Rubino S, Leori G, Rizzu P, Erre G, Colombo M M, Uzzau S, Masala G, Cappuccinelli P
Istituto di Microbiologia e Virologia, Università degli Studi di Sassari, Italy.
Infect Immun. 1993 May;61(5):1786-92. doi: 10.1128/iai.61.5.1786-1792.1993.
Salmonella abortusovis is a pathogenic bacterium highly specific to sheep, causing spontaneous abortion. In order to understand the role of genes involved in pathogenicity, we investigated S. abortusovis with the random mutagenic TnphoA transposon. A total of 95 S. abortusovis TnphoA mutants yielding alkaline phosphatase active fusion protein were obtained. In this way we created a bank of strains in order to identify any phenotypic modification which could affect the periplasmic and/or exported proteins involved in virulence. The TnphoA mutants were screened for the ability to adhere to epithelial cells: a total of 23 mutant strains lost this phenotypic feature. To detect the chromosomal TnphoA insertions, DNA was restricted by the enzyme EcoRV, which does not cleave the TnphoA sequence. Southern blotting analysis revealed the existence of four classes of integration. Colonies of adhesiveless mutants appear to be as smooth as the S. abortusovis wild type, and electrophoretic analysis indicates a normal lipopolysaccharide profile. To identify mutations affecting genes encoding for outer membrane proteins (OMPs), the alkaline phosphatase portion of the fusion proteins was revealed in TnphoA mutants by immunoblotting with specific antibodies. A mutation in OMPs was detected in seven mutants. Restriction analysis identified in four mutants a common region of 2 kb where alterations in genes coding for OMPs occur. We suggested that this region is involved in pathogenicity in mice, since a group of mutant strains has shown reduced virulence in mice and one mutant is completely avirulent. Furthermore, after mice were exposed orally to these mutants, significant protection against oral challenge with the parental virulent strain resulted.
羊流产沙门氏菌是一种对绵羊具有高度特异性的致病细菌，可导致自然流产。为了了解参与致病性的基因的作用，我们用随机诱变的TnphoA转座子对羊流产沙门氏菌进行了研究。共获得了95个产生碱性磷酸酶活性融合蛋白的羊流产沙门氏菌TnphoA突变体。通过这种方式，我们构建了一个菌株库，以鉴定可能影响参与毒力的周质和/或分泌蛋白的任何表型改变。筛选TnphoA突变体对上皮细胞的黏附能力：共有23个突变菌株丧失了这种表型特征。为了检测染色体上TnphoA的插入，用不切割TnphoA序列的EcoRV酶对DNA进行酶切。Southern印迹分析揭示了存在四类整合。无黏附性突变体的菌落看起来与羊流产沙门氏菌野生型一样光滑，电泳分析表明脂多糖谱正常。为了鉴定影响外膜蛋白（OMPs）编码基因的突变，通过用特异性抗体进行免疫印迹在TnphoA突变体中揭示融合蛋白的碱性磷酸酶部分。在7个突变体中检测到OMPs的突变。限制性分析在4个突变体中鉴定出一个2 kb的共同区域，其中发生了编码OMPs的基因改变。我们认为该区域与小鼠的致病性有关，因为一组突变菌株在小鼠中显示出毒力降低，且有一个突变体完全无毒。此外，在用这些突变体经口感染小鼠后，对亲本毒力菌株的经口攻击产生了显著的保护作用。
