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与革兰氏阴性细菌病原体在宿主呼吸道定植有关的毒力因素。胸膜肺炎放线杆菌（App）是猪胸膜肺炎的病原。我们利用转座子诱变产生了一系列App的脂多糖（LPS）突变体。我们已经证明，App LPS参与了猪呼吸道的定植，并且LPS外核中特定残基的缺失导致App毒力的减弱。我们假设LPS参与了App的生物膜形成，并且生物膜形成是一个复杂而精细的过程，在发病机制中起重要作用。本研究计划的短期目标是：(1)表征LPS与生物膜形成之间的关系。我们将首先描述我们生成的App LPS突变体的特征，并确定它们形成生物膜的能力。我们将在微滴板中使用标准的静态生物膜测定，以及更能代表肺部空气-液体界面的滴流式细胞。我们将使用噬菌体展示技术来选择对纯化LPS分子具有强亲和力的肽。然后，我们将使用表面等离子体共振来量化这些肽的亲和力，并确定这些肽是否会干扰生物膜的形成或LPS的其他生物活性。(2)利用基因组学（转座子诱变）和转录组学（DNA微阵列）方法鉴定参与App生物膜形成的其他基因。更好地了解App的表面多糖和生物膜形成过程中表达的基因，将有助于开发有效的感染控制策略，例如可以阻断重要毒力因子活性或抑制App生物膜形成的新分子，并可能抑制农场动物的其他病原体。

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.