本文選題：白念珠菌 + 鈣調(diào)神經(jīng)磷酸酶　； 參考：《第二軍醫(yī)大學(xué)》2013年博士論文

【摘要】：在白念珠菌（Candida albicans）中，由于越來越多的菌株對(duì)藥物產(chǎn)生了耐藥性，導(dǎo)致臨床上用于治療念珠菌感染的一線藥物氟康唑（Fluconazloe,FLC）的治療產(chǎn)生失敗。我們實(shí)驗(yàn)室前期的研究發(fā)現(xiàn)：在體外，RTA2基因編碼的蛋白參與了由白念珠菌鈣調(diào)神經(jīng)磷酸酶（calcineurin,CaN）信號(hào)通路介導(dǎo)的對(duì)氟康唑的耐藥。在本研究中，通過藥物敏感性實(shí)驗(yàn)考察不同基因背景的RTA2基因相關(guān)菌株對(duì)氟康唑的敏感性差異，發(fā)現(xiàn)：在野生型菌株（cnaΔ/Δ::CNA，crz1Δ/Δ::CRZ1，rta2Δ/Δ::RTA2）中加入1mM CaCl2可以激活CaN信號(hào)通路，使菌株對(duì)唑類藥物氟康唑的敏感性顯著降低（最低抑菌濃度由0.5μg/ml升高為16μg/ml和64μg/ml）；加入1mM CaCl2激活CaN信號(hào)通路，RTA2基因缺失菌（rta2Δ/Δ,cnaΔ/Δ::CNA；rta2Δ/Δ, crz1Δ/Δ::CRZ1）和鈣調(diào)神經(jīng)磷酸酶CNA基因缺失菌（cnaΔ/Δ,rta2Δ/Δ::RTA2）以及CaN信號(hào)通路轉(zhuǎn)錄因子CRZ1基因缺失菌（crz1Δ/Δ, rta2Δ/Δ::RTA2）一樣，對(duì)氟康唑的敏感性無顯著變化。說明在體外鈣離子激活的CaN信號(hào)通路通過RTA2p降低白念珠菌對(duì)氟康唑的敏感性。通過透射電鏡實(shí)驗(yàn)考察不同濃度的氟康唑?qū)Σ煌虮尘暗腞TA2基因相關(guān)菌株細(xì)胞膜超微結(jié)構(gòu)的影響，我們發(fā)現(xiàn)：在體外，，鈣離子激活鈣調(diào)神經(jīng)磷酸酶通路后，可以通過Rta2p來減弱氟康唑?qū)?xì)胞膜的損傷，從而顯著的降低了白念珠菌對(duì)氟康唑的敏感性。此外，我們構(gòu)建了小鼠深部真菌感染模型，考察基因RTA2對(duì)模型動(dòng)物生存時(shí)間及腎臟荷菌量的影響，發(fā)現(xiàn)：RTA2基因本身并不能影響白念珠菌的毒力；但RTA2基因缺失后，感染小鼠接受氟康唑治療后，生存率顯著提高；相反，異位表達(dá)RTA2基因顯著的降低了氟康唑在宿主中對(duì)白念珠菌的療效。綜上所述，我們認(rèn)為在體內(nèi)體外鈣離子激活的鈣調(diào)神經(jīng)磷酸酶通路，通過Rta2p來降低白念珠菌對(duì)氟康唑的敏感性。 在白念珠菌中，基因RTA2是鈣調(diào)神經(jīng)磷酸酶通路調(diào)控的一個(gè)下游基因，但是其調(diào)控機(jī)制并不明確。本課題采用海腎熒光素酶報(bào)告基因（Renilla Luciferase，RLUC）系統(tǒng)進(jìn)行研究：將不同長度的RTA2基因啟動(dòng)子片段連接到報(bào)告基因載體中，再將構(gòu)建好的載體分別轉(zhuǎn)入菌株DJY201（Δ/Δcna,Δ/Δrta2）、MJY201（Δ/Δcrz1,Δ/Δrta2）、JXM101（Δ/Δrta2）中，考察加入鈣離子前后熒光活性的變化倍數(shù)。進(jìn)一步證明RTA2基因是CaN信號(hào)通路的靶基因，其表達(dá)水平受到轉(zhuǎn)錄因子Crzlp的調(diào)控；發(fā)現(xiàn)Crzlp的DNA結(jié)合序列—鈣調(diào)神經(jīng)磷酸酶依賴性應(yīng)答元件（calcineruin-dependent responseelement,CDRE），可能位于RTA2基因啟動(dòng)子上游-973bp~-920bp的區(qū)間內(nèi)，通過生物信息學(xué)分析推測其序列可能為GATGT。 我們實(shí)驗(yàn)室前期研究發(fā)現(xiàn)：在臨床白念珠菌中，外源性加入1mMCaCl2激活CaN信號(hào)通路，可以不同程度的上調(diào)RTA2基因的表達(dá)水平。通過小鼠深部真菌感染模型發(fā)現(xiàn)，鈣離子誘導(dǎo)的RTA2基因高表達(dá)的菌株感染的小鼠接受氟康唑治療后，生存率顯著低于鈣離子誘導(dǎo)的RTA2基因非高表達(dá)的菌株感染的小鼠。本研究通過體外藥物敏感性實(shí)驗(yàn)和實(shí)時(shí)定量（Real-Time） PCR實(shí)驗(yàn)發(fā)現(xiàn)：鈣離子誘導(dǎo)的菌株對(duì)氟康唑敏感性降低和RTA2基因的表達(dá)水平密切相關(guān)。在鈣離子誘導(dǎo)的RTA2基因高表達(dá)的臨床菌株0511522中，敲除了RTA2基因。通過體外藥物敏感性實(shí)驗(yàn)發(fā)現(xiàn)， RTA2基因缺失菌RTA2N4（△/△rta2）無法產(chǎn)生鈣離子介導(dǎo)的對(duì)氟康唑的敏感性降低；通過小鼠深部真菌感染模型發(fā)現(xiàn)，敲除RTA2基因并不影響菌株的毒力；但是RTA2基因缺失后，感染動(dòng)物接受氟康唑治療以后，生存率顯著升高。綜上所述，在體內(nèi)體外鈣離子通過激活鈣調(diào)磷酸酶通路誘導(dǎo)RTA2基因高表達(dá)從而降低臨床菌株對(duì)氟康唑的敏感性。
[Abstract]:In Candida albicans, as more and more strains are resistant to drugs, the treatment of Fluconazloe (FLC), a first-line drug used to treat Candida infection, has failed. Our early laboratory study found that the protein encoded by the RTA2 gene was involved in calcium Candida albicans in vitro. Calcineurin (CaN) signaling pathway mediated resistance to fluconazole. In this study, the sensitivity of RTA2 related strains with different gene backgrounds was investigated by drug sensitivity experiments. It was found that 1mM CaCl2 could be added to the wild type strains (CNA Delta / Delta: CNA, Crz1 Delta / Delta: CRZ1, rta2 Delta / Delta: RTA2). Activation of CaN signaling pathway significantly reduced the sensitivity of the strain to fluconazole (the minimum inhibitory concentration increased from 0.5 mu g/ml to 16 mu g/ml and 64 mu g/ml), and 1mM CaCl2 activated CaN signaling pathway, RTA2 gene deletion bacteria (rta2 Delta / Delta, CNA Delta / Delta: CNA; rta2 Delta / Delta, Delta / delta) and calcineurin gene deletion bacteria Delta / Delta, rta2 Delta / Delta: RTA2), as well as the CaN signaling pathway transcription factor CRZ1 gene deletion bacteria (Crz1 Delta / Delta, rta2 Delta / Delta: RTA2), the sensitivity to fluconazole was not significantly changed. It indicated that the CaN signaling pathway activated by calcium ions in vitro reduced the sensitivity of Candida albicans to fluconazole through