| Haemophilus influenzae is a major cause of chronic infections in chronic obstructive pulmonary disease (COPD) patients. It penetrates between epithelial cells into the underlying tissues of the respiratory tract, where the bacteria persist. The bacteria survive despite antibiotic treatment. When the bacteria are located in epithelial cell layers between the cells, they are insensitive to antibody-mediated killing and antibiotics present in the extracellular fluid. Thus, this property of entering epithelial cell layers is of pathogenic significance. Recently, we have identified a gene in H. influenzae involved in the passage of the bacteria between the epithelial cells (paracytosis). When the gene is cloned into Escherichia coli, the E. coli shows paracytosis. Mutants of the gene HI638 lost the property to pass between the epithelial cells. We named the gene paracytin. The aim of this project is to purify and characterise the paracytin protein of H. influenzae, to characterise its interaction with epithelial cell layers with respect to epithelial cell layer permeability, signalling and cytokine induction, and finally to determine how conserved the protein is among clinical isolates of H. influenzae. Specific questions are: 1. Is paracytin surface exposed in H. influenzae or secreted by the bacteria? Is it expressed in vivo in patients and during interaction with epithelial cells? 2. Is the paracytin gene present in all clinical isolates of H. influenzae from COPD patients, and how conserved is its protein sequence? 3. Does the purified paracytin permeabilise epithelial cell layers? Is protein kinase C involved (like ZOT protein from Vibrio cholerae)? 4. Does paracytin induce the production of inflammatory cytokines interleukin (IL)-6 and IL-8 by epithelial cells? Are cytoskeleton rearrangements involved? The paracytin gene will be overexpressed in E. coli in a vector suitable for one-step purification of the protein by affinity chromatography. Antibodies will be raised against the purified protein to identify where the protein is present in H. influenzae (surface or excreted), to determine whether the protein is detectable in sputum samples of COPD patients with H. influenzae infections and during interaction of the bacteria with epithelial cells in culture. The presence of the paracytosis gene among clinical isolates will be analysed by polymerase chain reaction (PCR) and the expression of the gene, by whole-cell enzyme-linked immunosorbent assay (ELISA) using antibodies specific for the protein. Functional studies on the protein will be performed in two directions. A direct enzymatic effect of paracytin on epithelial cells will be analysed by testing for protease, phospholipase and glycosidase activity, and the direct effect of the protein on epithelial cell permeability in a model system using confluent cell layers of H292 epithelial cells. The second approach assumes that paracytin may activate cellular processes (especially cytokine production), resulting in increased permeability of the tight junctions, since the interaction of H. influenzae with epithelial cells results in the production of the pro-inflammatory cytokines IL-6 and IL-8. The understanding of the mechanisms involved in the paracytosis of H. influenzae offers possibilities to block this process, which is an essential step in the persistence of the bacteria. Thereby, the vicious circle of infection and inflammation can be broken, and the conditions of patients with chronic, non-specific lung diseases can be improved. |
