Lin Ching-Ting, Lin Tien-Huang, Wu Chien-Chen, Wan Lei, Huang Chun-Fa, Peng Hwei-Ling
School of Chinese Medicine, China Medical University, Taichung, Taiwan, Republic of China.
Division of Urology, Taichung Tzu Chi Hospital, The Buddhist Tzu Chi Medical Foundation, Taichung, Taiwan, Republic of China.
PLoS One. 2016 Sep 15;11(9):e0162884. doi: 10.1371/journal.pone.0162884. eCollection 2016.
Klebsiella pneumoniae is the predominant pathogen isolated from liver abscesses of diabetic patients in Asian countries. However, the effects of elevated blood glucose levels on the virulence of this pathogen remain largely unknown. Type 3 fimbriae, encoded by the mrkABCDF genes, are important virulence factors in K. pneumoniae pathogenesis. In this study, the effects of exogenous glucose and the intracellular cyclic AMP (cAMP) signaling pathway on type 3 fimbriae expression regulation were investigated. The production of MrkA, the major subunit of type 3 fimbriae, was increased in glucose-rich medium, whereas cAMP supplementation reversed the effect. MrkA production was markedly increased by cyaA or crp deletion, but slightly decreased by cpdA deletion. In addition, the mRNA levels of mrkABCDF genes and the activity of PmrkA were increased in Δcrp strain, as well as the mRNA levels of mrkHIJ genes that encode cyclic di-GMP (c-di-GMP)-related regulatory proteins that influence type 3 fimbriae expression. Moreover, the activities of PmrkHI and PmrkJ were decreased in ΔlacZΔcrp strain. These results indicate that CRP-cAMP down-regulates mrkABCDF and mrkHIJ at the transcriptional level. Further deletion of mrkH or mrkI in Δcrp strain diminished the production of MrkA, indicating that MrkH and MrkI are required for the CRP regulation of type 3 fimbriae expression. Furthermore, the high activity of PmrkHI in the ΔlacZΔcrp strain was diminished in ΔlacZΔcrpΔmrkHI, but increased in the ΔlacZΔcrpΔmrkJ strain. Deletion of crp increased the intracellular c-di-GMP concentration and reduced the phosphodiesterase activity. Moreover, we found that the mRNA levels of multiple genes related to c-di-GMP metabolism were altered in Δcrp strain. These indicate that CRP regulates type 3 fimbriae expression indirectly via the c-di-GMP signaling pathway. In conclusion, we found evidence of a coordinated regulation of type 3 fimbriae expression by the CRP-cAMP and c-di-GMP signaling pathways in K. pneumoniae.
肺炎克雷伯菌是亚洲国家糖尿病患者肝脓肿中分离出的主要病原体。然而，血糖水平升高对该病原体毒力的影响在很大程度上仍不清楚。由mrkABCDF基因编码的3型菌毛是肺炎克雷伯菌致病过程中的重要毒力因子。在本研究中，研究了外源性葡萄糖和细胞内环磷酸腺苷（cAMP）信号通路对3型菌毛表达调控的影响。在富含葡萄糖的培养基中，3型菌毛主要亚基MrkA的产量增加，而补充cAMP则逆转了这种影响。cyaA或crp缺失使MrkA产量显著增加，而cpdA缺失使其略有下降。此外，Δcrp菌株中mrkABCDF基因的mRNA水平和PmrkA的活性增加，以及编码影响3型菌毛表达的环二鸟苷酸（c-di-GMP）相关调节蛋白的mrkHIJ基因的mRNA水平也增加。此外，ΔlacZΔcrp菌株中PmrkHI和PmrkJ的活性降低。这些结果表明CRP-cAMP在转录水平上下调mrkABCDF和mrkHIJ。在Δcrp菌株中进一步缺失mrkH或mrkI会减少MrkA的产生，表明MrkH和MrkI是CRP调节3型菌毛表达所必需的。此外ΔlacZΔcrpΔmrkHI中ΔlacZΔcrp菌株中PmrkHI的高活性降低，但在ΔlacZΔcrpΔmrkJ菌株中增加。crp缺失增加了细胞内c-di-GMP浓度并降低了磷酸二酯酶活性。此外，我们发现Δcrp菌株中多个与c-di-GMP代谢相关的基因的mRNA水平发生了改变。这些表明CRP通过c-di-GMP信号通路间接调节3型菌毛表达。总之，我们发现了肺炎克雷伯菌中CRP-cAMP和c-di-GMP信号通路对3型菌毛表达进行协同调节的证据。
