A requirement of TolC1 for effective survival, colonization and pathogenicity of Actinobacillus pleuropneumoniae.

To establish infection in the host, pathogens have evolved sophisticated systems to cope with environmental conditions and to protect cells against host immunity. TolC is the outer membrane channel component of type 1 secretion systems and multidrug efflux pumps that plays critical roles during the infection process in many pathogens. However, little is known about the exact roles of TolC1 in the pathogenicity of A. pleuropneumoniae, an etiological agent of the porcine contagious pleuropneumoniae that causes severe respiratory disease. In this study, deletion of tolC1 causes apparent ultrastructural defects in A. pleuropneumoniae cell examined by transmission electron microscopy. The tolC1 mutant is hypersensitivity to oxidative, osmotic and acid challenges by in vitro stress assays. Analysis on secreted proteins shows that the excretion of ApxIIA and an ApxIVA-like protein, ApxIVA-S, is abolished in the absence of TolC1. This result confirms the essential role of TolC1 in the secretion of Apx toxins and this is the first identification of an ApxIVA-like protein in in vitro culture of A. pleuropneumoniae. Besides, disruption of TolC1 leads to a significant attenuation of virulence in mice by an intraperitoneal route of A. pleuropneumoniae. The basis for the attenuation is further investigated using a mouse intranasal infection model, which reveals an impaired ability to colonize and induce lesions in the lungs for the loss of TolC1 of A. pleuropneumoniae. In conclusion, our findings demonstrate significant roles of TolC1 in facilitating bacterial survival in hostile conditions, maximum colonization as well as pathogenicity during the infection of A. pleuropneumoniae.

T cell-specific immune response induced by bacterial ghosts.

BACKGROUND: Bacterial ghosts, genetically inactivated Gram-negative bacterial pathogens, possess significant advantages over commonly used vaccination technologies. The autolysis of the bacteria, by the expression of a cloned viral gene, results in empty bacterial envelopes through the expulsion of cytoplasmic content. Immunostimulatory properties are generally presented through the targeting of professional antigen-presenting cells (APCs), such as macrophages and dendritic cells (DCs). MATERIAL/METHODS: This study investigated the interactions between porcine antigen-presenting cells and bacterial ghosts derived from the bacterial pathogen Actinobacillus pleuropneumoniae. The maturation process of DCs and their generation of immune responses to bacterial ghosts was shown by the expression of activation markers on their surface, as well as in the functional tests. RESULTS: A population of porcine APCs was generated from PBS by incubation with rpo-GMCSF and rh-IL-4. The cells expressed SWC3, MIL-2, CD80/86 molecules, as well as a high level of MSA3 molecules. The internalization of bacterial ghosts by the cells resulted in increased expression of MSA3 molecules. The capacity of T cells to proliferate when induced by bacterial ghosts was 4 times higher in the cultures including APCs than in cultures stimulated with bacterial ghosts only. CONCLUSIONS: We found that antigen-presenting cells have the capacity to stimulate specific T cells after the internalization and processing of Actinobacillus ghosts, as demonstrated by a strong specific T-cell response generated against the ghost antigens.