Hufnagel David A, Evans Margery L, Greene Sarah E, Pinkner Jerome S, Hultgren Scott J, Chapman Matthew R
Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Michigan, USA.
Department of Molecular Microbiology and Center for Women's Infectious Disease Research, Washington University School of Medicine, St. Louis, Missouri, USA.
J Bacteriol. 2016 Nov 18;198(24):3329-3334. doi: 10.1128/JB.00652-16. Print 2016 Dec 15.
The extracellular matrix protects Escherichia coli from immune cells, oxidative stress, predation, and other environmental stresses. Production of the E. coli extracellular matrix is regulated by transcription factors that are tuned to environmental conditions. The biofilm master regulator protein CsgD upregulates curli and cellulose, the two major polymers in the extracellular matrix of uropathogenic E. coli (UPEC) biofilms. We found that cyclic AMP (cAMP) regulates curli, cellulose, and UPEC biofilms through csgD The alarmone cAMP is produced by adenylate cyclase (CyaA), and deletion of cyaA resulted in reduced extracellular matrix production and biofilm formation. The catabolite repressor protein (CRP) positively regulated csgD transcription, leading to curli and cellulose production in the UPEC isolate, UTI89. Glucose, a known inhibitor of CyaA activity, blocked extracellular matrix formation when added to the growth medium. The mutant strains ΔcyaA and Δcrp did not produce rugose biofilms, pellicles, curli, cellulose, or CsgD. Three putative CRP binding sites were identified within the csgD-csgB intergenic region, and purified CRP could gel shift the csgD-csgB intergenic region. Additionally, we found that CRP binded upstream of kpsMT, which encodes machinery for K1 capsule production. Together our work shows that cAMP and CRP influence E. coli biofilms through transcriptional regulation of csgD IMPORTANCE The catabolite repressor protein (CRP)-cyclic AMP (cAMP) complex influences the transcription of ∼7% of genes on the Escherichia coli chromosome (D. Zheng, C. Constantinidou, J. L. Hobman, and S. D. Minchin, Nucleic Acids Res 32:5874-5893, 2004, https://dx.doi.org/10.1093/nar/gkh908). Glucose inhibits E. coli biofilm formation, and ΔcyaA and Δcrp mutants show impaired biofilm formation (D. W. Jackson, J.W. Simecka, and T. Romeo, J Bacteriol 184:3406-3410, 2002, https://dx.doi.org/10.1128/JB.184.12.3406-3410.2002). We determined that the cAMP-CRP complex regulates curli and cellulose production and the formation of rugose and pellicle biofilms through csgD Additionally, we propose that cAMP may work as a signaling compound for uropathogenic E. coli (UPEC) to transition from the bladder lumen to inside epithelial cells for intracellular bacterial community formation through K1 capsule regulation.
细胞外基质可保护大肠杆菌免受免疫细胞、氧化应激、捕食及其他环境压力的影响。大肠杆菌细胞外基质的产生受转录因子调控，这些转录因子会根据环境条件进行调整。生物膜主要调节蛋白CsgD可上调卷曲菌毛和纤维素的表达，这两种物质是致病性大肠杆菌（UPEC）生物膜细胞外基质中的两种主要聚合物。我们发现，环磷酸腺苷（cAMP）通过CsgD调节卷曲菌毛、纤维素及UPEC生物膜的形成。警报素cAMP由腺苷酸环化酶（CyaA）产生，敲除cyaA会导致细胞外基质产生减少和生物膜形成受阻。分解代谢物阻遏蛋白（CRP）正向调节csgD转录，从而导致UPEC菌株UTI89中卷曲菌毛和纤维素的产生。葡萄糖是一种已知的CyaA活性抑制剂，添加到生长培养基中时会阻断细胞外基质的形成。突变菌株ΔcyaA和Δcrp不会产生皱缩生物膜、菌膜、卷曲菌毛、纤维素或CsgD。在csgD - csgB基因间区域内鉴定出三个假定的CRP结合位点，纯化后的CRP可使csgD - csgB基因间区域发生凝胶迁移。此外，我们发现CRP结合在kpsMT的上游，kpsMT编码K1荚膜产生所需的机制。我们的研究共同表明，cAMP和CRP通过对csgD的转录调控影响大肠杆菌生物膜的形成。重要性分解代谢物阻遏蛋白（CRP）-环磷酸腺苷（cAMP）复合物影响大肠杆菌染色体上约7%的基因转录（D. Zheng、C. Constantinidou、J. L. Hobman和S. D. Minchin，《核酸研究》32:5874 - 5893，2004，https://dx.doi.org/10.1093/nar/gkh908）。葡萄糖抑制大肠杆菌生物膜的形成，ΔcyaA和Δcrp突变体的生物膜形成受损（D. W. Jackson、J. W. Simecka和T. Romeo，《细菌学杂志》184:3406 - 3410，2002，https://dx.doi.org/10.1128/JB.184.12.3406 - 3410.2002）。我们确定cAMP-CRP复合物通过CsgD调节卷曲菌毛和纤维素的产生以及皱缩和菌膜生物膜的形成。此外，我们提出cAMP可能作为致病性大肠杆菌（UPEC）的信号化合物，通过K1荚膜调节从膀胱腔向上皮细胞内转变以形成细胞内细菌群落。
