Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, Florida, United States of America.
PLoS One. 2013;8(1):e54498. doi: 10.1371/journal.pone.0054498. Epub 2013 Jan 23.
The transcription factors MglA and SspA of Francisella tularensis form a heterodimer complex and interact with the RNA polymerase to regulate the expression of the Francisella pathogenicity island (FPI) genes. These genes are essential for this pathogen's virulence and survival within host cells. In this study, we used a small molecule screening to identify quinacrine as a thermal stabilizing compound for F. tularensis SCHU S4 MglA and SspA. A bacterial two-hybrid system was used to analyze the in vivo effect of quinacrine on the heterodimer complex. The results show that quinacrine affects the interaction between MglA and SspA, indicated by decreased β-galactosidase activity. Further in vitro analyses, using size exclusion chromatography, indicated that quinacrine does not disrupt the heterodimer formation, however, changes in the alpha helix content were confirmed by circular dichroism. Structure-guided site-directed mutagenesis experiments indicated that quinacrine makes contact with amino acid residues Y63 in MglA, and K97 in SspA, both located in the "cleft" of the interacting surfaces. In F. tularensis subsp. novicida, quinacrine decreased the transcription of the FPI genes, iglA, iglD, pdpD and pdpA. As a consequence, the intramacrophage survival capabilities of the bacteria were affected. These results support use of the MglA/SspA interacting surface, and quinacrine's chemical scaffold, for the design of high affinity molecules that will function as therapeutics for the treatment of Tularemia.
弗氏志贺菌的转录因子 MglA 和 SspA 形成异二聚体复合物，并与 RNA 聚合酶相互作用，以调节弗朗西斯氏菌致病岛（FPI）基因的表达。这些基因对该病原体在宿主细胞内的毒力和存活至关重要。在这项研究中，我们使用小分子筛选法确定了吖啶橙是弗氏志贺菌 SCHU S4 MglA 和 SspA 的热稳定化合物。细菌双杂交系统用于分析吖啶橙对异二聚体复合物的体内影响。结果表明，吖啶橙影响 MglA 和 SspA 之间的相互作用，表现为β-半乳糖苷酶活性降低。进一步的体外分析，使用大小排阻层析法，表明吖啶橙不会破坏异二聚体的形成，但是圆二色性证实了其改变了α螺旋含量。基于结构的定点突变实验表明，吖啶橙与 MglA 中的 Y63 氨基酸残基和 SspA 中的 K97 氨基酸残基相互作用，这两个残基都位于相互作用表面的“裂缝”中。在弗氏志贺菌亚种 novicida 中，吖啶橙降低了 FPI 基因 iglA、iglD、pdpD 和 pdpA 的转录。因此，细菌在巨噬细胞内的生存能力受到影响。这些结果支持使用 MglA/SspA 相互作用表面和吖啶橙的化学支架来设计高亲和力的分子，作为治疗土拉热的治疗药物。
