臺灣博碩士論文加值系統

肺炎鏈球菌是全球單一致死率最高的病原菌之一，其感染能造成廣泛的疾病，包含了鼻竇炎、中耳炎、肺炎、菌血症以及腦膜炎等。在臨床上即使抗生素成功的清除病原菌本身，肺炎鏈球菌感染所造成的死亡與其所引起免疫反應的失衡有很大的關係。所有已被鑑定的肺炎鏈球菌株中，都能夠表現唾液酸酶(NanA)在細菌的表面，我們先前的結果顯示肺炎鏈球菌NanA能夠藉由移除表現在相同細胞上Siglec受體的順配體進而加劇白血球的發炎反應。免疫系統的細胞表面填充了各式各樣的凝集素與聚醣，而Siglec受體的辨認位置通常會被同個細胞表面其他唾液酸聚醣的配體覆蓋；而先天免疫系統上唾液酸廣泛且大量的表現以及其相對應具有免疫抑制模組受體的重要性，因此唾液酸通常被認為是self-associated molecular patterns來調控免疫細胞啟動的閾值。類鐸受體為模式識別受體(pattern recognition receptors)的一員，被廣泛地認為是對抗病原菌的第一道防線，當感染時，免疫細胞表面的模式識別受體感應外在的刺激，並藉由細胞內的訊息傳導來整合並引發適當的免疫反應來對抗外在的感染源，在本篇研究中我們將探討肺炎鏈球菌NanA誘導之唾液酸化對於Siglec與模式識別受體之間交互作用的影響。我們假設在肺炎鏈球菌感染情況下，NanA誘導的去唾液酸化會破壞類鐸受體以及Siglec之間的交互作用，使得類鐸受體下游的訊息傳遞失衡而產生大量的發炎物質。我們的結果顯示野生型的肺炎鏈球菌相比NanA缺失的肺炎鏈球菌更能引起腫瘤壞死因子-α的表現，而腫瘤壞死因子-α表現的增加仰賴部分細胞表面脂筏的構造以及一些參與在MAPK、PI3K/AKT以及NF-кB訊息路徑中分子的活化；這樣細胞活化過程，可能來自於類鐸受體-2與Siglec-5交互作用以及Siglec-5相關調控分子受到影響的結果。除了探討肺炎鏈球菌NanA誘導的發炎反應中，抑制性Siglec所扮演的角色外，我們也發現當人類巨噬細胞以及單核球細胞表現促進性Siglec在表面時，也會影響肺炎鏈球菌NanA誘導發炎反應的結果。

Streptococcus pneumoniae (SPN) is the single most deadly bacterial infection globally, capable of causing a wide spectrum of disease, including sinusitis, otitis media, pneumonia, bacteremia and meningitis. The mortality caused by pneumococcal disease is sometimes linked to imbalanced hyper-inflammatory responses of the host despite of successful pathogen clearance. All SPN strains identified so far produce a surface-associated sialidase (NanA) and we recently demonstrated that leukocyte inflammatory responses are aggravated by pneumococcal NanA through removing the cis-lignads of Siglec expressed on the same myeloid cells. Cell surface in the immune system are richly equipped with a complex mixture of various lectins and glycans. Siglec binding sites are typically ‘masked’ by cis interactions with other sialoglycan ligands expressed on the same cell. Given the ubiquitous and abundant expression of host Sias and the prominence of cognate ITIM-bearing receptors on innate immune cells, Sia is proposed to act as “self-associated molecular patterns” to control the activation threshold of immune cells. Toll-like receptor (TLR) family, a member of pattern recognition receptors (PRRs), are generally thought to be a first-line defense to recognize pathogens. Upon infection, immune cells sense the environment through their various PRRs, processing and integrating this external information through the intracellular signaling cascade and induce appropriate immune responses to combat infectious microbial agents. Thus, this study we aimed to further investigate how pneumococcal NanA-mediated cell desialylation affects the crosstalk between Siglec and PRR signaling pathway. We hypothesized NanA-mediated host cell desialylation may disrupt the interaction between TLR and Siglec, dysregulate the inflammatory signals downstream of TLR and cause excessive pro-inflammatory substance upon pneumococcal infection. Our data suggested that WT SPN was able to induce stronger TNF-α production that partially rely on lipid rafts structure on cell membrane and activation of signaling molecules involved in MAPK, PI3k/Akt and NF-кB pathways compared to ∆nanA SPN. This pneumococcal NanA-mediated cell activation may result from affecting TLR-2-Siglec-5 interaction and Sigelc-5-associated signaling molecules. In addition to dissect the role of inhibitory Siglec in NanA-mediated pneumococcal inflammation, we also found that expression of an activating Siglec, Siglec-14, on human primary macrophages and THP-1 cells also affects host immune responses upon cell surface desialylation by pneumococcal NanA.

口試委員審定書 i
致謝 ii
中文摘要 iii
Abstract iv
Content v
I. Introduction 1
A. Streptococcal neuraminidase 1
B. Sialic acid (Sia)-binding immunoglobulin-like lectins (Siglecs) 2
C. Siglec-5 and Siglec-14 paired receptors and their roles in immunity 3
D. Cell surface receptors synergize to cellular responses 4
E. Hypothesis 4
II. Materials and methods 6
1. Materials 6
A. Cell lines and culture conditions 6
B. Bacterial strains and culture conditions 6
C. Antibiotics 6
D. Antibodies 6
E. Antibodies for micro-western array 8
F. ELISA (enzyme-linked immunosorbent assay) kit 10
G. Real-time quantitative polymerase chain reaction (qRT-PCR) primers 10
2. Methods 10
A. Analysis of TNF-α mRNA expression in THP-1 cells upon SPN infection 10
B. Analysis of TNF-α protein secretion in THP-1 cells upon SPN infection 10
C. Measurement of THP-1 surface desialylation upon SPN infection 11
D. Sample preparation and validation for Micro-western array 11
E. Cholesterol depletion – methyl-β-cyclodextrin treatment 13
F. Lipid raft isolation – sucrose gradient ultra-centrifugation 13
G. Immunoprecipitiation and immunoblotting 13
H. Examination of direct interaction between pneumococcus and recombinant Siglec-5 or Siglec-14 14
I. Statistics 14
III. Results 15
IV. Discussion 22
V. Figures 27
VI. Appendix 44
VII. References 47

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