O1群E1 Tor型霍乱弧菌分型噬菌体919TP抗性机制研究及噬菌体ZP66的发现

发布时间：2018-04-20 20:14

本文选题：霍乱弧菌 + 溶原性噬菌体　；参考：《中国疾病预防控制中心》2016年博士论文

【摘要】：霍乱是由霍乱弧菌引起的急性肠道传染病,据历史记载已有七次霍乱全球大流行,目前仍未停息的第七次霍乱大流行,是由01群El Tor生物型引起。高守一等建立的“噬菌体-生物分型”方案,根据菌株的溶原性、对溶原噬菌体的敏感性、山梨醇发酵试验和溶血试验等4个生物学试验,将El Tor型菌株分成12个(a-l)生物型,再结合噬菌体分型将埃尔托型菌株分为流行株和非流行株,为我国的霍乱防控做出了突出贡献。对噬菌体-生物分型中的溶原性测定和对溶原性噬菌体的敏感性两个分型指标的机制进行研究,有助于我们从表型差异入手,分析霍乱弧菌的分型机理、生物学变异和基因组变异。本研究首先对噬菌体-生物分型中对溶原性噬菌体的敏感性指标中所用的溶原性噬菌体919TP进行了全基因组测序和一般生物学特性分析。噬菌体919TP属于肌尾噬菌体科,其最佳感染复数为1, 919TP感染宿主菌SM6的潜伏期约为60 min,爆发期约为60min,爆发量为4。噬菌体919P基因组为线性双链DNA,长度为33,133bp,GC含量为48.92 %。经过基因预测注释及全基因组比对,确定了噬菌体919TP为K139家族噬菌体。接着我们对噬菌体919TP的抗性机制进行了研究,选取01群El Tor型霍乱弧菌116株,检测这些菌株对噬菌体919TP的敏感性,同时检测这些菌株是否具有溶源化的919TP,菌株的LPS基因簇是否完整,最终发现在检测菌株中,部分菌株LPS基因簇存在突变,但并不影响其对噬菌体919TP的敏感性;大多数菌株含有溶源化的919TP,利用超感染免疫对噬菌体919TP产生抗性,实验数据还证实这种免疫发生在噬菌体与细菌受体的结合之后;部分菌株由于LPS的突变从而导致噬菌体919TP与菌株不吸附,从而对919TP不感染;尚有部分菌株既不含有溶源化的919TP噬菌体基因组,又不含有突变的LPS序列,可能含有未知的抗噬菌体感染的机制而造成对噬菌体919TP的不感染,具体对噬菌体感染的机制尚需进一步研究。再者我们对溶原性测定的机理进行了研究,结果显示01群El Tor型溶原性测定为阳性的菌株,其释放的噬菌体主要为K139家族噬菌体,具有溶原化的K139噬菌体基因组,并对溶原性噬菌体919TP的敏感性为阴性;对溶原性噬菌体919TP的敏感性为阳性的01群El Tor型菌株,溶原性测定全部为阴性,基因组中不含有K139噬菌体;6株01群古典型菌株溶原性检测为阳性,但产生的噬菌体不是K139类;0139群溶原性测定为阳性的菌株能检测到溶原性K139噬菌体基因组,并且所有0139菌株都不能被919TP感染。最终确定了溶原性测定指标主要检测的是菌株释放K139类噬菌体的能力,即是否有K139噬菌体的溶原化并释放子代,“对溶原性噬菌体的敏感性”检测的是菌株能否被K139家族噬菌体(919TP)感染,生物分型中“溶原性测定”与“对溶原性噬菌体的敏感性”两个指标是相关联的。最后我们在霍乱弧菌古典株浙66菌株中发现并分离了一株新的溶原性噬菌体ZP66。通过对ZP66进行基因组测定和一般特性分析,发现噬菌体ZP66属于肌尾噬菌体科,基因组为线性双链DNA,大小为33,499 bp, GC含量49.85 %,共预测到49个ORFs,覆盖了94.78%的噬菌体全基因组序列。经过基因预测注释及蛋白序列比对,判定噬菌体ZP66为Mu类噬菌体。对噬菌体ZP66尾鞘蛋白基因进行聚类分析显示,噬菌体ZP66与霍乱弧菌中的同源基因聚为一个分支,并与其它弧菌和单胞菌聚为一个大的分支,这个聚类结果与细菌的分类相吻合。对噬菌体ZP66在霍乱弧菌中的宿主谱和流行情况进行了分析,发现噬菌体ZP66只能感染O1群菌株,并且只在01群古典生物型中发现有ZP66基因组的存在,但只含有部分ZP66基因组的古典株,同样能被噬菌体ZP66感染。通过对N16961、N16961-LPS缺失株和回补株对ZP66敏感性的检测,推断噬菌体ZP66的受体是01血清群的LPS。通过本研究,我们确定了噬菌体-生物分型中“溶原性测定”和“对溶原性噬菌体的敏感性”两个指标的分型机理,分析了霍乱弧菌天然分离株对噬菌体919TP的抗性机制,为我们了解噬菌体-生物分型机理提供了重要的理论基础,同时发现了一株新的溶原性噬菌体ZP66,为首次在霍乱弧菌分离到Mu类噬菌体。
[Abstract]:Cholera is an acute intestinal infectious disease caused by Vibrio cholerae. According to history, seven cholera global pandemics have been recorded, and the seventh cholera pandemic has not been stopped at present. It is caused by the 01 group of El Tor biotypes. The "phage biotyping" scheme established by Gao Shouyi, etc., is sensitive to the lylyphage phage sensitivity according to the lysin of the strain. 4 biological tests, such as sorbitol fermentation test and hemolysis test, divided the El Tor strain into 12 (A-L) biotypes, and then combined phage typing to divide the erto strains into epidemic and non epidemic strains, which made outstanding contributions to the prevention and control of cholera in China. The study of the mechanism of the two sensibility indexes helps us to analyze the genotyping mechanism, biological variation and genomic variation of Vibrio cholerae from the phenotypic difference. First, the whole genome sequencing of the lysoluble phage 919TP used in the sensitivity index of phage biotype to lytic phage was first sequenced. General biological characteristics analysis. Phage 919TP belongs to the myocutaneous phage family, the best infection complex number is 1, the incubation period of the 919TP infected host SM6 is about 60 min, the outbreak period is about 60min, the explosive amount of 4. phage 919P genome is linear double chain DNA, the length is 33133bp, and the GC content is 48.92%. The bacteriophage 919TP was identified as K139 family phage. Then the resistance mechanism of phage 919TP was studied. 