Interaction of Actinobacillus pleuropneumoniae with host cells

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）是造成全球养猪业经济损失的猪胸膜肺炎病原体，在我的实验室被用作呼吸道病原体的模式生物。我们发现App很容易在非生物（塑料）和生物（SJPL细胞系）表面形成生物膜，并且通过共聚焦显微镜在感染猪的肺部观察到大的生物膜样聚集体。在研究SJPL细胞的细菌-病毒共感染时，我们意外地发现App培养上清对一种重要的猪病毒PRRSV（猪繁殖与呼吸综合征病毒）具有很强的抗病毒活性。我们假设，通过使用体外感染或共感染模型更好地了解细菌病原体与其宿主之间的相互作用，可以开发新的感染控制策略。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。我们将采用ELISA和qRT-PCR方法比较App浮游细胞和生物膜细胞刺激猪肺泡巨噬细胞（PAMs）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，我们将通过质谱分析脂质A的组成，并确定诸如棕榈酸酰基链的掺入等修饰是否发生在生物膜细胞的脂质A中。(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不同生长条件下的App细胞上清液或裂解细菌细胞来提高这些分子的产量。为了鉴定活性分子，我们将改进提取方案，并获得纯度更高的最终产品，其数量与高分辨率质谱和核磁共振兼容。用PRRSV感染SJPL细胞和PAMs，并通过免疫荧光或qPCR确认上清液及其组分的抗病毒活性。一旦分子被识别出来，就有可能使编码它们的App基因失活，并测试突变菌株的抗病毒活性。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分子或靶向生物膜的分子。这些策略将有助于控制影响猪和其他农场动物的病原体，也可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.