RTA2p. The fluorine of different concentrations was investigated by transmission electron microscopy. The effect of conazole on the ultrastructure of the cell membrane of RTA2 gene related strains with different gene background was found. We found that in vitro, after calcium activation of calcineurin pathway, calcium ion can reduce the damage of fluconazole to the cell membrane through Rta2p, thus significantly reducing the sensitivity of Candida albicans to fluconazole. The effect of gene RTA2 on the survival time and kidney load of the model animal was investigated. It was found that the RTA2 gene itself did not affect the virulence of Candida albicans, but after the deletion of the RTA2 gene, the survival rate of the infected mice was significantly increased after the treatment of fluconazole; on the contrary, the ectopic expression of RTA2 gene significantly reduced the fluorine. The effect of conazole on Candida albicans in the host. In summary, we believe that calcium activated calcineurin pathway in vivo and in vivo reduces the sensitivity of Candida albicans to fluconazole by Rta2p.
In Candida albicans, the gene RTA2 is a downstream gene regulated by calcineurin pathway, but its regulatory mechanism is not clear. The subject uses the Renilla Luciferase (RLUC) system of the sea kidney luciferase reporter gene (RLUC) system to connect the RTA2 gene fragment of different lengths to the reporter gene carrier and then construct it. The good carriers were transferred to strain DJY201 (delta / delta cna, Delta / delta rta2), MJY201 (delta / delta Crz1, Delta / delta rta2), JXM101 (delta / delta rta2), and JXM101 (delta / delta rta2). The variation of fluorescence activity before and after the addition of calcium ions was investigated. The RTA2 gene was further proved to be the target gene of the CaN signaling pathway, and its expression was regulated by the Crzlp of the transcription factor. The calcineurin dependent response element (calcineruin-dependent responseelement, CDRE) may be located in the range of -973bp~-920bp in the upstream of the RTA2 gene promoter. It is presumed that the sequence of the RTA2 gene promoter may be GATGT..
Previous studies in our laboratory found that in clinical Candida albicans, exogenous addition of 1mMCaCl2 activates the CaN signaling pathway to increase the expression level of RTA2 genes in varying degrees. The survival rate of mice infected by the calcium induced RTA2 gene highly expressed strain infected mice after the treatment of fluconazole was found. Mice infected with the non high expression of RTA2 gene were significantly lower than calcium ion induced strain. This study found that the decrease of fluconazole induced by calcium ion induced strain was closely related to the expression level of RTA2 gene by drug sensitivity test in vitro and real-time quantitative (Real-Time) PCR test. The high expression of RTA2 gene induced by calcium ion was expressed in this study. In clinical strain 0511522, the RTA2 gene was knocked out. The drug sensitivity test in vitro showed that the RTA2 gene deletion strain RTA2N4 (delta / delta rta2) could not produce calcium ion mediated susceptibility to fluconazole; through the deep fungal infection model in mice, it was found that the knockout of the RTA2 gene did not affect the virulence of the strain; however, the RTA2 gene was missing after the deletion of the RTA2 gene. When the infected animals were treated with fluconazole, the survival rate was significantly increased. In summary, calcium ions in the body and in vivo induced the high expression of RTA2 gene by activating the calcineurin pathway and thus reducing the sensitivity of the clinical strain to fluconazole.

【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類號(hào)】：R519.3

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本文編號(hào)：1840526