01 groups of El Tor type Vibrio cholerae were selected and 116 strains of Vibrio cholerae were selected to detect the sensitivity of these strains to phage 919TP. At the same time, it was detected whether these strains had dissolved 919TP, and the LPS gene cluster of the strain was complete and finally found. Among the detected strains, some strains of LPS gene cluster have mutations, but they do not affect their sensitivity to phage 919TP; most of the strains contain soluble 919TP and use superinfection immunity to produce resistance to phage 919TP. The experimental data also confirm that the immunization occurs after the combination of phage with bacterial receptor; some strains are due to LPS. The mutation leads to no adsorption of phage 919TP and strain, which does not infect 919TP; some strains do not contain both the 919TP phage genome and the mutation LPS sequence, which may contain unknown anti phage infection mechanism and cause no infection to phage 919TP, and the mechanism of phage infection is still specific. Further study was needed. Furthermore, we studied the mechanism of LYSOGENICITY determination. The results showed that 01 groups of El Tor type LYSOGENICITY were positive, the phage released mainly was K139 family phage, the lysogenizing K139 phage genome, and the sensibility of the lysogen 919TP was negative, and the lysogen phage 91 was negative. The 01 group of El Tor strains with positive sensitivity were negative, and the lylygenicity was all negative. The genomes did not contain K139 phage; 6 strains of 01 groups of classical strains were positive, but the phage produced by the phage was not K139; 0139 groups of lylygenicity tested positive for the detection of the lytic K139 phage genome, and all 0139 The strains could not be infected by 919TP. Finally, the lyse determination index was determined mainly by the ability of the strain to release K139 phage, that is, whether there is the lylygenization of the K139 phage and release the progeny. "The sensitivity of the lytic phage" is that the strain can be infected by the K139 family phage (919TP), and the biotype "lytic" Sex determination "is associated with two indexes" sensitivity to lysogen phage. Finally, we found and separated a new lysogen phage ZP66. in the classical strain of Vibrio cholerae, Zhejiang 66, and found that the phage ZP66 belongs to the myocutaneous phage, and the genome is the genomes. Linear double stranded DNA, 33499 BP in size and 49.85% for GC, was predicted to 49 ORFs and covered 94.78% of the whole genome sequence of phage. The phage phage ZP66 was identified as Mu phage through gene prediction annotation and protein sequence alignment. The phage ZP66 tail sheath protein gene analysis showed that phage ZP66 and Vibrio cholerae were the same The source gene is clustered into one branch and is clustered with other Vibrio and monomonas sp. as a large branch. This clustering result is in accordance with the classification of bacteria. The host spectrum and epidemic situation of phage ZP66 in Vibrio cholerae are analyzed. It is found that phage ZP66 can only infect O1 group strains, and only found ZP66 in 01 groups of classical biologic forms. The existence of the genome, but the classical strain containing only part of the ZP66 genome, can also be infected by phage ZP66. By detecting the ZP66 sensitivity of N16961, N16961-LPS deletion and remedial plants, it is concluded that the receptor of phage ZP66 is the LPS. of the 01 serogroup, and we have determined the "lytic determination" in the phage biotyping and the "lytic determination" in the phage biotyping. The mechanism of two index typing for the sensitivity of lysogen phage was analyzed. The resistance mechanism of the natural isolates of Vibrio cholerae to phage 919TP was analyzed, which provided an important theoretical basis for us to understand the mechanism of phage biotyping, and a new lysogen phage ZP66 was discovered for the first time to isolate the Mu class from Vibrio cholerae. Phage.

【学位授予单位】：中国疾病预防控制中心
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R378

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