Interaction of Actinobacillus pleuropneumoniae with host cells

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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.