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研究生： 王靖淳
Wang, Ching-Chun
論文名稱： 藉由阻斷溶血素 Ahh1 介導的細胞損傷防止達卡產氣單胞菌感染造成有害後果：活體內外之證據
Blockage of hemolysin Ahh1 mediated cell injury prevents detrimental consequences of Aeromonas dhakensis infection: in vitro and in vivo evidence
指導教授： 陳柏齡
Chen, Po-Lin
學位類別： 碩士
Master
系所名稱： 醫學院 - 微生物及免疫學研究所
Department of Microbiology & Immunology
論文出版年： 2021
畢業學年度： 109
語文別： 英文
論文頁數： 42
中文關鍵詞： 達卡產氣單胞菌 、溶血素 、NLRP3發炎小體 、白介素-1β 、壞死性筋膜炎
外文關鍵詞： Aeromonas dhakensis, Hemolysin, NLRP3 inflammasome, IL-1β, Necrotizing fasciitis
相關次數： 點閱：177  下載：0 
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    • 研究背景
    　　達卡產氣單胞菌是台灣南部重要的人類致病菌，可以引起多種感染，例如壞死性筋膜炎，一種嚴重的軟組織感染。 達卡產氣單胞菌攜帶著許多毒力基因，例如由ahh1和aeroA譯成的穿孔毒素蛋白（PFTs）；alt和ast譯成的腸毒素；以及ascV、aexT、ascF和ascG譯成的 T3SS 相關蛋白。 然而，達卡產氣單胞菌的發病機制仍然未被完全了解。 發炎小體是一種先天免疫反應的調控系統，可藉由穿孔毒素蛋白引起的細胞膜破壞所誘導活化，促進成熟的促炎細胞因子白介素1β（IL-1β）和白介素18（IL-18）的分泌，並且引導細胞焦亡以防禦細菌的感染。 在先前的研究中，我們發現達卡產氣單胞菌的一種穿孔毒素蛋白，溶血素Ahh1，對秀麗隱桿線蟲具有毒性。 在本次研究中，我們進一步在活體外和活體內探討了溶血素Ahh1的致病作用。
    方法與結果
    　　在人類單核球分化而成的巨噬細胞THP-1、秀麗隱桿線蟲和小鼠的感染模型中，我們發現了ahh1基因缺失突變（Δahh1）造成了達卡產氣單胞菌的毒力減弱，而ahh1基因回補（pahh1）可以使毒性得到恢復。此外，ΔaeroA突變株的毒力沒有顯著下降，暗示著ahh1在毒力方面比aeroA扮演著更重要的角色。由達卡產氣單胞菌感染造成的細胞損傷在剔除了nlrp3的THP-1細胞顯著的減少，這個現象同時與caspase-1、GSDMD-N和IL-1β的表現量減少結果一致。綜合上述，這些證據表明了ahh1在達卡產氣單胞菌的感染中扮演著誘導NLRP3發炎小體活化的角色。此外，我們證實了THP-1細胞可以藉由抗溶血素Ahh1的抗體治療，來減弱溶血素Ahh1重組蛋白以劑量依賴性方式引起的損傷。在小鼠感染模型中，抗生素與抗溶血素Ahh1抗體或caspase-1抑製劑的組合治療顯著延長了小鼠的存活時間，並減輕了達卡產氣單胞菌感染造成的壞死性筋膜炎的嚴重程度。概括上述結果，我們在THP-1以及小鼠感染模型中呈現了藉由阻斷溶血素Ahh1或抑制溶血素Ahh1引起的的發炎小體活化可以減輕由達卡產氣單胞菌感染引起的傷害，進而防止產生災難性結果。

