Homologs of the LapD-LapG c-di-GMP Effector System Control Biofilm Formation by Bordetella bronchiseptica

Biofilm formation is important for infection by many pathogens. Bordetella bronchiseptica causes respiratory tract infections in mammals and forms biofilm structures in nasal epithelium of infected mice. We previously demonstrated that cyclic di-GMP is involved in biofilm formation in B. bronchiseptica. In the present work, based on their previously reported function in Pseudomonas fluorescens, we identified three genes in the B. bronchiseptica genome likely involved in c-di-GMP-dependent biofilm formation: brtA, lapD and lapG. Genetic analysis confirmed a role for BrtA, LapD and LapG in biofilm formation using microtiter plate assays, as well as scanning electron and fluorescent microscopy to analyze the phenotypes of mutants lacking these proteins. In vitro and in vivo studies showed that the protease LapG of B. bronchiseptica cleaves the N-terminal domain of BrtA, as well as the LapA protein of P. fluorescens, indicating functional conservation between these species. Furthermore, while BrtA and LapG appear to have little or no impact on colonization in a mouse model of infection, a B. bronchiseptica strain lacking the LapG protease has a significantly higher rate of inducing a severe disease outcome compared to the wild type. These findings support a role for c-di-GMP acting through BrtA/LapD/LapG to modulate biofilm formation, as well as impact pathogenesis, by B. bronchiseptica生物被膜（biofilm）的形成对于众多病原体的感染过程至关重要。支气管败血波氏杆菌（Bordetella bronchiseptica）可引发哺乳动物呼吸道感染，并在感染小鼠的鼻上皮细胞中形成生物被膜结构。我们此前已证实，环二鸟苷酸（cyclic di-GMP，c-di-GMP）参与支气管败血波氏杆菌的生物被膜形成过程。本研究基于此前在荧光假单胞菌（Pseudomonas fluorescens）中报道的功能，在支气管败血波氏杆菌基因组中鉴定出三个可能参与c-di-GMP依赖型生物被膜形成的基因：brtA、lapD和lapG。通过微孔板测定法、扫描电子显微镜与荧光显微镜分析缺失这些蛋白的突变体的表型，遗传学分析证实了BrtA、LapD和LapG在生物被膜形成中的作用。体内外实验表明，支气管败血波氏杆菌的蛋白酶LapG可切割BrtA的N端结构域，以及荧光假单胞菌的LapA蛋白，这提示两个物种间存在功能保守性。此外，尽管BrtA与LapG对感染小鼠模型中的细菌定植几乎无影响，但缺失LapG蛋白酶的支气管败血波氏杆菌菌株，相较于野生型菌株，引发严重疾病结局的比例显著升高。本研究结果证实，环二鸟苷酸可通过BrtA/LapD/LapG通路调控支气管败血波氏杆菌的生物被膜形成，并影响其致病过程。