Role of lipopolysaccharide of actinobacillus pleuropneumoniae in biofilm formation and pathogenesis

这项研究计划的长期目标是更好地了解革兰氏阴性细菌在宿主呼吸道的定植所涉及的毒力因素。猪胸膜肺炎的病原--胸膜肺炎放线杆菌(App)就是我们的模型。我们利用转座子诱变产生了一系列App的脂多糖(LPS)突变体。我们的研究表明，App-LPS参与了猪呼吸道的定植，而在LPS外核的特定残基的缺失会导致App的毒力减弱。我们推测，内毒素参与了APP生物膜的形成，生物膜的形成是一个复杂的、调控精细的过程，在发病机制中起着重要作用。本研究的短期目标是：(1)研究细菌内毒素与生物膜形成的关系。我们将首先鉴定我们产生的App LPS突变体的集合，并确定它们形成生物膜的能力。我们将在微量平板上使用标准的静态生物被膜分析，以及更能代表肺部发现的气液界面的滴流细胞。我们将利用噬菌体展示技术筛选与纯化的内毒素分子有很强亲和力的多肽。然后，我们将使用表面等离子激元共振来量化这些多肽的亲和力，并确定这些多肽是否可以干扰生物膜的形成或内毒素的其他生物学活性。(2)利用基因组学(转座子突变)和转录学(DNA微阵列)方法寻找与App生物膜形成相关的其他基因。更好地了解App的表面多糖和在生物膜形成过程中表达的基因将导致开发有效的感染控制策略，如新型分子可以阻断重要毒力因子的活性或抑制App和潜在的农场动物其他病原体的生物膜形成。

The long-term objective of this research program is to gain a better understanding of the virulence factors involved in the Gram-negative bacterial pathogens colonization of the host's respiratory tract. Actinobacillus pleuropneumoniae (App), the etiological agent of porcine pleuropneumonia, is our model. We have generated a collection of lipopolysaccharide (LPS) mutants of App using transposon mutagenesis. We have shown that App LPS is involved in colonization of porcine respiratory tract and that deletion of specific residues in the LPS outer core results in the attenuation of the virulence of App. We hypothesize that LPS are involved in biofilm formation in App and that biofilm formation is a complex and finely regulated process which plays an important role in pathogenesis. The short-term objectives of this research program are: (1) to characterize the relation between LPS and biofilm formation. We will first characterize the collection of App LPS mutants we have generated and determine their ability to form biofilms. We will use a standard static biofilm assay in microtiter plates as well as a drip-flow cell which is more representative of the air-liquid interface found in the lungs. We will use phage display technology to select peptides with a strong affinity for purified LPS molecules. We will then quantify the affinity of these peptides using surface plasmon resonance and determine whether these peptides can interfere with biofilm formation or with other biological activities of LPS. (2) to use genomic (transposon mutagenesis) and transcriptomic (DNA microarray) approaches to identify additional genes involved in App biofilm formation. A better understanding of surface polysaccharides of App and of the genes expressed during biofilm formation will lead to the development of effective infection-control strategies such as novel molecules that can block the activity of important virulence factors or inhibit biofilm formation of App and, potentially, other pathogens of farm animals.