Bröms Jeanette E, Lavander Moa, Sjöstedt Anders
Department of Clinical Microbiology, Clinical Bacteriology, Umeå University, SE-901 85 Umeå, Sweden.
J Bacteriol. 2009 Apr;191(8):2431-46. doi: 10.1128/JB.01759-08. Epub 2009 Feb 6.
Francisella tularensis harbors genes with similarity to genes encoding components of a type VI secretion system (T6SS) recently identified in several gram-negative bacteria. These genes include iglA and iglB encoding IglA and IglB, homologues of which are conserved in most T6SSs. We used a yeast two-hybrid system to study the interaction of the Igl proteins of F. tularensis LVS. We identified a region of IglA, encompassing residues 33 to 132, necessary for efficient binding to IglB, as well as for IglAB protein stability and intramacrophage growth. In particular, residues 103 to 122, overlapping a highly conserved alpha-helix, played an absolutely essential role. Point mutations within this domain caused modest defects in IglA-IglB binding in the yeast Saccharomyces cerevisiae but markedly impaired intramacrophage replication and phagosomal escape, resulting in severe attenuation of LVS in mice. Thus, IglA-IglB complex formation is clearly crucial for Francisella pathogenicity. This interaction may be universal to type VI secretion, since IglAB homologues of Yersinia pseudotuberculosis, Pseudomonas aeruginosa, Vibrio cholerae, Salmonella enterica serovar Typhimurium, and Escherichia coli were also shown to interact in yeast, and the interaction was dependent on preservation of the same alpha-helix. Heterologous interactions between nonnative IglAB proteins further supported the notion of a conserved binding site. Thus, IglA-IglB complex formation is clearly crucial for Francisella pathogenicity, and the same interaction is conserved in other human pathogens.
土拉弗朗西斯菌携带的基因与最近在几种革兰氏阴性细菌中鉴定出的编码VI型分泌系统（T6SS）组分的基因具有相似性。这些基因包括编码IglA和IglB的iglA和iglB，其同源物在大多数T6SS中是保守的。我们使用酵母双杂交系统研究土拉弗朗西斯菌LVS的Igl蛋白之间的相互作用。我们确定了IglA的一个区域，该区域包含33至132位氨基酸残基，对于与IglB的有效结合以及IglAB蛋白的稳定性和巨噬细胞内生长是必需的。特别是，103至122位氨基酸残基与一个高度保守的α螺旋重叠，发挥了绝对必要的作用。该结构域内的点突变在酿酒酵母中导致IglA-IglB结合出现适度缺陷，但显著损害巨噬细胞内复制和吞噬体逃逸，导致LVS在小鼠中的毒力严重减弱。因此，IglA-IglB复合物的形成显然对土拉弗朗西斯菌的致病性至关重要。这种相互作用可能对于VI型分泌是普遍存在的，因为也已证明假结核耶尔森菌、铜绿假单胞菌、霍乱弧菌、鼠伤寒沙门氏菌和大肠杆菌的IglAB同源物在酵母中相互作用，并且这种相互作用依赖于相同α螺旋的保留。非天然IglAB蛋白之间的异源相互作用进一步支持了保守结合位点的概念。因此，IglA-IglB复合物的形成显然对土拉弗朗西斯菌的致病性至关重要，并且相同的相互作用在其他人类病原体中是保守的。
