出版年:

2001

研究生:

羅月霞

研究生(英文姓名):

Yueh-Hsia Luo

論文名稱:

熱原性外毒素B在A群鏈球菌感染小鼠的模式中與組織病變、NO及cytokine產生的相關性

英文論文名稱:

Correlation of SPE B with histopathologic changes, NO, and cytokine production in a mouse model of Group A streptococcal infection

指導教授:

林以行

指導教授(英文姓名):

Yee-Shin Lin

學位類別:

碩士

校院名稱:

國立成功大學　

系所名稱:

微生物暨免疫學研究所

學號:

S46884032

學年度:

89

語文別:

中文

論文頁數:

87

關鍵詞:

鏈球菌致熱原性外毒素B ; 鏈球菌毒性休克症候群 ; 菌血症

英文關鍵詞:

SPE B ; streptococcal toxic shock syndrome ;
bacteremia ; IL-1 ; IL-6 ; TNF-a ; NO

被引用次數:

0

[ 摘要 ]

A群鏈球菌(group A streptococci)的感染會引起多種嚴重的疾病，臨床上所產生的嚴重併發症包括鏈球菌毒性休克症候群(streptococcal toxic shock syndrome) 及壞疽性肌膜炎(necrotizing fasciitis)。化膿性鏈球菌目前已知被認為與侵犯宿主有關的毒力因子包括鏈球菌致熱原性外毒素(streptococcal pyrogenic exotoxins; SPE) SPE A、SPE B、SPE C、SPE F與多種的M proteins等。所有的A群鏈球菌都具有speB基因，而且SPE B是A群鏈球菌分泌的蛋白分解脢種類中產量最多的，約佔95﹪。SPE B的活性與其Cys192有關所以被歸類為cysteine protease，其蛋白約為40 kDa為脢原(zymogen)型式，可能經由autocatalysis或其他的方式產生28 kDa具有蛋白分解脢活性的分子。根據我們實驗室先前以皮下氣囊的感染模式發現，SPE B在侵襲性A群鏈球菌的感染中是相當重要的毒力因子。本論文採用已建立之皮膚及軟組織的感染模式，探討在A群鏈球菌感染小鼠後SPE B對於小鼠器官的病理變化與細菌侵襲能力的影響，以及NO和cytokine的表現情形。小鼠感染野生型菌株後48、72 小時在小鼠氣囊回抽液與血清中可以測得明顯大量的SPE B的存在，皮下氣囊局部的菌量，與血液、器官的菌量可能與SPE B的產生有相關。而感染突變菌株的小鼠其氣囊回抽液中的菌量在12小時的菌量比感染野生型菌株的小鼠多，但隨著感染時間增加菌量有越來越少的現象。在感染野生型菌株後小鼠有菌血症的現象，並且可以在脾臟、腎臟和肝臟培養出細菌，而感染突變菌株的小鼠則沒有菌血症，也不能在臟器中可以培養出細菌。小鼠在感染野生型菌株24小時腎臟與肝臟有中度的病變而突變菌株則無此現象，感染野生型菌株48小時後小鼠的腎臟與肝臟病變的程度更加的嚴重，小鼠血清中AST、ALT與BUN與感染後肝臟、腎臟的受損有正相關的關係。小鼠在感染野生型菌株後12小時可測得血清中IL-1b、IL-6與TNF-a
有增高的現象，雖然突變菌株也會引起血清中IL-1b、IL-6與TNF-a增加，但是增加的量比野生型菌株所引起的低，血清中這些cytokines的量隨著時間逐漸減少。腎臟的腎小管在感染後12小時即表現TNF-a，相反的在感染突變菌株的小鼠中TNF-a的表現直到72小時才有少許出現。至於iNOS的表現，感染野生菌株的小鼠，在感染初期12、24 h腎臟有表現iNOS，但在48小時iNOS則沒有表現，在72小時又有少許表現，而感染突變菌株的小鼠一直到感染後期72小時才有iNOS的表現。而IL-1b與IL-6則是一直都沒有在腎臟表現。在初步的實驗發現感染野生菌株小鼠氣囊的回抽液中，48小時以後測到明顯大量的NO存在，而感染突變株SW510的小鼠檢體中皆沒有檢測到有NO存在，我們認為NO濃度增高的現象與感染後皮下氣囊抽出液中SPE B含量增高的時間相同，可能表示在A鏈球菌感染的過程中，NO可能與此菌引起的發炎與侵襲能力有關，但仍需更進一步的實驗證明。另外我們發現SPE B會改變小鼠皮下局部微血管的通透性並且造成大量的嗜中性白血球浸潤到局部組織，而不具蛋白脢活性的C192S沒有此現象。總括而言，A群鏈球菌產生的SPE B可以使細菌在局部組織繁殖而且造成組織嚴重的損傷，使細菌更進一步侵犯到深層組織，同時也造成血清中IL-1b、IL-6與TNF-a增高，進而導致小鼠產生菌血症與鏈球菌毒性休克的症狀。

