids increased with higher culture temperatures. The straight-chain fatty acids represented a minor constituent of fatty acids. The major fatty acids between them were iso-C15:0, iso-C17:0, anteiso-C15:0, and anteiso-C17:0. However, M. taiwanensis WR-220-W have higher ratio of normal-C16:0、normal-C18:1 and normal-C18:0. The different protein expression between them was identified by 2-DE and MALTI-Q-TOF. The higher expressed proteins in M. taiwanensis WR-220-R are chaperonin GroEL、elongation factor Tu、trigger factor、enolase、FeS assembly ATPase SufC、DNA-directed RNA polymerase subunit beta、proabable transaldolase、30S ribosomal protein S、inorganic pyrophosphatase and DNA-directed RNA polymerase subunit omega. The higher expressed protein in Meiothermus taiwanensis WR-220-W are chaperonin GroEL、elongation factor Tu、Candida agglutinin-like protein (ALS) and deoxyhypusine synthase.