本文關鍵詞： 銅綠假單胞菌 類脂A 生長 抗生素抗性 毒性　出處：《西北大學》2017年碩士論文　論文類型：學位論文

【摘要】：銅綠假單胞菌是一種常見的臨床機會致病菌,引起了全球約10%的臨床感染。感染該類病菌的患者通常免疫力低下,且致死率極高。另外在治療過程中,該類致病菌會迅速對抗生素類藥物產生抗性,針對該種病原菌感染的治療形勢不容樂觀。銅綠假單胞菌擁有多種抗性機制,其中外膜的低滲透性是其產生高耐藥性的主要因素之一。外膜外側的主要結構為脂多糖(LPS),該物質由類脂A、核心寡糖和O抗原構成,其中類脂A不僅是內毒素活性成分,同時還將脂多糖(LPS)緊密地錨定在外膜上,有效地維持了外膜的穩(wěn)定性與完整性。近年有關于類脂A的研究表明:該分子可影響多種病原菌的生長、抗生素抗性、毒性以及免疫原性等諸多生理特性。實驗室前期發(fā)現PA0011基因的缺失可顯著影響PAO1的抗生素抗性與毒性,據PAO1官方網站推測,該基因為類脂A�；D移酶HtrB同源基因。將HtrB與PAO1基因組進行比對發(fā)現另一個基因PA3242。該基因與HtrB的具有高達47%的同源性,預測其與PA0011共同參與PAO1中類脂A結構的二級�；揎�。本課題以PA3242為主要研究對象,深入探究了該基因對PAO1生長、抗生素抗性和毒性等諸多生理特性的影響,同時與PA0011進行了比較,對PAO1中�；D移酶的作用機理有了更為深刻的認識。PA3242對PAO1的生長影響極大,該基因的缺失導致突變體PAO1(△PA3242)在37℃的生長嚴重受限,且無法在溫度低于28℃的條件下存活,但PA0011的缺失卻對PAO1的生長沒有任何影響。PA3242的轉錄水平隨溫度降低而升高,其在28℃下的轉錄水平是37℃條件下的5倍。雖然PA0011在低溫下的表達也有所升高,但表達水平遠遠低于PA3242。以上結果表明:PA3242對于菌體在低溫下的生存起到更為重要的作用。另外,PAO1中類脂A翻轉酶基因PA4997的過表達可恢復突變體PAO1(△PA3242)的生長,表明PA3242很可能參與到類脂A的轉運。PA3242的缺失會嚴重破壞外膜的完整性與穩(wěn)定性并進一步影響PAO1的抗生素抗性。由實驗可知:雖然PA0011與PA3242的缺失均導致PAO1抗生素敏感性降低,但與PA0011相比,PA3242的缺失導致PAO1對更多種類的抗生素表現出更高的敏感性,同時對金屬螯合劑EDTA的耐受力也大大降低。PA3242還可顯著影響PAO1的毒性。與PAO1和PAO1(△PA0011)相比,PAO1(△PA3242)的細胞毒性和致病性都顯著減弱。實驗表明:PA3242的缺失導致PAO1外膜畸變并丟失所有菌毛結構,而突變體PAO1(△PA0011)仍保有部分菌毛并具有規(guī)則的外膜結構。進一步研究發(fā)現,突變體PAO1(△PA3242)喪失了叢動、泳動和蹭行能力,而突變體PAO1(△PA0011)僅從動能力部分受限。由以上結論可知:與PA0011相比,PA3242不僅能夠顯著影響PAO1抗生素抗性和毒性,它還極有可能參與到類脂A轉運過程,并進一步影響PAO1的生長、菌體形態(tài)和菌毛裝配。表明該基因在二級�；揎椷^程中起主導作用。以該基因為治療靶點可為治療銅綠假單胞菌感染提供許多新的思路。
[Abstract]:Pseudomonas aeruginosa is a common clinical opportunistic pathogen that causes about 10% clinical infections worldwide. Patients infected with Pseudomonas aeruginosa usually have low immunity and high mortality. This kind of pathogenic bacteria can quickly develop resistance to antibiotics, and the treatment of the pathogen infection is not optimistic. Pseudomonas aeruginosa has many resistance mechanisms. The low permeability of outer membrane is one of the main factors to produce high drug resistance. The main structure of outer membrane is lipopolysaccharide (LPS), which is composed of lipopolysaccharide, core oligosaccharide and O antigen. Lipids A is not only the active component of endotoxin, but also the lipopolysaccharide (LPS) is closely anchored on the outer membrane. It can effectively maintain the stability and integrity of the outer membrane. Recent studies on lipids A have shown that this molecule can affect the growth of many pathogens and antibiotic resistance. Toxicity, immunogenicity and many other physiological characteristics. The lack of PA0011 gene can significantly affect the antibiotic resistance and toxicity of PAO1, according to the PAO1 official website. This gene is based on the HtrB homologous gene of lipoacyltransferase. Another gene PA3242has been found by comparing the genome of HtrB and PAO1. The gene has a 47% homology with HtrB. Source. It is predicted that PA3242 and PA0011 are involved in the secondary acylation modification of liposome A structure in PAO1. In this study, PA3242 was used as the main research object to explore the growth of PAO1 by the gene. The effects of antibiotic resistance and toxicity on many physiological characteristics were also compared with those of PA0011. The effect of PA3242 on the growth of PAO1 was greatly influenced by a deeper understanding of the mechanism of acyltransferase in PAO1. The deletion of the gene caused the mutant PAO1 (PA3242) to grow at 37 鈩，

本文編號：1474552