口腔及血液中分離之血清c型轉糖鏈球菌的基因多樣性

Genetic diversity within oral and blood isolates of Streptococcus mutans serotype c

黃紋娟(Wen-Chung Huang)

指導教授 ： 賈景山

國立臺灣大學/醫學院/微生物學研究所/碩士(2004年)

https://doi.org/10.6342/NTU.2004.10345

摘要

不同的致病菌為適應不同的生活形態其生理和毒力特性反映出有不同的基因存在，近十年來，眾多的致病菌基因圖譜已被建構完成，加速了對細菌基因演化的了解。利用基因體比對，顯示參與細菌基因體演化的機轉主要可分為兩種：一是基因的獲得；另一則為基因的喪失。黴漿菌、披衣菌、分枝桿菌屬和立克次體等細胞內病原生物的基因會有缺失的情形，而一些感染性病原菌，則可從不同的跳躍因子獲得毒力基因。此外，越來越多的研究發現，不管是高度保留性或高度重組性的細菌，皆有大規模的基因體重組經由大片段基因互換所產生，且經常是沿著複製的起始點具有對稱性。
轉糖鏈球菌（Streptococcus mutans）為草綠色鏈球菌的一員，是人類齲齒、及感染性心內膜炎等疾病的病原菌。利用生化鑑定或血清型鑑定已證實該菌具有高度變異性存在，而同一血清型c的分離株間仍有變異性存在其中，例如產生bacteriocin，發酵melibiose和其他醣類的能力。本實驗室先前的研究已發現轉糖鏈球菌同一血清型c的分離株之營養需求不同，而且與致病力相關之細菌表面蛋白質的表現例如 gbpA的合成與否，也有差異。但至今尚未有系統性關於轉糖鏈球菌染色體中，是否具有大規模的基因重組或大片段的基因互換的研究。而另一方面，卻有新菌種不屬於已知任何之血清型的菌株，自心內膜炎的血液中分離出來。因此更加顯示利用基因體尋找分類及與致病力相關的因子之重要性及必要性。本實驗的目的是 (1) 利用分子生物學方法分析分別從健康人、高齟齒罹患者口腔當中，和心內膜炎患者血液中分離出之臨床菌株轉糖鏈球菌染色體的變異性，(2) 找到基因重組或大片段的基因互換的位置及分子機轉。
我們總共篩選了7株實驗室菌株，58株臨床菌株（50株口腔菌株，8株血液菌株），利用NotI和I-CeuI水解不同Streptococcus mutans菌株的DNA，利用PFGE分析其基因變異，並以已定序出來的S. mutans UA159當作比對的依據。比較實驗室菌株之間的差異性，設計不同的探針分別與實驗室菌株S. mutans UA159、GS-5(S)、和GS-5(R)不同的限制酵素水解片段雜合，顯示，GS-5(S)當中有一個大片段的基因互換在兩個相反方向的核醣體之間。而GS-5(R)中有約10 kb的基因缺失位於gbpA基因附近和I-CeuI 189 kb的水解片段，另外還有一段約8 kb的基因增加位於可能是穿膜蛋白的未知基因，和recA基因之間，且可能有IS序列參與在GS-5(R)的基因變異中。利用專一性的探針來偵測臨床菌株，顯示在臨床菌株中大片段基因互換頻率較實驗室菌株高，且臨床菌株中gbpA基因缺失的頻率約有12.3%。
我們選用PFGE分類法將S. mutans分類並以GelCompar軟體分析Streptococcus mutans菌株的PFGE形式，試圖將之分類，結果顯示NotI水解的片段過於複雜，難以將其分類。因此我們選用I-CeuI水解的PFGE形式分類，因其保留性高且水解片段較不複雜，依水解片段的缺失或增加分為四大類，第一類其三個I-CeuI水解的片段都比Streptococcus mutans UA159的高或是有一個特別的高，顯示可能有大片段的基因增加；第二類菌株其I-CeuI水解的片段跟S. mutans UA159位置相似，但其中有一至兩個片段比S. mutans UA159高一點，顯示可能有小片段基因的增加；第三類菌株其水解的片段都偏低且有一個片段特別低，顯示可能有大片段的基因缺失；第四類菌株水解的片段位置與S. mutans UA159相似，但其中有一至兩個片段比UA159低一點，顯示可能有小片段基因缺失。
利用統計分析不同來源的菌種之間，其外觀特性和基因變異性的比例是否有差異性，結果顯示，外觀上，大多數菌株都是粗糙、深藍色的菌落，在大片段的基因互換比率上，臨床菌株比實驗室菌株高。相反的，臨床菌株gbpA基因缺失的比率卻少很多。此外，分析實驗室菌株與臨床菌株I-CeuI水解片段，臨床菌株基因增加的比例比實驗室菌株的比例高，約為53.4%比14.3%，且從血液分離出來的菌株其基因缺失的比例較其他來源的菌株高一些，綜合以上的結果，我們發現在S. mutans臨床菌株中有高頻率的染色體互換（約41.4%）及各種不同長度的增加或缺失的基因片段散佈在基因複製起始點附近，顯示S. mutans具有很高的基因特異性。