    Background
    　　Aeromonas dhakensis is an important human pathogen in southern Taiwan and causes various infections in humans, such as necrotizing fasciitis, a severe soft-tissue infection. Many virulence genes have been discovered in this species, such as pore-forming toxins (PFTs) encoded by ahh1 and aeroA; enterotoxins encoded by alt and ast; T3SS-related proteins encoded by ascV, aexT, ascF and ascG. However, the pathogenesis of A. dhakensis remains unclear. The inflammasome is an innate immune response that can be induced by pore-forming toxin-caused membrane disruption, promotes the secretion of matured pro-inflammatory cytokines, interleukin 1β (IL-1β) and interleukin 18 (IL-18), as well as causes pyroptosis for defending the bacterial infection. In our previous studies, the hemolysin Ahh1, a pore-forming toxin in A. dhakensis, is toxic to Caenorhabditis elegans. In this study, we further investigated the pathogenic role of hemolysin Ahh1 in vitro and in vivo.
    Methods and Results
    　　The virulence of A. dhakensis ahh1 mutant (Δahh1) was attenuated and the toxicity of Δahh1 was restored by pAhh1 complementation in models of human monocyte-derived macrophage THP-1, C. elegans and mice infection model. In addition, the virulence of ΔaeroA mutant was not decreased, indicating ahh1 plays a more important role than aeroA in terms of virulence. Cell damage mediated by A. dhakensis was reduced in THP-1nlrp3-/-, in consistent with reduced expression of caspase-1, GSDMD-N, and IL-1β. Taken together, this evidence suggested that ahh1 induces NLRP3 inflammasome activation. In addition, Ahh1 caused THP-1 cell damage in a dose-dependent manner and the cytotoxicity was blunted by the anti-Ahh1 antibody. In the mice infection model, combination of antibiotic and anti-Ahh1 antibody or caspase-1 inhibitor treatment significantly prolonged mice survival and attenuated the necrotizing fasciitis severity after infection of A. dhakensis. In conclusion, blockage of hemolysin Ahh1 or inhibiting Ahh1-mediated inflammasome activation prevent catastrophic results caused by A. dhakensis infection.

    摘要 I 研究背景 I 方法與結果 I Abstract III Background III Methods and Results III 誌謝 V Contents VII Introduction 1 Materials and methods 4 Bacterial strains 4 Agar-based survival assay of C. elegans 4 Cell lines 5 Cytotoxicity assay 5 Western blotting 5 Mice survival and necrotizing fasciitis severity evaluation 7 Results 8 ahh1 of A. dhakensis is a virulence factor to C. elegans 8 Hemolysin Ahh1 is secreted by A. dhakensis and is cytotoxic to mammalian cell line 8 Hemolysin Ahh1 activates the NLRP3 inflammasome in A. dhakensis-infected macrophages 9 ahh1 is related to A. dhakensis-induced necrotizing fasciitis in mice model 10 Blockage of hemolysin Ahh1 or Ahh1-induced immune response attenuates the damage caused by A. dhakensis infection 11 Conclusion 13 Discussion 14 References 17 Figures 20 Figure 1. ahh1 in Aeromonas dhakensis is an important virulence factor to Caenorhabditis elegans. 21 Figure 2. ahh1 in Aeromonas dhakensis is virulent to mammalian cell lines. 22 Figure 3. Hemolysin Ahh1 is secreted by Aeromonas dhakensis and is cytotoxic to macrophages. 23 Figure 4. ahh1 induces cell damage mediated by inflammasome activation during Aeromonas dhakensis infection. 25 Figure 5. Hemolysin Ahh1 induces inflammasome activation in the THP-1. 26 Figure 6. Inflammasome activation and subsequent cell damage are impaired in THP-1nlrp3-/- cells during Aeromonas dhakensis infection. 28 Figure 7. ahh1 has a significant impact on the survival of mice infected with Aeromonas dhakensis. 29 Figure 8. ahh1 plays a pivotal role in Aeromonas dhakensis-necrotizing fasciitis in mice intramuscular infection model. 31 Figure 9. The anti-Ahh1 antibody attenuates the hemolysin Ahh1 mediated cell damage in Aeromonas dhakensis AAK1 infection cell model. 33 Figure 10. The anti-Ahh1 antibody treatment reduces activation of the NLRP3 inflammasome in the Aeromonas dhakensis AAK1 infection cell model. 35 Figure 11. The anti-Ahh1 antibody shows an additive effect in combination with cefepime. 37 Figure 12. Combination of anti-Ahh1 antibody and cefepime treatment attenuates the severity of necrotizing fasciitis in mice. 39 Figure 13. Combination of caspase-1 inhibitor and cefepime treatment improves the severity of necrotizing fasciitis in mice. 41

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