[ 英文摘要 ]

The group A streptococci (GAS) Streptococcus pyogenes can cause serious diseases in humans, including streptococcal toxic shock syndrome and necrotizing fasciitis. S. pyogenes secretes various provenor putative virulence factors, e.g., streptococcal pyrogenic exotoxin (SPE) A, SPE B, SPE C, SPE F,and various M proteins, that enable it to invade the host. SPE B, its gene is present in all isolates of S. pyogenes, is the predominant extracellular protein, accounting for up to 95﹪of total secreted proteins. SPE B, which is a cysteine protease, is secreted as a 40-kDa inactive precursor and subsequently cleaved to a 28-kDa molecule. Previous studies in our laboratory by using a mouse modelvia air pouch inoculation showed that SPE B appeared to be a critical virulence factor in invasive GAS infection. In this study, we further explore the role of SPE B in bacterial growth, invasion, and dissemination, as well as NO and cytokine production. After bacterial inoculation, high levels o
f SPE B expression were observed in both air pouch exudate and serum at 48 and 72 h. Bacterial growth and dissemination to blood, liver, and kidney were positively correlated with the presence of SPEB. Bacterial numbers in the air pouch exudates from NZ131-infected mice increased at 48 h, while those infected with the speB mutant SW510 were high at 12 h but continuously dropped afterward. Micemay develop bacteremia when infected with NZ131, and bacterial dissemination in spleen, kidney and liver was observed. Bacterial dissemination could not be detected when mice were inoculated with SW510. The histopathological changes in various organs were examined showing mild lesions in kidney and liver in some of the NZ131-infected but not the SW510-infected mice at 24 h. The lesions became more severe in a high percentage of NZ131-infected mice after 48 h. The elevation of BUN, AST and ALT in sera showed a positive correlation with the renal and liver impairment. The production of IL-1b, IL-6, and TNF-a were elevated in sera 12 h after bacterial inoculation then continuously decreased. These pro-inflammatory cytokines in the sera of NZ131-infected mice were higher than those in SW510-infected mice. TNF-a expression was observed in kidney 12 h after inoculation with wild-type bacteria while only low level of TNF-a was observed 72 h after inoculation with speB mutant strain. The expression of iNOS was observed at 12 and 24 h after wild-type bacteria inoculation, vanished at48 h, and appeared again in a low level at 72 h. The iNOS expression was not observed until 72 h after mutant-type bacteria inoculation. The expression of IL-1b and IL-6 could not be observed in the kidney after bacterial inoculation. Our preliminary results showed that the levels of nitric oxide (NO) in the exudates were elevated after wild-type bacteria inoculation, implicating the involvement of NO during bacterial invasion. Interestingly, subcutaneous injection of recombinant SPE B but not C192S mutant protein caused damage to local endothelial cells, increased vessel permeability, and massive neutrophil influx to local tissue in BALB/c mice. Taken together, SPE B produced during S.pyogenes infection plays a role in bacterial growth and dissemination. SPE B also causes elevation of pro-inflammatory cytokines in the acute phase of bacterial inoculation. These may lead to bactere