本文關(guān)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(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：銅綠假單胞菌是一種常見的臨床機(jī)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(chǎn)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(jī)制,其中外膜的低滲透性是其產(chǎn)生高耐藥性的主要因素之一。外膜外側(cè)的主要結(jié)構(gòu)為脂多糖(LPS),該物質(zhì)由類脂A、核心寡糖和O抗原構(gòu)成,其中類脂A不僅是內(nèi)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(guān)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(shí)驗(yàn)室前期發(fā)現(xiàn)PA0011基因的缺失可顯著影響PAO1的抗生素抗性與毒性,據(jù)PAO1官方網(wǎng)站推測,該基因?yàn)轭愔珹酰基轉(zhuǎn)移酶HtrB同源基因。將HtrB與PAO1基因組進(jìn)行比對發(fā)現(xiàn)另一個基因PA3242。該基因與HtrB的具有高達(dá)47%的同源性,預(yù)測其與PA0011共同參與PAO1中類脂A結(jié)構(gòu)的二級酰基化修飾。本課題以PA3242為主要研究對象,深入探究了該基因?qū)AO1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(jìn)行了比較,對PAO1中酰基轉(zhuǎn)移酶的作用機(jī)理有了更為深刻的認(rèn)識。PA3242對PAO1的生長影響極大,該基因的缺失導(dǎo)致突變體PAO1(△PA3242)在37℃的生長嚴(yán)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(zhuǎn)錄水平隨溫度降低而升高,其在28℃下的轉(zhuǎn)錄水平是37℃條件下的5倍。雖然PA0011在低溫下的表達(dá)也有所升高,但表達(dá)水平遠(yuǎn)遠(yuǎn)低于PA3242。以上結(jié)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(zhuǎn)酶基因PA4997的過表達(dá)可恢復(fù)突變體PAO1(△PA3242)的生長,表明PA3242很可能參與到類脂A的轉(zhuǎn)運(yùn)。PA3242的缺失會嚴(yán)重破壞外膜的完整性與穩(wěn)定性并進(jìn)一步影響PAO1的抗生素抗性。由實(shí)驗(yàn)可知:雖然PA0011與PA3242的缺失均導(dǎo)致PAO1抗生素敏感性降低,但與PA0011相比,PA3242的缺失導(dǎo)致PAO1對更多種類的抗生素表現(xiàn)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(xì)胞毒性和致病性都顯著減弱。實(shí)驗(yàn)表明:PA3242的缺失導(dǎo)致PAO1外膜畸變并丟失所有菌毛結(jié)構(gòu),而突變體PAO1(△PA0011)仍保有部分菌毛并具有規(guī)則的外膜結(jié)構(gòu)。進(jìn)一步研究發(fā)現(xiàn),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(jié)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(zhuǎn)運(yùn)過程,并進(jìn)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(dǎo)作用。以該基因?yàn)橹委煱悬c(diǎn)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