金黃色葡萄球菌之新的疫苗免疫及熱休克蛋白90差異表達下之免疫功能探討

A Novel Vaccine Immunization of Staphylococcus Aureus and Immune Function of Heat Shock Protein 90 under Differential Expression

羅智緯(Chih-Wei Lo)

指導教授 ： 黎耀基 ； 張大慈

國立清華大學/生命科學院/生物科技研究所/博士(2010年)

https://doi.org/10.6843/NTHU.2010.00022

摘要

Staphylococcus aureus (S. aureus) is one of Gram-positive bacteria and may cause diseases not only through tissue invasion but also through production of toxins. Antibiotic therapy was the most common way to cure S. aureus infection. However, non-specifically elimination of the major bacteria results in disruption of human microbiome homeostasis and development of resistant bacteria. Methicillin-resistant S. aureus (MRSA) is an increasingly common cause of nosocomial infections that leads to severe morbidity and mortality worldwide. Generating vaccines has been investigated for alternatively strategies recently. However, past efforts to generate vaccines against S. aureus have far been unsuccessful. Propionibacterium acnes (P. acnes), also a Gram-positive but anaerobic commensal bacteria, is widely distributed on human skin/body but absent on mouse. In this study, I found that the secretory Christie, Atkins, Munch-Peterson factor (CAMP factor) of P. acnes magnifies the hemolysis and cytolysis of S. aureus β-hemolysin, suggesting that S. aureus may utilize the secreted P. acnes CAMP factor 2 to intensify its virulence. Furthermore, P. acnes exacerbated S. aureus-induced skin lesions in vivo. The combination of CAMP factor 2 neutralization and β-hemolysin immunization cooperatively suppressed the skin lesions caused by co-infection of P. acnes and S. aureus. On the other hand, interest in the immune functions of heat shock proteins
(HSPs), until recently, has been investigated by the observations that these molecules can be released and are present in the extracellular environment under stressed conditions. They can elicit cytokine production by adhesion to antigen presenting cells (APCs), or they act as carrier molecules for immunogenic peptides that are presented on APCs through receptor-mediated interactions. Furthermore, HSP-derived peptides have been also regarded as immune effectors. In previous study, I found that hsp90α exhibited higher translation efficiency than hsp90β. I present evidences identifying the roles of leaky scanning and 5’-untranslated region (5’-UTR) sequence acts ad negative regulators in hsp90β mRNA. In addition, it was found that a small peptide (17 amino acid peptide) is translated from hsp90β mRNA owing to leaky scanning. According to the observations of HSP-derived peptides, the small peptide produced from leaky scanning may belong to one of antimicrobial peptide, which not only provides a protection but also helps to kill invaded bacteria. Taken together, a vaccine targeting to secretory β-hemolysin of S. aureus may create more effective protection for S. aureus infection. On the other hand, the role of HSP can be regarded as microbicidal molecules under pathogen infection.

關鍵字

金黃色葡萄球菌 ； 痤瘡桿菌 ； 溶血作用 ； 疫苗 ； 熱休克蛋白90 ； 轉譯調控

並列摘要

金黃色葡萄球菌﹙Staphylococcus aureus, S. aureus﹚屬於革蘭氏陽性菌，藉由入侵組織並釋放毒素而致病。過去，普遍以抗生素作為治療金黃色葡萄球菌感染之方法。然而，非專一性之抗生素卻導致大多數菌種被消滅而破壞人體體表菌落群的平衡與抗藥性菌種的產生。抗藥性金黃色葡萄球菌﹙methicillin-resistant S. aureus, MRSA﹚是日益普遍的院內感染導因，也造成全球的發病率與致死率。開發疫苗是近幾年間被研究以治療金黃色葡萄球菌之替代策略，然而，目前仍尚未成功應用於人體。痤瘡桿菌﹙Propionibacterium acnes, P. acnes﹚亦屬於革蘭氏陽性菌，但為厭氧菌，廣泛分佈於人體皮膚體表，卻不會寄生在老鼠身上。本研究中發現，痤瘡桿菌所釋放的毒素﹙CAMP factor﹚和金黃色葡萄球菌產生之溶血毒素﹙β-hemolysin﹚作用後，會強化溶血與細胞溶解之毒性，因此，推測金黃色葡萄球菌會利用痤瘡桿菌之CAMP factor 2而促進其毒性。此外，在動物實驗中﹙in vivo﹚亦發現痤瘡桿菌會加劇金黃色葡萄球菌所造成之皮膚傷口。若中和CAMP factor 2毒素並配合加以β-hemolysin抗原之免疫，可有效抑制而降低金黃色葡萄球菌與痤瘡桿菌共同感染下所造成之傷口。 另一方面，熱休克蛋白﹙heat shock proteins, HSPs﹚於免疫方面之功能為最近被研究的方向，此乃因為當受到壓力的刺激下，熱休克蛋白被發現會因細胞破裂被釋放而存在於細胞外的環境。熱休克蛋白本身可接觸抗原呈現細胞﹙antigen-presenting cell, APC﹚而激起細胞激素﹙cytokine﹚之產生，或者扮演保
護者護送引起抗原反應之胜肽﹙immunogenic peptides﹚，藉由受器傳達之作用﹙receptor-mediated interactions﹚，傳達至抗原呈現細胞。另外，熱休克蛋白衍生胜肽﹙HSP-derived peptide﹚已被視為免疫反應受動器﹙immune effector﹚。之前研究，我發現hsp90α之表達效率高於hsp90β。而本實驗中證實，leaky scanning和5’-UTR序列對於hsp90β之表達效率，扮演了負調控之作用。並且，一個短胜肽會因leaky scanning而從hsp90β被表達。根據熱休克蛋白衍生胜肽之研究，此短胜肽對於病菌感染，或許為一種抗微生物肽﹙antimicrobial peptide﹚，可抵禦並幫助毒殺侵入之病菌。 總結而言，針對金黃色葡萄球菌產生之溶血毒素﹙β-hemolysin﹚所設計之疫苗，可更有效預防金黃色葡萄球菌之感染。另外，面對外來病菌感染時，熱休克蛋白或可視為抑制病菌之分子。

並列關鍵字

Staphylococcus Aureus ； Propionibacterium acnes ； hemolysis ； vaccine ； heat shock protein 90 ； translation regulation

參考文獻

Ahmed R, Duncan RF. 2004. Translational regulation of Hsp90 mRNA. AUG-proximal 5'-untranslated region elements essential for preferential heat shock translation. J Biol Chem 279(48):49919-49930.

Amagai M, Matsuyoshi N, Wang ZH, Andl C, Stanley JR. 2000. Toxin in bullous impetigo and staphylococcal scalded-skin syndrome targets desmoglein 1. Nat Med 6(11):1275-1277. Babior BM. 2004. NADPH oxidase. Curr Opin Immunol 16(1):42-47.

Bertram J, Palfner K, Hiddemann W, Kneba M. 1996. Increase of P-glycoprotein-mediated drug resistance by hsp 90 beta. Anticancer Drugs 7(8):838-845.

Binder RJ, Vatner R, Srivastava P. 2004. The heat-shock protein receptors: some answers and more questions. Tissue Antigens 64(4):442-451.

Bowdish DM, Davidson DJ, Hancock RE. 2005. A re-evaluation of the role of host defence peptides in mammalian immunity. Curr Protein Pept Sci 6(1):35-51.