MrkH, a Novel c-di-GMP-Dependent Transcriptional Activator, Controls Klebsiella pneumoniae Biofilm Formation by Regulating Type 3 Fimbriae Expression

Klebsiella pneumoniae causes significant morbidity and mortality worldwide, particularly amongst hospitalized individuals. The principle mechanism for pathogenesis in hospital environments involves the formation of biofilms, primarily on implanted medical devices. In this study, we constructed a transposon mutant library in a clinical isolate, K. pneumoniae AJ218, to identify the genes and pathways implicated in biofilm formation. Three mutants severely defective in biofilm formation contained insertions within the mrkABCDF genes encoding the main structural subunit and assembly machinery for type 3 fimbriae. Two other mutants carried insertions within the yfiN and mrkJ genes, which encode GGDEF domain- and EAL domain-containing c-di-GMP turnover enzymes, respectively. The remaining two isolates contained insertions that inactivated the mrkH and mrkI genes, which encode for novel proteins with a c-di-GMP-binding PilZ domain and a LuxR-type transcriptional regulator, respectively. Biochemical and functional assays indicated that the effects of these factors on biofilm formation accompany concomitant changes in type 3 fimbriae expression. We mapped the transcriptional start site of mrkA, demonstrated that MrkH directly activates transcription of the mrkA promoter and showed that MrkH binds strongly to the mrkA regulatory region only in the presence of c-di-GMP. Furthermore, a point mutation in the putative c-di-GMP-binding domain of MrkH completely abolished its function as a transcriptional activator. In vivo analysis of the yfiN and mrkJ genes strongly indicated their c-di-GMP-specific function as diguanylate cyclase and phosphodiesterase, respectively. In addition, in vitro assays showed that purified MrkJ protein has strong c-di-GMP phosphodiesterase activity. These results demonstrate for the first time that c-di-GMP can function as an effector to stimulate the activity of a transcriptional activator, and explain how type 3 fimbriae expression is coordinated with other gene expression programs in K. pneumoniae to promote biofilm formation to implanted medical devices.肺炎克雷伯菌（Klebsiella pneumoniae）在全球范围内引发严重的发病率与死亡率，尤其在住院患者群体中造成高发危害。医院环境中该菌致病的核心机制在于生物膜（biofilm）的形成，且多发生于植入式医疗器械表面。本研究以临床分离株肺炎克雷伯菌AJ218构建转座子突变体库（transposon mutant library），旨在筛选参与生物膜形成的基因与通路。3株生物膜形成严重缺陷的突变株，其插入突变位点位于mrkABCDF基因簇——该基因簇编码3型菌毛（type 3 fimbriae）的主要结构亚基与组装机器。另有2株突变株的插入位点分别为yfiN与mrkJ基因，二者分别编码含GGDEF结构域与EAL结构域的环二鸟苷单磷酸（c-di-GMP）代谢酶。剩余2株突变株的插入突变使mrkH与mrkI基因失活：前者编码携带c-di-GMP结合PilZ结构域的新型蛋白，后者编码LuxR型转录调节因子。生化与功能实验结果显示，上述因子对生物膜形成的调控作用伴随3型菌毛表达水平的同步变化。本研究明确了mrkA的转录起始位点，证实MrkH可直接激活mrkA启动子的转录，且仅在c-di-GMP存在的条件下，MrkH才能强效结合mrkA的调控区域。进一步实验发现，MrkH假定的c-di-GMP结合结构域发生单点突变后，其作为转录激活因子的功能完全丧失。针对yfiN与mrkJ基因的体内功能分析证实，二者分别特异性发挥二鸟苷酸环化酶与磷酸二酯酶的活性。此外，体外酶活实验显示，纯化得到的MrkJ蛋白具有显著的c-di-GMP磷酸二酯酶活性。本研究首次证实，c-di-GMP可作为效应分子激活转录激活因子的活性，并阐明了肺炎克雷伯菌中3型菌毛的表达如何与其他基因表达程序协同，进而促进其在植入式医疗器械表面形成生物膜。