Interaction of Actinobacillus pleuropneumoniae with host cells

本研究计划的长期目标是更好地了解革兰氏阴性细菌病原体在宿主呼吸道定植的毒力因子。猪胸膜肺炎放线杆菌（Actinobacillus pleuropneumoniae，App）是引起猪胸膜肺炎的病原菌，在世界范围内给养猪业造成了巨大的经济损失，在我的实验室被用作呼吸道病原菌的模式生物。我们发现，应用程序很容易形成生物膜上的非生物（塑料）和生物（SJPL细胞系）表面，和大的，生物膜样聚集体被感染的猪肺共聚焦显微镜观察。在研究SJPL细胞的细菌-病毒共感染时，我们意外地观察到App培养物上清对一种重要的猪病毒--猪繁殖与呼吸综合征病毒（PRRSV）具有较强的抗病毒活性。我们假设，新的感染控制策略，可以通过更好地了解细菌病原体和它的主机之间的相互作用，在体外模型的感染或共感染。本研究计划的短期目标是：（1）了解生物膜细胞如何与宿主免疫细胞相互作用。我们将使用ELISA和qRT-PCR比较App的促炎细胞和生物膜细胞刺激猪肺泡巨噬细胞（PAM）中促炎细胞因子表达的能力。由于已知脂质A修饰会改变宿主细胞的脂多糖刺激水平，因此我们将通过质谱法分析脂质A的组成，并确定生物膜细胞的脂质A中是否发生修饰，例如棕榈酸酯酰基链的掺入。(2)进一步表征App培养上清液中存在的抗病毒分子。我们将首先尝试通过取样在不同生长条件下生长的App细胞的上清液或通过裂解细菌细胞来增加这些分子的产量。为了识别活性分子，我们将改进我们的提取方案，并获得与高分辨率质谱和核磁共振兼容的高纯度最终产品。将通过用PRRSV感染SJPL细胞和PAM评价上清液及其组分的抗病毒活性，并通过免疫荧光或qPCR确认。一旦分子被鉴定，将有可能克隆编码它们的App基因，并测试突变株的抗病毒活性。更好地了解单一或混合感染中病原体与其宿主之间的复杂相互作用将导致开发更有效的感染控制策略，例如具有抗病毒活性的新型分子或靶向生物膜的分子。这些策略对于控制影响猪和其他农场动物的病原体将是有意义的，并且也可以应用于控制人类病原体。

The long-term objective of this research program is to gain a better understanding of the virulence factors involved in the colonization of the host respiratory tract by Gram-negative bacterial pathogens. Actinobacillus pleuropneumoniae (App) is the agent of porcine pleuropneumonia responsible for economic losses in the swine industry worldwide and is used in my laboratory as a model organism for respiratory tract pathogens. We showed that App readily forms biofilms on abiotic (plastic) and biotic (SJPL cell line) surfaces, and large, biofilm-like aggregates were observed in the lung of infected pigs by confocal microscopy. While investigating bacterial-viral co-infection of SJPL cells we unexpectedly observed that the App culture supernatant has strong antiviral activity against PRRSV (porcine reproductive and respiratory syndrome virus), an important swine virus. We hypothesize that new infection-control strategies can be developed through a better understanding of the interactions between a bacterial pathogen and its host using in vitro models of infection or co-infection. The short-term objectives of this research program are: (1) to understand how biofilm cells interact with the host immune cells. We will compare the ability of planktonic and biofilm cells of App to stimulate pro-inflammatory cytokine expression in porcine alveolar macrophages (PAMs) using ELISA and qRT-PCR. Since lipid A modifications are known to alter the level of lipopolysaccharide stimulation of host cells, we will analyse the composition of lipid A by mass spectrometry and determine if modifications such as the incorporation of a palmitate acyl chain occurs in lipid A of biofilm cells. (2) To further characterize the antiviral molecules present in App culture supernatants. We will first try to increase the yield of these molecules by sampling supernatants of App cells grown in different growth conditions or by lysing the bacterial cells. To identify the active molecules, we will refine our extraction protocols and obtain end products with higher purity in quantities that are compatible with high-resolution mass spectrometry and nuclear magnetic resonance. The antiviral activity of the supernatants and their fractions will be evaluated by infecting SJPL cells and PAMs with PRRSV and confirmed by immunofluorescence or qPCR. Once the molecules are identified it will be possible to inactivate the App gene(s) coding for them and test the mutant strain for antiviral activity. A better understanding of the complex interactions between a pathogen, in single or mixed infections, and its host will lead to the development of more effective infection-control strategies such as novel molecules with antiviral activity or molecules targeting biofilms. These strategies will be of interest for the control of pathogens affecting swine and other farm animals, and could also be applied to the control of human pathogens.