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    On the role of Yersinia LcrV alleles in type III secretion and protective immunity.


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    Type III secretion is a mechanism used by many bacterial species to evade and subvert the host immune system, allowing the bacteria to survive and replicate. This system is utilized by three species of Yersinia that are pathogenic to humans, Yersinia enterocolitica, Yersinia pseudotuberculosis and Yersinia pestis. While the first two species generally cause gastrointestinal disease and are rarely lethal, Y. pestis is the causative agent of plague and as such has a high incidence of mortality associated with it. Additionally, due to the possibility of airborne transmission of the pneumonic form of the disease, there has been an increasing concern over illegitimate use of the bacteria as an agent of biological warfare. Efforts at understanding the pathogenesis of each species, as well as attempts at engineering successful vaccines against Y. pestis, have focused on the type III secretion system utilized by the bacteria to elude the host immune system, as loss of functionality of the type III secretion system renders the bacteria avirulent.;In each species, the genes necessary for production and activation of the type III secretion system are harbored on an approximately 70 kb virulence plasmid. After perception of several host signals, the bacteria fully assemble the type III secretion apparatus, which bears some resemblance to the flagellar system. This system essentially acts as a needle through which effector proteins are translocated from the cytoplasm of the bacteria to the cytosol of host cells, where they perform a variety of functions that ultimately result in escape from host immune responses. In chapter II, the transport and visualization of this process is examined by adapting a fluorescent technology to type III secretion in Y. enterocolitica. The ability of the bacteria to inject type III substrates containing a 12 amino acid motif that binds to a fluorophore was interrogated, as well as the ability of the bacteria to inject primary immune cells. Chapter III explores the effect of fusion of impassable reporter proteins to type III substrates, including how such hybrids affect both secretion and injection of other type III substrates. This study revealed that while such hybrids appear to block transport of other type III substrates, in actuality they are initiated into the type III pathway and are subsequently rejected. However, this initiation prevents the secretion of negative regulatory proteins, which accounts for the apparent blockade. Mutations in the negative regulators indeed relieve the perceived "blockade.";The contribution of heterologous genes to protective immunity is investigated in chapter IV. Current vaccine efforts focus largely on LcrV, a protein that resides at the tip of the type III needle and is absolutely essential for type III injection and virulence. Earlier work reported that antibodies against Y. pestis LcrV cannot block type III injection by other Yersinia species and suggested that polymorphisms in lcrV may provide for escape from LcrV-mediated plague immunity. This chapter demonstrates that while antibodies directed against Y. pestis LcrV are unable to prevent infection by Y. enterocolitica, expression of Y. enterocolitica lcrV in Y. pestis does not in fact provide for escape from LcrV-mediated protective immunity. A potential explanation for the protective effect afforded by LcrV-specific antibodies is brought to light in chapter V. These studies reveal that purified LcrV is able to interact with YopD secreted by Y. enterocolitica, and that particular regions of LcrV are required for such an interaction to occur. Additionally, it was shown that antibodies directed against LcrV are able to perturb the interaction with YopD, an interaction that is thought to be essential for type III injection to occur, providing a possible mechanism by which LcrV-specific antibodies generate protective immunity.
    機譯:III型分泌是許多細菌物種逃避和破壞宿主免疫系統所使用的機制,使細菌得以存活和復制。該系統被對人類有致病性的三種耶爾森菌,小腸結腸炎耶爾森氏菌,假結核耶爾森氏菌和鼠疫耶爾森氏菌利用。前兩個物種通常引起胃腸道疾病,很少致死,而鼠疫耶爾森氏菌是鼠疫的病原體,因此具有很高的死亡率。另外,由于該疾病的肺炎形式可能通過空氣傳播,因此人們越來越擔心將細菌非法用作生物戰劑。努力了解每種物種的發病機理,以及嘗試設計成功的抗鼠疫耶爾森氏菌疫苗的嘗試,都集中在細菌利用III型分泌系統逃避宿主免疫系統,因為III型分泌功能喪失在每個物種中,生產和激活III型分泌系統所需的基因都藏在大約70 kb的毒性質粒上。在感知到幾個宿主信號后,細菌完全組裝了III型分泌設備,這與鞭毛系統有些相似。該系統本質上起著針的作用,效應蛋白通過該針從細菌的細胞質轉移到宿主細胞的細胞質中,在那里它們執行多種功能,最終導致逃脫宿主免疫反應。在第二章中,通過使熒光技術適應小腸結腸炎耶爾森氏菌的III型分泌,研究了該過程的運輸和可視化。詢問了細菌注射含有結合至熒光團的12個氨基酸基序的III型底物的能力,以及細菌注射原代免疫細胞的能力。第三章探討了不可逾越的報告蛋白與III型底物融合的影響,包括這種雜種如何影響其他III型底物的分泌和注射。這項研究表明,盡管此類雜種似乎阻止了其他III型底物的運輸,但實際上它們開始進入III型途徑,隨后被拒絕。但是,這種啟動阻止了負調節蛋白的分泌,從而造成了明顯的封鎖。負調控因子的突變確實緩解了人們所感知的“封鎖”。第四章研究了異源基因對保護性免疫的作用。當前的疫苗工作主要集中在LcrV,LcrV是一種駐留在III型針尖的蛋白質,對于III型注射和毒力絕對必不可少。較早的工作報道抗鼠疫耶爾森氏菌LcrV的抗體不能阻斷其他耶爾森氏菌屬物種的III型注射,并建議lcrV中的多態性可避免LcrV介導的鼠疫免疫。本章表明,盡管針對鼠疫耶爾森氏菌LcrV的抗體無法預防腸球菌耶爾森氏菌的感染,但在鼠疫耶爾森氏菌中腸球菌耶爾森氏菌lcrV的表達實際上并不能逃避LcrV介導的保護性免疫。第五章揭示了LcrV特異性抗體提供的保護作用的潛在解釋。這些研究表明,純化的LcrV能夠與小腸結腸炎耶爾森氏菌分泌的YopD相互作用,而LcrV的特定區域是必需的。發生互動。另外,已表明針對LcrV的抗體能夠干擾與YopD的相互作用,該相互作用被認為對于III型注射的發生是必不可少的,從而提供了LcrV特異性抗體產生保護性免疫的可能機制。


    • 作者

      Miller, Nathan Charles.;

    • 作者單位

      The University of Chicago.;

    • 授予單位 The University of Chicago.;
    • 學科 Biology Microbiology.
    • 學位 Ph.D.
    • 年度 2010
    • 頁碼 153 p.
    • 總頁數 153
    • 原文格式 PDF
    • 正文語種 eng
    • 中圖分類 B9;
    • 關鍵詞

    • 入庫時間 2022-08-17 11:36:55


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