Genotypic and phenotypic analysis of Mycoplasma fermentans strains isolated from different host tissues.
Genotypic and phenotypic analysis of Mycoplasma fermentans strains isolated from different host tissues.
复制标题
从不同宿主组织分离的发酵支原体菌株的基因型和表型分析。
DOI：
10.1128/jcm.36.5.1371-1377.1998
复制
发表时间：
1998
影响因子：
9.4
通讯作者：
Watson,HL
中科院分区：
文献类型：
--
作者：
Campo,L;Larocque,P;LaMalfa,T;Blackburn,WD;Watson,HL
一种特定的支原体发酵表面抗原(Pra，蛋白酶抗性抗原)的表达与微生物从受感染宿主中分离的位置之间存在相关性。表达Pra的菌株最常与骨髓来源的细胞有关，而缺乏Pra的菌株最常从呼吸道、生殖道和关节炎关节(即上皮细胞表面)分离。PRA先前被证明能抵抗蛋白水解酶的降解，并被认为在生物体表面起保护作用，并可能影响生物体定植的宿主组织部位。用于这种表型方案的方法需要分离和生长足够数量的支原体，以便使用单抗进行免疫印迹分析。我们想要确定一种更快速且不那么麻烦的技术来补充这种直接在临床标本中确定Pra表型的方法。在这里，我们描述了用聚合酶链式反应研究先前确定的M的移动。发酵转导插入序列(IS)类元件。这些数据表明，特定的IS基因型与Pra+表型之间存在相关性。使用一组基于IS的引物生产160个碱基的产物与Pra的表达有关。通过Southern印迹分析确定了导致160bp产物的基因组IS位置，发现该插入位点是I型菌株特有的稳定插入位点。对IS插入位点内部和侧翼序列的进一步分析揭示了另一对导致450bp扩增子一致产生的聚合酶链式反应引物点。该酶切位点的稳定性取决于两个引物点之间是否存在类似IS的元件。该450个碱基的扩增产物与Pra突变表型相关，是II型菌株的特征。数据表明，IS内的序列可能是不稳定的，可靠的基因分型序列更容易在IS元件两侧的稳定基因组位置找到。
A correlation was found between the expression of a specificMycoplasma fermentanssurface antigen (Pra, proteinase-resistant antigen) and the site of isolation of the organism from the infected host. Strains which expressed Pra were most frequently associated with cells of bone marrow origin, and strains which lacked expression of Pra were most commonly isolated from the respiratory tract, genital tract, and arthritic joints, i.e., epithelial cell surfaces. Pra was previously shown to be resistant to degradation by proteinases and was hypothesized to play a protective role at the organism surface and perhaps to influence which host tissue site was colonized by the organism. The methods used for this phenotyping scheme required isolation and growth of the mycoplasma in quantities sufficient for immunoblot analysis using monoclonal antibodies. We wanted to determine a more rapid and less cumbersome technique to supplement this method for determining the Pra phenotype directly in clinical specimens. Here we describe PCR studies to investigate the movement of a previously identifiedM. fermentansinsertion sequence (IS)-like element. These data showed a correlation between a specific IS genotype and the Pra+phenotype. Production of a 160-bp product using a single set of IS-based primers was associated with expression of Pra. The genomic IS location resulting in the 160-bp product was determined by using Southern blot analysis and was found to be a stable insertion site characteristic of genotype I strains. Additional analyses of sequences within and flanking the IS insertion sites revealed another pair of PCR primer sites which resulted in the consistent production of a 450-bp amplicon. The stability of this site was dependent on the absence of the IS-like element between the primer sites. The production of this 450-bp amplicon correlated with the Pra mutant phenotype and was characteristic of genotype II strains. The data showed that the sequence within the IS may be unstable and that reliable genotyping sequences are more easily found in the stable genomic sites which flank the IS element.