關鍵字

轉糖鏈球菌 ； 基因互換

並列摘要

The broad spectrum of physiological and virulence properties of bacterial pathogens mirrors the existence of different subsets of genes enabling the pathogen’s different lifestyles. Whole genome sequence comparisons of related species had shown that specialization for an ecological niche is accompanied by the reorganization of the genomic structure. Close adaptation of parasites and intracellular pathogens (e.g. Mycoplasma spp., Chlamydia trachomatis, Mycobacterium leprae and Rickettsia prowazekii) to the physiologically stable environments of their host cells, a reductive genome evolution occurred that led to the loss of genes not essential for life within the host (a phenomenon termed ‘evolution by reduction’), but leaving genes responsible for functions needed for the performance of key metabolic steps and for interconversions of metabolites obtained from the host. The increasing number of available chromosome maps has prompted reconsideration of existing theories about the evolution of bacterial genome organization. There is increasing evidence that large genome rearrangement is not exceptional. The large inversions observed in bacterial genomes are symmetrical in relation to the origin of replication.
Streptococcus mutans is a member of mutans streptocci, which consist of 9 serotypes (a, b, c, d, e, f, g, and k). S. mutans serotype c is the most predominant clinical isolates from human oral cavity. S. mutans is a primary pathogen for human dental caries and also an opportunistic pathogen for infective endocarditis. Previous studies suggested that S. mutans might be a heterogeneous group of bacterium exhibited distinct phenotypical characteristics, such as colony morphology, sugar fermentation, etc. The increased use of molecular typing method, such as pulsed field gel electrophoresis (PFGE) of chromosomal DNA cut with a restriction enzyme that recognizes rare sites in the DNA, provides a useful tool for the classification of clinically relevant bacteria. The purposes of the present study are, firstly, to evaluate the genetic diversity of the S. mutans serotype c strains isolated from healthy donors, patients with dental caries or endocarditis; secondly, to identify possible molecular mechanisms and genetic elements involved in the generation of genetic diversity.
Comparative analysis of chromosomal macrorestriction polymorphism of the two closely related Streptococcus mutans strains UA159 and GS-5(S) revealed the presence of a large inversion covering half of the genome. To determine what kind of genetic element could be implicated in this rearrangement, three specific probes are designed. S. mutans GS-5 have large genomic rearrangements due to recombination at the ribosomal RNA (rrn) operons. In addition, comparative analysis of chromosomal macrorestriction polymorphism of Streptococcus mutans strains UA159 and GS-5(R) revealed the presence of gene insertions, and gbpA gene deletion. To determine what kind of genetic element could be implicated in this rearrangement, specific probes for insertion sequence are designed. Southern blot analysis suggested that ISs may be involved in the gene rearrangement.
In this study, we have examined 65 strains of S. mutans by PFGE with restriction enzymes NotI or I-CeuI, which cuts a 26-bp site in the rrl gene that has been shown to be highly conserved in eubacteria. Both the size and the number of NotI and I-CeuI fragments exhibit considerable variation among all tested strains, indicated that S. mutans is genetically heterogeneous even within strains belonging to the same serotype c group. Based on the PFGE band patterns of I-CeuI digested chromosomal DNA, four pulsotypes were found, with different fragment of DNA insertion or deletions, when compared with UA159 strain. Strains exhibit large insertions in the first pulsotype (20%), smaller insertions in the second pulsotype (29.2%), large deletions in the third pulsotype (1.5%), and smaller deletions in the fourth pulsotype (18.5%). The percentage of small deletion was identified at a higher frequency in isolates from blood than in oral cavity from either healthy or patients with dental caries (37.5 Vs 8 and 20 %; respectively). In addition, clinical isolates, regardless of their origin, exhibited higher frequency of insertions (large or smaller) than the laboratory strains (53.4% Vs 14.3 %). Based on these data, we concluded that S. mutans is genetically heterogeneous with a high frequency of large chromosomal inversion (41.4%) and different length of insertion and/or deletions scattering beside the putative chromosomal replication origin.

並列關鍵字

Streptococcus mutans ； PFGE

參考文獻

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