Department of Microbiology and Immunology, University of Illinois at Chicago, Chicago, Illinois, United States of America.
PLoS One. 2011;6(8):e23502. doi: 10.1371/journal.pone.0023502. Epub 2011 Aug 12.
Listeria monocytogenes is a food-borne intracellular bacterial pathogen capable of causing serious human disease. L. monocytogenes survival within mammalian cells depends upon the synthesis of a number of secreted virulence factors whose expression is regulated by the transcriptional activator PrfA. PrfA becomes activated following bacterial entry into host cells where it induces the expression of gene products required for bacterial spread to adjacent cells. Activation of PrfA appears to occur via the binding of a small molecule cofactor whose identity remains unknown. Electrostatic modeling of the predicted PrfA cofactor binding pocket revealed a highly positively charged region with two lysine residues, K64 and K122, located at the edge of the pocket and another (K130) located deep within the interior. Mutational analysis of these residues indicated that K64 and K122 contribute to intracellular activation of PrfA, whereas a K130 substitution abolished protein activity. The requirement of K64 and K122 for intracellular PrfA activation could be bypassed via the introduction of the prfA G145S mutation that constitutively activates PrfA in the absence of cofactor binding. Our data indicate that the positive charge of the PrfA binding pocket contributes to intracellular activation of PrfA, presumably by facilitating binding of an anionic cofactor.
李斯特菌是一种食源性病原体，能够引起严重的人类疾病。李斯特菌在哺乳动物细胞内的存活取决于许多分泌毒力因子的合成，其表达受转录激活因子 PrfA 的调节。PrfA 在细菌进入宿主细胞后被激活，随后诱导细菌扩散到邻近细胞所需的基因产物的表达。PrfA 的激活似乎是通过结合一种小分子辅因子发生的，但其身份仍然未知。对预测的 PrfA 辅因子结合口袋进行静电建模显示，该口袋具有高度正电荷区域，两个赖氨酸残基（K64 和 K122）位于口袋边缘，另一个（K130）位于口袋内部深处。这些残基的突变分析表明，K64 和 K122 有助于 PrfA 的细胞内激活，而 K130 的取代则使蛋白活性丧失。通过引入 prfA G145S 突变，可以绕过 K64 和 K122 对细胞内 PrfA 激活的要求，该突变在没有辅因子结合的情况下使 PrfA 持续激活。我们的数据表明，PrfA 结合口袋的正电荷有助于 PrfA 的细胞内激活，可能通过促进阴离子辅因子的结合来实现。
