Mintz Keith P
Department of Microbiology and Molecular Genetics, Rm 110 Stafford Hall, University of Vermont, Burlington, VT 05405, USA.
Microbiology (Reading). 2004 Aug;150(Pt 8):2677-2688. doi: 10.1099/mic.0.27110-0.
Actinobacillus actinomycetemcomitans is an aetiologic agent in the development of periodontal and some systemic diseases in humans. This pathogen localizes to the underlying connective tissue of the oral cavity in individuals with periodontal disease. The adhesion of A. actinomycetemcomitans to extracellular matrix components of the connective tissue prompted this study to identify gene products mediating the interaction of A. actinomycetemcomitans to these molecules. A transposon mutagenesis system was optimized for use in A. actinomycetemcomitans and used to generate an insertional mutant library. A total of 2300 individual insertion transposon mutants were screened for changes in the adhesion to collagen and fibronectin. Mutants were identified which exhibited the following phenotypes: a decrease in collagen binding; a decrease in fibronectin binding; a decrease in binding to both proteins; and an increase in binding to both collagen and fibronectin. The identification of mutants defective in adhesion to the individual proteins indicates that distinct adhesins are expressed by this organism. Molecular analysis of these mutants implicated 11 independent loci in protein adhesion. One gene, emaA, is likely to encode a direct mediator of collagen adhesion, based on predicted protein features homologous to the collagen-binding protein YadA of Yersinia enterocolitica. EmaA was localized to the outer membrane, as expected for an adhesin. Reduction in fibronectin adhesion appeared to be influenced by abrogation of proteins involved in molybdenum-cofactor biosynthesis. Several other loci identified as reducing or increasing adhesion to both collagen and fibronectin are suggested to be involved in regulatory cascades that promote or repress expression of collagen and fibronectin adhesins. Collectively, the results support the hypothesis that A. actinomycetemcomitans host colonization involves afimbrial adhesins for extracellular matrix proteins, and that the expression of adhesion is modulated by global regulatory mechanisms.
伴放线放线杆菌是人类牙周病和一些全身性疾病发病过程中的一种病原体。这种病原菌定位于患有牙周病个体口腔的下方结缔组织中。伴放线放线杆菌与结缔组织细胞外基质成分的黏附促使本研究去鉴定介导伴放线放线杆菌与这些分子相互作用的基因产物。一种转座子诱变系统被优化用于伴放线放线杆菌,并用于构建一个插入突变体文库。总共筛选了2300个单独的插入转座子突变体,以检测其对胶原蛋白和纤连蛋白黏附的变化。鉴定出了表现出以下表型的突变体:胶原蛋白结合减少;纤连蛋白结合减少;两种蛋白质的结合均减少;以及胶原蛋白和纤连蛋白的结合均增加。对黏附于单个蛋白质有缺陷的突变体的鉴定表明,该生物体表达了不同的黏附素。对这些突变体的分子分析表明有11个独立的基因座参与蛋白质黏附。基于与小肠结肠炎耶尔森菌的胶原蛋白结合蛋白YadA具有同源性的预测蛋白质特征,一个基因emaA可能编码胶原蛋白黏附的直接介导因子。正如对黏附素的预期,EmaA定位于外膜。纤连蛋白黏附的减少似乎受到参与钼辅因子生物合成的蛋白质缺失的影响。其他几个被鉴定为减少或增加对胶原蛋白和纤连蛋白黏附的基因座,被认为参与了促进或抑制胶原蛋白和纤连蛋白黏附素表达的调控级联反应。总的来说,这些结果支持了这样的假说,即伴放线放线杆菌在宿主体内的定殖涉及针对细胞外基质蛋白的非菌毛黏附素,并且黏附的表达受到全局调控机制的调节。